Genome Testing Results - GeneDX and Invitae - Crowdsourced Opinions Welcomed

[powerpadman]

My brother, 38-years-old, was diagnosed with ALS about 6 months ago. Even with just one known other family member with ALS, an aunt that passed 15 years ago, my brother's neurologists have said his ALS is likely familial ALS.

However, his initial Invitae DNA panel came back negative, which tested for roughly 30 genes, including the most common ones like C9orf72 and SOD1.

He then took a more comprehensive test through a company called GeneDX called XomeDx, or exome sequencing (ES), which also came back negative. I suppose this likely places my brother in the "unknown" grouping of familial ALS cases.

Here's some more info on the GeneDX test: The XomeDx test targets the protein-coding regions of the human genome, which represents ~20,000 genes and accounts for approximately ~2% of all human genetic material. These targeted regions of an individual’s genes, called exons, are captured and sequenced using massively parallel sequencing. An individual’s sequence is then compared to published reference sequences, other individuals from the affected individual’s family, and control individuals, and phenotype-driven gene lists are generated using Human Phenotype Ontology and HGMD gene-phenotype associations.

The XomeDx test is most effective when other family members (both biological parents, if available) are included in the analysis of the affected individual’s exome sequence. Several large studies have demonstrated that exome sequencing identifies a causal variant in 25-30% of cases, with a higher yield for cases that specifically include other family members. It is anticipated that approximately 98% of the targeted region of an affected individual’s exome will be assessed with the XomeDx test at a minimum of 10x coverage, the minimum read depth necessary to detect a variant.

Our parents also took the text and the only match with any significance was TIA1, which is labeled as a "variant of unknown significance." TIA1 has been hypothesized as associated with ALS, but the evidence is pretty weak. Thus it's not a gene variant that's yet connected to ALS.

So why post all this in this forum? I was wondering if anybody has any opinions or additional info that could be helpful to us or others. Additionally, perhaps posting this here could be helpful for others who are considering genetic testing.

Thanks in advance and I've posted the official test results below. All names and PII have been removed.



Test(s) Requested

Diagnostic Testing / XomeDxPlus / Clinical Exome Sequence Analysis

Clinical Indication

Individual with amyotrophic lateral sclerosis, foot drop, and motor neuron disease.

• Causative variant(s) in disease genes associated with reported phenotype: None Identified
• Variant(s) in disease genes possibly associated with reported phenotype: SEE INTERPRETATION
• ACMG Secondary Findings: None Identified
• mtDNA Test Results: Mitochondrial DNA sequencing and deletion results are reported separately.

Variant(s) in Disease Genes Possibly Associated with Reported Phenotype:

Interpretation

The variant(s) of uncertain significance in the TIA1 gene(s) do not establish a molecular diagnosis.

Recommendation(s)

• Genetic counseling is recommended to discuss the implications of these results.
• Correlation of these findings with the clinical features of this proband is recommended.
• As these results are not diagnostic, this proband and at-risk relatives should receive clinical evaluation and management based on medical and family history. Predictive testing for variants of uncertain significance is not recommended for family members.
• Targeted testing of the parents of this proband and/or other family members, if available, may help determine if the variant in the TIA1 gene occurred de novo or segregates with the phenotype and may assist in further interpretation.

TIA1 - GENE SUMMARY

The TIA1 gene encodes an RNA-binding protein that serves as a key component of stress granules, which are cytoplasmic foci that sequester untranslated mRNAs during different types of cellular stress (PMID: 23401021). The E384K founder mutation in the TIA1 gene causes Welander distal myopathy, an autosomal dominant late onset muscular dystrophy in the Nordic population characterized by initial weakness and atrophy of the fingers with slow progression of symptoms to include involvement of the small hand muscles and lower legs. Histopathology demonstrates myopathic changes and prominent rimmed vacuoles in distal muscles (PMID: 23401021). Electromyographic findings include myopathic and unspecific changes with fibrillations and complex potentials, accompanied by normal nerve conduction studies and normal to slightly elevated creatine kinase levels (PMID: 23348830, 28221306). Individuals who are homozygous for the E384K founder mutation have been reported to be more severely affected with earlier onset, faster progression and proximal muscle involvement (PMID: 23401021). Multiple unrelated individuals with a predominant distal myopathy with rimmed vacuoles have also been identified to be heterozygous for a pathogenic variant in the SQSTM1 gene and also heterozygous for the N357S variant in the TIA1 gene, suggesting possible digenic inheritance for this form of myopathy (PMID: 26627873, 29599744). In addition, an individual with myofibrillar myopathy with hypercapnic respiratory insufficiency and distal weakness was identified to be heterozygous for variants in both SQSTM1 and TIA1; the authors suggest the TIA1 variant may act as a modifier for the SQSTM1 variant (PMID: 29599744). The TIA1 gene has also been proposed as a candidate for amyotrophic lateral sclerosis (ALS)-frontotemporal dementia (FTD), but some of the reported variants have also been found in the general population, suggesting further research is needed to clarify the possible link between variants in TIA1 and ALS-FTD (PMID: 28817800, 29886022).

