Part 9. Disorders of Nucleic Acids, Heme Metabolism, and Metals Transport

The porphyrias are a group of inherited and acquired metabolic disorders, each resulting from the deficient activity of a specific enzyme in the heme biosynthetic pathway.1,2 These enzyme deficiencies are inherited as autosomal dominant or recessive traits, with the exception of porphyria cutanea tarda (PCT), which usually is sporadic. These disorders are classified as either hepatic or erythropoietic, depending on the primary site of overproduction and accumulation of the porphyrin precursor(s) or porphyrin(s) (Table 167-1). Although some have overlapping features, manifestations of the hepatic porphyrias are neurological, including abdominal pain, neuropathy, and mental disturbances, whereas the erythropoietic porphyrias characteristically cause cutaneous photosensitivity. The neurological involvement in the hepatic porphyrias, which typically presents after puberty, results from the hepatic production of a neurotoxic metabolite, as liver transplantation ameliorated the frequent attacks in a patient with acute intermittent porphyria (AIP).3 Cutaneous sensitivity to sunlight may occur in infancy because of the excitation of excess porphyrins in the skin by long-wave ultraviolet light, which leads to cell damage, scarring, and deformation. Steroid hormones, drugs, and nutrition influence the production of porphyrin precursors and porphyrins, thereby precipitating or increasing the severity of some porphyrias.

Rare homozygous variants of the autosomal dominant hepatic porphyrias have been identified and usually manifest clinically before puberty. The symptoms in these patients are usually more severe and occur earlier than those of patients with the respective autosomal dominant porphyria (see below).1,4 Thus, the porphyrias are actually ecogenic disorders in which environmental, physiological, and genetic factors interact to cause disease.

Many symptoms of the porphyrias are nonspecific, and diagnosis is often delayed. Laboratory testing can confirm or exclude the diagnosis of a porphyria. Table 167-1 summarizes the major metabolites that accumulate in each porphyria. Urinary 5′-aminolevulinic acid (ALA) and porphobilinogen (PBG) are easily quantitated by chemical methods, and the urinary porphyrin isomers can be separated and quantitated by high-performance liquid chromatography. However, a definite diagnosis requires demonstration of the specific enzyme or gene defect. The isolation and characterization of the genes encoding all the heme biosynthetic enzymes have permitted the identification of the molecular lesions that cause each porphyria.1,2,5 Such molecular analyses make it possible to provide precise heterozygote or homozygote identification and prenatal diagnosis in families with known mutations.

The American Porphyria Foundation (www.porphyriafoundation.com) and the European Porphyria Initiative (www.porphyria-europe.org) sponsor informative and up-to-date Web sites. An extensive list of unsafe and safe drugs for individuals with porphyria is given at the Drug Database for Acute Porphyrias (www.drugs-porphyria.com).

Heme Biosynthesis

Heme biosynthesis involves eight enzymatic steps in the conversion of glycine and succinyl coenzyme A (CoA) to heme (Fig. 167-1 and Table 167-2). The first and last three enzymes in the heme biosynthetic pathway are located in the mitochondrion, whereas the other four are in the cytosol (see Fig. 167-1). The first enzyme, 5′-aminolevulinate synthase (ALA synthase), catalyzes the condensation of glycine, activated by pyridoxal phosphate and succinyl coenzyme A, to form ALA. This rate-limiting enzyme can be induced in the liver by a variety of drugs, steroids, and other chemicals. Distinct erythroid-specific and housekeeping forms of ALA synthase are encoded by separate genes located on chromosome 3p21.1 (ALAS1) and Xp11.2 (ALAS2). Defects in the X-linked erythroid gene ALAS2 cause X-linked sideroblastic anemia (see Chapter 168).

The second enzyme, 5’-aminolevulinate dehydratase (ALA-dehydratase), catalyzes the condensation of two molecules of ALA to form PBG. Four molecules of PBG condense to form the tetrapyrrole uroporphyrinogen III by a two-step process catalyzed by the third enzyme in the pathway, hydroxymethylbilane (HMB) synthase (also known as PBG-deaminase or uroporphyrinogen III synthase). HMB synthase catalyzes the head-to-tail condensation of four PBG molecules by a series of deaminations to form the linear tetrapyrrole (HMB) hydroxymethylbilane. URO synthase catalyzes the rearrangement and rapid cyclization of HMB to form the asymmetric, physiological, octacarboxylate porphyrinogen uroporphyrinogen III isomer.

The fifth enzyme in the pathway, uroporphyrinogen decarboxylase (URO-decarboxylase), catalyzes the sequential removal of the four carboxyl groups from the acetic acid side chains of uroporphyrinogen III to form coproporphyrinogen III, a tetracarboxylate porphyrinogen. This compound then enters the mitochondrion via a membrane transporter,6 where coproporphyrinogen (COPRO) oxidase, the sixth enzyme, catalyzes the decarboxylation of two of the four propionic acid groups; this then forms the two vinyl groups of protoporphyrinogen IX, a dicarboxylate porphyrinogen. Next, protoporphyrinogen (PROTO) oxidase oxidizes protoporphyrinogen IX to protoporphyrin IX by removing six hydrogen atoms. The product of the reaction is a porphyrin (oxidized form), in contrast to the preceding tetrapyrrole intermediates, which are porphyrinogens (reduced forms). Finally, ferrous iron is inserted into protoporphyrin IX to form heme, a reaction catalyzed by the eighth enzyme in the pathway, ferrochelatase (FECH, also known as heme synthetase or protoheme ferrolyase).

Regulation of Heme Biosynthesis

Regulation of heme synthesis differs in the two major heme-forming tissues, the liver and erythron. About 85% of the heme produced in the body is synthesized in erythroid cells to provide heme for hemoglobin; most of the remainder is produced in hepatocytes, primarily for synthesizing cytochromes. In the liver, the biosynthetic pathway is under negative feedback control. “Free” heme in the liver regulates the synthesis and mitochondrial translocation of the housekeeping form of ALA synthase (ALAS1). Heme represses the synthesis of the ALAS1 mRNA and interferes with the transport of the enzyme from the cytosol into mitochondria. Hepatic ALAS1 is inducible by many of the same chemicals that induce the cytochrome P450 enzymes in the liver’s endoplasmic reticulum. Because most of the heme in the liver is used for synthesizing cytochrome P450 enzymes, hepatic ALA synthase and the cytochrome P450 enzymes are regulated in a coordinated fashion.

Different regulatory mechanisms control production of heme for hemoglobin. The erythroid-specific ALA synthase (ALAS2) gene encoded on the X-chromosome is expressed at higher levels than the hepatic enzyme, and an erythroid-specific control mechanism regulates iron transport into erythroid cells. During erythroid differentiation, the activities of the heme biosynthetic enzymes are increased.

The Hepatic Porphyrias

The acute hepatic porphyrias are characterized by the rapid onset of neurological manifestations. During the acute attack, individuals have markedly elevated plasma and urinary concentrations of the porphyrin precursors ALA and PBG, which originate in the liver.

ADP is a rare autosomal recessive trait that has been described in only a few patients.7,8 Onset and severity of the disease are variable, presumably depending on the amount of residual ALA dehydratase activity. All ADP patients had significantly elevated levels of plasma and urinary ALA and markedly decreased ALA dehydratase activity. Treatment and prevention of neurological complications are the same as for other acute porphyrias.

Clinical Features

The first reported cases were in two unrelated German men who had clinical onset during adolescence of abdominal pain and neuropathy, resembling AIP. A Swedish infant presented with failure to thrive and required transfusions and parenteral nutrition. Presumably, the earlier age of onset and more severe manifestations reflect a more severe enzyme deficiency. At age 63, a Belgian man developed an acute motor polyneuropathy that was associated with a myeloproliferative disorder. This patient was heterozygous for an ALA dehydratase mutation that presumably was present in erythroblasts, which underwent clonal expansion due to the bone marrow malignancy.

Diagnosis

Patients have increased urinary levels of ALA and coproporphyrin. ALA dehydratase activity in erythrocytes is less than 5% of normal. Lead or succinylacetone (which accumulates in hereditary tyrosinemia and is structurally similar to ALA) can inhibit ALA dehydratase and increase urinary excretion. This may cause manifestations that resemble those of the acute porphyrias, lead intoxication (see Chapter 120) and hereditary tyrosinemia (fumarylacetoacetase deficiency; see Chapter 155). These conditions should therefore be considered in the differential diagnosis of ALA dehydratase–deficient porphyria. Immunologic studies in the reported cases demonstrated the presence of nonfunctional enzyme proteins that cross-reacted with anti-ALA–dehydratase antibodies. DNA analysis revealed different mutations (see the Human Gene Mutation Database, www.hgmd.org).

Heterozygotes are clinically asymptomatic and do not excrete increased levels of ALA, but they can be detected by demonstrating intermediate levels of erythrocyte ALA dehydratase activity or by demonstrating a specific mutation in the ALA dehydratase gene. Prenatal diagnosis of this disorder is possible by determining the ALA dehydratase activity or the family’s specific gene mutation(s) in cultured chorionic villi or amniocytes.

Treatment

Treatment is similar to that of AIP.

This hepatic porphyria is an autosomal dominant condition resulting from the half-normal level of HMB synthase (also termed PBG deaminase) activity.1,9 The disease is widespread but is especially common in Scandinavia and Great Britain. The enzyme deficiency can be demonstrated in most heterozygous individuals, but clinical expression is highly variable. Activation of the disease is related to ecogenic factors, such as drugs, diet, and steroid hormones, which can precipitate the manifestations. Attacks can be prevented by avoiding known precipitating factors. Rare cases of homozygous AIP have been reported in children.10

Clinical Features

Most heterozygotes remain clinically asymptomatic (latent) unless exposed to factors that increase porphyrin production. Endogenous and exogenous gonadal steroids, porphyrinogenic drugs, alcohol ingestion, and low-calorie diets (usually instituted for weight loss) are common precipitating factors. Table 167-3 lists the major drugs that are harmful in AIP (and in hereditary coproporphyria [HCP] and variegate porphyria [VP]) and some drugs and anesthetic agents known to be safe. More extensive lists of drugs considered harmful or safe are available at the Drug Database for Acute Porphyrias (www.drugs-porphyria.com) and at www.porphyriafoundation.com and www.porphyria-europe.com, but information is incomplete for many drugs. Attacks also can be provoked by infections and by surgery.

Because the neurovisceral symptoms rarely occur before puberty and are often nonspecific, a high index of suspicion is required to make the diagnosis. The disease can be disabling but is rarely fatal. Abdominal pain, the most common symptom, is usually steady and poorly localized but may be cramping. Ileus, abdominal distention, and decreased bowel sounds are common. However, increased bowel sounds and diarrhea may occur. Abdominal tenderness, fever, and leukocytosis are usually absent or mild, because the symptoms are neurological rather than inflammatory. Nausea; vomiting; constipation; tachycardia; hypertension; mental symptoms; muscle weakness; sensory loss; dysuria; urinary retention; and pain in the limbs, head, neck, or chest are characteristic. Tachycardia, hypertension, restlessness, tremors, and excess sweating are due to sympathetic overactivity.

The peripheral neuropathy is the result of axonal degeneration (rather than demyelination) and affects primarily motor neurons. Significant neuropathy does not occur with all acute attacks; abdominal symptoms are usually more prominent. Motor neuropathy initially affects the proximal muscles, more often in the shoulders and arms. The course and degree of involvement are variable. Deep-tendon reflexes may be normal or hyperactive but are usually decreased or absent with advanced neuropathy. Motor weakness can be asymmetric and focal and may involve cranial nerves. Sensory changes such as paresthesias and loss of sensation are less prominent. Progressive muscle weakness can lead to respiratory and bulbar paralysis and death when diagnosis and treatment are delayed. Sudden death may result from sympathetic overactivity and cardiac arrhythmia.

Mental symptoms, such as anxiety, insomnia, depression, disorientation, hallucinations, and paranoia, can occur in acute attacks. Seizures can be caused by neurological effects or by hyponatremia. Treatment of seizures is difficult, because virtually all antiseizure drugs (except bromides) may exacerbate AIP (clonazepam may be safer than phenytoin or barbiturates). Hyponatremia results from hypothalamic involvement and inappropriate secretion of antidiuretic hormone, or from electrolyte depletion due to vomiting, diarrhea, poor intake, or excess renal sodium loss. Persistent hypertension and impaired renal function may occur. When an attack resolves, abdominal pain may disappear within hours, and paresis begins to improve within days and may continue to improve over several years.

Diagnosis

ALA and PBG levels are increased in plasma and urine during acute attacks.9 Although the diagnosis of an acute attack is based on clinical findings and not the absolute level of these porphyrin precursors, the increase is expected to be substantial. PBG excretion is usually 50 to 200 mg/24 hr (220–880 mmol/24 hr; normal, 0–4 mg/24 hr [0–18 mmol/24 hr]), and urinary ALA excretion is 20 to 100 mg/24 hr (150–760 mmol/24 hr; normal, 1–7 mg/24 hr [8–53 mmol/24 hr]). The excretion of these compounds generally decreases with clinical improvement, particularly after hematin infusions. A normal urinary PBG level effectively excludes AIP as a cause for current symptoms. Fecal porphyrins are usually normal or minimally increased in AIP, in contrast to HCP and VP. Most asymptomatic (“latent”) heterozygotes with HMB synthase deficiency have normal urinary excretion of ALA and PBG. Therefore, measuring HMB synthase in erythrocytes may be useful to confirm the diagnosis and to screen asymptomatic family members.

