Advances in genetic diagnosis of neurological disorders

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1 Acta Neurol Scand 2014: 129 (Suppl. 198): DOI: /ane John Wiley & Sons A/S. Published by John Wiley & Sons Ltd ACTA NEUROLOGICA SCANDINAVICA Review Article Advances in genetic diagnosis of neurological disorders Toft M. Advances in genetic diagnosis of neurological disorders. Acta Neurol Scand: 2014: 129 (Suppl. 198): John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Neurogenetics has developed enormously in recent years, and the genetic basis of human disorders is being unravelled rapidly. Many neurological disorders are Mendelian disorders, caused by mutations in genes involved in normal function of the brain, spinal cord, peripheral nerves or muscles. Due to high costs and time-consuming procedures, genetic tests have normally been performed late in the diagnostic process, when clinical examination and other tests have indicated a specific gene as the likely disease cause. Many neurological phenotypes are genetically very heterogeneous, and testing of all possible disease genes has been impossible. As a result, many patients with genetic neurological disorders have remained without a specific diagnosis, even when the disease is caused by mutations in known disease genes. Recent technological advances, in particular nextgeneration DNA sequencing techniques, have resulted in rapid identification of genes involved in Mendelian disorders and provided new possibilities for diagnostic genetic testing. The development of methods for coupling targeted capture and massively parallel DNA sequencing has made it possible to examine a large number of genes in a single reaction. Diagnostic genetic testing can today be performed by the use of gene panels and exome sequencing. This allows a more precise diagnosis of many neurological disorders, and genetic testing should now be considered earlier in the diagnostic procedure. M. Toft Department of Neurology, Oslo University Hospital Rikshospitalet, Oslo, Norway Key words: diagnostic test; genetic; sequencing M. Toft, Department of Neurology, Oslo University Hospital Rikshospitalet, P.O. Box 4950 Nydalen, N-0424 Oslo, Norway Tel.: Fax: mtoft@ous-hf.no Accepted for publication September 12, 2013 Introduction Many neurological disorders are caused by single mutations in genes involved in normal function of the brain, spinal cord, peripheral nerves or muscles. In addition to this, a large number of neurological disorders are so-called complex disorders, caused by a presumed interplay between several genetic and environmental factors. Over the past few years, the field of neurogenetics has developed rapidly and enabled a much greater understanding in the aetiology of many neurological disorders. Recent technological developments have led to an enormous increase in the identification of disease-related genes. These advances have allowed the determination of whole-genome structure and variation and examination of its impact on human phenotypes. Genome-wide association studies have provided information on how common genetic variability influences risk for the development of complex diseases. Furthermore, identification of rare disease-causing mutations has been followed by the discovery of novel biological pathways involved in disease pathogenesis. The purpose of this review is to give an introduction into the field of neurogenetics, and to discuss some of the recent advances in genetic technology. I will also discuss the implications these developments have on the use of genetic testing in the diagnostic process of neurological conditions. Mutations and genes In 1953, Crick and Watson identified the structure of DNA and showed that genetic information exists in the sequence of nucleotides on two 20

2 Genetics of neurological disorders strands of DNA (1). A gene is a sequence of DNA located on a chromosome, and the human genome contains more than 20,000 genes. The sequence of nucleotides in a gene is translated to a chain of amino acids, which in turn spontaneously fold into proteins. Before the human genome sequence was published in 2001, a limited number of disease-related genes had been identified and genetic testing was only performed for a very limited number of disorders. However, the completion of the Human Genome Project provided the necessary basis for the identification of disease genes as it provided an almost complete map of human genes (2). Only a little more than 1% of the human genome encodes genes. The remaining sequence has regulatory or largely unknown functions. Several million variants in the genome sequence exist in the human population. Despite this, more than 99.9% of two individual s genetic sequence is identical. The information in a gene is not always identical in two individuals, these alternative forms are called alleles. Spontaneous changes in genes, referred