-It is great to see how our solutions are impacting your real-life clinical research and streamlining your decision-making when it matters the most. On the implementation side, how was your experience with the Setup program for SOPHiA DDM^TM in terms of easiness, time spent on validation, and time to routine?

-In our experience, the Setup program for SOPHiA DDM^TM was very fast and easy. In fact, after the training with SOPHiA customer support, we spent just a working for validating the entire process and personalizing the information flow before introducing it in the routine analysis. This is very important and efficient for the users.

-And how does your experience with the SOPHiA GENETICS customer support look like?

-I am extremely satisfied with SOPHiA GENETICS customer support because they are efficient and proactive, answering all our queries and addressing our issues in a very short time and with great competence and kindness. I would like to thank you on behalf of the entire team for that.

We would like to warmly thank Dr. Davide Martorana for joining us in this customer spotlight.

Are you interested in exploring how the New Generation SOPHiA DDM Platform can revolutionize your workflows? Check out our recent blog post here! 

The term « SOPHIA » used by the speaker refers to SOPHiA GENETICS and its products. The opinions expressed during this presentation are these of the speaker and may not represent the opinions of SOPHiA GENETICS. SOPHiA GENETICS does not provide support in the validation of custom products for clinical use. SOPHiA DDM™ Dx Homologous Recombination Deficiency Solution is available as a CE-IVD product for In Vitro Diagnostic Use in the European Economic Area (EEA), the United Kingdom and Switzerland. SOPHiA GENETICS products are for Research Use Only and not for use in diagnostic procedures unless specified otherwise.

What is cascade testing?

Cascade testing is the practice of offering genetic testing to relatives of known carriers of pathogenic variants associated with autosomal dominant conditions. In oncology, cascade testing is performed in families affected by hereditary cancer syndromes¹. The most common include hereditary breast and ovarian cancer syndrome (HBOC), Lynch syndrome (LS), familial adenomatous polyposis syndrome, hereditary pancreatic cancer syndrome and gastric cancer syndrome^2,3. Testing for variants associated with HBOC and LS belongs to the so-called Tier 1 testing, i.e., genomic applications, the implementation of which is supported by robust evidence⁴.

Cascade testing starts with first-degree relatives (parents, siblings, children) of index cases (i.e., the family member in whom a pathogenic variant was identified) and then proceeds to second- (grandparents/grandchildren, aunts/uncles, nieces/nephews, half-siblings) and third-degree relatives (great-grandparents/great-grandchildren, first cousins)¹.

Most hereditary cancer syndromes follow the autosomal dominant inheritance pattern. Therefore, the first-, second- and third-degree relatives have, respectively, a 50%, 25%, and 12.5% probability of inheriting the predisposition to develop cancer (see Figure 1)¹. For some pathogenic variants of genes associated with hereditary cancer syndromes, such as BRCA1/2, the penetrance is high⁵. Establishing accurate estimates of penetrance and relative risk for genes implicated in hereditary cancer syndromes is an ongoing task³.

Figure 1. Heritable pathogenic variants increase the risk of developing cancer at a younger age (left). Cascade genetic testing is the practice of testing the relatives of known carriers (right)¹.

Why is cascade testing important?

At the level of an individual and their family, cascade testing has two important goals. The first goal is to identify relatives that carry the familial pathogenic variant and require personalized cancer risk management¹. The second goal is to exclude the non-carriers from intensive cancer surveillance and prevention interventions¹. The detection of pathogenic variants in individuals at a reproductive age may lead to decisions of assisted reproduction or prenatal diagnosis. In the case of actionable monogenic conditions, cascade testing may reduce adverse health outcomes in cohorts of relatives¹.

At the societal level, cascade testing has important clinical and research implications for oncology. It can further our knowledge of hereditary cancers and is a cost-effective way of identifying unaffected individuals at-risk, thus, providing important information to plan long-term resources necessary to cope with hereditary cancers. Moreover, today’s testing is needed to tailor future approaches in cascade testing¹.

Figure 2. Cascade testing involves genetic counseling before and after the test, risk estimation and management, and has treatment implication⁷.

