Wallace, Stephanie E. MD; Bird, Thomas D. MD. Molecular genetic testing for hereditary ataxia: What every neurologist should know. Neurology: Clinical Practice. 8(1), February 2018, p 27–32.
Purpose of review: Because of extensive clinical overlap among many forms of hereditary ataxia, molecular genetic testing is often required to establish a diagnosis. Interrogation of multiple genes has become a popular diagnostic approach as the cost of sequence analysis has decreased and the number of genes associated with overlapping phenotypes has increased. We describe the benefits and limitations of molecular genetic tests commonly used to determine the etiology of hereditary ataxia.
Recent findings: There are more than 300 hereditary disorders associated with ataxia. The most common causes of hereditary ataxia are expansion of nucleotide repeats within 7 genes: ATXN1, ATXN2, ATXN3, ATXN7, ATXN8, CACNA1A (spinocerebellar ataxia type 6), and FXN (Friedreich ataxia). Recent reports describing the use of clinical exome sequencing to identify causes of hereditary ataxia may lead neurologists to start their clinical investigation with a less sensitive molecular test providing a misleading “negative” result.
Summary: The majority of individuals with hereditary ataxias have nucleotide repeat expansions, pathogenic variants that are not detectable with clinical exome sequencing. Multigene panels that include specific assays to determine nucleotide repeat lengths should be considered first in individuals with hereditary ataxia.
From the article
Multigene panels including a variety of genes associated with ataxia are clinically available. These panels may or may not include specific assays to determine nucleotide repeat lengths within the genes most commonly associated with hereditary ataxia. Therefore, pathogenic mutations in these common ataxia genes may be missed. Neurologists should be careful to choose the best set of tests most likely to identify the genetic cause of ataxia in any given patient.
Hereditary ataxias are a group of more than 100 genetic disorders primarily characterized by slowly progressive incoordination of gait. Additional features often include poor coordination of the upper extremities, abnormal eye movements, and dysarthria.
Ataxia also occurs in hundreds of additional genetic disorders not considered primary hereditary ataxias. Multiple inheritance patterns occur in this large group of disorders, including autosomal dominant, autosomal recessive, X-linked, and mitochondrial. Specific treatments are beneficial in individuals with a few of the known hereditary ataxias including ataxia with vitamin E deficiency, Refsum disease, cerebrotendinous xanthomatosis, and CoQ10 deficiency. Establishing a diagnosis in an individual with ataxia can clarify recurrence risk and lead to specific treatments for some individuals.
Distinguishing clinical features or a positive family history of ataxia can suggest a specific diagnosis in some individuals. However, the number of genes associated with hereditary ataxia continues to increase. Clinicians are faced with the challenge of trying to identify a diagnosis in an individual presenting with ataxia as our understanding of the clinical spectrum of many hereditary ataxias is expanding. Results of molecular genetic testing have broadened the phenotypes to include mildly affected individuals and individuals with clinical findings that differ from previously established diagnostic criteria. Simultaneous interrogation of multiple genes using clinical exome sequencing or a large multigene panel is increasingly reported as an efficient method for identifying a diagnosis in individuals with ataxia.3–5 However, the most common causes of hereditary ataxia are due to nucleotide repeat expansions that would not be identified by these sequencing techniques.
A nucleotide repeat is a sequence of nucleotides repeated a number of times in tandem; nucleotide repeats can occur within or near a gene. The size of nucleotide repeats varies. Smaller numbers of repeats are common and not often associated with phenotypic abnormalities. Abnormally large numbers of repeats may be associated with phenotypic abnormalities and are classified as (in increasing order of size) mutable normal alleles, premutations, reduced-penetrance alleles, and full-penetrance alleles.
Nucleotide repeats increase the risk of DNA replication errors, which can lead to an expansion or contraction of the number of repeats. Larger nucleotide repeats are associated with increased severity of symptoms (full-penetrance alleles). When the size of a nucleotide repeat increases from one generation to the next, anticipation is observed. Anticipation is the tendency in certain genetic disorders for individuals in successive generations to present at an earlier age or with more severe manifestations. Larger nucleotide repeats can also expand during cell division, leading to variable nucleotide repeat sizes in neighboring cells.
Molecular genetic testing used to sequence a nucleotide repeat is more difficult than sequencing nonrepetitive regions of the exome because many of the known nucleotide repeats contain a higher proportion of guanine and cytosine nucleotides compared to adenine and thymine nucleotides. Regions with high guanine and cytosine content are more difficult to amplify by PCR. In addition, repetitive regions do not align uniquely; thus, the length and therefore the pathogenicity of the repeated sequence cannot be determined.
Specific assays are required to analyze each nucleotide repeat of interest. DNA containing smaller nucleotide repeats can be amplified by PCR. The amplified segments of DNA are then separated by gel or capillary electrophoresis to determine repeat length. Highly expanded nucleotide repeats may not be detected by PCR-based assays due to difficulty in aligning the sequence to a unique genomic position. Additional testing (e.g., Southern blot analysis or triplet repeat primed PCR) may be required to determine the length of highly expanded nucleotide repeats.