The enzyme deficiency is detectable in erythrocytes from most AIP heterozygotes (classic AIP). Note that the activity is higher in young erythrocytes and may increase into the normal range in AIP when erythropoiesis is increased due to a concurrent condition. However, patients with the rare erythroid form of AIP (erythroid or variant AIP) have normal enzyme levels in erythrocytes and deficient activity in nonerythroid tissues. The erythroid and housekeeping forms of HMB synthase are encoded by a single gene that has two promoters. One promotes transcription of a messenger RNA for the housekeeping form found in all tissues, and the other promotes formation of the erythroid-specific transcript found only in erythroid cells. Over 250 mutations causing AIP have been identified (see the Human Gene Mutation Database, www.hgmd.org). Mutations that cause erythroid AIP variants with half-normal enzyme in nonerythroid tissues but normal activity in erythrocytes include point mutations in the initiation methionine codon (which prevent translation) or in the 5'-donor splice site of intron 1 (which causes abnormal splicing of the HMB synthase transcript).

Identifying the gene in an index case enables detection of latent family members and prenatal diagnosis of an at-risk fetus using cultured amniotic cells or chorionic villi. However, this is seldom done, because the prognosis of individuals with HMB synthase mutations is generally favorable.

Treatment

During acute attacks, narcotic analgesics are usually required for abdominal pain, and phenothiazines are useful for nausea, vomiting, anxiety, and restlessness.9 Chloral hydrate can be given for insomnia, and benzodiazepines in low doses are probably safe if a minor tranquilizer is required. Carbohydrate loading, usually with intravenous glucose (at least 300 g/dL), may be effective in milder acute attacks of porphyria (without paresis, hyponatremia, etc). Because intravenous hemin is more effective and the response slower if treatment is delayed, it is no longer recommended that hemin therapy for a severe attack be started only after an unsuccessful trial of intravenous glucose for several days. Hemin should be used initially for severe attacks and for mild attacks that do not respond to carbohydrate loading within 1 to 2 days. The standard regimen is 3 to 4 mg of heme in the form of lyophilized hematin (Ovation Pharmaceuticals), heme albumin (hematin reconstituted with human albumin) or heme arginate (Orphan Europe), infused daily for 4 days.9,11 Heme arginate and heme albumin are chemically stable and are less likely than hematin to produce phlebitis or an anticoagulant effect. The rate of recovery from an acute attack depends on the degree of neuronal damage and may be rapid (1 to 2 days) with prompt therapy. Recovery from severe motor neuropathy may continue for months or years. Identifying and avoiding inciting factors can hasten recovery from an attack and prevent future attacks. Multiple inciting factors may contribute to a symptomatic episode. Frequent, clear-cut cyclical attacks occur in some women and can be prevented with a gonadotropin-releasing hormone analogue (which prevents ovulation and progesterone production).

An allogeneic liver transplant was performed on a 19-year-old female AIP heterozygote who had 37 acute attacks in the 29 months prior to transplantation.3 Post-transplantation, her elevated urinary ALA and PBG levels returned to normal in 24 hours, and she did not experience acute neurological attacks for more than 18 months. Liver transplantation is a high-risk procedure and should not be considered as an established treatment for acute porphyrias.

Homozygous Dominant Aip

Homozygous dominant AIP is a rare form of porphyria presenting in infancy. Patients inherit HMB synthase mutations from each of their heterozygous parents; therefore, they have very low (< 2%) enzyme activity.10 In these homozygous-affected patients, disease manifestations included failure to thrive, developmental delay, bilateral cataracts, or hepatosplenomegaly. Acute attacks did not occur. Urinary ALA and PBG were markedly elevated. Studies of brain MRIs of children with homozygous AIP have suggested damage primarily in white matter that was myelinated postnatally, while tracks that myelinated prenatally were normal.10 These findings suggest that a neurotoxic endogenous product, such as ALA or PBG, present in large amounts postnatally (rather than heme deficiency) caused nervous tissue damage. Prenatally, excess amounts of ALA and PBG cross the placenta and are excreted in the mother’s urine. Most children with homozygous AIP die at an early age.

Porphyria cutanea tarda (PCT), the most common of the porphyrias, can be sporadic (type I) or familial (types II and III) and can develop after exposure to halogenated aromatic hydrocarbons. Hepatic URO decarboxylase is deficient in all types of PCT and for clinical manifestations must be substantial (< 20% of normal). Generation of a URO decarboxylase inhibitor, specifically in the liver in the presence of iron and under conditions of oxidative stress, presumably causes this decrease.12 In type I PCT, URO decarboxylase activity is normal in erythrocytes. In type II PCT, an autosomal dominant trait, the enzyme is systemically deficient. In type III PCT, which clusters in families, deficiency of the enzyme activity is limited to the liver. Deficient hepatic URO decarboxylase and a porphyrin pattern resembling PCT can be produced by exposure of normal individuals to a number of halogenated aromatic hydrocarbons. Hepatoerythropoietic porphyria (HEP) is an autosomal recessive form of porphyria, as affected individuals inherited an URO decarboxylase mutation from each parent and have severe systematic deficiency of URO decarboxylase activity. HEP usually presents in childhood.

Clinical Features

Cutaneous photosensitivity is the major clinical feature. Neurological manifestations are not observed. Fluid-filled vesicles and bullae develop on sun-exposed areas such as the face, the dorsa of the hands and feet, the forearms, and the legs. The skin in these areas is friable, and minor trauma may lead to the formation of bullae. The appearance of small white plaques, termed milia, may precede or follow vesicle formation. Bullae and denuded skin heal slowly and are subject to infection. Other features include hypertrichosis and hyperpigmentation, especially of the face, and thickening, scarring, and calcification resembling the cutaneous changes of systemic sclerosis.

Several factors contribute to the development of hepatic URO decarboxylase deficiency, including excess alcohol, iron, and estrogens. Recently, the importance of excess hepatic iron as a precipitating factor was documented by finding an increased prevalence of the common hemochromatosis-causing mutations, HFE C282Y and H63D, in types I and II PCT patients.13 PCT is strongly associated with hepatitis C in southern Europe and the United States. PCT also can be induced by various chemicals; an epidemic of PCT occurred in eastern Turkey in the 1950s, from the consumption of wheat contaminated with the fungicide hexachlorobenzene. This chemical produces a disorder similar to PCT and induces hepatic URO decarboxylase deficiency in animals. PCT in humans has occurred after exposure to other chemicals, including di- and trichlorophenols and 2,3,7,8-tetrachlorodibenzo-(p)-dioxin (TCDD, dioxin). Patients with PCT characteristically have liver damage and are at risk for hepatocellular carcinoma (see Chapter 458). These carcinomas do not produce porphyrins.

Hepatoerythropoietic porphyria (HEP) resembles congenital erythropoietic porphyria (CEP) and usually presents with blistering skin lesions, hypertrichosis, scarring, and red urine in infancy or childhood.

Diagnosis

Porphyrin levels are increased in the liver, plasma, urine, and stool. The urinary ALA level may be slightly increased, but the PBG level is normal. Urinary porphyrins consist mostly of uroporphyrin and 7-carboxylate porphyrin, with lesser amounts of coproporphyrin and 5- and 6-carboxylate porphyrins. Plasma porphyrins also are increased in a pattern that resembles that in urine. Isocoproporphyrins are increased in feces and sometimes in plasma and urine. The finding of increased isocoproporphyrins is diagnostic for a deficiency of hepatic URO decarboxylase.

Type II PCT and HEP can be diagnosed by finding decreased URO decarboxylase activity in erythrocytes or by indentifying mutations in the URO decarboxylase gene. URO decarboxylase activity in liver, erythrocytes, and cultured skin fibroblasts in type II PCT is approximately 50% of normal in affected individuals and in family members with latent disease. In HEP, the URO decarboxylase activity is markedly deficient, with typical levels of 3% to 10% of normal. Over 70 mutations have been identified in the URO decarboxylase gene from unrelated type II PCT and HEP patients (see the Human Gene Mutation Database, www.hgmd.org).

Treatment

Alcohol, estrogens, iron supplements, and, if possible, any drugs that may exacerbate the disease should be discontinued, but this step does not always lead to improvement. A complete response can almost always be achieved by repeated phlebotomy to reduce hepatic iron. A unit (450 mL) of blood can be removed every 1 to 2 weeks. Because iron overload is not marked in most cases, remission may occur after only five or six phlebotomies. PCT patients with hemochromatosis may require many more phlebotomies. Hemoglobin levels or hematocrits and serum ferritin should be followed closely to prevent development of iron deficiency and anemia. After remission, continued phlebotomy may not be needed, even if ferritin levels return to normal. Relapses are treated again by phlebotomy.

PCT also can be treated with chloroquine or hydroxychloroquine, both of which complex with the excess porphyrins and promote their excretion. Small doses (eg, 125 mg chloroquine phosphate twice weekly) should be given, because standard doses can induce transient, sometimes marked, increases in photosensitivity and hepatocellular damage. Hepatic imaging can diagnose or exclude complicating hepatocellular carcinoma. Treating PCT in patients with end-stage renal disease is facilitated by administering erythropoietin.

Hepatoerythropoietic Porphyria (Hep)

Hepatoerythropoietic porphyria (HEP), which is the homozygous form of familial (type 2) PCT, resembles CEP clinically.4 Excess porphyrins originate mostly from the liver, with a pattern consistent with severe URO decarboxylase deficiency. There also is a substantial increase in erythrocyte zinc protoporphyrin in HEP, as in homozygous dominant forms of the acute porphyrias, ADP, and some cases of CEP.

HEP usually presents with blistering skin lesions, hypertrichosis, scarring, and red urine in infancy or childhood. Sclerodermoid skin changes are sometimes prominent. Unusually mild cases have been described. Concurrent conditions that affect liver function may alter disease severity. For example, hepatitis A caused the disease to manifest in a 2-year-old child and then improved with recovery from this viral infection.

HEP is readily distinguished from CEP by increases in both uroporphyrin and heptacarboxyl porphyrin in urine and in isocoproporphyrins in stool. In most cases of CEP, the excess erythrocyte porphyrins are predominantly URO I and COPRO I rather than zinc protoporphyrin. EPP is readily distinguished by its nonblistering photosensitivity and normal urine porphyrins and by demonstration that the excess erythrocyte protoporphyrin is free and not complexed with zinc. As in CEP, avoiding sunlight is most important in managing this disease. The outlook depends on the severity of the enzyme deficiency and may be favorable if sunlight can be avoided. Phlebotomy has shown little or no benefit.

Hereditary coproporphyria (HCP) is an autosomal dominant hepatic porphyria that results from half-normal levels of COPRO oxidase activity. Photosensitivity may occur. Cases of homozygous dominant HCP have been reported.

Clinical Features

HCP is influenced by the same factors that cause attacks in AIP. The disease is latent before puberty, and symptoms are more common in women. Neurovisceral symptoms and other manifestations are virtually identical to those of AIP. Photosensitivity may resemble that in PCT and VP. Cutaneous lesions may begin in childhood in the rare homozygous dominant cases.

Homozygous Dominant Hcp and Harderoporphyria

Individuals with mutations in both their COPRO oxidase alleles and markedly decreased COPRO oxidase activities have been described; these mutations cause homozygous dominant HCP or a rare variant form called harderoporphyria.4 Homozygous HCP presents early in childhood with symptoms of growth retardation, hypertrichosis, and skin hyperpigmentation.1,4 Later, these patients can have acute porphyric attacks.

Individuals with harderoporphyria, which is a biochemical and clinical variant form of HCP in which hemolysis and erythropoietic features are prominent, usually present in early childhood with jaundice, hemolytic anemia, hepatosplenomegaly, and skin photosensitivity.1,4 However, the symptoms may be variable; acute attacks do not occur.

Diagnosis

Coproporphyrin concentrations are markedly increased in the urine and feces of HCP patients when the disease is symptomatic and sometimes when there are no symptoms. Urinary ALA and PBG levels are increased during acute attacks but may return to normal when symptoms resolve. Although the diagnosis can be confirmed by measuring COPRO oxidase activity, these assays are not widely available and require cells other than erythrocytes. COPRO oxidase gene mutations are diagnostic, and over 25 have been reported (see the Human Gene Mutation Database, www.hgmd.org). Patients with homozygous HCP have markedly increased fecal coproporphyrin III due to markedly deficient (<10%) COPRO oxidase activity. In patients with harderoporphyria, urinary coprotoporphyrin III, fecal porphyrins (66–90% harderoporphyrin), and zinc protoporphyrin are increased. All harderoporphyria patients reported to date are either homoallelic or heteroallelic for the K404E missense mutation.

Treatment

Neurological symptoms are treated as in AIP. Phlebotomy and chloroquine are ineffective when cutaneous lesions are present.

Variegate porphyria (VP), a hepatic porphyria that results from the deficient activity of PROTO oxidase, is inherited as an autosomal dominant trait and can present with neurological symptoms, photosensitivity, or both. Homozygous VP is rare and presents in early childhood.1,4

Clinical Features

Neurovisceral signs and symptoms develop after puberty and are similar to those of AIP or HCP. Attacks are provoked by the same drugs, steroids, and nutritional factors that are detrimental in AIP. Skin manifestations are more common than in HCP but usually occur apart from the neurovisceral symptoms. Because the skin lesions in VP, HCP, and PCT are not distinguishable by clinical examination or biopsy, these conditions must be diagnosed by assay of porphyrins and porphyrin precursors in blood, urine, and feces or by demonstration of PROTO oxidase gene mutations.

