Chapter 21. Genetic Testing For Neurological Disorders

Many of the neurological disorders discussed in this book have a genetic etiology that can be diagnosed and/or confirmed by chromosomal or molecular testing. Due to advances in genetic technology, the range of testing options has increased dramatically in the last few years. Deciding which test to perform and how to interpret the results can be daunting. Accordingly, in this chapter we review the different types of clinically available genetic tests. These can be employed for general screening or for confirmation of a clinically diagnosed condition. The general portfolio of such testing is illustrated in Table 21-1, and this chapter will review these types of tests.

A routine chromosome study (karyotype) involves evaluating the chromosomes during the metaphase period of cell mitosis, and the test is usually performed on blood cells. Lymphocytes are grown in culture and stained with a dye such as Giemsa to obtain a distinct pattern of light and dark bands. This banding pattern is unique to each of the 46 chromosomes, and helps to identify them on microscopic examination (Figure 21-1). A routine chromosome study can identify extra or missing chromosomes (eg, Down [47, XY+21] and Klinefelter [47, XXY] syndromes) as well as detect large inversions, translocations, deletions, duplications, and extra fragments (eg, marker chromosomes). The visual resolution of this study, however, is only at the 10 to 20 million base pair level (Table 21-2). Higher-resolution tests are available (as discussed below in Array-Based Comparative Genomic Hybridization) that detect much smaller changes in the chromosome.

Individuals who present with developmental delay and/or mental retardation should prompt the clinician to consider ordering at least a routine chromosome study. A chromosome study is especially indicated when a developmentally delayed child has any of these associated conditions:

• Single or multiple malformations
• Seizures
• Dysmorphic facial features
• Short stature
• Autism
• Family history of mental retardation

Fluorescence in-situ hybridization (FISH) detects the presence or absence of a large DNA sequence involving a particular gene or chromosome region. In this technique, test DNA is first separated into single strands and then hybridized with a fluorescent-labeled DNA probe (usually 0.3-1.0 Mb in size) that is complementary to the region of interest. If the area under study is present, the labeled DNA will bind to it. This can be visualized as a fluorescent spot under the microscope, as illustrated in Figure 21-2 for the 1p36.3 microdeletion condition. This deletion is usually not detected on a routine chromosome study.1 During the last decade, the development of FISH led to the identification of various "microdeletion syndromes" (Table 21-3).2-4 However, to be able to judiciously order the appropriate FISH study, one must be aware of the clinical phenotypes of the specific microdeletion syndromes. To overcome this limitation, FISH screening panels have been developed to test simultaneously for multiple microdeletion syndromes.5 An example of this is the subtelomere FISH panel that can detect minute rearrangements in the subtelomeric regions of all the chromosomes. Subtelomeric deletions or duplications are responsible for some cases of unexplained developmental delay that were otherwise not diagnosed by a routine karyotype.6

Array-based comparative genomic hybridization (array-CGH) can detect alterations in copy number of DNA sequences for specific chromosome regions.7 In this technique, the DNA of the patient and a normal control are labeled with fluorescent dyes (green and red, respectively) and both DNAs are hybridized to normal cloned DNA fragments embedded in a silicon target chip. The exact locations of these DNA fragments on the chromosomes are known. The DNA sequences from both sources compete for their targets on the chip, and images of both fluorescent signals are then captured. Regions equally represented in both samples appear yellow (because both the red and green fluorochromes are detected), deleted regions appear red, and amplified regions are green (Figure 21-3). In this way, a global overview of gains and losses throughout the entire test genome is obtained.8 Microarray analysis can identify deletions and duplications of the loci represented on the microarray chip, with the resolution being a function of the number and size of target fragments used and the genomic distance between each of the fragments.9 Using array-CGH, detection rates for chromosome abnormalities range from 5% to 17% in individuals with developmental delay who have had prior routine cytogenetic testing.9-11Figure 21-4 illustrates the utility of this test in a case of identical twins that otherwise had normal chromosome study and extensive FISH testing. Because of the high sensitivity of array-CGH, false positives can occur due to the presence of genomic regions of duplications and deletions that are evolutionarily derived and are of no clinical consequence (often termed copy number variants). In such cases, the parents must be tested to see if they too carry the copy number variation. Array-CGH will not be able to detect balanced translocations because in these situations there is no loss or gain of chromosome material.

