C9orf72 and the Care of the Patient With ALS or FTD

Current Status of Testing and Counseling

Clinical testing for the C9orf72 HRE has been available since 2012.¹⁴ Currently available commercial assays include repeat-primed PCR (RP-PCR), fluorescent amplicon length analysis, and Southern blot. United States ALS management guidelines do not address the offer of genetic testing, whereas European guidelines specify that testing be offered to patients with familial disease.^15,16 Several published clinician surveys suggest a growing consensus to offer genetic testing to patients with familial ALS, but the offer of testing to the typical ALS patient with sporadic disease remains inconsistent.¹⁷ Table 1 summarizes published clinician practice surveys.

Patient experience and attitudes toward genetic testing in ALS have been investigated in several studies. In a 2015 survey of US ALS patients, 33.4% of respondents (n = 449) reported being offered genetic testing, including 68.9% of those with familial and 27.5% of those with sporadic ALS. Respondents expressed positive attitudes toward genetic testing and perceived benefits to ALS patients, families, the medical community, and society; this was most pronounced in those who reported a positive family history of ALS. Among those who had testing, those who received positive and negative results reported equally favorable experiences, suggesting that those who get “bad news” maintained a favorable outlook about the benefits of testing.^18,19 Attitudes toward presymptomatic ALS testing have also been studied; in a study of family members of persons with ALS, 60% indicated they would have presymptomatic testing if offered.²⁰

Challenges in Laboratory Detection and Interpretation of the HRE

PCR-based assays for detection of the HRE are widely used in commercial laboratories, but their accuracy has been questioned. In a 2014 blinded study, only 5/14 laboratories reported PCR-based C9orf72 results in complete concordance with the reference Southern blot result, and both false negative and false positive results were identified.²¹ Insertions and deletions adjacent to the repeat have been shown to interfere with detection of the expansion using PCR-based assays.²² Southern blot has been advocated as the gold standard for clinical testing.⁶ Currently, it is the only commercial test capable of sizing large repeats, but few laboratories offer it, and it may not detect intermediate repeats or small expansions.²³ In the future, alternative methods for detecting and sizing the HRE may be commercially available, such as short- and long-read sequencing techniques that are currently used in the research setting.

In addition, laboratory interpretation and reporting of the HRE is inconsistent. Published surveys of commercial laboratory tests reveal that cutoffs for normal, intermediate, and expanded alleles varied widely. Cutoffs for intermediate alleles ranged from 20 to several hundred; cutoffs for pathogenic alleles ranged from >23 to >700 (figure). Intermediate or small expansions could therefore be interpreted differently by different laboratories, and such cases have been reported.^23,24 There is currently no validated cutoff that differentiates between pathogenic and nonpathogenic alleles.

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Figure Variable Interpretations of the C9orf72 Hexanucleotide Repeat Number Among Commercial Laboratories

Finally, the C9orf72 HRE shows somatic and germline instability further complicating result interpretation. For example, expansion sizes as measured in blood may differ from those in relevant neural tissues.²⁵ Intermediate alleles identified in patients with ALS may be incidental or could be associated with larger, pathogenic expansions in the brain.⁶ The size of the HRE as measured in blood may change over time in the same individual,²⁶ although this has not been observed to increase from the normal to pathogenic range. Repeat instability also occurs in the germline, but evidence does not support the common occurrence of genetic and clinical anticipation as seen in other repeat expansion disorders such as myotonic dystrophy type 1 and Fragile X syndrome.²⁶ Contraction of the HRE in successive generations has recently been demonstrated, particularly with paternal transmission.^26,27

Unanswered Questions Regarding Disease Expression

When patients with C9orf72 HRE present with symptoms of motor neuron disease, the phenotype is often indistinguishable from classic ALS. Although the most common cognitive phenotype is behavioral variant FTD, a range of cognitive-behavioral presentations are observed, including psychotic features such as delusions and hallucinations. Patients presenting with neuropsychiatric changes may not be recognized and diagnosed with C9orf72-related disease.⁶ Repeat number has not been robustly shown to correlate with age at onset, disease duration, or clinical presentation (ALS and/or FTD),^25,28 although this remains an active area of investigation.

