The genetic diagnosis of different spinocerebellar ataxia subtypes is based on different technical approaches. Three main methods can be used: fragment length analysis, sequencing, and deletion/duplication testing.
Note: for the latest update on all SCA subtypes known to date, please read here.
FRAGMENT LENGTH ANALYSIS (FLA)
Fragments lengths analysis (with or without associated repeat primed assay) can be used to screen for repeat expansions. Several genetic subtypes of autosomal dominantly inherited SCAs are caused by a repeat expansion in a particular gene. Fragment length analysis (FLA) is based on amplification by PCR of the gene region including the repeats and subsequent capillary electrophoresis on a Sanger sequencer. FLA is relatively easy to perform, yet quite operator-dependent in the interpretation. The identification of normal and expanded alleles is easy, but extremely expanded alleles may not be caught by the technique. Indeed, if the expansion is very large, the amplification of the expanded allele will not take place and the final result will be homozygosity for the normal, non-pathogenic allele (the only one that the PCR primers are able to amplify). Therefore, especially for those subtypes of SCAs where very largely expanded alleles have been reported, it is essential to always go for further investigations in case of homozygous results. For this purpose, some labs are offering Southern blot analysis, some other ones the so-called repeat primed assay (RPA). By Southern blotting or RPA it is possible to ultimately say if the patient is a real homozygote or not, thus the diagnosis can be finally confirmed or excluded.
SEQUENCING
Many other subtypes of SCAs are caused by a point mutation or a small deletion or insertion of few nucleotides. These mutations can be detected by sequencing (either Sanger sequencing or Next Generation Sequencing). Most of these mutations are located within the coding region of the genes or at the exon/intron boundaries. Sequencing usually shows a very high sensitivity and specificity. Therefore, if the patient really carries a mutation in the coding region, this will be almost surely detected by the test. However, since routine sequencing does not analyze deep intronic
regions, it is not known how many patients missing a genetic diagnosis
may carry deep intronic mutations.
DELETION/DUPLICATION TESTING
Some of the SCA subtypes described to be caused by point mutations can also be caused, in some patients, by large deletions or duplications encompassing part of or the entire gene. These mutations are in a heterozygous state in autosomal dominant SCAs and usually in a homozygous state in autosomal recessively inherited SCAs. Large homozygous deletions are usually detectable by failed amplification before sequencing, whereas large homozygous duplications or large heterozygous deletions and duplications are not detectable by sequencing. A clear example is given by ITPR1 mutations, which are known to cause autosomal dominant SCA15 or SCA29. ITPR1 mutations described date are consistent with a large heterozygous deletion in all but very few pedigrees, in which a point mutation was described. It is therefore clear that sequencing is not a recommended approach for the first screening of ITPR1 mutations in a patient with suspected SCA15 or SCA29. Actually the first approach to ITPR1 analysis should deletion/duplication testing by qPCR.
Deletion/duplication testing can be done either by multiplex ligation probe amplification (MLPA) or by quantitative polymerase chain reaction (qPCR). MLPA is usually preferred if the commercial kit (MRC-Holland) is available. Whenever MLPA kits are not available, the laboratory can establish a custom qPCR assay.