What are the barriers to cascade testing? 

Despite the advantages of cascade testing, its uptake is low. The reported rates of uptake of cascade testing in HBOC and LS equals ~50% and the underutilization of testing results in missed opportunities of cancer prevention¹. In a recent Swiss study, there was a 25-50% response rate to invitations to cascade testing and at least one-in-three individuals at risk did not undergo testing. An index case possesses an average of 10 relatives eligible for testing, while the average rate of genetic tests per index case is only 1.5¹.

There are several barriers to cascade testing^6,7. These include ineffective family communication of genetic risk information, low knowledge of cascade testing among index cases and primary care providers, and geographic barriers to receiving genetic services. Cascade testing uptake is also lower among male than female relatives and in distant compared to first-degree relatives. A facilitator of adherence to cascade testing is the parents’ desire to understand their children’s risk⁶. “Dear family” letters, digital chatbots (a technology-based simulated conversations), and direct contact programs have been shown to be effective in motivating cascade testing⁸.

Several initiatives exist to promote cascade testing. One such enterprise is the Cascade Resources Network, an independently run, non-profit organization that offers access to genetic testing, genetic counseling, variant interpretation, screening guidelines, and forums and support. It was developed by Memorial Sloan Kettering Cancer Center (MSK) fellows, Ryan Kahn and Sushmita Gordhandas. The network was created to increase the rate of genetic testing among relatives of patients with inherited cancer risk variants to help identify cancer early in families and, ultimately, to prevent future cancers. Similarly, the Swiss Cancer Genetic Predisposition Cascade Screening Consortium was established in 2016 to foster research related to the hereditary cancer predisposition. In particular, the Consortium promotes the CASCADE cohort, a family-based open-ended cohort targeting HBOC and LS variant-harboring families to elicit factors that enhance adherence to testing (NCT03124212).

Analyze genetic predisposition to cancer with the SOPHiA DDM™ Platform

Multi-gene testing is an efficient, affordable, and guideline-recommended⁹ approach to cascade testing as it allows for comprehensive assessment of biologically relevant hereditary cancer genes. The SOPHiA DDM™ Platform supports various next generation sequencing (NGS)-based Hereditary Cancer Applications to help clinician researchers characterize the complex mutational landscape associated with hereditary cancer disorders. 

Powered by advanced analytics, users can detect challenging variants in a streamlined sample-to-report workflow, including:

• Single nucleotide variants (SNVs), insertions and deletions (Indels) and copy number variations (CNVs)
• Boland inversions
• Alu insertions
• PMS2 and PMS2CL variants

Variant pathogenicity levels are assigned using machine learning complemented by guideline-driven ranking, helping to prioritize relevant variants and reduce interpretation time. Furthermore, deeper variant exploration is supported by Alamut™ Visual Plus, a full-genome browser that integrates numerous curated genomic and literature databases, guidelines, missense and splicing predictors.

To learn more about SOPHiA DDM™ for Hereditary Cancers, explore here or request a demo here.

References

1. Sarki M, et al. Cancers (Basel) 2022;14:1636.

2. Brown GR, et al. JAAPA 2020;33(12):10-16.

3. Mighton C, Lerner-Ellis JP. Genes Chromosomes Cancer 2022;61(6):356-381.

4. Dotson WD, et al. Clin Pharmacol Ther 2014;95(4):394-402.

5. Chen S, Parmigiani G. J Clin Oncol 2007;25(11):1329-1333.

6. Roberts MC, et al. Health Aff (Millwood) 2018;37(5):801-808.

7. O'Neill SC, et al. Hered Cancer Clin Pract 2021;19(1):40.

8. Campbell-Salome G, et al. (2022) Transl Behav Med 2022;12(7):800–809.

9. Daly MB, et al. J Natl Compr Canc Netw. 2021 Jan 6;19(1):77-102.

The HGVS nomenclature guidelines are used worldwide for genetic variant interpretation but can seem complicated and difficult to understand and apply. That is why we have created this beginner’s guide to mutation nomenclature using the HGVS recommendations, with clear visual examples that break down the process into bitesize pieces.