Early family studies suggested that nearly all HRE carriers develop disease by the age of 80 years.^9,29 However, the identification of the HRE in 0.2%–0.6% of unaffected controls and the presence of elderly, neurologically healthy obligate HRE carriers in affected families support incomplete penetrance.^5,25,29 Penetrance has not been established to correlate with repeat size.²⁶ A recent study modeled HRE penetrance in a large cohort drawn from published literature, concluding that lifetime penetrance is incomplete (estimated at ∼90% by the age of 80 years).²⁹ Oligogenic inheritance has been proposed to contribute to the variable penetrance of the HRE and other pathogenic ALS variants; however, evidence in support of this model is lacking.³⁰

Incomplete Knowledge of C9orf72 HRE Prevalence

HRE frequencies are highest in Scandinavian countries^9,31 and lowest in Asian populations.^{9,10,12,13,31} HRE frequencies in other populations, however, including non-White or admixed populations in the United States and Europe have not been studied extensively. Tables e-1 and e-2 (links.lww.com/NXG/A345) summarize published frequencies by geoancestry.

All Persons With ALS and/or FTD of European Geoancestry Are Candidates for HRE Testing

Many persons with ALS and/or FTD wish to know as much as possible about why they developed the condition and the risk that family members could also be affected. A genetic diagnosis enables clinicians to provide personalized, specific answers to these questions, and affected persons and their relatives may feel empowered by the knowledge and the opportunity to act, whether for reproductive planning, presymptomatic testing, or research participation. Testing of persons with ALS and/or FTD, but without a known family history of either condition, identifies the HRE in up to 10%.³² HRE testing should be offered to any patient who meets clinical diagnostic criteria for either condition.³³ The current availability of clinical trials targeting HRE pathology in ALS underscores the importance of HRE testing in ALS management. Although pathogenic variation in more 30 other genes has been established to cause ALS and/or FTD, these account for a smaller proportion of cases in populations of European descent.³⁴ In addition, uncertain or indeterminate results are rare with C9orf72 HRE testing, occurring in less than 1% cases; most patients get a clear positive or negative result.

Amplicon Length Analysis With Bidirectional RP-PCR Is Suitable for Large Scale HRE Testing

The RP-PCR assays widely used in commercial testing for the HRE are cost-effective, rapid, and identify patients with an expanded allele by revealing a “saw-tooth pattern” when a significant repeat expansion is present. However, as noted previously, interpretation of C9orf72 RP-PCR results is not always straightforward. Amplicon length analysis, however, is a reliable method for sizing intermediate and smaller HREs and may be helpful in resolving cases in which sequence variants interfere with RP-PCR results. A combination of amplicon-length analysis and bidirectional 5′ and 3′ RP-PCR is recommended to obtain the highest level of sensitivity and specificity.^21,22 The use of 2 RP-PCR assays from both the 3′ and 5′ end of the repeat region and 2 nonoverlapping primer sets for amplicon length analysis buffers against potential allele drop out due to neighboring sequence variants and aids in determining zygosity in patients with HRE (Luke Drury, PhD, written communication, August 20, 2020). Although this approach cannot size large expansions, there are currently no established correlations between expansion size and disease expression. This combined technique currently represents the best approach to the clinical detection of the C9orf72 HRE on a large scale.

Southern blot or advanced sequencing techniques may be necessary in rare cases, such as those in which RP-PCR results are discrepant. Southern blot may also be considered if PCR detects only one allele, particularly with a rare repeat size, to determine whether the individual is truly homozygous or whether an HRE is present that failed to amplify.³⁵ Recently published testing guidelines for myotonic dystrophy, type 2, include performing Southern blot in all apparently homozygous cases.³⁶ Although up to 34% of individuals are expected to have homozygous C9orf72 repeat sizes, rare homozygous repeats (3, 7, 11, and 12) are found in less than 1% (Matthew Harms, MD, written communication, April 1, 2020); such cases could be targeted for additional testing. Southern blot and other methods capable of sizing the HRE remain important tools in the research setting and may be clinically necessary in the future if HRE size is clearly shown to affect disease expression.