to as mutations, can give rise to new alleles with different properties. These new alleles can either be recessive or dominant. An individual s allele at a specific locus is called this person s genotype. A mutation is a permanent change in the nucleotide sequence or DNA structure. The consequence of a mutation on the function or amount of proteins is normally the molecular cause of a genetic disease. The vast majority of normally occurring genetic variants in the genome, so-called single-nucleotide polymorphisms (SNPs), are occurring in intronic regions that are not encoding amino acids. These variants therefore do not cause changes of protein sequence, but can influence regulatory elements and gene promoters and thus be involved in disease susceptibility. The most common form of mutations is point mutations caused by the substitution of a single nucleotide. On the protein level, this can cause a substitution of a single amino acid (missense mutation) or the introduction of a premature stop codon (nonsense mutation). Point mutations can also change splicing of exons or influence regulatory regions affecting gene expression. Structural mutations that are adding or deleting small sequences of nucleotides or large parts of chromosomes also exist. In addition, expansions of repeated sequence can also alter gene function and cause neurological disorders. Mutations can cause disease through different mechanisms. A common mechanism is that the mutation leads to a reduced or completely abolished function of the protein. This is typical for autosomal recessive disorders caused by defects of enzymes. Alternatively, the mutation causes disease by a gain of function, with increased protein activity or altered protein function for example in the form of protein aggregation. The genetics of neurological disorders The central nervous system is very complex, and normal function of neurons is depending on the correct performance of thousands of genes and their products. A large number of genetic disorders are therefore affecting functions of the nervous system. There are several different types of inherited genetic disorders, and neurological disease can be caused by all these different mechanisms. Chromosomal disorders This group of disorders is caused by structural mutations of one or several chromosomes. Chromosome abnormalities impair cell functions because of missing or extra chromosomal material (either whole chromosome or segments of chromosomes) or because a structural rearrangement interrupts a gene (3). Chromosome abnormalities are a relatively common cause of developmental disorders, occurring in 1 in 200 individuals. Chromosomal disorders are common syndromes with a complex phenotype and frequently involve intellectual disability. Thus, these syndromes are mostly diagnosed in childhood. However, neurologists are involved in the treatment for symptoms persisting into adulthood as epilepsy and spasticity. A diagnosis of a chromosomal disorder was traditionally based on cytogenetic testing using karyograms and fluorescent in situ hybridization (FISH). Due to recent advances in diagnostic techniques, including the development of arraybased comparative genomic hybridization (acgh), genomic copy number variations can be detected at a much higher resolution level than through traditional methods. This had led to the identification of a large number of chromosomal disorders in the last years. As an example, acgh has been applied to identify the molecular causes of several types of epilepsy (4). Mendelian disorders These disorders are caused by a mutation in a single gene and are therefore also known as monogenic disorders. In Mendelian disorders, 21

3 Toft one copy (for dominant genes) or two copies (for recessive genes) of the mutant gene inevitably lead to the development of the disease. Diseases with such simple inheritance patterns are each relatively rare and constitute a small proportion of all cases of neurological disease. However, the total frequency of all inherited neurological disorders is considerable. There are a many Mendelian neurological disorders, including neuropathies, myopathies, epilepsies, ataxias and other degenerative disorders of the brain and spinal cord. In 1993, one of the first molecular causes of a Mendelian disorder was found when an international consortium identified the gene causing Huntington s disease (5). Huntington s disease is caused by an expansion of a CAG trinucleotide repeat. A number of repeat disorders are caused by abnormal length of a repeated section within a gene, including Friedreich s ataxia, several spinocerebellar ataxias and myotonic dystrophy. Interestingly, a hexanucleotide repeat within the C9orf72 gene has recently been identified in a substantial proportion of both familial and sporadic forms of frontotemporal dementia and amyotrophic lateral sclerosis (6). This indicates that repeat