VP is particularly common in South Africa, where 3 in 1000 whites have the disorder. Most are descendants of a couple who emigrated from Holland to South Africa in 1688. Homozygous-dominant VP is associated with photosensitivity, neurological symptoms, and developmental disturbances, including growth retardation in infancy or childhood; all cases had increased erythrocyte levels of zinc protoporphyrin, a characteristic finding in all homozygous porphyrias so far described.

Homozygous-Dominant VP

Affected individuals with homozygous-dominant VP have mutations affecting both PROTO oxidase alleles, resulting in very low enzyme activity levels.1,4,14 These patients generally develop cutaneous symptoms, including photosensitivity and hypertrichosis, before the age of 2 years. Scarring and deformities of the face and digits may be prominent. Most patients do not have acute attacks. Neurological symptoms in some patients include mental retardation, convulsions, growth retardation, and nystagmus. Laboratory findings include elevated erythrocyte zinc protoporphyrin levels, as in other homozygous-dominant porphyrias. Mutations have been identified in most homozygous VP patients. Expression studies have indicated that these mutations have residual activity.

Diagnosis

Urinary ALA and PBG levels are increased during acute attacks but may return to normal more quickly than in AIP. Increases in fecal protoporphyrin and coproporphyrin III and in urinary coproporphyrin III are more persistent. Plasma levels of porphyrins are increased, particularly when there are cutaneous lesions. VP can be distinguished rapidly from all other porphyrias by examining the fluorescence emission spectrum of porphyrins in plasma at neutral pH. This test is particularly useful for differentiating VP from PCT.

Assays of PROTO oxidase activity in cultured fibroblasts or lymphocytes are not widely available. Some latent cases of VP can be diagnosed by measuring fecal porphyrins in relatives of VP patients. The diagnosis can be confirmed by demonstrating a PROTO oxidase gene mutation.

Treatment

As in AIP, acute attacks are treated with hematin. Other than avoiding sun exposure, there are few effective measures for treating the skin lesions. Beta-carotene, phlebotomy, and chloroquine are not helpful.

The Erythropoietic Porphyrias

In the erythropoietic porphyrias, porphyrins from bone marrow erythrocytes and plasma are deposited in the skin and lead to cutaneous photosensitivity.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder, also known as Gunther disease, that results from the markedly deficient activity of URO synthase and is associated with hemolytic anemia and severe cutaneous photosensitivity.1,15 CEP is characterized by accumulation of uroporphyrin I and coproporphyrin I isomers.

Clinical Features

Severe cutaneous photosensitivity begins in early infancy. The disease may be recognized in utero as a cause of nonimmune hydrops fetalis. The skin over sun-exposed areas is friable, and bullae and vesicles are prone to rupture and infection. Skin thickening, focal hypo- and hyperpigmentation, and hypertrichosis of the face and extremities are characteristic. Secondary infection of the cutaneous lesions can disfigure the face and hands. Porphyrins are deposited in teeth and bones. Consequently, the teeth are reddish brown and fluoresce on exposure to long-wave ultraviolet light. Hemolysis is probably due to the marked increase in erythrocyte porphyrins and leads to splenomegaly. Adults with a milder form of the disease have been described.

Diagnosis

Uroporphyrin and coproporphyrin (mostly type I isomers) accumulate in the bone marrow, erythrocytes, plasma, urine, and feces. The diagnosis should be confirmed by demonstration of markedly deficient URO synthase activity and/or URO synthase gene mutations.15,16 The disease can be detected in utero by measuring porphyrins in amniotic fluid and URO synthase activity in cultured amniotic cells or chorionic villi. Over 35 URO synthase mutations have been reported (see the Human Gene Mutation Database, www.hgmd.org).

Treatment

The transfusion of sufficient blood to suppress erythropoiesis is effective but results in iron overload. Splenectomy may reduce hemolysis and decrease transfusion requirements. Protection from sunlight is essential, and minor skin trauma should be avoided. Beta-carotene may be of some value. Complicating bacterial infections should be treated promptly. Recently, bone marrow or cord-blood transplantation has proven effective in several transfusion-dependent children, providing the rationale for stem-cell gene therapy.

Erythropoietic protoporphyria (EPP) is an inherited disorder resulting from the markedly deficient activity of FECH activity, the last enzyme in the heme biosynthetic pathway. EPP is the most common erythropoietic porphyria and is the most common porphyria in children and the second most common in adults. EPP patients have FECH activities as low as 15% to 25% in lymphocytes and cultured fibroblasts. In most patients, a disabling (ie, causative) mutation in one FECH allele is combined with a relatively common intron 3 (IVS3) single nucleotide alteration (IVS3-48T ↔ C) in the other allele, which results in decreased amounts of the normal enzyme. The C allele results in expression of an aberrantly spliced mRNA that is degraded by a nonsense-mediated decay mechanism, thus decreasing the steady-state level of normal FECH mRNA transcribed from the C allele. The low expression allele is found in approximately 10% of the normal Caucasian population. In several studies, over 90% of symptomatic EPP patients had a causative FECH mutation and the C allele, while no asymptomatic relatives carrying only one causative mutation did. In a few families, two FECH causative mutations and a pattern of autosomal recessive inheritance has been found.

Clinical Features

Skin photosensitivity usually begins in childhood. The skin manifestations differ from those of other porphyrias. Vesicular lesions are uncommon. Redness, swelling, burning, and itching can develop within minutes of sun exposure and can resemble angioedema. Symptoms may seem out of proportion to the visible skin lesions. Sparse vesicles and bullae occur in 10% of cases. Chronic skin changes may include lichenification, leathery pseudovesicles, labial grooving, and nail changes. Severe scarring is rare, as are pigment changes, friability, and hirsutism.

The primary source of excess protoporphyrin is the bone marrow reticulocyte. Erythrocyte protoporphyrin is free (not complexed with zinc) and is mostly bound to hemoglobin. In plasma, protoporphyrin is bound to albumin. Hemolysis and anemia are usually absent or mild.

Liver function is usually normal, but up to 20% of EPP patients may have minor abnormalities of liver function, and in some patients (~5%), accumulation of protoporphyrin causes chronic liver disease that can progress to liver failure and death. The hepatic complications are often preceded by increasing levels of erythrocyte and plasma protoporphyrin and probably result, in part, from protoporphyrin accumulation in the liver. Protoporphyrin is insoluble; it forms crystalline structures in liver cells and can decrease hepatic bile flow. Some patients have gallstones that are at least partially composed of protoporphyrin.

Diagnosis

A substantial increase in erythrocyte protoporphyrin, which is predominantly free and not complexed with zinc, is the hallmark of this disease. Protoporphyrin levels are also variably increased in bone marrow, plasma, bile, and feces. Plasma and fecal porphyrins are less increased than in most other cutaneous porphyrias and are sometimes normal. Therefore, measuring erythrocyte protoporphyrin is important for diagnosis. Since erythrocyte protoporphyrin concentrations are increased in other conditions, it is important to confirm the diagnosis by an assay that distinguishes free and zinc-complexed protoporphyrin. Erythrocytes in EPP exhibit red fluorescence when examined by fluorescence microscopy at 620 nm. Urinary levels of porphyrins and porphyrin precursors are normal. FECH activity in cultured lymphocytes or fibroblasts is decreased, but such assays are not widely available. FECH mutation analysis is recommended to detect the causative mutation and, in most affected families, the presence of the IVS3-48T↔C alteration in the other FECH allele. To date, over 90 mutations have been identified in the FECH gene (see the Human Gene Mutation Database; www.hgmd.org).

Treatment

Avoiding sunlight exposure and wearing clothing designed to provide protection for individuals with chronic photosensitivity are essential. Oral beta-carotene improves tolerance to sunlight in many patients. The dosage may need to be adjusted to maintain serum carotene levels in the recommended range of 10 to 15 μmol/L (600–800 μg/dL). Mild skin discoloration due to carotenemia is the only significant side effect. The beneficial effects of beta-carotene may involve the quenching of singlet oxygen or free radicals. Unfortunately, this drug is less effective in other forms of porphyria associated with photosensitivity.

Treating hepatic complications is difficult. However, cholestyramine and other porphyrin absorbents, such as activated charcoal, may interrupt the enterohepatic circulation of protoporphyrin and promote its fecal excretion, leading to some improvement. Splenectomy may be helpful when the disease is accompanied by hemolysis and significant splenomegaly or secondary hypersplenism. Caloric restriction and drugs or hormones that may induce the heme pathway or impair hepatic excretory function should be avoided. Iron deficiency should be prevented or treated. Transfusions or intravenous heme therapy may suppress erythroid and hepatic protoporphyrin production and are sometimes beneficial. Liver transplantation has been performed in some patients with severe liver complications. However, liver disease often eventually recurs in the transplanted liver because of continued bone marrow production of excess protoporphyrin. Post-transplantation treatment with hemin and plasmapheresis may help prevent recurrence of this complication. Bone marrow transplantation, which has been successful in human EPP and has prevented liver disease in a mouse model, should be considered after the liver transplantation if a suitable donor can be found.

This work was supported in part by grants from the National Institutes of Health, including a research grant (5 R01 DK026824); the General Clinical Research Center Programs at the Mount Sinai School of Medicine (5 M01 RR00071); and the National Center for Research Resources.

Metabolic Pathways

Purines comprise bases, nucleosides in association with ribose or deoxyribose, and nucleotides with one or more added phosphate groups. Purine nucleotides are essential cellular constituents. They are the building blocks of the polynucleotides, DNA and RNA, and, under the form of mononucleotides or of nucleosides, also intervene in numerous cellular functions. Among these are energy transfer (eg, by adenosine triphosphate [ATP]), metabolic regulation (eg, by guanosine triphosphate [GTP]), and signaling (eg, by adenosine). Purine metabolism can be divided into three pathways (Fig. 168-1):

1. 1. The biosynthetic pathway, starts with the formation, often termed de novo, of the high-energy compound phosphoribosyl pyrophosphate (PRPP) and leads in 10 steps to the synthesis of the nucleoside monophosphate inosine monophosphate (IMP). From IMP, two reactions lead to the formation of adenosine monophosphate (AMP). Subsequently, the nucleoside di- and triphosphates ADP and ATP and their deoxy counterparts are synthesized. Two other reactions convert IMP into GMP, from which GDP, GTP, and their deoxy counterparts are formed.

   2. The purine catabolic pathway starts from the nucleoside monophosphates GMP, IMP, and AMP and produces uric acid, a poorly soluble compound that tends to crystallize once its plasma concentration increases above 6.5 to 7.0 mg/dl (0.38–0.47 mmol/l).

   3. The purine salvage pathway utilizes the purine bases guanine, hypoxanthine, and adenine, which are provided by food intake or the catabolic pathway, and reconverts them by phosphoribosylation into GMP, IMP, and AMP, respectively. It also utilizes the purine nucleosides adenosine, guanosine, and their deoxy counterparts by phosphorylation into the corresponding monophosphates, catalyzed by kinases.

Inborn errors of purine metabolism comprise errors of purine nucleotide synthesis, of purine catabolism, and of purine salvage. They should be considered in patients with hyper- or hypouricemia, kidney stones, and a variety of muscle, neurological, and other symptoms (Table 168-1). The deficiencies of adenosine deaminase and purine nucleoside phosphorylase, two purine catabolic enzymes, cause severe combined immunodeficiency (SCID; see Chapter 188). Adenosine deaminase superactivity causes hemolytic anemia. The deficiency of deoxyguanosine kinase causes a mitochondrial disease. The deficiency of thiopurine methyltransferase is of importance in pharmacogenetics.

Phosphoribosylpyrophosphate Synthetase Superactivity

Phosphoribosylpyrophosphate (PRPP) synthetase superactivity (OMIM 311850), one of the few known examples of a hereditary anomaly that enhances the activity of an enzyme, was initially reported by Sperling and colleagues1 in a patient with early adult-onset gout. Since then, about two dozen families have been identified in which excessive production of uric acid could be traced to this disorder.2,3

Clinical Presentation

The disorder presents mostly in young adult males with uric acid lithiasis or gouty arthritis. The high incidence of renal calculi composed of uric acid potentially leads to renal insufficiency. A few patients present in infancy with clinical signs of uric acid overproduction accompanied by neurological abnormalities, mainly sensorineural deafness (particularly for high tones) but also hypotonia, locomotor delay, ataxia, and autistic features. The disorder is X-linked, but hemizygous females with gout are also found.4 Uricemia can be very high, reaching 10 to 15 mg/dl (0.60–0.90 mmol/l; normal is 2.9–5.5 mg/dl, or 0.17–0.32 mmol/l). Affected individuals synthesize purines at an excessive rate, manifested by uric acid excretion ranging from 1.0 to 2.4 g per 24 hours (normal 0.5–0.8 g) while maintained on a purine-free diet.

Metabolic Derangement

PRPP synthetase catalyzes the formation (from ribose-5-phosphate and ATP) of PRPP, the first intermediate in the de novo synthesis of purine nucleotides (Fig. 168-1). Three distinct isoforms of PRPP synthetase—PRS 1, 2, and 3—have been identified. They encode highly homologous polypeptides, which nevertheless differ by several physical and kinetic properties. PRPP synthetases are subjected to complex regulation, including stimulation by Pi and feedback inhibition by the nucleotides produced by PRPP utilizing pathways such as ADP and GDP. PRPP synthetase superactivity involves the PRS1 isoform. It can be caused by various kinetic anomalies2,3: (1) regulatory defects, such as increased sensitivity to the stimulator of the enzyme Pi or decreased sensitivity to its inhibitors ADP and GDP; (2) catalytic defects with increased Vmax owing to overproduction of the enzyme protein; (3) combined regulatory and catalytic defects; and (4) increased affinity for the substrate ribose-5-phosphate. Since physiological concentrations of PRPP are not saturating for PRPP amidotransferase, (which is the first rate-limiting enzyme of the de novo pathway), the first rate-limiting enzyme of the de novo pathway increased synthesis of PRPP will result in increased production of uric acid.