Genetic Counseling Should Be Provided to All Affected Persons

All persons with ALS or FTD should be provided genetic counseling, irrespective of whether they have a positive family history of ALS or FTD or are seeking genetic testing. A discussion of ALS and/or FTD genetics should introduce genetic heterogeneity, dominant transmission, disease expression, and penetrance, leading to the offer of C9orf72 HRE testing for persons with European geoancestry.

Develop Evidence-based Guidelines for ALS and FTD Genetic Testing and Counseling

There is currently no consistent approach to the offer of testing for the typical ALS or FTD patient, who has no family history of the disease. Many patients who desire testing and counseling are not offered these services.¹⁸ A recent survey of ALS clinicians revealed that 74% would be more likely to offer ALS genetic testing if testing guidelines were published.¹⁷ Genetic testing guidelines have been published for other neurodegenerative diseases such as Huntington and Alzheimer disease^40,41 and successfully incorporated into patient care. Consistent genetic testing practices would also support the identification of appropriate patients for gene-targeted trials and therapies.

Although further study is needed regarding the minimum HRE size necessary to cause disease, commercial laboratories are in urgent need of guidance to ensure consistent interpretations of HRE sizes. Currently, intermediate or smaller expansions could be interpreted differently at different laboratory tests, potentially leading to discrepant and/or inaccurate results for patients or family members undergoing diagnostic or presymptomatic testing.^23,24 Laboratories, clinicians, and patients could benefit from guidelines establishing consistent HRE cutoffs, which will likely require updating as new information becomes available. Given that some affected HRE carriers have been found to carry pathogenic variants in other genes, it is unclear whether additional genetic should be offered after a patient tests positive for the HRE. Guidance is also needed to arm genetic counselors and other clinicians with a consistent message regarding penetrance of the C9orf72 HRE. The development of evidence-based guidelines that encompass HRE test indication, methodology, interpretation, and genetic counseling would support clinicians and laboratories in providing consistent and equitable testing and care.

Prioritize C9orf72 as a Target for Clinical Trials

Recent data from clinic-based cohorts has shown that the relative contribution of the C9orf72 HRE, as compared to other ALS genes, is even greater than initial estimates suggested. Among more than 500 ALS patients tested via the ALS Genetic Access program of the Northeast ALS Consortium, the HRE accounted for 75% of all pathogenic or likely pathogenic variants identified in the 6 genes tested (unpublished data). Similarly, the HRE comprised 79% of pathogenic or likely pathogenic variants detected in a Midwestern clinic cohort; testing included comprehensive multigene panel testing for familial or early onset cases. The incidence of pathogenic or likely pathogenic variants in SOD1 and in genes other than C9orf72 in clinic-based ALS cases in the United States may be lower than that reported in research cohorts.³⁹ Accordingly, research and industry should prioritize the C9orf72 HRE as a target for therapy. Carriers of the HRE are present in every ALS and FTD clinic, and with consistent testing practices, they will be increasingly identified. They represent a rich opportunity to study the natural history of the condition, identify early biomarkers of disease, and ultimately, develop gene-targeted therapies and neuroprotective interventions. With coordinated effort from the scientific community, the next generation of HRE carriers may stand to benefit.

Engage Diverse Patients in Research

Further study is needed to better understand the incidence of the HRE in diverse populations. Our current understanding of the familial and genetic basis of ALS and FTD is based on the study of White individuals, and data thus derived (such as published empiric recurrence risks) may not be applicable to other populations.³⁷ Although the HRE has been identified in persons with ALS in Europe, North and South America, and East and South Asia,⁹ incidence has not been extensively studied in many populations. Data on the genetic profile of sporadic and familial ALS and FTD in persons of diverse geoancestry are critical to determine the need for population-specific testing approaches. It is currently unclear, for example, whether the universal offer of HRE testing is warranted in affected persons of non-European descent. Likewise, for familial ALS or FTD cases of non-European descent, it is uncertain whether the first step of testing should be multigene panel testing or HRE testing. The success of ALS and FTD research hinges on successfully recruiting and retaining patients of diverse backgrounds. Table 4 summarizes practice recommendations and goals for future research.