Invitae Amyotrophic Lateral Sclerosis with C9orf72 Panel

Test description

The Invitae Amyotrophic Lateral Sclerosis with C9orf72 Panel analyzes genes that are associated with amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative condition involving loss of upper and lower motor neurons. The genetic heterogeneity associated with these conditions can make it difficult to use phenotype as the sole criterion to select a definitive cause. These genes were curated based on the available evidence to date in order to provide analysis for ALS. This panel includes repeat expansion analysis testing for the hexanucleotide repeat expansion in the C9orf72 gene. Pathogenic expansions in the C9orf72 gene are the most common genetic causes of ALS and frontotemporal dementia. Given the clinical overlap of ALS, broad panel testing allows for an efficient evaluation of several potential genes based on a single clinical indication. Some genes in this test may also be associated with additional unrelated disorders, which are not included in the list of disorders tested. Genetic testing of these genes may help confirm a clinical diagnosis, predict disease prognosis and progression, facilitate early detection of symptoms, inform family planning and genetic counseling, or promote enrollment in clinical trials.

Genes tested

ALS2, ANG, ANXA11, CHCHD10, DCTN1, ERBB4, FUS, HEXA, KIF5A, OPTN, PFN1, SETX, SOD1, SPG11, SQSTM1, TARDBP, TBK1, TFG, UBQLN2 ,VAPB, VCP, C9orf72*

*C9orf72: testing is limited to repeat expansion analysis only, and does not include read-through or CNV analysis. Sizing accuracy is expected to be +/-1 for GGGGCC repeat alleles less than or equal to 31 repeat units. The reported size is corrected for the presence of insertions/deletions (indel) in the downstream variant region. Rarely, certain indel events may negatively impact analytical validity. If the two GGGGCC repeat counts listed are the same, this may indicate that both alleles are the same size (presumed homozygosity) or that one allele went undetected by the assay (allelic dropout). Due to somatic mosaicism, repeat size identified in DNA isolated from peripheral blood, buccal cells, or saliva may not reflect the repeat size in untested tissues (e.g. brain). In addition, a negative result does not definitively rule out the presence of an expansion in the mosaic state, as the current test is not validated to detect low-level mosaic variants. 

DISORDERS TESTED

Gene

Condition

ALS2 Juvenile primary lateral sclerosis (JPLS), juvenile amyotrophic lateral sclerosis (ALS2)
ANG Amyotrophic lateral sclerosis 9 (ALS9)
ANXA11 Amyotrophic lateral sclerosis 23 (ALS23)
C9orf72 Amyotrophic lateral sclerosis and/or frontotemporal dementia 1 (FTDALS1)
CHCHD10 Frontotemporal dementia and/or amyotrophic lateral sclerosis 2 (FTDALS2) 
DCTN1 Perry syndrome, distal hereditary motor neuropathy type VIIB  (HMN7B), amyotrophic lateral sclerosis 1 (ALS1) 
ERBB4 Amyotrophic lateral sclerosis 19 (ALS19)

FUS Amyotrophic lateral sclerosis 6 with or without frontotemporal dementia (ALS6)

HEXA Tay-Sachs disease, beta-hexosaminidase A (HEXA) deficiency
KIF5A Amyotrophic lateral sclerosis 25 (ALS25)
OPTN Amyotrophic lateral sclerosis 12 (ALS12)

PFN1 Amyotrophic lateral sclerosis 18 (ALS18)
SETX Amyotrophic lateral sclerosis 4 (ALS4)
SOD1 Amyotrophic lateral sclerosis 1 (ALS1)
SPG11 Juvenile amyotrophic lateral sclerosis 5 (ALS5)
SQSTM1 Paget disease of bone (PDB3), neurodegeneration with ataxia, dystonia and gaze palsy (NADGP)
TARDBP Amyotrophic lateral sclerosis 10 with or without frontotemporal dementia (ALS10) 
TBK1 Frontotemporal dementia and/or amyotrophic lateral sclerosis 4 (FTDALS4)
TFG Hereditary motor and sensory neuropathy, Okinawa type (HMSNO)
UBQLN2 Amyotrophic lateral sclerosis 15 with or without frontotemporal dementia (ALS15)
VAPB Amyotrophic lateral sclerosis 8 (ALS8)
VCP Inclusion body myopathy with early-onset Paget disease and  frontotemporal dementia 1 (IBMPFD1), amyotrophic lateral  sclerosis 14, with or without frontotemporal dementia (ALS14)

CLINICAL DESCRIPTION

Amyotrophic lateral sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a clinically and genetically heterogeneous neurodegenerative condition involving the loss of upper and lower motor neurons1,2. Signs and symptoms of ALS typically manifest in the fourth or fifth decade of life, and the condition is typically fatal within three to four years after diagnosis1,2,3. The majority of individuals present with weakness of the lower extremities; however, some individuals experience bulbar onset (i.e. difficulty with speech or swallowing), while others present with trunk or respiratory involvement1,4. As neuronal loss progresses, clinical findings eventually include spasticity, weakness, muscle wasting, brisk deep tendon reflexes, and eventually respiratory failure1. Rate of disease progression varies and may be dependent on initial clinical manifestations and/or genotype5,6. Clinical management guidelines for ALS can be found at www.aan.com.