Genetics

The three isoforms of PRPP synthetase are encoded by separate genes, PRPS1, PRPS2, and PRPS1L1. PRPS1 and PRPS2 map to the long and short arms of the X chromosome (Xq22q-q24 and Xp22.2-p22.3, respectively) and are expressed in all tissues. The expression of PRPS1L1, an intronless gene located on chromosome 7, is restricted to testes. PRPP synthetase superactivity is caused by gain-of-function mutations of the PRPS1 gene. In six families with purine nucleotide feedback resistant PRPP synthetase, distinctive point mutations were found that, when expressed, displayed kinetic properties comparable to those found in cells from the respective affected individual.5 In contrast, in patients with catalytically overactive PRPP synthetase, a two- to fourfold selective acceleration of transcription of a normal PRPS1 isoform was identified.6 The genetic basis of this acceleration remains unresolved, since normal- and patient-derived genes have identical nucleotide sequences up to 850 base pairs 5' to the consensus transcription initiation site.

Diagnostic Tests

Diagnosis of PRPP synthetase superactivity requires extensive kinetic studies of the enzyme that can be performed on erythrocytes or on cultured skin fibroblasts in a few specialized laboratories. The disorder should be differentiated from partial hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency, which gives similar clinical signs (see below). Familial juvenile hyperuricemia7 should also be considered.

Treatment and Prognosis

Patients should be treated with the xanthine oxidase inhibitorallopurinol. This compound is oxidized by xanthine oxidase into oxypurinol, which has a very high affinity for the reduced molybdenum site of this enzyme, resulting in essentially irreversible inhibition of its activity. Inhibition of xanthine oxidase, the last enzyme of purine catabolism (Fig. 168-1), decreases the production of uric acid and its replacement by hypoxanthine, which is about ten times old more soluble, and by xanthine, which is slightly more soluble than uric acid. Initial allopurinol dosage is 10 to 20 mg/kg per day in children and 2 to 10 mg/kg per day in adults. It should be adjusted to the minimum required to maintain normal uric acid levels in plasma and should be reduced in subjects with renal insufficiency. In rare patients with a considerable increase in de novo synthesis, xanthine calculi can be formed during allopurinol therapy.8 Additional measures to prevent crystallization are thus recommended. These include a low purine diet (free of organ meats, sardines, dried beans and peas); high fluid intake; and, since uric acid and xanthine are more soluble at alkaline than at acid pH, administration of sodium bicarbonate, potassium citrate, or citrate mixtures to bring urinary pH to 6.0 to 6.5. Adequate control of the uricemia prevents gouty arthritis and urate nephropathy but does not correct the neurological symptoms.

Phosphoribosylpyrophosphate Synthetase Deficiency

A severe deficiency of PRPP synthetase has been reported by Wada and colleagues9 in a Japanese boy with hypouricemia, mental retardation, convulsions, increased urinary excretion of orotic acid, and megaloblastic bone-marrow changes. He showed a remarkable and unexplained recovery of enzyme activity after treatment with adrenocorticotrophic hormone. His disease has, however, not been proven genetic.

Recently, loss-of-function mutations of the PRPS1 gene have been identified in several patients with X-linked disorders characterized by peripheral neuropathy, hearing impairment, and optic atrophy; these disorders mainly included Arts syndrome (OMIM 301835),10 Rosenberg-Chutorian syndrome, and Charcot-Marie Tooth disease CMTX5 (OMIM 311070).11 PRPP synthetase activity was markedly decreased, although not completely deficient. There were no reductions of serum and urinary uric acid. It is speculated that the defect could be compensated by synthesis of ATP via the S-adenosylmethionine pathway and that administration of the latter compound could be beneficial.

Adenylosuccinate Lyase Deficiency

The deficiency of adenylosuccinate lyase (ADSL, also known as adenylosuccinase; OMIM 103050) is the first enzyme defect reported in humans to affect the de novo pathway of purine synthesis.12 Over 60 patients have been identified so far.13,14

Clinical Presentation

Patients present with widely variable associations of psychomotor retardation, epilepsy, autistic features (failure to make eye-to-eye contact, repetitive behavior, temper tantrums, autoagressivity), hypotonia, and secondary feeding problems. Several clinical pictures can be distinguished. In the first reported presentation, often referred to as type I, patients display moderate to severe psychomotor retardation, frequently accompanied by epilepsy after the first years and various behavior disturbances. In some patients, intractable convulsions start within the first days to weeks of life15-17 and are sometimes associated with severe muscular hypotonia necessitating mechanical ventilation.18 Impaired intrauterine growth, microcephaly, fetal hypokinesia, and a lack of fetal heart rate variability have also been observed.18

Rare patients, referred to as type II, are strikingly less retarded as compared with the other cases.19 One patient suffered from profound muscle hypotonia accompanied by slightly delayed motor development.20 Finally, a few patients display mainly autistic features.14,21,22 Dysmorphic features are usually absent, but a suggestive facial dysmorphism, including brachycephaly, anteverted nostrils, long smooth philtrum, and thin upper lip, has been reported.23 Fundoscopy; auditory, somatosensory, and visual evoked responses; nerve conduction velocities; and electromyography are normal, the latter also being true for patients with muscular hypotonia and wasting. Computed tomography and brain MRI often show hypotrophy or hypoplasia of the cerebellum, particularly of the vermis.

Metabolic Derangement

ADSL catalyzes two steps in the synthesis of purine nucleotides (Fig. 168-1): (1) the conversion of succinylaminoimidazolecarboxamide ribotide (SAICAR) into aminoimidazolecarboxamide ribotide (AICAR), the eighth step of the de novo pathway, and (2) the formation of AMP from adenylosuccinate (S-AMP), the second step in the conversion of IMP into AMP. The reaction is a nonhydrolytic cleavage of the C-N bond linking the succinate moiety of the two substrates to their nucleotide part, similar to that catalyzed by the urea cycle enzyme argininosuccinate lyase. ADSL is composed of four subunits of about 50 kDa. The enzyme of Thermotoga maritima has been crystallized,24 and, based on its structure, homology models of the human and Bacillus subtilis enzyme have been constructed.25,26

ADSL deficiency results in the accumulation of succinylaminoimidazole carboxamide (SAICA) riboside and succinyladenosine (S-Ado) in urine, cerebrospinal fluid (CSF), and to a minor extent plasma. These succinylpurines, the hallmarks of the disorder, are the products of the dephosphorylation by 5'-nucleotidase(s) of SAICAR and adenylosuccinate (S-AMP). In profoundly retarded type I patients, CSF concentrations of both succinylpurines are 100 to 200 μmol/l, and the S-Ado/SAICA-riboside ratios are between 1 and 2. In ADSL-deficient patients with milder clinical pictures, the CSF concentrations of SAICA-riboside are in the same range, but those of S-Ado tend to be higher. This results in S-Ado/SAICA-riboside ratios around or above 2, even reaching 4 to 5 in the strikingly less retarded type II patients. In urine, the concentrations of the succinylpurines can reach up to 5 μmol per mg of creatinine, and their ratio reflects that in CSF. Comparison of the activities of ADSL with both S-AMP and SAICAR as substrates showed that both are lost in parallel, to about 30% of normal in fibroblasts of profoundly retarded type I patients. In contrast, in fibroblasts of a mildly retarded type II patient, activity with S-AMP was reduced to 3% of normal, whereas that with SAICAR was 30% of control. These findings provide an explanation for the markedly higher S-Ado/SAICA-riboside ratio in the body fluids of the latter patient.

Two main hypotheses can be put forward to explain the symptoms of ADSL deficiency: impaired synthesis of purine nucleotides and toxic effects of the accumulating succinylpurines. Although ADSL deficiency might be expected to lead to decreased synthesis of purine nucleotides, normal levels of the latter were measured in freeze-clamped liver, kidney, and muscle of patients. This suggests that residual ADSL activity remains sufficient to allow flux through the pathway of purine synthesis, as evidenced by the finding that in fibroblasts of the mildly retarded patient, in whom ADSL activity with S-AMP was only 3% of normal, conversion of S-AMP into IMP remained possible. In addition, the ADSL defect could be circumvented by supply of purines from nonaffected cell types via purine salvage. It is therefore more likely that the symptoms of ADSL deficiency are due to toxic effects of the accumulating succinylpurines. Moreover, the observation of generally less severe mental retardation of patients with similar SAICA-riboside levels, but with S-Ado/SAICA-riboside ratios above 2, suggests that SAICA-riboside is the offending compound and that S-Ado could protect against its toxic effects.

Positron emission tomography revealed a marked decrease in the uptake of [18F]-labeled 2-fluoro-2-deoxyglucose in the cortical areas of ADSL-deficient patients, suggesting interference with glucose metabolism.27 However, the mechanism of this effect remains unexplained. Owing to the structural analogy of the succinylpurines with adenosine and N-methyl-D-aspartate (NMDA), the possibility that they might interfere with the receptors of these compounds was investigated. Interferences could not be evidenced, but SAICA-riboside induced a significant degeneration of the pyramidal neurons of rat hippocampus28 and was toxic to undifferentiated neurons.29

Genetics

The adenylosuccinate lyase (ADSL) gene has been mapped to chromosome region 22q13.1-13.2 in humans. The gene is 23 kb in length and comprises 13 exons.30 Correction of a first 1337 nucleotide sequence31 has shown that the open reading frame of the human cDNA comprises 1452 nucleotides and encodes a protein that contains 484 amino acids (E.A. Fon, EMBL database, accession number X65867).30,32 Analysis of the first family with ADSL deficiency had revealed a T-to-C substitution, resulting in an S413P change,31 now labeled S438P.30,32 To date, in accordance with the variability of the clinical symptoms, about 50 missense mutations have been reported in apparently unrelated sibships13,14 (Adenylosuccinate Lyase Mutation Database, http://www.icp.ucl.ac.be/adsldb). In about half of the families, the patients are compound heterozygotes. The most frequently encountered mutation is R426H, previously identified as R401H; this accounts for about one third of the alleles investigated. A splicing error that leads to deletion of amino acids 206-218,32 two nonsense mutations (R337X30 and R190X), and a mutation in the 5'UTR of the ADSL gene (-49T/C33), have also been identified. The latter was found in three unrelated patients, suggesting that mutation in a regulatory region of the gene might be an unusually frequent cause of ADSL deficiency.

Nearly all mutations identified hitherto are distant from the fumarate lyase signature of the enzyme (amino acids 288–298, encoded by nucleotides 862–894) and do not affect the conserved histidyl 159 residue believed to function in catalysis. Expression studies of these mutations show that they provoke thermolability and decrease Vmax of ADSL with its two substrates in parallel.34 Studies of mutant Bacillus subtilis enzymes corresponding to human ADSL deficiencies are consistent with these observations.22,25 A single mutation, R303C, located five amino acids distal from the fumarate lyase signature and found in two independent, mildly retarded type II patients, does not affect enzyme stability; however, when expressed, it displays markedly less activity with S-AMP than with SAICAR, in accordance with the findings in fibroblasts.

Diagnostic Tests

The marked clinical heterogeneity of ADSL deficiency justifies systematic screening for the disorder in unexplained psychomotor retardation (profound or mild), accompanied or not with autistic features, and in neurological disease with convulsions or hypotonia. Diagnosis is based on the presence of SAICA-riboside and succinyl adenosine (S-Ado) in urine and CSF, which are normally nearly undetectable. For systematic screening, a modified Bratton-Marshall test, performed on urine, appears most practical.35 However, false-positive results may be observed in patients who receive antiepileptic medications or antibiotics, particularly sulfonamides, because the test was originally devised for measurement of sufonamides. The succinylpurines can also be detected by other techniques, including thin-layer chromatography.36 Final diagnosis requires HPLC with UV spectral analysis12 or proton NMR spectroscopy.37

Although not required for diagnosis, measurements of ADSL’s activity with S-AMP as substrate show markedly decreased (even undetectable) activities in liver and kidney of all patients investigated. In muscle, activity is within normal range in some patients but is reduced in those presenting with growth retardation and muscular wasting. In peripheral blood lymphocytes, ADSL activity is about 40% of normal. The activity of erythrocyte ADSL is normal in some patients but is decreased in others. As a rule, a partial deficiency of ADSL is measured in cultured fibroblasts.

Prenatal diagnosis of ADSL deficiency has been performed twice on chorion villi of the mother of a patient who had a C5T mutation on the maternal allele and a C1185A mutation on the paternal allele.38

Treatment and Prognosis

With the aim to replenish hypothetically decreased concentrations of adenine nucleotides in adenylosuccinate lyase (ADSL)-deficient tissues, some patients have been treated for several months with oral supplements of adenine (10 mg/kg per day) and allopurinol (5–10 mg/kg per day). Adenine can be incorporated into the adenine nucleotides via adenine phosphoribosyltransferase (APRT), whereas allopurinol is required to prevent xanthine oxidase from converting adenine into minimally soluble 2,8-dihydroxyadenine, which forms kidney stones (see below). No clinical or biochemical improvement was recorded, with the exception of some acceleration of growth. More recently, oral administration of ribose (10 mmol/kg per day) has been reported to reduce seizure frequency in an ADSL-deficient girl,39 although not in a sustained way. Uridine (2 mmol/kg per day) also had a slight beneficial effect.40

The prognosis for survival of ADSL-deficient patients varies greatly. Several of those presenting with early epilepsy have died within the first months of life. In markedly retarded patients, further evolution is characterized by absent or minimal progression of psychomotor development and persistence of autistic behavior, except for occasional improvement of eye contact. Mildly retarded patients have reached adult age and are able to work in a protected environment.