Molecular genetic testing reviewed herein includes tests to detect mutations, gene deletions, abnormal number of nucleotide repeats, abnormal DNA methylation, and uniparental disomy. A mutation in a gene can have different effects, and some of the common changes are illustrated in Figure 21-5. A pathogenic mutation either eliminates the protein that the gene codes for, or affects the structure and ultimately the function of the translated protein. Gene mutations can be identified either by targeted mutation analysis (searching for common known mutations) or by sequence analysis of the entire coding region of the gene. These can be achieved by using techniques such as Southern blot analysis or polymerase chain reaction (PCR). In Southern blot analysis, restriction fragment enzymes are used to break the DNA into fragments of different size. The DNA fragments are loaded onto an agarose gel and separated by applying an electric current. If the test DNA has mutations, bands with abnormal lengths are formed. PCR is a method that allows the DNA region of interest to be amplified in an exponential manner using a DNA polymerase enzyme and an amplification reaction that is repeated over and over again, producing multiple copies of the DNA region of interest (eg, 20 cycles will produce over 100,000 copies). The PCR product can be analyzed by several methods. For sequence analysis of the gene, nucleotides tagged to fluorescent dyes are added to the PCR reaction, and a laser within an automated DNA sequencing machine is used to analyze the DNA for sequence changes.

DNA sequencing can provide unequivocal diagnosis when a known pathogenic mutation is identified, as illustrated in the case of Sotos syndrome (Figure 21-6), in which the child is found to have a missense mutation. The mutation is determined to be pathogenic because this sequence variant has also been identified in other cases of Sotos syndrome. However, some missense mutations can be normal variants, so extra care is needed in interpreting this type of mutation change. This caveat becomes especially important when multiple genes are sequenced as part of a test panel. For example, the Charcot-Marie-Tooth group of disorders (ie, the hereditary sensory neuropathies) is caused by mutations in many different genes.12 Genetic screening for these genes can detect several missense mutations of unknown significance, and unless the missense mutation has previously been shown to be pathogenic, the significance of these mutations may be difficult to determine. Furthermore, it should be stressed that gene sequencing may not be able to detect a complete gene deletion, as only the normal allele present would be amplified and sequenced. Accordingly, in certain disorders, studies to detect gene deletions should also be pursued when the sequence testing does not identify a mutation (Table 21-4). For example, in the Sotos syndrome, 10% of individuals can have a complete gene deletion but this can be diagnosed using a FISH probe for the NSD1 gene region.13,14

Normally, some genes have stable repeating sequences within their nucleotide structure. Up to a certain number of repeats is considered normal; beyond this, the individual is at risk of either developing disease or passing an expansion on to his or her offspring that will cause disease in them. There are over 40 neurological disorders that have been linked to what are called "unstable" repeat expansions.15 Fragile X syndrome, which causes mild to severe mental retardation (MR), is caused by an unstable trinucleotide expansion in the FMR1 gene (Figure 21-7). The diagnosis of fragile X syndrome should be considered in boys with MR or autism who have nonspecific physical examination findings. In premutation carrier females, the number of triplet repeats can expand during gametogenesis and can cause transmission of a full mutation to an offspring. Full mutation causes moderate MR in males and can cause mild MR in females. Premutation carrier females can develop premature ovarian failure, and carrier males, in late adulthood, can develop fragile X-associated tremor/ataxia characterized by late-onset, progressive cerebellar ataxia and intention tremor.16 Fragile X syndrome and other repeat expansion disorders can be diagnosed using PCR or Southern blotting. See Table 21-5 for a list of common pediatric repeat expansion disorders.

Genomic imprinting is a phenomenon in which expression of a gene on a particular chromosome depends on the parent of origin of the chromosome. This effect is achieved by silencing the gene derived from one parent. This silencing is associated with changes in DNA methylation of the imprinted gene/gene region during gametogenesis. A clinical disorder can occur if the child does not inherit the active copy of the gene. In such cases, the active gene is typically disrupted by a microdeletion. This disruption can be caused by other mechanisms such as uniparental disomy. Such absence is detected by an abnormal DNA methylation pattern for the gene of interest (Fig. 21-8).