disorders might be even more frequent than appreciated. The most common causes of Mendelian neurological disorders are however missense and nonsense mutations within the coding region of a gene. Many neurological phenotypes are genetically heterogeneous. Different mutations within a single gene can cause the same disease (allelic heterogeneity), and mutations in several different genes can be related to the same clinical expression (locus heterogeneity). For example, a large number of genes have been identified in neuropathies (7), myopathies (8) and ataxias (9). In addition to these more traditional genetic disorders, Mendelian forms of disorders that are normally occurring sporadically have also been identified. As an example, a proportion of patients with Parkinson s disease have inherited mutations in one of several disease genes (10). Even more complex is the aetiology of epilepsies, where a number of environmental causes are also known. In addition to improved diagnostic possibilities, these forms of disease have identified pathways that are involved in disease pathogenesis, providing information that is applicable also for the more common sporadic forms. Complex disorders Complex disorders are presumably caused by interplay between a large number of genetic, environmental and stochastic factors. Genome-wide association studies (GWAS) have identified large numbers of loci that contribute to the genetic basis of complex traits. For example, a large international study of multiple sclerosis, which included Norwegian participants, identified 29 new risk loci in addition to the more than 20 previously known loci (11). Many of the identified genes are involved in immune response, in particular differentiation of T-helper cells, further emphasizing the role of the immune system in the pathogenesis of multiple sclerosis. Despite the identification of many susceptibility genes for complex disorders, these genes collectively contribute very little to disease risk of each individual patient. We recently confirmed a number of disease associations in a large Scandinavian study of Parkinson s disease (12). However, the results from risk-profile analysis showed that the group of individuals with the largest number of risk alleles only had an about three times higher disease risk than individuals with few risk alleles. Thus, the most important contribution of genetic association studies is increased general insights, rather than individual predictions. There is therefore currently a very limited role for genetic testing of complex disorders. Nevertheless, detailed genetic and molecular characterizations might in the future translate into personalized and better treatment options for patients with these common disorders (13). Genetic testing today The neurological diagnostic process starts with the anamnesis followed by a clinical neurological and general examination. This is followed by a variable number of biochemical laboratory test, MRI or other imaging techniques, neurophysiological examinations and other diagnostic tests. Diagnostic genetic tests are being carried out by polymerase chain reaction (PCR) followed by analysis of known frequent mutations, capillary sequencing of known disease genes, or by testing of multiplications, deletions and repeat expansions within these genes (Fig. 1). An updated list of diagnostic genetic tests provided by Norwegian laboratories is available on Diagnostic genetic tests are relatively time-consuming and expensive. Thus, such tests have normally been performed late in the diagnostic process, when the clinical examination and other performed tests have indicated a mutation in a specific gene as the likely disease cause (Fig. 2A). As previously mentioned, many neurological phenotypes are genetically heterogeneous, and complex 22

4 Genetics of neurological disorders A B C Figure 1. Detection of genetic mutations. Different types of mutations have to be tested using different techniques. (A) Gel electrophoresis to separate DNA of different size, for example heterozygous trinucleotide repeat expansions. (B) DNA capillary sequencing to detect nucleotide exchanges. A homozygous point mutation is marked in the electropherogram with an asterix. (C) Semiquantitative PCR to detect deletions and multiplications. A heterozygous deletion in the two samples in the middle is demonstrated by a reduction in gene product by around 50%. A B Figure 2. Current and new strategies for genetic testing in myopathies. (A) Genetic testing of single mutations or genes is currently being performed late in the diagnostic process. (B) With the use of gene panels and exome sequencing, genetic testing of genetically heterogeneous phenotypes could be performed earlier in the diagnostic process. diagnostic algorithms have been proposed, as testing of all possible disease genes has been impossible (Fig. 3) (14). As a result, many