Aica-Ribosiduria (Atic Deficiency)

This disorder41 (OMIM 608688), identified on the de novo pathway of purine synthesis, was diagnosed in a female infant presenting with profound mental retardation, marked dysmorphic features (prominent forehead and metopic suture, brachycephaly, wide mouth with thin upper lip, low-set ears, and prominent clitoris due to fused labia majora), and congenital blindness. Urine analysis showed a positive Bratton-Marshall test. This led to the identification of a massive excretion of 5-amino-4-imidazolecarboxamide (AICA) riboside, the dephosphorylated counterpart of AICAR (Fig. 168-1), also termed ZMP. Analysis of the patient’s erythrocytes revealed accumulation of ZMP and of the di- and triphosphates, ZDP and ZTP, respectively. Incubation of her fibroblasts with AICA-riboside led to accumulation of AICAR (not observed in control cells), indicating impairment of the final steps of de novo purine biosynthesis. Assay of AICAR transformylase/IMP cyclohydrolase (ATIC), the bifunctional enzyme catalyzing the two last steps of the pathway, revealed a profound deficiency of the transformylase and a partial deficiency of the cyclohydrolase. Sequencing of the ATIC gene (gene map locus 2q35) showed a K426R change in the transformylase region in one allele and a frameshift in the other. The recombinant enzyme carrying mutation K426R completely lacks AICAR transformylase activity. The discovery of this novel inborn error of purine synthesis reinforces the necessity to perform a Bratton-Marshall test in all cases of unexplained mental retardation or neurological symptoms.

Myoadenylate Deaminase Deficiency

The deficiency of myoadenylate deaminase (OMIM 102770), the muscle-specific isoenzyme of AMP deaminase (AMPD), was first reported by Fishbein and colleagues.42 Since then, the defect has been found in 1% to 2% of the Caucasian population, but most deficient individuals are asymptomatic.43

Clinical Presentation

Two distinct forms of myoadenylate deaminase deficiency are known. The first form, primary myoadenylate deficiency, was initially detected in young, otherwise healthy adults; however, later on, a wide variability was observed with respect to the age (1.5–70 years) of patient at the onset of symptoms.44,45 The defect is associated with isolated muscular weakness, fatigue, cramps, or myalgia following moderate to vigorous exercise, sometimes accompanied by an increase in serum creatine kinase and minor electromyographic abnormalities. Rhabdomyolysis and myoglobinuria may also occur. Muscular wasting or histological abnormalities are usually absent. The defect was also detected in patients with hypotonia or cardiomyopathy. Later on, identifying the deficiency in asymptomatic family members of affected subjects, finding that approximately 2% of muscle biopsies taken for diagnostic purposes are AMPD deficient, and studies of the AMPD gene led to the conclusion that the defect is present in 1% to 2% of the Caucasian population, most being asymptomatic.

The second form of the disorder, secondary myoadenylate deaminase deficiency, is found in several neuromuscular disorders, including amyotrophic lateral sclerosis, fascioscapulohumeral myopathy, Kugelberg-Welander syndrome, polyneuropathies, Werdnig-Hoffmann disease, and myophosphorylase deficiency. The symptoms of these disorders dominate those of myoadenylate deaminase deficiency, although some synergism may be observed.46

Metabolic Derangement

AMP deaminase catalyzes the conversion of AMP into IMP with liberation of ammonia. Four isoenzymes of human AMPD are known. Isoenzyme M, or myoadenylate deaminase, is found only in skeletal muscle; isoenzyme L predominates in liver and brain; and isoenzymes E1 and E2 are found in erythrocytes. The muscle enzyme is closely associated with the contractile apparatus and is highly regulated. Kinetic studies suggest that myoadenylate deaminase is nearly inactive in resting muscle but becomes highly active during contraction due to modifications of the ATP/ADP and GTP/GDP ratios; of the concentrations of K+, H+, Pi, and creatine phosphate; and of myofibrillar binding. This increased activity accounts for the production of ammonia during muscle contraction. AMPD, together with adenylosuccinate synthetase and adenylosuccinate lyase, forms the purine nucleotide cycle (eFig. 168.1). Several functions have been proposed for the purine nucleotide cycle in muscle: (1) removing AMP formed during exercise to favor the regeneration of ATP from ADP by myokinase (adenylate kinase); (2) releasing IMP and ammonia, both stimulators of glycolysis and hence of energy production; (3) producing fumarate, an intermediate of the citric acid cycle, and hence also a provider of energy. It has therefore been suggested that the muscle dysfunction observed in primary myoadenylate deaminase deficiency is caused by impairment of energy production required for muscle contraction. Paradoxically, complete and partial myoadenylate deficiency did not affect intermediates of the citric acid cycle or the cellular energy charge during exhaustive exercise.47 Studies in adenylate kinase knockout mice have corroborated that it is not essential that AMP deaminase operates during intense muscle contraction.48 Together with the identification of numerous asymptomatic myoadenylate deaminase-deficient individuals, these findings suggest that myoadenylate deaminase deficiency is not a primary cause of muscle dysfunction but might have a triggering or synergistic effect in association with other, hitherto unidentified disorder(s).

Genetics

Three AMPD genes exist: AMPD1, which produces isoenzyme M (myoadenylate deaminase); AMPD2 isoenzyme L; and AMPD3 isoenzyme E. The AMPD1 gene is located on chromosome 1 in the region p13-p21. Most Caucasian and African American subjects with documented primary myoadenylate deaminase deficiency have been found homozygotes for a single mutant AMPD1 allele with C-to-T transitions at nucleotide 34 in exon 2 and at nucleotide 143 in exon 3. The mutation at nucleotide 34 produces a nonsense mutation, Q12X, which severely truncates myoadenylate deaminase and accounts for its lack of activity. The mutation at nucleotide 143 results in a missense mutation, P48L, which does not influence enzyme activity. Population studies show that this mutant allele is found in 10% to 14% of Caucasians. This accords with the finding that about 2% of diagnostic muscle biopsies are AMPD deficient and suggests that the mutation arose in a remote western European ancestor.

More recently, other, more rare mutations of the AMPD1 gene have been identified in myoadenylate deaminase deficient individuals. A Japanese patient with myopathy was identified as a compound heterozygote for two missense mutations, producing R388W and R425H changes.49 Ten Caucasian patients with exercise-induced myalgia were found compound heterozygotes for the C34-T/C143T mutation and a G468T mutation, the latter resulting in a Q156H substitution enzyme with labile catalytic activity.50

Interestingly, a protection against heart failure has been repeatedly documented in heterozygous subjects for mutant AMPD1.51-53 This could be explained by increased catabolism of AMP to cardioprotective adenosine rather than to IMP. Nevertheless, a systemic increase in adenosine following exhaustive exercise could be observed in homozygous but not in heterozygous subjects for mutant AMPD1.54 Individuals with a complete but totally asymptomatic deficiency of erythrocyte AMPD have been identified in Japan, Korea, and Taiwan.55

Diagnostic Tests

Screening for myoadenylate deaminase deficiency can be performed by a semi-ischemic exercise test. A sphygmomanometer cuff is inflated around the upper arm to the mean arterial pressure. Upon squeezing a hand manometer for 2 minutes, the several-fold elevation of venous plasma ammonia, recorded in normal subjects, is absent in myoadenylate deaminase deficiency. Final diagnosis is established by histochemical or biochemical assay in a muscle biopsy. In the primary form, the activity of AMPD is mostly below 2% of normal, and as a rule, little or no immunoprecipitable enzyme is found. In the secondary form, the residual activity is 2% to 15% of control, appreciable immunoreactivity is usually present, and progressive declines of AMPD activity have been documented. AMPD activity is deficient in approximately 2% of all muscle biopsies taken for a wide variety of indications, making this defect the most common metabolic disorder of skeletal muscle.

Treatment and Prognosis

Symptomatic myoadenylate deaminase deficient patients may display a gradual worsening of their symptoms, which may lead to marked handicap. They should be advised to exercise with caution to prevent rhabdomyolysis and myoglobinuria. Administration of ribose (2–60 g per day orally in divided doses) has been reported to improve muscular strength and endurance in some patients56but not in others. After ribose is converted into PRPP, it could increase the synthesis of purine nucleotides. In one patient, xylitol, which is converted into ribose, proved beneficial at the dose of 15 to 20 g/day orally.57

Adenosine Deaminase Deficiency

Adenosine deaminase (ADA) catalyzes the deamination of adenosine and its deoxy counterpart deoxyadenosine. A gross deficiency of ADA was first reported by Giblett and colleagues58 in two unrelated children with profound impairment of both cellular (T-cell) and humoral (B-cell) immunity, known as severe combined immunodeficiency disease (SCID). Since that time, several hundred patients with ADA deficiency have been diagnosed. The disorder is discussed in Chapter 188.

Adenosine Deaminase Superactivity

A hereditary, approximately fiftyfold elevation of erythrocyte ADA has been shown to cause nonspherocytic hemolytic anemia.59 It is explained by an enhanced catabolism of the adenine nucleotides, including ATP, owing to the increased activity of ADA.

Purine Nucleoside Phosphorylase Deficiency

Their finding of ADA deficiency prompted Giblett and colleagues to search for other defects of purine and pyrimidine metabolism in patients with immune disorders. This resulted in the discovery of purine nucleoside phosphorylase (PNP) deficiency in a child with an isolated defect of T-cell function.60 The disorder is much less frequent than ADA deficiency, with about 50 patients reported. It is discussed in Chapter 188.

Xanthine Oxidase Deficiency

The deficiency of xanthine oxidase (also termed xanthine dehydrogenase and xanthine oxidoreductase, or XOR), characterized by xanthinuria, was the first enzyme defect reported in human purine metabolism.61 Originally described as a benign disorder, it was subsequently found that it could also occur in combination with a devastating disorder similar to isolated sulfite oxidase deficiency.62 The combination is caused by inability to synthesize a molybdenum cofactor common to three oxidases: XOR, aldehyde oxidase, and sulfite oxidase. Accordingly, three types of XOR deficiency are now recognized: (1) type I, isolated XOR deficiency (OMIM 278300); (2) type II, combined XOR and aldehyde oxidase deficiency (OMIM 603592), which causes equally benign xanthinuria; and (3) combined deficiency of XOR, aldehyde, and sulfite oxidase (OMIM 252150), provoking a lethal disease.

Clinical Presentation

Type I and type II XOR deficiency can be completely asymptomatic, although in about one third of the cases, kidney stones are formed. Most often, they are not visible on X-ray, and they may appear at any age and cause a variety of symptoms, including hematuria, renal colic, and even acute renal failure. Myopathy may be present, precipitated by strenuous exercise and associated with crystalline xanthine deposits in muscle. In combined XOR and sulfite oxidase deficiency, the clinical picture of sulfite oxidase deficiency (which is also found as an isolated defect63 overrides that of XOR deficiency. The symptoms include neonatal feeding difficulties and intractable seizures, myoclonia, increased or decreased muscle tone, eye lens dislocation, and severe mental retardation.

Metabolic Derangement

XOR catalyzes the conversion of hypoxanthine into xanthine and that of xanthine into uric acid (Fig. 168-1). Under physiological conditions, the enzyme is a dehydrogenase that utilizes NAD+ as the electron acceptor and yields NADH. In several in vitro and in vivo situations, particularly glutathione depletion and proteolysis, the enzyme converts into an oxidase that utilizes molecular oxygen as the electron acceptor and produces superoxide and hydrogen peroxide. This conversion plays a role in ischemia-reperfusion damage.64 XOR is composed of two subunits, each of which has a molybdenum center that contains molybdenum under the form of a molybdenum cofactor.

The deficiency of XOR results in the near total replacement of uric acid by hypoxanthine and xanthine as the end products of purine catabolism (Fig. 168-1). This alteration is clearly manifested in urine, in which over 90% of oxypurine excretion is normally uric acid. Generally, plasma hypoxanthine is not elevated (or is minimally elevated) in the various forms of XOR deficiency, owing to efficient reutilization by hypoxanthine-guanine phosphoribosyltransferase. Plasma xanthine, normally below 1 μM, may rise to 10 to 40 μM, which is way below the normal uric acid level because of the markedly higher renal clearance of xanthine. Concentrations of hypoxanthine and xanthine are approximately 10 times higher than normal in muscle of xanthinuric subjects. The limited solubility of xanthine may explain the numerous birefringent crystals observed in the muscles of patients complaining of pain and stiffness. The role of hypoxanthine, which increases during vigorous exercise in both normal and xanthinuric subjects, is unclear, since it is much more soluble than xanthine.