For this gene, 100% of the coding region was covered at a minimum of 10x by this test. There is no indication of a multi-exon deletion/duplication involving this gene in the sequencing data.

p.(Val294Met) (GTG>ATG): c.880 G>A in exon 11 of the TIA1 gene (NM_022173.2)

The proband's mother (GeneDx #XXXXXXX) is heterozygous for the p.(V294M) variant in the TIA1 gene. The proband's father (GeneDx #XXXXXXX) does not harbor the p.(V294M) variant in the TIA1 gene.

Identified heterozygous in a patient with amyotrophic lateral sclerosis in published literature (PMID: 28817800) In silico analysis supports that this missense variant does not alter protein structure/function

Observed in large population cohorts (gnomAD; internal data)

We interpret this as a Variant of Uncertain Significance.

Additional Analysis Comments

This analysis takes into consideration family structure, reported phenotype, and provided clinical and/or differential diagnosis. Independent analysis of submitted relative samples is not performed unless ordered separately. As this is a phenotype-driven test of a very large number of genes, reported results are focused on pathogenic and likely pathogenic variants in genes related to the phenotype of the proband. Variants of uncertain significance may be reported at the discretion of the laboratory.

Variants identified by previous testing that do not meet the reporting threshold for this test may be included below. A variant is unlikely to be included if any of the following apply: this analysis does not provide additional information (e.g. parental inheritance), the provided nomenclature is illegible or incorrect, or the variant is classified as likely benign or benign, is outside the covered regions of this test, falls within the limitations of testing, or is a single variant in a gene only associated with recessive disease inheritance and no second variant was identified.

ACMG Secondary Findings Limitations

Pathogenic and/or likely pathogenic variants in genes on the ACMG secondary findings v3.2 list are reported for the proband, in accordance with the reporting structure recommended by the ACMG (PMID: 37347242). Only pathogenic and likely pathogenic variants associated with the disorders and inheritance specified by the ACMG are reported (PMID: 34012069, 25741868). Reporting the presence or absence of the proband's identified secondary findings is available for relatives who underwent analysis as part of the proband's test. ACMG secondary findings are not reported for relatives that did not request analysis for ACMG secondary findings. Variants that may be present in a relative, but are not present in the proband, would not be detected and therefore are not reported. The absence of reportable secondary findings for any particular gene does not mean there are no pathogenic, likely pathogenic, or other variants in that gene that may confer susceptibility to the associated disorder.

Additional Samples Submitted

Sample(s) submitted for analysis by next generation sequencing: father (GeneDx #XXXXXXX), mother (GeneDx #XXXXXXX).

Quality Metrics

The above values represent metrics from this test evaluation. 1Mean depth of coverage refers to the mean number of sequence reads obtained across the whole exome; specifically the coding exons and splice junctions of protein-coding RefSeq genes that are captured by massively parallel (NextGen) sequencing. 2The quality threshold refers to the percentage of the test, which is covered by at least 10 sequence reads (10x coverage). This is the analysis threshold level that permits high quality exome variant base calling, annotation and evaluation. Average quality thresholds may range from >90-95% of the targeted region, indicating a small portion of the target region may not be covered with sufficient depth or quality to confidently call variant positions.

Copy Number Variant (CNV) Detection

CNV analysis was performed using the NGS data for this proband. This analysis identifies most clinically relevant deletions and duplications of three or more exons. Only CNVs confirmed by a validated confirmatory method at this laboratory are reported.

Methods

Genomic DNA was extracted directly from the submitted specimen or, if applicable, from cultured fibroblasts. The DNA was enriched for the complete coding regions and splice site junctions for most genes of the human genome using a proprietary capture system developed by GeneDx for next-generation sequencing with CNV calling (NGS-CNV). The enriched targets were simultaneously sequenced with paired-end reads on an Illumina platform. Bi-directional sequence reads were assembled and aligned to reference sequences based on NCBI RefSeq transcripts and human genome build GRCh37/UCSC hg19. Using a custom-developed analysis tool (XomeAnalyzer), data were filtered and analyzed to identify sequence variants and most deletions and duplications involving three or more coding exons (PMID: 25356966). Smaller deletions or duplications may not be reliably identified. Reported variants were confirmed, if necessary, by an appropriate orthogonal method in the proband and, if submitted, in selected relatives. Sequence variants are reported according to the Human Genome Variation Society (HGVS) guidelines. Copy number variants are reported based on the probe coordinates, the coordinates of the exons involved, or precise breakpoints when known. Reportable variants include pathogenic variants and likely pathogenic variants. Variants of uncertain significance, likely benign and benign variants, if present, are not routinely reported. Available evidence for variant classification may change over time and reported variant(s) may be reclassified according to the ACMG/AMP Standards and Guidelines (PMID: 25741868), which may lead to issuing a revised report.

Disclaimer

Genetic testing using the methods applied at GeneDx is expected to be highly accurate. Normal findings do not rule out the diagnosis of a genetic disorder since some genetic abnormalities may be undetectable by this test. Unless otherwise indicated, the methods used cannot reliably detect deletions of 20bp to 500bp in size, or insertions of 10bp to 500bp in size. Sequencing cannot detect low-level mosaicism. Copy number assessment methods cannot reliably detect mosaicism and cannot identify balanced chromosome aberrations. Regions of certain genes have inherent sequence properties (for example: repeat, homology, or pseudogene regions, high GC content, rare polymorphisms) that yield suboptimal data, potentially impairing accuracy of the results. Inaccurate results may occur in the setting of allogeneic bone marrow/stem cell transplantation, active or chronic hematologic conditions, recent blood transfusion, suboptimal DNA quality, or in other rare circumstances. DNA extracted at other laboratories may negatively affect test performance. Rarely, incidental findings of large chromosomal rearrangements outside the gene of interest may be identified. As the ability to detect genetic variants and naming conventions can differ among laboratories, rare false negative results may occur when no positive control is provided for testing of a specific variant identified at another laboratory. In addition, the chance of an erroneous result due to laboratory errors incurred during any phase of testing cannot be completely excluded. Interpretations are made with the assumption that any clinical information provided, including family relationships, are accurate. Consultation with a genetics professional is recommended for interpretation of results.

This test was developed and its performance characteristics determined by GeneDx. This test has not been cleared or approved by the U.S. Food and Drug Administration. The FDA has determined that such clearance or approval is not necessary. The test is used for clinical purposes and should not be regarded as investigational or for research. The laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA) as qualified to perform high-complexity clinical testing.

Variant Table

This supplement provides evidence to support the classification of each reportable variant in the attached result report. This information is provided as a resource. It is not inclusive of all available information used by GeneDx for variant classification, and individual data elements may be weighted differently to derive at the classification. This information is subject to change and may differ from what is currently available. Results should always be interpreted in the context of the patient's clinical presentation. Blank fields indicate that no data were available at time of analysis.

• dbSNP - NCBI repository for single base nucleotide substitutions and short deletion and insertion polymorphisms https://www.ncbi.nlm.nih.gov/snp/
• The Genome Aggregation Database (gnomAD) combines exome and genome sequencing data from a variety of large-scale sequencing projects, including approximately 15,000 genomes and 123,000 exomes, including individuals recruited for disease-specific studies such as cancer and cardiovascular diseases. (PMID 32461654).
• gnomAD_Freq - variant allele frequency (in percent) from approximately 15,000 genomes and 123,000 exomes in the Genome Aggregation Database. Select ancestries include: gnomAD_AMR (Admixed American/Latino); gnomAD_AFR (African); gnomAD_EAS (East Asian); gnomAD_FIN (Finnish of European ancestry); gnomAD_NFE (non-Finnish of European ancestry); gnomAD_SAS (South Asian); gnomAD_ASJ (Ashkenazi Jewish). gnomAD_Hom - number of individuals homozygous for the variant.
• gnomAD_AMR- variant frequency (in percent) for individuals of Latino ancestry
• PROVEAN (Protein Variation Effect Analyzer) - predicts whether an amino acid substitution or indel affects the biological function of a protein using a delta alignment score from
• -14 to +14 (< or = -2.5, predicted deleterious; >-2.5, predicted neutral).
• Other published in silico algorithms, including those that predict splicing impact, may be considered for variant analysis. In silico scores may change. In silico models use algorithms that predict the effect a variant may have on the protein. Thus, predictions should be interpreted with caution and only be used in combination with other available evidence to support the classification of any variant (PMID 23056405).
• ClinVar - Classification of variant in ClinVar database, an NCBI archive of human variants with supporting evidence of phenotypic association. https://www.ncbi.nlm.nih.gov/clinvar/ (PMID 26582918).

Resources

These clinician and family resources are of an informational nature only. While GeneDx makes every effort to ensure its accuracy, this information is not meant to endorse a particular group, be a complete list, nor serve as medical advice. GeneDx presents this opportunity as a courtesy to clinicians and families who may wish to obtain more details. All GeneDx genetic test reports include this Resource page, no matter the positive or negative result.

General support and advocacy

Child Neurology Foundation www.ChildNeurologyFoundation.org (859) 551-4977

Global Genes www.GlobalGenes.org (949) 248-7273

National Organization for Rare Disorders www.RareDiseases.org

(617) 249-7300

Information exchange and connection

GenomeConnect: www.GenomeConnect.org MyGene2: www.MyGene2.org

 

[Nikki J]

So the aunt presumably had no testing. Your brother who has ALS does not carry this variant. Your mother who does carry it does not have ALS or a related condition like FTD. Is this all accurate? It is what it says a variant of unknown significance and if your brother doesn’t have it then it can’t be contributing to his disease. I hope this finding isn’t causing your mother undue worry.

These are called vus because current science can’t say anything more. Associated with ALS can be and often turns out to be just coincidence. We know your brother’s ALS has nothing to do with this variant because he doesn’t have it but if he had inherited it it would have become a data point for an association.

It is likely that as you say there is some undiscovered genetic factor here. It might be causative with decreased penetrance because your parent and their parents don’t/ didn’t have ALS but if there is a mutation then one of the grandparents had to have it. It is also possible there is a susceptibility mutation not yet discovered. I recently heard Dr Garret of MGH differentiate causative mutations like sod1 and c9 from susceptibility ones like ATXN2 and UNC13A which only increase risk slightly (1-2%). It is also not impossible it is a miserable coincidence. One of my c9 relatives who indubitably got his c9 from his mother had a paternal cousin with ALS

 

[powerpadman]

Yes that is correct. Aunt didn't get tested, perhaps because it wasn't as common back then (2009) and because there wasn't any family history, despite lots of offspring up the family tree one notch. Mother is presumably a carrier because her sister (our aunt) died from ALS. One clarifying point: my brother and mother both have the TIA1 gene, the variant of uncertain significance.

So the most likely scenario is that our grandmother was a carrier (and even though she was one of 8 children, neither her nor her siblings had ALS). And then at least 2 of her 4 children were carriers, our aunt, who passed from ALS, and our mother, who doesn't have ALS. And then the mother passed it to at least my brother. I am his only sibling and I haven't had signs of ALS.

Can you explain or link more about susceptibility mutations? I'd be curious to dig in more on that.

Thanks for your guidance!

 

[Nikki J]

Why grandmother and not grandfather? Just curious

Mark Garret is the only person I have heard make this distinction though I have seen others reference it in passing. He gave two talks that were I think pretty much the same. One was an everythingals a month or two ago and the other around the same time was a healy platform webinar. Everything has a youtube channel. Healey platform webinars are archived on the Mgh healey center website. Mark is the doctor working on DIALS at Mgh. It is a study for carriers and potential carriers

 

[powerpadman]

Ok thank you. I will dig in on Everything ALS's YouTube videos. I've watched their videos before.

Good callout - we're not 100% sure it's our grandmother, but it's thought to potentially be a higher likelihood with her over my grandfather. This is because my grandfather has a brother with multiple children and grandchildren and nobody on that side has ALS.