Frontotemporal dementia (FTD)

Frontotemporal dementia (FTD) is a neurodegenerative condition characterized by progressive behavioral and cognitive impairment. Symptom onset typically occurs in the 50s or 60s and is initially characterized by behavioral and personality changes including apathy, restlessness, disinhibition and hyperorality, with later progression to stereotypies, non-fluent aphasia, mutism and dystonia7,8. Individuals often display a marked lack of insight into their symptoms7,8. Disease duration is highly variable; the average duration is approximately 10 years8.

Inheritance

Amyotrophic lateral sclerosis can be inherited in an autosomal dominant, autosomal recessive, or X-linked pattern.

Clinical sensitivity

The clinical sensitivity of this test is dependent on the individual’s underlying genetic condition. For each condition, the table below shows the percentage of clinical cases in which a pathogenic variant is expected through analysis of the genes on this panel. Amyotrophic lateral sclerosis (ALS) is genetically heterogeneous. For conditions not listed in the table, the clinical sensitivity is unknown or not well-established, but these rare disorders represent a small proportion of all ALS.

Disorder

Gene

% of Disorder Attributed to Gene

Amyotrophic lateral sclerosis C9orf72
SOD1
FUS
TARDBP
CHCHD10, MATR3, OPTN, VCP, UBQLN2, SQSTM1
~40% (familial) and ~6% (sporadic)
~12% (familial), ~1-2% (sporadic)
~ 4% (familial), ~1% (sporadic)
~ 4% (familial), ~1% (sporadic)
Rare
Frontotemporal dementia C9orf72
GRN
MAPT
VCP, CHMP2B, TARDBP, FUS, TBK1, SQSTM1, UBQLN2
~18% (familial) and ~6% (sporadic)
5-20%
5-20%
Rare

Prevalence

The annual incidence of ALS is estimated to be 4-8 per 100,000 individuals. The estimated prevalence is approximately 5 per 100,000 in the United States; approximately 30,000 Americans currently have the condition. Up to 17% of all cases of ALS are hereditary. 

Considerations for testing

This test may be appropriate for individuals who present with any of the following:
• A clinical diagnosis or strong clinical suspicion of ALS, especially in those with a family history of ALS or related conditions.
• Clinical features of both ALS and frontotemporal dementia (FTD)
 No personal history of disease (unaffected individuals), but who are known to be at risk for a hereditary form of ALS because of family history

In addition to meeting one of the above criteria, individuals considering genetic testing for hereditary forms of ALS should first receive thorough pre-test genetic counseling from a professional qualified to provide such counseling regarding the implications of testing for neurodegenerative disorders that have no known treatment or cure at this time. 

References

1. Kiernan MC, Vucic S, Cheah BC, Turner MR, Eisen A, Hardiman O, Burrell JR, Zoing MC. Amyotrophic lateral sclerosis. Lancet. 2011 Mar 12;377(9769):942-55. doi: 10.1016/S0140-6736(10)61156-7. Epub 2011 Feb 4. PMID: 21296405.

2. Riva N, Agosta F, Lunetta C, Filippi M, Quattrini A. Recent advances in amyotrophic lateral sclerosis. J Neurol. 2016 Jun;263(6):1241-54. doi: 10.1007/s00415-016-8091-6. Epub 2016 Mar 30. PMID: 27025851; PMCID: PMC4893385.

3. Talbott EO, Malek AM, Lacomis D. The epidemiology of amyotrophic lateral sclerosis. Handb Clin Neurol. 2016;138:225-38. doi: 10.1016/B978-0-12-802973-2.00013-6. PMID: 27637961.

4. Turner MR, Scaber J, Goodfellow JA, Lord ME, Marsden R, Talbot K. The diagnostic pathway and prognosis in bulbar-onset amyotrophic lateral sclerosis. J Neurol Sci. 2010 Jul 15;294(1-2):81-5. doi: 10.1016/j.jns.2010.03.028. Epub 2010 May 10. PMID: 20452624.

5. Yates E, Rafiq MK. Prognostic factors for survival in patients with amyotrophic lateral sclerosis: analysis of a multi-centre clinical trial. J Clin Neurosci. 2016 Oct;32:51-6. doi: 10.1016/j.jocn.2015.12.037. Epub 2016 Jul 9. PMID: 27401224.

6. Knibb JA, Keren N, Kulka A, Leigh PN, Martin S, Shaw CE, Tsuda M, Al-Chalabi A. A clinical tool for predicting survival in ALS. J Neurol Neurosurg Psychiatry. 2016 Dec;87(12):1361-1367. doi: 10.1136/jnnp-2015-312908. Epub 2016 Jul 4. PMID: 27378085; PMCID: PMC5136716.