Genetics

The inheritance of type I and type II XOR deficiency and of combined XOR and sulfite oxidase deficiency is autosomal recessive. Studies of the XOR gene, localized on chromosome 2p23-p22, have led to the identification in hereditary xanthinuria type I of two mutations, resulting respectively, in a nonsense substitution and a termination codon.65 Xanthinuria type II is caused by mutations of a molybdenum cofactor sulfurase gene.66,67More than 30 different mutations in three different molybdenum cofactor biosynthetic genes (MOCS1, MOCS2, and GEPH) have been identified in combined XOR and sulfite oxidase deficiency.68

Diagnostic Tests

Both in isolated and combined XOR deficiency, plasma concentrations of uric acid below 1 mg/dl are measured; they may decrease to virtually undetectable values on a low-purine diet. Urinary uric acid is reduced to a few percent of normal and is replaced by hypoxanthine and xanthine. In combined XOR and aldehyde oxidase deficiency (xanthinuria type II), patients are unable to oxidize allopurinol. In combined XOR, aldehyde, and sulfite oxidase deficiency, the urinary changes of xanthinuria are accompanied by an excessive excretion of sulfite and other sulfur-containing metabolites, such as S-sulfocysteine, thiosulfate, and taurine. The enzymatic diagnosis requires liver or small intestine mucosa, the only human tissues that normally contain substantial amounts of xanthine oxidase. Sulfite oxidase and the molybdenum cofactor can be assayed in liver and fibroblasts. Mutant XOR has been investigated in a few patients. Despite immeasurably low activity, immunoreactive protein remains detectable in the duodenal mucosa of some patients but not of others.6

Treatment and Prognosis

Isolated XOR deficiency is mostly benign. A low purine diet should be prescribed and fluid intake increased. The prognosis of combined XOR and sulfite oxidase deficiency is very poor. So far, all therapeutic attempts, including low-sulfur diets, the administration of sulfate and molybdenum, and trials to bind sulfite with thiol-containing drugs, have been unsuccessful.

Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency

In 1967, Seegmiller and coworkers discovered a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT or HGPRT) in children with the Lesch-Nyhan syndrome (OMIM 300322), an incapacitating neurological disorder that, with few exceptions, is limited to males.69 It is characterized by choreoathetosis, spasticity, compulsive self-mutilation manifested by biting away lips and tongue and ends of the fingers, and three- to fourfold normal uric acid levels in plasma and urine.70 That same year, a partial deficiency of HPRT was described in patients with milder presentations, sometimes termed Kelley-Seegmiller syndrome or now more often called Lesch-Nyhan variants (OMIM 300323); this disorder involves overproduction of uric acid that leads to severe gouty arthritis in early adult life and involves minor or no neurological dysfunction.71 This led to the identification of a continuous spectrum of HPRT deficiencies, ranging from gout to a devastating neurological syndrome.72

Clinical Presentation

At birth, children with complete HPRT deficiency (Lesch-Nyhan disease) appear entirely normal. The first indication of the disease may be the presence of brownish to red-orange sand in the diapers, particularly when the baby becomes dehydrated. Some of the infants are very irritable, with episodes of screaming, suggesting the possibility of renal colic. Most infants show normal development during the first 4 to 6 months of life. During the second part of the first year, impaired motor development becomes evident by the inability to support the head, generalized muscle hypotonia, and voluntary movements of both athetoid and choreiform types with spasticity. Recently, the motor syndrome of complete HPRT deficiency has been reclassified as a severe action dystonia, superimposed to a baseline hypotonia.73 By 1 year of age, pyramidal symptoms consist of an increase in muscle tone, an increase in deep tendon reflexes, a sustained ankle clonus, and a scissoring of the lower extremities, and extensor plantar responses are usually present. An increased incidence of dislocation of the hips and club feet may be related to the early hypotonia. Severely affected children are never able to walk.

Over the years, the patients develop a compulsive aggressiveness and self-mutilation that involves biting away lips, tongue, and ends of the fingers if given the opportunity. Because it is painful, children are fearful of the biting. Biting can, in some cases, begin with the eruption of incisors. In other patients, it may be delayed until early adolescence. In any given patient, biting can be highly variable, with patients going through periods when they show extensive self-mutilation and other periods when this is no longer a problem. Self-mutilation tends to be correlated, at least in some cases, with emotional stress. In addition, some older children develop opisthotonic spasms, which appear to be at least semivoluntary. An accompanying laryngeal spasm and stridor sometimes produces a temporary cyanosis. If the patient’s head is in range of a hard object at the time of a spasm, he or she may injure it. Children also sometimes throw themselves from the bed if left unattended or may injure themselves on sharp edges of wheelchairs that are left unpadded.

Aggressive acts against others are also included in the bizarre behavior of these children. This can take the form of biting, hitting, spitting, or kicking. Eyeglasses are common targets for their aggression. Patients often pinch or strike attendants in areas of sexual significance, and become verbally aggressive, often with a remarkably shocking vocabulary. Projectile vomiting is also used by older children as a weapon, especially when the child becomes upset emotionally. Although this peculiar behavior would seem to alienate them from others, these children are often favorite patients of ward personnel, because they are charming and smile and laugh easily. They are very responsive to their environment and show a remarkably good sense of humor. They have a characteristic dysarthric speech, although they can usually make themselves understood to those who are caring for them.

The first descriptions of Lesch-Nyhan syndrome included mental retardation. However, reevaluation with specific tests for their motor difficulties has shown attention deficits but preservation of nonverbal intelligence, and some patients display normal intelligence.74-76

Partial HPRT deficiency is found in rare patients with gout. Most of them or normal on neurological examination, but occasionally spasticity, dysarthria, and a spinocerebellar syndrome are found.77 Some but not all of the patients with less severe deficiencies of the HPRT enzyme with resulting minimal neurological dysfunction also show a compulsive behavior. Whereas most subjects with Lesch-Nyhan syndrome do not develop gouty arthritis, this finding is common in partial HPRT deficiency.

Metabolic Derangement

HPRT is normally expressed in all cells of the body. Early studies reported that its specific activity is highest in the basal ganglia, which correlates with the brain area that can cause analogous movement disorders. However, later investigations have not confirmed these findings. HPRT activity is also high in testes, which accords with failure of sexual maturation and atrophic testes found in some patients with the most severe enzyme deficit. Alterations in the kinetic properties of the enzymes in patients comprise, besides reduction of the maximal velocity up to its complete loss, reduced affinities for phosphoribosyl pyrophosphate (PRPP) and the purine bases, which have been shown to correlate with the gene lesions (see below).

The considerably excessive production of uric acid in HPRT deficiency is explained by an enhancement of the purine synthesis rate, which is caused by the increased amount of PRPP that accumulates from its underutilization (caused by the HPRT mutation). Fibroblasts cultured from affected patients show a two- to threefold accumulation of PRPP, and their erythrocytes show a tenfold accumulation over normal values. Since PRPP is a rate-limiting, normally not saturating substrate for the first, presumably rate-determining reaction of purine biosynthesis de novo catalyzed by the enzyme PRPP glutamine amidotransferase, its elevation will increase uric acid production. Further support for this concept comes from the correlation of increased intracellular PRPP in fibroblasts and excessive rates of purine synthesis in PRPP synthetase superactivity (see above).

Theoretically, the deficiency of HPRT should provoke accumulation of its substrates (hypoxanthine, guanine, and its deamination product xanthine) and of PRPP and depletion of its products (GMP and the other guanine nucleotides and IMP). Owing to the conversion of IMP into AMP (Fig. 168-1), the synthesis of the latter and the other adenine nucleotides could also be decreased. Hypoxanthine and xanthine are elevated in body fluids of HPRT-deficient patients, but their toxicity is ruled out by the absence of neurological symptoms in isolated xanthine oxidase deficiency (see above) and the absence of worsening neurological symptoms upon allopurinol treatment. Because allopurinol lowers uric acid but does not influence the neurobehavioral abnormalities, uric acid toxicity is ruled out. PRPP toxicity is also unlikely, since its elevation in PRPP synthetase superactivity (see above) is not associated with the neurological symptoms of HPRT deficiency. Similarly, accumulation of AICAR, an intermediate of de novo purine biosynthesis, and its further phosphorylation products (collectively termed Z-nucleotides) is unlikely to be involved, since it has also been recorded in disorders without neurobehavioral abnormalities.

Depletion of guanine or adenine nucleotides is also proposed as an explanation for the neurological symptoms of HPRT deficiency. ATP depletion might result in impairment of energy production, DNA repair,78 and metabolic defense against oxidant stress. GTP depletion, which can be shown in HPRT-deficient cells under certain conditions,79 has been proposed to interfere with the function of G proteins and the formation of tetrahydrobiopterin (BH4), a cofactor in dopamine synthesis. However, other studies80 have invalidated the latter hypothesis, which might have provided an explanation for the abnormalities of the dopaminergic system observed in Lesch-Nyhan patients.

Multiple studies have shown abnormalities of the dopaminergic neurotransmitter system in Lesch-Nyhan disease. Autopsy studies81,82 showed a reduction in the basal ganglia (to 10–30% of control) of dopamine; its metabolite homovanillic acid; and tyrosine hydroxylase, the dopamine synthetizing, BH4-dependent enzyme. In vivo PET studies of the brains of Lesch-Nyhan subjects have revealed large reductions in dopaminergic terminals not only in the basal ganglia, but also in other brain areas.83,84 Dopamine deficiency is also found in HPRT knockout mice.85 How HPRT deficiency impairs the dopaminergic system remains an open question. One possibility could be an increased sensitivity to dopamine-associated oxidant stress, as thought to play a role in Parkinson disease. Dopamine and other catecholamines are toxic to many cell types, probably through various mechanisms, including spontaneous and metal-catalyzed oxidation that produces reactive oxidant species such as superoxide radicals.86 Studies in HPRT knockout mice provide evidence for an increased oxidant stress, which is not affected by overexpression of superoxide dismutase indicating that other mechanisms intervene.85

Genetics

The human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene spans approximately 45 kb on the long arm of chromosome X at Xq26-q27.2 and contains nine exons.87 It encodes a tetrameric protein composed of four identical subunits of 219 amino acids with a mass of 25 kDa. The three-dimensional structure of the human enzyme has been resolved by X-ray crystallography.88 A single restriction fragment length polymorphism with three alleles is known. A rare histidine-to-arginine mutation at codon 60 reduces HPRT activity by approximately 40% but does not provoke symptoms89 and might thus be a rare polymorphism. The HPRT gene on the inactivated X-chromosome is rendered transcriptionally silent by methylation of the majority of its CpG clusters.90

Over 250 mutations causing HPRT deficiency have been reported,91 and new mutations are regularly identified.92,93 Although with some overlap, they can be divided into three categories, based on clinical phenotype: (1) full-fledged Lesch-Nyhan disease; (2) intermediate with some neurological dysfunction but no self-injurious behavior; and (3) mild, with uric acid overproduction without neurological symptoms. As reviewed by Jinnah and colleagues,91 single base substitutions account for approximately 65% of all mutations; deletions account for 25%; and insertions, duplications, and complex mutations account for the remaining 10%. About 60% of the point mutations causing amino acid substitutions, nearly all those causing a premature stop, and 80% of those leading to splicing errors provoke Lesch-Nyhan disease. As expected, 90% of intermediate and mild cases have point mutations leading to amino acid substitutions. Mutations are found throughout the HPRT gene, but several hot spots seem to exist. Although some mutations that alter HPRT kinetics implicate the binding site of its substrates, most are located outside, which suggests that they provoke conformational changes in the active site. Overall, location of the HPRT mutations seems to have a less significant influence on phenotype than the different amino acid changes that can be found at the same codon. Structural and functional analysis with modeling methods has also shown that most mutations can be explained by the predicted effect on protein structure and function.94 With rare exceptions,95 genotype/phenotype correlations are thus quite apparent in HPRT deficiency.

Although Lesch-Nyhan disease is an X-linked disorder, a small number of females with the disorder have been identified.96–99 This can be explained by the coexistence of a nonsense mutation or deletion on one of the parents’ alleles, with a nonrandom inactivation of the other parent’s allele.

Diagnostic Tests

The excessive excretion of uric acid forms the basis for a relatively simple screening test in which the ratio of uric acid to creatinine (both expressed as mg/100 ml in the morning urine sample) is measured.100 Lesch-Nyhan patients with the full syndrome show ratios between 2.0 to 5.3, as opposed to 0.2 to 0.6 for normal individuals. However, some overlap with the normal may be found in the first few months or years of life because of high ratios observed at birth in normal infants, followed by a marked age-related decline. Although most patients also show an elevation of serum urate—up to 18 mg/dl (1 mmol/l), particularly in later stages of the disease—this measurement cannot be used to rule out Lesch-Nyhan disease, as around 5% to 10 % may show a normal serum urate, particularly in early life.

Measuring HPRT is most easily performed in red blood cells or skin fibroblasts. In patients with the most severe enzyme defect, HPRT activity is virtually absent in erythrocyte lysates. In patients with less severe deficiencies, HPRT activity may range from less than 0.1 to 10% to 20% of normal. In rare kinetic mutations (see below), near-normal activities can be measured when saturating substrate concentrations are used.

Carrier detection of Lesch-Nyhan disease has long been accomplished, most practically by assaying individual hair follicles, which are advantageous because they are largely clonal in females. Prenatal diagnosis of Lesch-Nyhan disease can be made by measuring HPRT activity in cultured amniocytes and chorionic villi. These techniques are now largely supplanted by molecular diagnosis in families in which the mutation is known.101

Treatment and Prognosis

Treatment comprises controlling the uric acid overproduction, the motor syndrome, and the behavioral manifestations. Allopurinol, at doses up to 20 mg/kg of body weight per day, prevents the damage to kidneys caused by the excessive amounts of uric acid excreted in the urine. Although it produces a marked decrease in both urine and serum uric acid, it fails to decrease de novo purine synthesis: Hypoxanthine and xanthine merely replace uric acid in urine. Established renal calculi can thereby be substantially reduced in size. Further general measures for treatment are those of gout, including high fluid intake (at least 50 ml/kg per day). This will diminish the occurrence of urinary concretions composed of xanthine that have been noted occasionally in children treated with allopurinol.8 Adequate nutrition should also be prescribed, since many of these children take a very long time to eat and may actually be malnourished in an understaffed institution.