Uniparental disomy (UPD) means that both chromosomes of a pair are derived from the same parent.17 For example, in Prader-Willi syndrome, about 30% of cases are caused by maternal UPD for chromosome 15. UPD can be detected by PCR using parent-specific short tandem repeat markers and more recently single nucleotide polymorphism (SNP) microarrays.18 Parental blood samples must be tested along with the child's sample, and the UPD is thus confirmed when a child fails to inherit alleles from one parent for one specific chromosome.19

In oxidative phosphorylation, ATP is produced by the enzymes of the mitochondrial respiratory chain. The respiratory chain is arranged in five complexes that are embedded in the inner mitochondrial membrane (Figure 21-9). The genes that code for the various components of the mitochondrial respiratory chain are present on the nuclear as well as the mitochondrial genome. Of the 87 protein components of the respiratory chain, 13 are encoded by genes in the mitochondrial genome. Respiratory chain dysfunction often leads to symptoms in multiple organs or systems, and this reflects the ubiquitous importance of ATP.20

In a neonate or young infant, the neurological presentation of a disorder of mitochondrial function can be one of poor feeding, muscle hypotonia, decreased arousal, abnormal movements, and seizures. Laboratory testing often shows impressive lactic acidosis. Presentations in later childhood include gaze paralysis, external ophthalmoplegia (from brainstem involvement), weakness, ataxia, seizures, myoclonus, and stroke-like episodes. Other types of multiorgan involvement can occur. Many individuals display a cluster of symptoms described as clinical syndromes, such as MELAS (mitochondrial encephalopathy with lactic acidosis), MERRF (myoclonic epilepsy with ragged red ribers), and NARP (neurogenic weakness with ataxia and retinitis pigmentosa).20

Homoplasmy is a state in which thousands of copies of mitochondrial DNA (mtDNA) in each of our cells are identical, as would be expected for someone having a normal mitochondrial genome. However, most individuals with mitochondrial disorders due to mutations in the mtDNA have a mixture of normal and abnormal mtDNA in their cells; this is termed heteroplasmy. This may be an explanation for the varied clinical phenotypes seen in individuals with the same mtDNA abnormality.21

Genetic testing for mitochondrial disorders includes molecular testing to look for mutations in genes present on the mitochondrial or nuclear genome. For molecular testing for mtDNA, Southern blot analysis will detect deletions or duplications, while targeted mutation analysis looks for known common mutations. Sequencing of the entire mtDNA (18,000 bps) can also be performed. If an abnormality is found, it will confirm the diagnosis, but if no abnormality is found the diagnosis cannot be excluded.

The child in the Figure 21-10 was found to have a point mutation in the gene coding for one of the subunits of complex V (ATP synthase) in 65% of his mtDNA in blood, which indicates that he is heteroplasmic in blood for this mutation. He has mental retardation, ataxia, and muscle weakness, and had been diagnosed with cerebral palsy before the mtDNA mutation was discovered. Mutation analysis in his case tested only for a panel of common mtDNA mutations (eg, mutations associated with NARP, MELAS, and MERRF), and a point mutation was indeed identified. Currently, the entire mtDNA can be sequenced, although in clinical practice this is rarely done. There is also limited testing for nuclear gene mutations.

Electron transport function (ETF) studies are usually performed on fresh or flash-frozen muscle or liver tissue. ETF studies measure the consumption of oxygen by mitochondria in the presence of various substrates.

• Complex I: Impaired respiration with NADH-producing substrates
• Complex II: Impaired respiration with FADH2-producing substrates
• Complex III and IV: Impaired respiration with NADH and FADH2 substrates
• Complex V: Impaired respiration with various substrates and correction with addition of an uncoupling agent, suggesting abnormality in phosphorylation20

ETF studies provide evidence for either focal or general impairment of the respiratory chain. However, the ETF enzymatic phenotype (eg, a 50% reduction in complex I and III activities) is usually not sufficient to confirm the clinical suspicion of a mitochondrial disorder. In such cases, additional mitochondrial or nuclear DNA testing is needed and careful clinical correlation is required.