1. What is HGVS nomenclature?
2. How to read mutation nomenclature: Breaking down the variant description
2.1 Reference sequence e.g., NM
2.2 Description of variant e.g., c.4375C>T
2.3 Predicted consequence e.g., p.(Arg1459*)
3. The 3 prime rule for mutation
4. Final thoughts and helpful tool

1. What is HGVS nomenclature?

The Human Genome Variation Society (HGVS) nomenclature standard was developed to prevent the misinterpretation of variants in DNA, RNA, and protein sequences. The HGVS nomenclature standard is used worldwide, especially in clinical diagnostics, and is authorized by the Human Genome Organisation (HUGO).^1,2

HGVS General Terminology Recommendations¹

HGVS follow recognized standards for the nomenclature of DNA and RNA nucleotides, the genetic code, amino acid descriptions, and cytogenetic band position in chromosomes.³

2. How to read mutation nomenclature: Breaking down the variant description

The HGVS recommendations for mutation nomenclature state that the format of a complete variant description should first include the reference sequence, followed by the variant description, and then the predicted consequence in parentheses. For example, NM-004006.2:c.4375C>T p.(Arg1459*) (Figure 1).

2.1. How to read mutation nomenclature: Reference Sequence

The HGVS nomenclature recommendations for sequence variants state that a complete variant description should begin with the reference sequence.¹ The reference sequence accession number begins with a two-letter abbreviation (explained in Table 1), followed by a multi-digit number, and finally a version number.

Table 1. Meaning of the two-letter abbreviation at the beginning of a reference sequence accession number.

2.2. How to read mutation nomenclature: Description of variant

The variant description begins by depicting the type of reference sequence used (c = coding DNA sequence, g = genomic reference sequence). When a protein-coding reference sequence is used (c), the nucleotide numbering begins with a 1, which represents the first position in the protein-coding region (the A of the translation-initiating ATG), and ends at the last position of the stop codon. Thus, if you divide the position number by 3, you can identify the affected amino acid in the protein sequence e.g., using the same example as above, 4375/3 = 1459, indicating that the predicted consequence affects amino acid 1459, which is an arginine. Different variants are indicated using different notations (explained in Table 2).

Table 2. HGVS notation and examples for the most common types of mutations²

2.3. How to read mutation nomenclature: Predicted consequence

When only DNA has been analyzed, the RNA- and protein-level consequences of the variant can only be predicted, and should thus be reported in parentheses e.g., p.(Arg1459*) is the predicted effect at protein level (p) for the example described above.

3. The 3 prime rule for mutation nomenclature

For all variant descriptions using HGVS nomenclature, the nucleotide at the most 3’ position of the variation in the reference sequence is arbitrarily assigned to have changed (see how to apply this rule in Figure 2).⁴ The exception is for deletions/duplications around exon junctions for which shifting the variant 3’ would place it in the next exon.⁵

4. Final thoughts and helpful tool

Although the HGVS recommendations can be difficult to understand and might take a bit of getting used to, if you break them down and refer to the examples in this guide, you are on the road to success!

If you want to accelerate your variant annotation and interpretation, Alamut™ Visual Plus is a comprehensive, full genome browser for efficient and user-friendly variant interpretation. The software accelerates the complex and time-consuming assessment of variants thanks to its user-friendly interface and integrated features for variant annotation and analysis.​

Find out how Alamut™ Visual Plus applies the HGVS nomenclature recommendations to ensure that variant annotation follows the universally applied standards for variant analysis, interpretation, and reporting in our dedicated Technical Note.

Alamut™️ Visual Plus is for Research Use Only. Not for use in diagnostic procedures.

References

1. https://varnomen.hgvs.org/bg-material/basics/
2. http://varnomen.hgvs.org/bg-material/simple/
3. http://varnomen.hgvs.org/bg-material/standards/
4. http://varnomen.hgvs.org/recommendations/general/
5. http://varnomen.hgvs.org/bg-material/numbering/#DNAc

At the American Society of Human Genetics (ASHG) Annual Meeting this year, our esteemed speakers shared the ins and outs of how the SOPHiA DDM™ Platform, in combination with Alamut™ Visual Plus, adapted to their laboratories’ needs to provide sample-to-report workflows that streamlined the identification and interpretation of nuclear and mitochondrial variants associated with rare and inherited diseases, including hereditary cancers.

All SOPHiA GENETICS™️ products discussed in this article are for Research Use Only – not for use in diagnostic procedures. SOPHiA GENETICS™ does not facilitate and does not accept any liability for any validation of SOPHiA GENETICS™ products for clinical use by a third party.

Exome sequencing with combined mitochondrial genome sequencing for the detection of nuclear and mitochondrial DNA variants

Jessica Van Ziffle, PhD, FACMG Associate Clinical Professor, Pathology at University of California, San Francisco, California, United States

In the Pathology laboratory at the University of California, the proportion of positive/probably positive variants detected in pediatric and prenatal cases analyzed using the SOPHiA DDM™ Custom Whole Exome Solution (optimized for the Illumina NovaSeq 6000) was consistent with the literature (see charts). Approximately 70% of the positive pediatric cases were associated with autosomal dominant inheritance. Most reported variants were missense single nucleotide variants (SNVs), with the positive cases fairly equally split between frameshift, nonsense, and missense variants.

Proportion of positive/probably positive findings for pediatric and prenatal exome cases
Figure sourced from Jessica Van Ziffle’s presentation

With the goal of increasing the proportion of positive findings, Jessica and the team at UCSF explored what additional variants could be identified by exome sequencing to potentially solve the ∼10% of inconclusive cases and ∼65% of negative cases. First, the team investigated the impact of calling copy number variants (CNVs), specifically contiguous gene changes, whole gene changes, and exon-level changes. UCSF worked closely with SOPHiA GENETICS™ to optimize their exome sequencing to ensure high and even coverage for accurate CNV detection, and indeed doubled their sequencing depth to 80M reads to ensure the sensitive detection of CNVs 1-2 exons in size.

Next, UCSF wanted to be able to simultaneously call variants in mitochondrial DNA, which is especially relevant for metabolic diseases. Different cell types have different numbers of mitochondria, and each mitochondrion has its own genome that can have different variants in it (heteroplasmy). The UCSF team, therefore, wanted to assess the lower limit of detection for mitochondrial heteroplasmy through a mixing study, which concluded that exome testing could detect variants down to 5% variant allele fraction with high sensitivity.

In conclusion, the custom SOPHiA DDM™ workflow for exome sequencing successfully increased positive/probable positive findings at UCSF to 25-30% by integrating both CNV and mitochondrial variant detection.

Streamlining clinical implementation of hereditary cancer analysis and reporting with a custom application

Hong Wang, PhD, FCCMG, FACMG, DABMGG Laboratory Geneticist at North York General Hospital, Toronto, Ontario, Canada
Andrea Vaags, PhD, FCCMG Discipline Co-Lead and Laboratory Geneticist at Trillium Health Partners – Credit Valley Hospital, Mississauga, Ontario, Canada

Drs Wang and Vaags provided a step-by-step overview of how they developed a brand new hereditary cancer panel to meet the Ontario Health - Cancer Care Ontario criteria for hereditary cancer testing.

Laboratory and clinical working groups were established to evaluate evidence and identify key genes and non-coding variants to include in a cutting-edge custom hereditary cancer panel. Furthermore, genetic testing eligibility criteria were co-developed with the Hereditary Cancer Clinical Eligibility Working Group. The laboratory working group used an evidence-based framework to design a standardized 76-gene panel, organized into 13 larger disease site-linked panels, and 25 single/small gene panels. After designing the panel, the Ontario group worked with SOPHiA GENETICS™ to expeditiously develop and implement the custom SOPHiA DDM^TM Hereditary Cancer application in academic community hospitals (see timeline).

Timeline of SOPHiA DDM™ Hereditary Cancer panel implementation in Ontario hospitals
Figure sourced from the Ontario hospital responsible for validating this product

Thanks to the streamlined end-to-end SOPHiA GENETICS™ workflow, hereditary cancer testing approximately doubled, according to Dr Vaags.

Implementing the custom automated SOPHiA GENETICS™ Hereditary Cancer Testing workflow saved 8 hours hands-on time for wet work, and 13 hours hands-on time for data analysis and reporting.

The workflow for each batch of 70 samples (plus one control) in the Ontario group laboratories, consists of DNA preparation, automated 3-day library preparation using the SOPHiA GENETICS program on the Hamilton STARlet, and sequencing on a NextSeq® 550 using mid-output. Sequencing data are automatically uploaded to the SOPHiA DDM™ cloud for processing ahead of analysis. For additional time savings, genes requiring special consideration due to the presence of pseudogenes are flagged with a warning in the SOPHiA DDM™ Platform and the SOPHiA GENETICS™ support team is on hand to answer queries on unusual findings. The cloud-based software for data management enables the laboratories to streamline data access, storage, and archiving back-up. Dr Wang shared that the time saved through this workflow has been instrumental in maintaining turnaround times, especially with significant understaffing during challenging periods.

By applying Virtual Panels and custom filters, the teams can analyze from as little as a single variant to as many as 76 genes using a single workflow. The high analytical sensitivity and specificity enable the laboratories to pick up unusual findings, such as Alu insertions, Boland inversions, and low-level mosaicism of copy number changes. And finally, the one-step secondary and tertiary analysis for concurrent detection of SNVs and CNVs allows the teams to significantly speed up their analysis, and the pseudogene pipeline enables the laboratories to minimize reflex testing. In summary, the custom SOPHiA DDM™ Hereditary Cancer application provides the Ontario laboratories with a one-size-fits-all solution.

Screening for genetic variants in hereditary cancer syndromes using the end-to-end SOPHiA DDM™ workflow

Mark Williams, FHGSA – Chief Scientist at Genomic Diagnostics, Heidelberg, Victoria, Australia

Speaker Mark Williams began his talk by highlighting that a key goal of the Genomic Diagnostics lab is to facilitate equal access to hereditary cancer testing. To do this, the lab set multiple criteria that were highly important to them when developing a new hereditary cancer application. Employing the complete SOPHiA GENETICS™ workflow for Hereditary Cancer allowed the team at Genomic Diagnostics to successfully meet these testing criteria.

In collaboration with Genomic Diagnostics, the custom SOPHiA DDM™ Hereditary Cancer application was designed to include genes that align with current practice guidelines. It was important to the lab that the pipeline could detect SNVs, Indels, and copy number variations (CNVs) in a single workflow. Mark confirmed that the resultant application effectively detects CNVs and is scalable, with turnaround times that meet their needs, even with testing volumes increasing year-on-year. In addition, the solution provides high-quality, consistent results, a full record of curation, visualization of BAM files, and is easily accessible and usable by all laboratory staff.

Like numerous other SOPHiA GENETICS™️ customers, Mark concluded that the SOPHiA DDM™ Platform offers a robust, automated, and secure bioinformatics pipeline that meets Australia’s privacy regulations. In addition, the software is extremely user-friendly, from its visual interface to the detailed QC metrics, annotation information, and links to databases. All laboratory personnel can effectively use the end-to-end solution, even without prior bioinformatics expertise.

The integrated workflow and affordable price allowed Genomic Diagnostics to expand access to the custom SOPHiA DDM™ Hereditary Cancer application (see chart), meeting Genomic Diagnostics’ goal of facilitating equal access to hereditary cancer testing.

Increasing access to hereditary cancer genomic testing over time
Figure sourced from Mark Williams’ presentation

The custom SOPHiA DDM™ Hereditary Cancer Application provides affordable, accessible, and quality genetic testing.

We thank all our speakers for sharing their research stories at our ASHG symposium this year. We’re delighted to hear how their integrated SOPHiA DDM™ workflows are reducing workloads, expanding access, and continuing to discover new variants associated with rare diseases and hereditary cancers.