Although children with the Lesch-Nyhan syndrome seem to be incapable of learning from punishment, they do respond to positive experiences, and some success in behavior modification has been achieved by simply turning away from the child when they display aberrant behavior. Partial improvement in self-mutilation has also been reported from using both positive and negative conditioning programs. Patients should also be made more comfortable by appropriate restraints. Hands can be kept away from the mouth and yet be left free for use by constructing loose-fitting wraparound fabric splints for the elbows. Chronic irritation in the mouth from sharp teeth edges can be eliminated by lip guards and even tooth extraction.

Despite trials with a variety of agents, no successful pharmacological treatment of the motor syndrome and the behavioral manifestations has yet been found. Children treated with diazepam (Valium) are less spastic. Levodopa with or without a decarboxylase inhibitor has been reported to improve, worsen, or not influence the dystonia. Hydroxytryptophan has been reported to reduce self-mutilation but only transiently and is hardly used anymore. Risperdal and carbamazepine may be useful. Gabapentin has been suggested to improve self-injurious behavior.102 Disappearance of self-mutilating behavior with bilateral chronic stimulation of the globus pallidus internus has been reported.103,104 Recently, local injection of botulinum toxin has also been advocated.105 Lesch-Nyhan disease was one of the first disorders proposed for gene therapy and continues to serve as a model for its development. Bone marrow transplantation has been performed in a small number of Lesch-Nyhan patients without obvious benefit and with significant morbidity, since the majority of the patients died shortly thereafter.

Adenine Phosphoribosyltransferase Deficiency

The deficiency of adenine phosphoribosyltransferase (APRT; OMIM 102600) was first identified in heterozygotes with a partial enzyme defect.106 Later, Cartier and Hamet107 reported a homozygous 4-year-old with urinary crystals and stones. Approximately 300 patients have been diagnosed worldwide, but up to 50% of APRT-deficient subjects may be asymptomatic.

Clinical Presentation

The deficiency may clinically manifest in childhood, even from birth, but may also remain silent for several decades.108 Symptoms include urinary passage of gravel, small stones, and crystals, frequently accompanied by abdominal colic, dysuria, hematuria, and urinary tract infection. Some patients may present with acute anuric renal failure, while others have developed chronic renal failure requiring dialysis and transplantation. The urinary precipitates are composed of 2,8-dihydroxyadenine (2,8-DHA) and radiotranslucent.

Metabolic Derangement

The APRT deficiency results in the loss of adenine salvage (Fig. 168-1), provided by food and by the polyamine pathway. Consequently, adenine is oxidized by xanthine oxidase into 2,8-DHA. Symptoms are caused by the very poor solubility of 2,8-DHA. Its solubility in urine, at pH 5 and 37°C, is about 0.3 mg/dl, considerably less than that of 15 mg/dl for uric acid.

Two types of APRT deficiency are known. Patients with type I deficiency have no detectable activity in erythrocyte lysates. In patients with type II deficiency, significant residual activity is found, reaching 5% to 25% of normal when measurements are made at supraphysiological, saturating concentrations of PRPP. However, kinetic studies reveal that the S0.5 value for PRPP, which is about 3 μM for the normal enzyme, is increased to 50 to 80 μM. This decreased affinity for PRPP results in near inactivity under physiological conditions. Consequently, APRT activity is not detectable in intact cells such as erythrocytes or fibroblasts. Confirmatory diagnosis of type II deficiency is performed on viable T cells.109 To date, type II patients have been found only in Japan, where they account for approximately 80% of the affected subjects.110 Determining the three-dimensional structure of human APRT111 has provided insights in the mechanisms by which many mutations hamper normal enzyme activity.

GeneticS

The human adenine phosphoribosyltransferase gene has been mapped to chromosome 16q24.3. Both in Caucasian and Japanese subjects, numerous polymorphisms are observed. In type I deficiency, more than 30 mutations have been identified.108 About half of these are single base changes. T insertion mutations, resulting in frameshift, and splicing errors are also frequent. The observation of several more common mutations suggests founder effects. All the type II Japanese patients carry the same c.2069T- > C substitution in exon 5, resulting in an M136T change. Approximately 80% are homozygous. Another W98X nonsense mutation, and a CCGA insertion resulting in a frameshift, account for nearly all the other cases.112

Diagnostic Tests

The presence of brownish spots on diapers or the finding of round, brownish crystals in urine under a light microscope suggest the presence of 2,8-DHA. Notwithstanding its very low solubility, 2,8-DHA can also be identified by HPLC. Confirming its presence requires complex analyses, including UV and infrared spectrography, mass spectrometry, X-ray crystallography, and capillary electrophoresis. It is therefore usually easier to measure adenine phosphoribosyltransferase (APRT) activity. As said under “Metabolic Derangement,” based on the level of residual enzyme activity in red blood cell lysates, two types of APRT deficiency are recognized. In both types, APRT activity is absent in intact cells.

Treatment and Prognosis

In patients with symptoms, allopurinol should be given (as detailed under treatment of PRPP synthetase superactivity) to inhibit the formation of 2,8-DHA. Both in patients with stones and in those without symptoms, dietary purine restriction and high fluid intake are recommended. Alkalinization of the urine is, however, not advised: unlike that of uric acid, the solubility of 2,8-DHA does not increase up to pH 9. Shock-wave lithotripsy has been beneficial in a small number of patients.

Ultimate prognosis depends on renal function at the time of diagnosis: late recognition may result in irreversible renal insufficiency requiring chronic dialysis, and early treatment may result in prevention of stones. Kidney transplantation has been followed by recurrence of microcrystalline deposits and subsequent loss of graft function.

Deoxyguanosine Kinase Deficiency

In three large consanguineous kindreds of Druze origin with the hepatocerebral form of mitochondrial DNA depletion syndrome (characterized by early progressive liver failure, neurological abnormalities, hypoglycemia, and increased lactate), homozygosity mapping was used to search for the causative gene defect.113 It led to the identification of a mitochondrial deoxyguanosine kinase deficiency. This enzyme phosphorylates the deoxy counterpart of guanosine (Fig. 168-1) into deoxy GMP. The disorder is discussed in Chapter 176.

Thiopurine Methyltransferase Deficiency

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of several synthetic pharmacological purine analogs that contain a thiol group such as 6-mercaptopurine, 6-thioguanine, and azathioprine that is converted to 6-mercaptopurine in vivo. These drugs are used to treat various diseases, including cancers, rheumatoid arthritis, and other autoimmune disorders, and immunosuppressants after organ transplantation. They are converted via phosphoribosylation by hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase into active thionucleotides, which exert their therapeutic action by incorporation into DNA and RNA. Their oxidation by xanthine oxidase and S-methylation by TPMT results in inactivation.

The wide variations in therapeutic response and occurrence of toxic side effects in some patients receiving thiopurines led to the identification of TPMT as a determining factor in this variability.114,115Approximately 90% of individuals in various ethnic populations have high TPMT activity, about 10% have intermediate activity, and 1 in 300 lack activity. Patients with no or less efficient methylation of thiopurines have more extensive conversion to active thionucleotides that leads to severe, potentially fatal myelosuppression. Therefore, determining the TPMT status prior to treatment with thiopurines is now recommended in predictive pharmacogenetics.116

Metabolic Pathways

Similar to that of the purine nucleotides, the metabolism of the pyrimidine nucleotides can be divided into three pathways (Fig. 168-3):

1. 1. The biosynthetic pathway, also often termed de novo, starts with the formation of the high-energy compound carbamoyl phosphate by cytosolic carbamoyl phosphate synthetase (CPS II). This enzyme is different from mitochondrial CPS I, which catalyzes the first step of ureogenesis. Formation of carbamoyl phosphate is followed by the synthesis of the nucleoside monophosphates uridine monophosphate (UMP), cytidine monophosphate (CMP), their deoxy counterparts, and thymidine monophosphate (TMP).

   2. The pyrimidine catabolic pathway originates from CMP, UMP, and TMP, and after deamination of cytidine into uridine, it yields β-alanine and β-aminoisobutyrate, which are converted into intermediates of the citric acid cycle.

   3. The pyrimidine salvage pathway, which is composed of kinases, converts the pyrimidine nucleosides, cytidine, uridine, and thymidine (which are provided by food intake or the catabolic pathway) into the corresponding nucleotides CMP, UMP, and TMP.

Inborn errors of pyrimidine metabolism comprise errors of pyrimidine nucleotide synthesis, pyrimidine catabolism, and pyrimidine salvage. They cause mostly neurological symptoms, including developmental disability, and muscle wasting.

Hereditary Orotic Aciduria (Ump Synthase Deficiency)

Hereditary orotic aciduria, the first inborn error discovered in pyrimidine metabolism,117 is caused by a deficiency of the last two steps of de novo pyrimidine synthesis—orotate phosphoribosyltransferase (OPRT) and orotidine 5′-monophosphate decarboxylase (ODC). Both steps are catalyzed by a single bifunctional polypeptide called uridine monophosphate (UMP) synthase (Fig. 168-3). Hereditary orotic aciduria is exceedingly rare, with about 20 cases published over nearly five decades. Early recognition of the disorder is mandatory, since it can be easily treated with apparently very good results.

Dihydropyrimidine Dehydrogenase Deficiency

The deficiency of dihydropyrimidine dehydrogenase (DPD; OMIM 274270) was first identified in a patient with developmental problems and overexcretion of uracil and thymine.118 Later on, the defect was also found in subjects with very variable clinical symptoms, including asymptomatic relatives of deficient patients, and in subjects presenting with toxic reactions to 5-fluorouracil.119 This imparted some doubt on the causal relationship between the deficiency, which might be quite common, and the symptoms.

Clinical Presentation

Two forms of DPD deficiency occur. The first is found in children. Approximately half of these display epilepsy, motor retardation, and mental retardation, often accompanied by generalized hypertonia; hyperreflexia; growth retardation; dysmorphic features, including microcephaly; ocular abnormalities; and autistic features.120,121In these patients, the deficiency of DPD is complete or nearly complete. Nevertheless, the severity of the disorder is highly variable, and even asymptomatic cases have been identified. The second clinical picture is found in adults who receive the pyrimidine analog 5-fluorouracil, a classic treatment of various cancers, including breast, ovary, and colon.122 It is characterized by severe life-threatening toxicity, manifested by profound neutropenia; stomatitis; diarrhea; and neurological symptoms, including ataxia, paralysis, and stupor. In these patients, DPD deficiency is as a rule partial and revealed only by 5-fluorouracil therapy.

Metabolic Derangement

DPD catalyzes the first step in the degradation of the pyrimidine bases uracil and thymine, namely their conversion into dihydrouracil and dihydrothymine, respectively (Fig. 168-3). Why a profound DPD deficiency becomes manifest in some pediatric patients but not in others remains an open question and suggests that other factors contribute to the clinical manifestations of the disorder. How the defect leads to neurological symptoms also remains elusive, but reducing the concentration of β-alanine, a neuromodulator that can block the reuptake of GABA, may play a role. A depletion of β-aminoisobutyrate may also play a role. The marked potentiation of the action and thus the toxicity of the anticancer drug 5-fluorouracil, is explained by an impairment of the inactivation of this pyrimidine analog via DPD, which normally accounts for approximately 80% of its catabolism.

Genetics

The human dihydropyrimidine dehydrogenase (DPD) gene has been mapped to chromosome 1p22. About 20 mutations have been identified in DPD deficiency.123–126 Most frequent (approximately 50%) is a G to A mutation in the GT5' splicing recognition sequence of intron 14, IVS14+1G>A (DPYD*2A), which results in a 165-bp deletion corresponding to exon 14 in the DPD mRNA. Strikingly, patients who carry the same mutation may display widely variable clinical symptoms. About 2% of the Dutch population was found heterozygous for the IVS14+1G>A mutation,127 and approximately 25% of the patients with the adult form of DPD deficiency, characterized by 5'-fluorouracil toxicity, are heterozygotes for this mutation.122 A threefold higher prevalence of DPD deficiency has been reported in African Americans as compared with Caucasians.128 The crystal structure of recombinant pig DPD has been resolved.129 Analysis of the DPD crystal structure indicates that several mutations interfere with cofactor binding or electron transport or destabilize the protein.125

Diagnostic Tests

The pyrimidine catabolites can be detected by various techniques, including HPLC, GC-MS, analysis of amino acids in urine before and after acid hydrolysis, and proton NMR spectroscopy.37 Patients excrete high amounts of uracil (56–683 mmol/mol creatinine, as compared to 3–33 in control urine) and thymine (7–439 mmol/mol creatinine, as compared to 0–4 in control urine). Elevations of uracil and thymine in plasma and cerebrospinal fluid are also recorded.120 Excretion of both compounds may be less elevated in patients with high residual DPD activity.

The enzyme defect can be demonstrated in the patient’s fibroblasts, liver, and blood cells, with the exception of erythrocytes.120,122 In pediatric patients, DPD deficiency is complete or nearly complete; in the adult cancer patients experiencing acute 5-fluorouracil toxicity, it is partial, with residual enzyme activities ranging from 3% to 30%.

Treatment and Prognosis

No treatment is available for pediatric patients. Symptoms usually remain the same, but death in early infancy of a more severely affected child has been reported. In the adult cancer patients, discontinuation of 5-fluorouracil results in slow resolution of the toxic symptoms.119,123 Pharmacogenetic testing of patients before administration of 5-fluorouracil should be considered.

Dihydropyrimidinase Deficiency

Deficiency of dihydropyrimidinase (DHP; OMIM 222748) was first reported in a single male baby of consanguineous parents, presenting with convulsions and metabolic acidosis.130 Several other patients have been diagnosed since then.120,121,131 As in dihydropyrimidine dehydrogenase (DPD) deficiency, completely asymptomatic individuals have been identified,132 including siblings of deficient patients.131

In children, complete or nearly complete DHP deficiency presents features similar to those of PDP deficiency described above.

Clinical Presentation

As with DPD deficiency, the clinical picture is very variable and may include severe psychomotor retardation with epilepsy, delay in speech development, growth retardation, dysmorphic features including microcephaly, and white matter abnormalities. Increased sensitivity to 5-fluorouracil, leading to severe toxicity, has also been reported.133

Metabolic Derangement

DHP catalyzes the second step in the degradation of the pyrimidine bases uracil and thymine, namely the cleavage of dihydrouracil and dihydrothymine into β-ureidopropionate and β-ureidoisobutyrate, respectively (Fig. 168-3). Its deficiency provokes accumulation of dihydrouracil and dihydrothymine. The reasons for the appearance and the mechanisms of the symptoms remain unexplained. As in DPD deficiency, reduced concentrations of the neuromodulator β-alanine may play a role. Also as in DPD deficiency, the increased sensitivity to 5-fluorouracil is explained by DHP’s role in the catabolism of this pyrimidine analog.

Genetics

Studies of the DHP gene, localized on chromosome 8q22, have led to the identification of various mutations in symptomatic and asymptomatic individuals.131,134 Enzyme expression showed no significant difference in residual activity between the mutations of the symptomatic and the asymptomatic individuals.

Diagnostic Tests

Urinary dihydrouracil and dihydrothymine, normally found in small amounts, may reach 400 to 600 mmol/mol creatinine.120 They can be measured by the techniques listed above for uracil and thymine in DPD deficiency, with the exception of HPLC analysis with UV detection. Indeed, since dihydropyrimidinase (DHP) catalyzes the opening of the pyrimidine ring, its products, dihydrouracil and dihydrothymine, are not UV-detectable. Nevertheless, moderate (two to twentyfold) elevations of uracil and thymine are also found in the urine of DHP-deficient individuals and may provide a diagnostic clue. Enzyme assay requires liver biopsy, since more accessible tissues do not possess DHP activity.120

Treatment and Prognosis

There is no therapy, and prognosis seems unpredictable. The first reported patient recovered completely and apparently displays normal physical and mental development.130 In contrast, another patient had a progressive neurodegenerative clinical course.135

Ureidopropionase Deficiency

Ureidopropionase deficiency (OMIM 606673) was first identified in a 17-month-old girl of consanguineous parents. She presented with muscle hypotonia, dystonic movements, and severe developmental delay.136 To date, less than 10 patients have been diagnosed.122,137,138

Clinical Presentation

Symptoms of the disorder are widely variable. They include early onset dystonic movement disorder, severe developmental delay with marked impairment of visual responsiveness combined with severely delayed myelination, optic atrophy, pigmentary retinopathy, and cerebellar hypoplasia.139 Another patient presented at 4 months of age with an acute life-threatening febrile status epilepticus.140 At 11 months, he showed severe psychomotor retardation with muscular hypotonia, extremely limited visual contact, and poorly controlled epilepsy. MRI revealed large subdural hematomata and global supratentorial atrophy. Another patient showed congenital urogenital and colorectal anomalies but reached normal developmental milestones.138

Metabolic Derangement

Ureidopropionase (also termed β-alanine synthase) catalyzes the last step of the pyrimidine degradative pathway, the conversion of β-ureidopropionate and β-ureidoisobutyrate into β-alanine and β-aminoisobutyrate, respectively (Fig. 168-3). Its deficiency provokes elevations of ureidopropionic acid (also called ββ) and ureidoisobutyric acid (also called ββ). Studies with cultured chick neurons suggest that β-ureidopropionate may act as a neurotoxin by inhibiting mitochondrial energy metabolism.141 Also, as in DPD and DHP deficiency, a reduction of the concentration of β-alanine may play a role.

Genetics

Genetic analysis of the human β-ureidopropionase gene, mapped to chromosome 22q11.2, has revealed the presence of two splice-site and one missense mutation in the first four patients with β-ureidopropionase deficiency.142

Diagnostic Tests

As with dihydrouracil and dihydrothymine, ureidopropionic acid and ureidoisobutyric acid are not UV-detectable but can be easily detected with HPLC-electrospray tandem mass spectrometry.137 Assay of β-ureidopropionase in liver confirms the diagnosis.122,137

Treatment and Prognosis

A therapeutic trial with β-alanine over 1.5 years has shown no convincing clinical improvement.139

Pyrimidine 5′-Nucleotidase Deficiency

Purine and pyrimidine nucleoside monophosphates are catabolized to their corresponding nucleosides (Figs. 168-1 and 168-3) by a variety of cytosolic and membrane 5′-nucleotidases that may be more specific for one or another type of nucleoside monophosphate. Several pyrimidine 5′-nucleotidases (P5N) exist that dephosphorylate CMP, UMP, their deoxy counterparts, and TMP to the corresponding nucleosides (Fig. 168-3). The deficiency of one these, P5N-1, a 52 kDa an isozyme restricted to erythrocytes that catalyzes the dephosphorylation of UMP and CMP but not of TMP, was identified in patients with severe chronic hemolytic anemia.143

Cytosolic 5′-Nucleotidase Superactivity

This disorder was first identified in the cultured skin fibroblasts of a 3-year-old girl with frequent infections, seizures, developmental delay, severe language deficit, hyperactivity with short attention span, and poor interaction with other children.144 Later on, three additional unrelated patients were diagnosed,145 and at least five other patients have reportedly been identified. Routine laboratory tests were unremarkable except for persistent hypouricosuria. Biochemical studies in the patients’ fibroblasts showed six- to tenfold elevations of the activity of cytosolic 5′-nucleotidase, measured either with a pyrimidine (UMP) or a purine (AMP) as substrate. Electrophoretical abnormalities were also observed.146 The hypouricosuria accompanying the syndrome has been tentatively explained by a 30% to 50% lower concentration of PRPP, as found in patients’ fibroblasts; this is caused by increased salvage of the purine bases derived from accelerated nucleotide catabolism. Decreased PRPP would result in decreased de novo purine synthesis and hence of uric acid production. Based on the possibility that increased catabolism might cause a deficiency of pyrimidine nucleotides, the patients were treated with oral uridine at the dose of 200 to 1000 mg/kg per day. Remarkable developmental improvement and a decrease in frequency of seizures and infections were recorded. Upon replacement of uridine by placebo, all patients rapidly reverted to their pretreatment states.

Thymidine Phosphorylase Deficiency

Thymidine phosphorylase (TP) catalyzes the phosphorolytic cleavage of thymidine (eFig. 168.1 and Fig. 168-3) and deoxyuridine to thymine and uracil, respectively. Deficiency of this enzyme was found147 to cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease associated with multiple deletions and depletion of skeletal muscle mitochondrial DNA (mtDNA). MNGIE is characterized by ptosis, progressive external ophthalmoplegia, severe gastrointestinal dysmotility, cachexia, myopathy, peripheral neuropathy, and lactic acidosis.

Thymidine Kinase Deficiency

Purine and pyrimidine nucleosides can be salvaged to their corresponding nucleoside monophosphates (Figs. 168-1 and 168-3) by a variety of kinases with overlapping substrate specificities. Thymidine kinase 2 (TK2), a key mitochondrial enzyme, phosphorylates thymidine (Fig. 168-3), deoxycytidine, and deoxyuridine into TMP, dCMP, and dUMP, respectively. In several independent patients who developed very severe isolated myopathy in infancy and depletion of muscular mtDNA, reduced activity of TK2 and mutations of its gene were identified.148-151

Metals are indispensable elements of cell biology. They function as cofactors in many specific proteins and are involved in all major metabolic pathways. Their metabolism and implications in inborn errors of metabolism are still not well known, but the number of inherited metabolic disorders involving the absorption, transport, or metabolism of metals is rapidly growing. Clinical presentations are very diverse and can involve all organs and systems, including the liver and the central nervous system (Table 169-1). Deficiency in metals results in metabolic abnormalities due mostly to loss of function of metal-dependent proteins. On the other hand, excess of metals can result in the unregulated oxidation of proteins, lipids, and other cellular components, causing subsequent tissue injury. Some inherited metal disorders are treatable by chelating drugs or by daily supplementation of the missing metal at pharmacological doses.

Copper is readily available in the diet. After absorption through the stomach and duodenum, it is rapidly removed from the portal circulation by hepatocytes. Biliary excretion is the only physiological mechanism of copper elimination, and at steady state, the amount of copper excreted into the bile is equivalent to that absorbed from the intestine.1Ctr1 is a plasma membrane protein essential for early embryonic development, intestinal copper uptake, and transportation from the plasma into the brain.2 Ninety percent of plasma copper is bound to ceruloplasmin. Intracellular copper metabolism is dependent on the copper transport ATPases, ATP7a, and ATP7b. Intracellular trafficking also requires proteins called metallochaperones (superoxide dismutase, Atox1, CCS, Cox 17, Sco1, and Sco2), which direct copper to specific cellular pathways.3 Wilson disease or hepatolenticular degeneration is due to mutations in the ATP7B gene, which encodes a copper-transport protein required for exporting copper from the liver into the bile. Menkes disease is due to mutations in the ATP7A gene, which encodes a copper-transport protein required for the efflux of copper from cells (see Chapter 573).

Ferrous ion (Fe2+) is taken up into cells by the divalent membrane transporter DMT1, whereas ferric iron (Fe3+) enters cells (including brain cells) via endocytosis of the transferrin receptor following binding to transferrin.4 Iron requirements far exceed the gastrointestinal absorption capacity; thus, almost all the iron utilized each day is continuously recycled from internal stores.5 Steap3 is a ferrireductase critical for transferring-mediated iron release into the cell. Ferritin is the predominant storage protein consisting of heavy chains with ferroxidase activity and light chains. Ferroportin is the only cellular iron exporter, and ceruloplasmin is a ferroxidase that mediates efficient cellular iron release. Iron homeostasis is regulated by hepcidin, a circulating peptide that binds to ferroportin, mediating uptake and degradation of this exporter.

Iron enters mitochondria via mitoferrin, and frataxin is a mitochondrial protein mediating Fe-S cluster formation and heme biosynthesis. Although many of the proteins essential for systemic iron homeostasis are expressed within the brain, genetic disorders resulting in loss of functions of these proteins rarely result in brain iron overload or deficiency or neurological disease. Conversely, the concentration of iron in the brain is greatest in the basal ganglia and in astrocytes; this supports the idea that these cells function in iron storage and regulation. Aceruloplasminemia, neuroferritinopathies due to ferritin light chain gene mutations, and neurodegeneration with brain iron accumulation are recent examples of this brain iron metabolism defect6 (Table 169-1; see Chapter 573).

Zinc is a cofactor for over 100 enzymes and as such is involved in all major metabolic pathways. It is also essential for nucleic acid metabolism and protein synthesis and their regulation through zinc-finger proteins. In humans, about 1% of the total body zinc content is replenished daily by the diet.7 This is accomplished principally by tight control of two systems: absorption from the intestine and endogenous loss via pancreatic and other intestinal secretions. The zinc transporter Znt proteins lower intracellular zinc by mediating zinc efflux from cells or influx into intracellular vesicles. SLC30A mutations are responsible for zinc deficiency in breast-fed babies.8 The Zip proteins promote zinc transport from the extracellular fluid or from intracellular vesicles into the cytoplasm. SLC39A mutations are responsible for acrodermatitis enteropathica.9 Zinc deficiency, either hereditary or acquired, has major detrimental effects, whereas high serum zinc has few, probably because of binding to albumin or a2-macroglobulin.

Magnesium is the second most important intracellular cation and plays an essential role in many biochemical processes and in neuromuscular excitability. Its homeostasis is regulated by the interplay between intestinal absorption and renal excretion.

Physiology of manganese homeostasis remains poorly understood, but it seems certain that concentrations in subcellular compartments are closely controlled despite quite large variations in dietary manganese intake (eg, it is higher in vegetarians). Manganese plays an important role in many biological processes. It acts as a cofactor for various enzymes in the cytoplasm, Golgi apparatus, and mitochondria. However, high cytoplasmic concentrations of manganese are cytotoxic. Two Ca2+-ATPases are known to be involved in Mn2+ transport: the Golgi-associated secretory pathway (SPCA) and the sarco/endoplasmic reticulum (SERCA)Ca2+ ATPases.10,11They transport both calcium and manganese. Mutations in ATP2C1, a gene encoding for SPCA1, are responsible for Hailey-Hailey disease11 (OMIM 169600). ATP2C2, which encodes for SPCA2 (a protein thought to be the main candidate for manganese uptake and detoxification via the secretory pathway), was excluded in the cirrhosis/dystonia syndrome.12 Three human SERCA pumps—SERCA1, SERCA2, and SERCA3—encoded by ATP2A1, ATP2A2, and ATP2A3 genes are known. Mutations in the ATP2A1 gene are responsible for Brody disease13 (OMIM 601003), and mutations in the ATP2A2 gene are known to cause Darier disease14 (OMIM 124200). Several of the proteins involved in manganese uptake and transport are shared with those involved in iron intake and transport.15 It is tempting to speculate that similar mechanisms of iron regulation by hepcidin could also exist for manganese homeostasis.12