7. Skibinski G, Parkinson NJ, Brown JM, Chakrabarti L, Lloyd SL, Hummerich H, Nielsen JE, Hodges JR, Spillantini MG, Thusgaard T, Brandner S, Brun A, Rossor MN, Gade A, Johannsen P, Sørensen SA, Gydesen S, Fisher EM, Collinge J. Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nat Genet. 2005 Aug;37(8):806-8. doi: 10.1038/ng1609. Epub 2005 Jul 24. PMID: 16041373.

8. Isaacs AM, Johannsen P, Holm I, Nielsen JE; FReJA consortium. Frontotemporal dementia caused by CHMP2B mutations. Curr Alzheimer Res. 2011 May;8(3):246-51. doi: 10.2174/156720511795563764. PMID: 21222599; PMCID: PMC3182073.

Assay

Assay and technical information

Invitae is a College of American Pathologists (CAP)-accredited and Clinical Laboratory Improvement Amendments (CLIA)-certified clinical diagnostic laboratory performing full-gene sequencing and deletion/duplication analysis using next-generation sequencing technology (NGS).

Based on review of current medical guidelines and peer-reviewed publications, our sequence analysis covers clinically important regions of each gene, including coding exons +/- 10 base pairs of adjacent intronic sequence in the transcript listed below. Any variants that fall outside these regions are not analyzed unless otherwise noted. Unless explicitly guaranteed, sequence changes in the promoter, non-coding exons, and other non-coding regions are not covered by this assay. Specific limitations in the analysis of these genes are included in the list of gene-specific limitations.

Based on validation study results, this assay achieves >99% analytical sensitivity and specificity for single nucleotide variants, insertions and deletions <15bp in length, and exon-level deletions and duplications. Invitae's methods also detect insertions and deletions larger than 15bp but smaller than a full exon but sensitivity for these may be marginally reduced. Invitae’s deletion/duplication analysis determines copy number at a single exon resolution at virtually all targeted exons. However, in rare situations, single-exon copy number events may not be analyzed due to inherent sequence properties or isolated reduction in data quality. Certain types of variants, such as structural rearrangements (e.g. inversions, gene conversion events, translocations, etc.) or variants embedded in sequence with complex architecture (e.g. short tandem repeats or segmental duplications), may not be detected. This test is intended to detect germline variants only. It may not be possible to fully resolve certain details about variants, such as mosaicism, phasing, or mapping ambiguity. This report reflects the analysis of an extracted genomic DNA sample. In very rare cases (such as circulating hematolymphoid neoplasm, bone marrow transplant, recent blood transfusion, or maternal cell contamination), the analyzed DNA may not represent the patient's constitutional genome.

This test is also designed to detect and categorize hexanucleotide (GGGGCC) repeat units in intron 1 of the C9orf72 gene. Analysis of other variant types or other regions is not performed for this gene. Specific limitations in the analysis of this gene is included in the list of gene-specific limitations.

The number of hexanucleotide repeat units in intron 1 of the C9orf72 gene is assessed by repeat-primed PCR (RP-PCR) with fluorescently labeled primers followed by fragment size analysis of the resulting amplicons via capillary electrophoresis (CE). This assay detects and appropriately accounts for the vast majority of indels directly downstream of the repeat region that occur in approximately 3% of the general population and 20-25% of individuals with an expansion in this region (PMID: 27936955). A secondary round of RP-PCR + CE, utilizing a non-overlapping set of primers to amplify the locus from the opposite direction, is used to confirm the initial call in the case of suspected allele sizes of 22 or more repeat units. This test can accurately (+/-1 repeat unit) determine repeat size up to 31 repeat units (Pathogenic full mutation cut-off), but is not designed to determine the exact number of repeat units over 31.

Based on validation study results and previous literature, the C9orf72 assay is expected to achieve >95% analytical sensitivity and specificity for hexanucleotide repeat expansion detection. In rare cases, an individual may harbor sequence variation (insertions/deletions) in the region downstream of the GGGGCC repeat expansion locus. Although this assay detects and appropriately accounts for the vast majority of indels directly downstream of the repeat region, in rare cases we may not be able to PCR amplify both alleles, and a false negative result may be reported in which only a single allele size is called (presumed homozygosity). Due to somatic mosaicism, repeat size identified in DNA isolated from peripheral blood, buccal cells, or saliva may not reflect the repeat size in untested tissues (e.g. brain). In addition, a negative result does not definitively rule out the presence of an expansion in the mosaic state, as the current test is not validated to detect low-level mosaic variants. In very rare cases (such as circulating hematolymphoid neoplasm, bone marrow transplant, recent blood transfusion, or maternal cell contamination), the analyzed DNA may not represent the patient’s constitutional genome. This test does not assess for methylation status.

Detected variants are interpreted using Invitae's Sherloc variant classification framework and reported in our standard diagnostic report format. Benign and Likely Benign variants are not included in the report but are available upon request.

Detected repeat expansions in C9orf72 are interpreted using a modified version of Invitae's Sherloc variant classification framework. Repeat size ranges- Benign (Normal Range): <25 repeat units, Uncertain: 25-30 repeat units, Pathogenic (Full Mutation): >=31 repeat units. Repeat lengths for normal and uncertain alleles are sized and included in the report. The exact size of expansion alleles over 30 repeats (full mutations) are not reported.