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Improving diagnosis of spinocerebellar ataxias

Jorge Sequeiros
IBMC – Institute for 
Molecular and Cell Biology, and ICBAS; University of Porto, Portugal

Joanne Martindale
Sheffield Diagnostic Genetics Service, Sheffield Children’s NHS Foundation Trust, Sheffield, UK

Sara Seneca
Center for Medical 
Genetics, UZ Brussel, 
Brussels, Belgium

The spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of autosomal dominant, neurodegenerative disorders.1 The defect is chromosomally mapped or molecularly characterised for about half of them; it involves a (CAG)n expansion for all of the most common forms, but other repeat configurations, deletions and point mutations are also known.2 The diagnostic tests available and laboratories offering them can be found in Orphanet.3

Prevalence of the different types of SCA varies considerably, according to geographic region and population;1 this must be kept in mind by physicians and diagnostic laboratories. Clinical diagnosis of a specific SCA can often be suspected in larger families with several patients exhibiting, altogether,  the full picture of the disease. Some clinical features, when present, may help in the differential diagnosis. This is the case, e.g., for:

Very slow saccadic eye movements in SCA2
Ophthalmoparesis, bulging eyes, and face and tongue fasciculations in MJD/SCA3
Retinopathy in SCA7
Epilepsy in SCA10
Chorea and dementia in SCA17
Myoclonic epilepsy and dementia in DRPLA.

Phenotypic overlap, however, is still considerable, and there may be ample variation within each form and even in a single family. Patients with shorter disease duration, no family history, atypical presentation or age-at-onset, end-stage cases and small families with few patients are more difficult to diagnose. Anticipation of age-at-onset may further challenge a diagnosis. Genotyping is thus required to resolve genetic heterogeneity and provide a definitive diagnosis, which may be important for clinical management and is critical for genetic counselling.

The EMQN quality assessment scheme
The European Molecular Genetics Quality Network (EMQN) is a non-profit organisation committed to improving performance of molecular genetics laboratories throughout Europe and beyond. EMQN administers several external quality assessment (EQA) schemes for a variety of genetic diseases and methodologies.5 Regular participation in EQA helps laboratories to achieve and maintain quality testing and is essential for accreditation under ISO 151898.1

An EQA scheme for the SCAs was started in 2004 and its findings published after the first three years.2  Gross genotyping errors (3% overall) were seen every year, with 30% of the reports showing other minor errors, and 56% showed problems with interpretation and reporting of results. The need was felt for widespread information on appropriate methodologies, standardisation and improvement of accurate repeat sizing, and updated reference ranges for normal and pathogenic alleles. This called for a best practice (BP) meeting, to update BP guidelines for molecular diagnosis of the SCAs.

The best practice meeting and guidelines
A pre-meeting survey was circulated to gain evidence that might inform that process.1 The BP meeting took place in 2007,4 bringing together 32 people from 19 countries, mostly molecular geneticists, but also clinicians and patient representatives. After introductory lectures, small-group discussions and plenary sessions, participants were asked to indicate their major conclusions, which were considered for the drafting of the guidelines. An electronic discussion group then focused on that draft, which was revised and circulated until a consensus document was finalised in 2008. This was submitted to the EMQN board for approval, and then harmonised with BP guidelines existing or being developed for other ‘repeat diseases’.5

The EMQN endorsed BP guidelines were recently published,3 back to back with a discussion paper about the consensus obtained and controversies still existing.1

Preparing for SCA testing
Laboratories should follow the OECD Guidelines for Quality Assurance of Molecular Genetic Testing, 2007,6  as well as the EMQN guidelines for internal quality control and for reporting.5
In addition, the BP guidelines state that all diagnostic laboratories offering molecular genetic testing for the SCAs should participate annually in EQA schemes and are encouraged to seek accreditation.

Also, it is crucial that both in-house developed assays and commercial kits are validated before use. Laboratories must be knowledgeable concerning primers in use, reference allelic ranges and formulae for repeat counting. Routine testing should only be performed when appropriate positive controls are available at the laboratory.

SCA1, SCA2, MJD/SCA3, SCA6 and SCA7 are the minimum panel for laboratories offering genotyping in the SCAs. Each laboratory should then add other loci, based on regional frequency of the various SCA types, including usually rarer forms as DRPLA and SCA17 (particularly in some populations), but also other diseases such as Friedreich’s ataxia, Huntington’s disease and the fragile-X tremor-ataxia syndrome.

Pre-test requirements
Laboratories need to have clear criteria for sample acceptance. The clinical question must be clearly restated, and clinical presentation and family history should be provided. The testing context (diagnostic, presymptomatic, prenatal) must be unequivocal; lack of clinical information may lead to an inadvertent presymptomatic test (PST). PST and prenatal diagnosis (PND) should be requested by a clinical geneticist; other familial samples may be required. Diagnostic testing of minors must be undertaken with great care and PST should not be performed, as it brings no benefit to them.

For PST and PND, the laboratory should ensure that pre-test counselling took place and proper consent was obtained. For PST, the minimum requirement is that DNA is extracted on two separate occasions, but two blood samples are preferred. For direct PND, a sample from the affected parent must be available and maternal contamination excluded.