Biochemical testing often includes serum amino acids, lactate, pyruvate, and acylcarnitine profile determinations. Urine screening can include testing for organic acids, mucopolysaccharides, and oligosaccharrides. Selected neurotransmitters can be measured on CSF, but these tests are not generally included under the rubric of metabolic testing. Serum amino acid, acylcarnitine profile, and urine organic acid screening tests provide a reasonable first approach for evaluation of a child presenting with episodic illness or failure to thrive. These will detect most biochemical disorders associated with defects in amino acid, organic acid, and fatty acid metabolism.

When a particular disorder is suspected based on an abnormal metabolic profile, enzyme assays can be performed to confirm the diagnosis. However, in some cases the clinician may decide to proceed to confirmatory DNA testing or may elect to first perform DNA testing prior to enzyme confirmation. Identification of the mutation(s) in the affected individual then allows other family members to be tested for affected or carrier status. Enzyme analysis is not usually the best method to detect carrier status, as the carrier can produce enough enzyme activity so as to overlap the range of activity for normal controls.

Lysosomal storage diseases are usually neurodegenerative diseases and include Niemann–Pick disease, mucopolysaccharidoses, Krabbe disease, Gaucher disease, and metachromatic leukodystrophy. When a lysosomal enzyme is significantly reduced, its substrate accumulates in the cell, leading to the disorder. Enzyme panels are available to screen for various lysosomal disorders, and based on clinical features and other supporting tests, individual patients are screened for selected ones. The panel usually includes a list of enzymes and many of them can be ruled out clinically.

The clinical evaluation involves obtaining a detailed family history to identify familial inheritance patterns. Recognizing a specific inheritance pattern can help focus the clinical differential diagnosis and guide clinical testing. For example, the hereditary spastic parapareses (SPG disorders) can be inherited in an autosomal dominant, autosomal recessive, or X-linked recessive manner, and each has a different set of associated genes.22 Identifying the inheritance pattern can thus help focus the diagnosis and limit the extent of genetic testing.

The most important inheritance patterns are illustrated in Figure 21-11 and summarized below.

• Autosomal dominant: Only one abnormal allele is required to cause disease. With every pregnancy, the affected parent has a 50% chance of transmitting the abnormal allele to the offspring.
• Autosomal recessive: Two abnormal alleles are required to cause disease. Parents of the affected child are usually unaffected and are carriers. With each pregnancy, the parents have a 25% chance of having an affected child and a 50% chance that the child will be a carrier.
• X-linked recessive: Caused by a mutation in a gene on the X chromosome. All males who carry the mutation express the phenotype. Carrier females do not generally express the phenotype. Due to the process of X chromosome inactivation, carrier females could have varying degrees of clinical expression. The probability that a carrier female can transmit the mutated gene to her children is 50% at each pregnancy. The females who inherit the mutation will be carriers and males who inherit it will be affected.
• Mitochondrial inheritance: The mitochondrial genome is different from the nuclear genome because it is only transmitted by the mother to her offspring. A mother carrying the mutation will transmit it to 100% of her offspring, but they may or may not be symptomatic depending usually on the percentage of mutant mtDNA and the age of the individual.
• Polygenic inheritance: Also known as multifactorial inheritance, and probably caused by the influence of multiple genes that act in confluence with environmental factors. Examples of such inheritance include cleft lip with or without cleft palate and spina bifida. Although polygenic traits disorders can occur in multiple family members, no recognizable pattern of inheritance is typically observed. A risk of 3% to 10% can usually be assessed for first-degree relatives (eg, siblings, children) of an affected individual.

There are excellent online resources that provide current information about genetic disorders. Two important ones are listed here:

• OMIM (Online Mendelian Inheritance in Man): www.ncbi.nlm.nih.gov/omim/ This is a database of genetic disorders and their genes that show a Mendelian inheritance pattern.
• Genetests: www.ncbi.nlm.nih.gov/sites/GeneTests/?db=Gene Tests This site contains disease reviews and a directory of genetic testing laboratories.