patients with genetic neurological disorders have remained without a specific diagnosis, even when the disease is caused by mutations in known disease genes. Technological developments New genomic technologies developed in the last decade have provided unprecedented opportunities to perform large-scale analyses of an individual s genome. Microarray-based techniques as acgh and genome-wide SNP arrays have largely replaced cytogenetic testing. Recently, next-generation DNA sequencing platforms have become widely available, reducing the cost of DNA sequencing by several orders of magnitude compared with traditional capillary sequencing. The development of methods for coupling targeted capture and massively parallel DNA sequencing has made it possible to examine a large number of genes (gene panels) or nearly all of the coding parts of genes (exome sequencing) in a single reaction. Also, whole-genome sequencing is gradually becoming routine, but requires more bioinformatic resources. These advances have resulted in a rapid identification of genes involved in 23

5 Toft bioinformatics analyses than tests using a gene panel, and it increases the likelihood of findings that are unrelated to the neurological disorder. Thus, this technique has several additional ethical issues that must be addressed. Figure 3. Proposed diagnostic algorithm for Charcot Marie Tooth disease (CMT). In patients with suspected autosomal dominant neuropathy, this diagnostic algorithm has been proposed (modified from Li, Semin Neurol 2012). Similar algorithms have been proposed for autosomal recessive neuropathy. Mendelian diseases and provided new possibilities for diagnostic genetic testing (15). Genetic testing in the future The recent tremendous advances in sequencing technology now allow for much broader testing of disease genes than in the past. Several laboratories offer testing of gene panels, where a large number of genes related to a specific phenotype are tested in a single reaction. This is particularly interesting for genetically heterogeneous disorders, especially if other diagnostic tests are cumbersome and expensive. An example is myopathies, in which neurophysiological examinations and muscle biopsies are frequently being performed. For these disorders, genetic testing using gene panels should be considered earlier in the diagnostic process (Fig. 2B). This might result in a molecular diagnosis preventing the use of further diagnostic tests. A recent study using a gene panel to examine patients with hereditary spastic paraparesis identified the genetic cause in 25% of patients, even though the most common genetic form had already been excluded (16). In some patients with unknown familial disorders, complex phenotypes or atypical manifestations exome sequencing might be the test of choice. In exome sequencing, nearly all coding regions of genes are tested, and thus also previously unknown disease genes can be found using this method. Exome sequencing requires more Conclusions Today, diagnostic genetic testing of neurological disorders is normally performed late in the diagnostic process. Due to recent developments in sequencing technology, new genetic disease causes are identified almost on a daily basis. These technological advances also enable comprehensive genetic testing of large panels of candidate genes or the whole exome. This allows a more precise diagnosis of many neurological disorders, and genetic testing should be considered earlier in the diagnostic procedure. Acknowledgments Genetic studies in the author s research group are supported by grants from the Research Council of Norway and the South-Eastern Norway Regional Health Authority. Conflict of interest The author reports no financial conflict of interest. References 1. WATSON JD, CRICK FHC. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature 1953;4356: LANDER ES, LINTON LM, BIRREN B et al. Initial sequencing and analysis of the human genome. Nature 2001; 409: BIESECKER LG, SPINNER NB. A genomic view of mosaicism and human disease. Nat Rev Genet 2013;14: MULLEY JC, MEFFORD HC. Epilepsy and the new cytogenetics. Epilepsia 2011;52: The Huntington s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington s disease chromosomes. Cell 1993;72: VAN LANGENHOVE T, VAN DER ZEE J, VAN BROECKHOVEN C. The molecular basis of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum. Ann Med 2012;44: BRAATHEN GJ. Genetic epidemiology of Charcot-Marie- Tooth disease. Acta Neurol Scand Suppl 2012;193:iv MERCURI E, MUNTONI F. Muscular dystrophies. Lancet 2013;381: HERSHESON J, HAWORTH A, HOULDEN H. The inherited ataxias: genetic heterogeneity, mutation databases, and future directions in research and clinical diagnostics. Hum Mutat 2012;33: HOULDEN H, SINGLETON AB. The genetics and neuropathology of Parkinson s disease. Acta Neuropathol 2012;124:

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