Analytical methods
Preferred methodologies. Any genotyping for the main SCA loci should be able to detect pathogenic repeat expansions – when present – discriminate alleles one repeat unit apart and size both alleles accurately. Capillary electrophoresis is the method preferred, but other methods suiting those minimal requirements are acceptable. Amplicons must be kept as small as possible to maximise resolution.

Internal controls. Using sequenced alleles as internal controls is highly recommended for accurate repeat sizing. A polymerase chain reaction (PCR) blank and a very large expansion are essential, but an allelic ladder is highly recommended – for example, with a normal heterozygote with alleles one repeat apart, alleles of intermediate size and near the borderlines between size ranges.

Repeat sizing and allele ranges. Exact sizing may be difficult due to mosaic templates and amplification biases; the highest peak or strongest band should be chosen and margin errors defined. Laboratories should size exactly (e.g. by sequencing) normal unstable, intermediate and low penetrance alleles, as well as those where interruptions may be relevant for phenotype or stability (e.g. in SCA1). A margin of ±1 for normal alleles and of ±3 for larger expansions is acceptable for most loci (±1 only for SCA6 expansions). Sizing by standard size ladders may not be accurate, as this is not necessarily a linear conversion.

Other limitations. Sequence variations may lead to non-amplification of normal or expanded alleles. PCR may fail to amplify very large expansions, which is particularly important to bear in mind when testing juvenile or infantile-onset cases. The sensitivity of assays based on triplet repeat sizing may be less than 100% when testing diseases caused also by point mutations (e.g. SCA6).

Homoallelism. Cases of two normal alleles with the same size, particularly an uncommon one, should be confirmed by appropriate methods (e.g., triplet primed-PCR or Southern blotting) and/or typing other relatives, mainly in earlier-onset cases. If homoallelism cannot be confirmed, the possibility of having missed a large expansion must be considered and discussed in the report (particularly important for SCA2 and SCA7, and in any infantile/juvenile cases).

Interpretation and reporting
The Human Genome Variation Society (HGVS) nomenclature is not appropriate to report repeat expansions, but should be used for any point mutations. Both alleles should always be mentioned and reports should assign them to known ranges: (1)normal, (2) large normal or unstable, (3) pathogenicity uncertain, (4) incomplete or (5) full penetrance. Methods used and their limitations should always be included in the report.

Confirmation of a diagnosis has implications that must be discussed, so the need for counselling, offer to test family members and availability of PND should be clearly stated. The report should comment on instability of expanded and intermediate alleles known to undergo further large expansion (e.g. SCA7) and on increased risk with gender of the transmitting parent. If no mutation is detected after a diagnostic request, the report should recommend clinical reassessment and further testing, where appropriate. If a mutation was not found in a PST and a molecular diagnosis had not been confirmed in the family, this must be clearly discussed.

Major difficulties and ongoing debates
The variable motif is the logical part to measure, but defining a repeat is not so easy and is done differently for different SCAs. The repeat motif is a pure (CAG)n in SCA6 and SCA7; however, in SCA1, SCA2, MJD/SCA3 and SCA17 the repeat tract contains variant triplets that are included when counting repeat units. The original reference must be used to ensure consistency and comparability. The significance of interspersions should be well known, as phenotype or pathogenicity may depend on the pure or interrupted pattern of certain repeats.

Testing for other loci should depend on the physician’s request and the clinical and family information provided. Multiple testing will increase false positives and the chance of disclosing undesired information which has not been requested. CAG repeat sizing should not be offered as a diagnostic test for SCA8, unless an expansion proves to segregate with the disease in a large pedigree. Even then, due to its high frequency, finding a SCA8 expansion in a patient does not exclude other causative mutations and this must be discussed in the report.

Laboratories are expected to determine allele sizes; reporting actual allele sizes and margins of error in all cases is much preferred, but still varies with local and regional policies.

Validated, standard and certified reference materials (CRMs) are unavailable currently and need be developed and used for SCA assays. If lymphoblastoid cell lines are to be the source of CRMs, there will be the need to validate each new culture batch.

Additional information
A list of SCA loci and mutations, population frequency and founder effects, definition of the repeat tracts to be measured and of formulae to count repeat units, a list of primers and reference ranges for allele sizes at the main SCA loci are kept and updated at SCAbase2 – an evidence-based online resource in the field of the spinocerebellar ataxias, together with the relevant bibliographic references (


Sequeiros J et al. Consensus and controversies in best practices for molecular genetic testing of spinocerebellar ataxias. Eur J Hum Genet 18: 1188-1195, 2010
SCAbase: an evidence-based online resource in the field of the spinocerebellar ataxias. Available at:
ORPHANET: The portal for rare diseases and orphan drugs. Available at:
Seneca S et al. Experience and outcome of 3 years of a European EQA scheme for genetic testing of the spinocerebellar ataxias. Eur J Hum Genet 2008;16:913–920.
Sequeiros J et al. on behalf of EMQN. EMQN Best Practice Guidelines for molecular genetic testing of SCAs. Eur J Hum Genet 18: 1173-1176, 2010
EuroGentest: Harmonizing genetic testing across Europe. Available at:
European Molecular Genetics Quality Network (EMQN). Available at:
International Organization for Standardization: ISO 15189:2007 – Medical laboratories: Particular requirements for quality and competence. Available at:
OECD/OCDE – Organisation for Economic Co-operation and Development: OECD Guidelines for Quality Assurance in Genetic Testing. Available at: