Non-coding variants are a rare cause of recessive developmental disorders in trans with coding variants.

PURPOSE: Identifying pathogenic non-coding variants is challenging. A single protein-altering variant is often identified in a recessive gene in individuals with developmental disorders (DD), but the prevalence of pathogenic non-coding 'second hits' in trans with these is unknown. METHODS: In 4,073 genetically undiagnosed rare disease trio probands from the 100,000 Genomes project, we identified rare heterozygous protein-altering variants in recessive DD-associated genes. We identified rare non-coding variants on the other haplotype in introns, untranslated regions (UTRs), promoters, and candidate enhancer regions. We clinically evaluated the top candidates for phenotypic fit, and performed functional testing where possible. RESULTS: We identified 3,761 rare heterozygous loss-of-function or ClinVar pathogenic variants in recessive DD-associated genes in 2,430 probands. For 1,366 (36.3%) of these, we identified at least one rare non-coding variant in trans. Bioinformatic filtering and clinical review, revealed seven to be a good clinical fit. After detailed characterisation, we identified likely diagnoses for three probands (in GAA, NPHP3, and PKHD1) and candidate diagnoses in a further three (PAH, LAMA2, IGHMBP2). CONCLUSION: We developed a systematic approach to uncover new diagnoses involving compound heterozygous coding/non-coding variants and conclude that this mechanism is likely to be a rare cause of DDs.

Potential clinical applications of advanced genomic analysis in cerebral palsy.

Cerebral palsy (CP) has historically been attributed to acquired insults, but emerging research suggests that genetic variations are also important causes of CP. While microarray and whole-exome sequencing based studies have been the primary methods for establishing new CP-gene relationships and providing a genetic etiology for individual patients, the cause of their condition remains unknown for many patients with CP. Recent advancements in genomic technologies offer additional opportunities to uncover variations in human genomes, transcriptomes, and epigenomes that have previously escaped detection. In this review, we outline the use of these state-of-the-art technologies to address the molecular diagnostic challenges experienced by individuals with CP. We also explore the importance of identifying a molecular etiology whenever possible, given the potential for genomic medicine to provide opportunities to treat patients with CP in new and more precise ways.

Population screening shows risk of inherited cancer and familial hypercholesterolemia in Oregon.

The Healthy Oregon Project (HOP) is a statewide effort that aims to build a large research repository and influence the health of Oregonians through providing no-cost genetic screening to participants for a next-generation sequencing 32-gene panel comprising genes related to inherited cancers and familial hypercholesterolemia. This type of unbiased population screening can detect at-risk individuals who may otherwise be missed by conventional medical approaches. However, challenges exist for this type of high-throughput testing in an academic setting, including developing a low-cost high-efficiency test and scaling up the clinical laboratory for processing large numbers of samples. Modifications to our academic clinical laboratory including efficient test design, robotics, and a streamlined analysis approach increased our ability to test more than 1,000 samples per month for HOP using only one dedicated HOP laboratory technologist. Additionally, enrollment using a HIPAA-compliant smartphone app and sample collection using mouthwash increased efficiency and reduced cost. Here, we present our experience three years into HOP and discuss the lessons learned, including our successes, challenges, opportunities, and future directions, as well as the genetic screening results for the first 13,670 participants tested. Overall, we have identified 730 pathogenic/likely pathogenic variants in 710 participants in 24 of the 32 genes on the panel. The carrier rate for pathogenic/likely pathogenic variants in the inherited cancer genes on the panel for an unselected population was 5.0% and for familial hypercholesterolemia was 0.3%. Our laboratory experience described here may provide a useful model for population screening projects in other states.

ONT long-read WGS for variant discovery and orthogonal confirmation of short read WGS derived genetic variants in clinical genetic testing.

Technological advances in Next-Generation Sequencing dramatically increased clinical efficiency of genetic testing, allowing detection of a wide variety of variants, from single nucleotide events to large structural aberrations. Whole Genome Sequencing (WGS) has allowed exploration of areas of the genome that might not have been targeted by other approaches, such as intergenic regions. A single technique detecting all genetic variants at once is intended to expedite the diagnostic process while making it more comprehensive and efficient. Nevertheless, there are still several shortcomings that cannot be effectively addressed by short read sequencing, such as determination of the precise size of short tandem repeat (STR) expansions, phasing of potentially compound recessive variants, resolution of some structural variants and exact determination of their boundaries, etc. Therefore, in some cases variants can only be tentatively detected by short reads sequencing and require orthogonal confirmation, particularly for clinical reporting purposes. Moreover, certain regulatory authorities, for example, New York state CLIA, require orthogonal confirmation of every reportable variant. Such orthogonal confirmations often involve numerous different techniques, not necessarily available in the same laboratory and not always performed in an expedited manner, thus negating the advantages of "one-technique-for-all" approach, and making the process lengthy, prone to logistical and analytical faults, and financially inefficient. Fortunately, those weak spots of short read sequencing can be compensated by long read technology that have comparable or better detection of some types of variants while lacking the mentioned above limitations of short read sequencing. At Variantyx we have developed an integrated clinical genetic testing approach, augmenting short read WGS-based variant detection with Oxford Nanopore Technologies (ONT) long read sequencing, providing simultaneous orthogonal confirmation of all types of variants with the additional benefit of improved identification of exact size and position of the detected aberrations. The validation study of this augmented test has demonstrated that Oxford Nanopore Technologies sequencing can efficiently verify multiple types of reportable variants, thus ensuring highly reliable detection and a quick turnaround time for WGS-based clinical genetic testing.

Discordance between germline genetic findings and abnormal tumor immunohistochemistry staining of mismatch repair proteins in individuals with suspected Lynch syndrome.

Background and Aims: Tumor immunohistochemical staining (IHC) of DNA mismatch repair (MMR) proteins is often used to guide germline genetic testing and variant classification for patients with suspected Lynch syndrome. This analysis examined the spectrum of germline findings in a cohort of individuals showing abnormal tumor IHC. Methods: We assessed individuals with reported abnormal IHC findings and referred for testing with a six-gene syndrome-specific panel (n=703). Pathogenic variants (PVs) and variants of uncertain significance (VUS) in MMR genes were designated expected/unexpected relative to IHC results. Results: The PV positive rate was 23.2% (163/703; 95% confidence interval [CI], 20.1%-26.5%); 8.0% (13/163; 95% CI, 4.3%-13.3%) of PV carriers had a PV in an unexpected MMR gene. Overall, 121 individuals carried VUS in MMR genes expected to be mutated based on IHC results. Based on independent evidence, in 47.1% (57/121; 95% CI, 38.0%-56.4%) of these individuals the VUSs were later reclassified as benign and in 14.0% (17/121; 95% CI, 8.4%-21.5%) of these individuals the VUSs were reclassified as pathogenic. Conclusions: Among patients with abnormal IHC findings, IHC-guided single-gene genetic testing may miss 8% of individuals with Lynch syndrome. In addition, in patients with VUS identified in MMR genes predicted to be mutated by IHC, extreme caution must be taken when the IHC results are considered in variant classification.

Perspectives on Consumer and Clinical Genetic Testing Education among Medical Students in West Texas.

There is a popular trend for genetic test companies to market genetic screenings to identify detrimental mutations directly to consumers. As a result, there has been an increase in the number of patients wanting to discuss the results of their personal genetic tests with their physician. In turn, the medical education community has recognized a need to provide medical staff and patients with resources to interpret and act on personal genetic data. Most medical students and residents lack the requisite knowledge to interpret and manage patient-provided individualized genetic testing. This has prompted a growing interest among medical students in learning more about genetic testing and how to counsel their patients on this information. Consequently, early educational exposure to clinical genetic testing and counseling would benefit medical education training programs. In this study, we examine the perceptions of medical students in West Texas with regard to clinical genetic testing.

Risks and Function of Breast Cancer Susceptibility Alleles.

Family history remains one of the strongest risk factors for breast cancer. It is well established that women with a first-degree relative affected by breast cancer are twice as likely to develop the disease themselves. Twins studies indicate that this is most likely due to shared genetics rather than shared epidemiological/lifestyle risk factors. Linkage and targeted sequencing studies have shown that rare high- and moderate-penetrance germline variants in genes involved in the DNA damage response (DDR) including BRCA1, BRCA2, PALB2, ATM, and TP53 are responsible for a proportion of breast cancer cases. However, breast cancer is a heterogeneous disease, and there is now strong evidence that different risk alleles can predispose to different subtypes of breast cancer. Here, we review the associations between the different genes and subtype-specificity of breast cancer based on the most comprehensive genetic studies published. Genome-wide association studies (GWAS) have also been used to identify an additional hereditary component of breast cancer, and have identified hundreds of common, low-penetrance susceptibility alleles. The combination of these low penetrance risk variants, summed as a polygenic risk score (PRS), can identify individuals across the spectrum of disease risk. However, there remains a substantial bottleneck between the discovery of GWAS-risk variants and their contribution to tumorigenesis mainly because the majority of these variants map to the non-protein coding genome. A range of functional genomic approaches are needed to identify the causal risk variants and target susceptibility genes and establish their underlying role in disease biology. We discuss how the application of these multidisciplinary approaches to understand genetic risk for breast cancer can be used to identify individuals in the population that may benefit from clinical interventions including screening for early detection and prevention, and treatment strategies to reduce breast cancer-related mortalities.

Non-coding region variants upstream of MEF2C cause severe developmental disorder through three distinct loss-of-function mechanisms.

Clinical genetic testing of protein-coding regions identifies a likely causative variant in only around half of developmental disorder (DD) cases. The contribution of regulatory variation in non-coding regions to rare disease, including DD, remains very poorly understood. We screened 9,858 probands from the Deciphering Developmental Disorders (DDD) study for de novo mutations in the 5' untranslated regions (5' UTRs) of genes within which variants have previously been shown to cause DD through a dominant haploinsufficient mechanism. We identified four single-nucleotide variants and two copy-number variants upstream of MEF2C in a total of ten individual probands. We developed multiple bespoke and orthogonal experimental approaches to demonstrate that these variants cause DD through three distinct loss-of-function mechanisms, disrupting transcription, translation, and/or protein function. These non-coding region variants represent 23% of likely diagnoses identified in MEF2C in the DDD cohort, but these would all be missed in standard clinical genetics approaches. Nonetheless, these variants are readily detectable in exome sequence data, with 30.7% of 5' UTR bases across all genes well covered in the DDD dataset. Our analyses show that non-coding variants upstream of genes within which coding variants are known to cause DD are an important cause of severe disease and demonstrate that analyzing 5' UTRs can increase diagnostic yield. We also show how non-coding variants can help inform both the disease-causing mechanism underlying protein-coding variants and dosage tolerance of the gene.

Pathways, Processes and Protections: Australia's Clinical and Direct-to-Consumer Genetic Testing Spaces.

Health-related genetic testing, once exclusively within the medical space, is now available within the commercial space. This paradigm shift from medical to consumer presents challenges to regulators, health care professionals and individuals. This section reports on pathways, processes and protections afforded to Australians whether genetic test results are provided by medical professionals or commercial players. While a complex web of protections is available within Australia's medical space, those accessing commercial testing rely on the same protections afforded all consumers in all marketplace transactions. There is also potential for these two initially bifurcated pathways to merge, either by business model or individual choice. Individuals pursuing commercial options obtain personal genetic information, which they self-interpret and, if they choose, share with family, medical professionals and online. While this section focuses on health-related genetic testing, it gives insight into what will undoubtedly be continued incursions into the medical space by commercial players.

The role of clinical response to treatment in determining pathogenicity of genomic variants.

PURPOSE: The 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants provide a framework to standardize terminology in the classification of variants uncovered through genetic testing. We aimed to assess the validity of utilizing clinical response to therapies specifically targeted to a suspected disease in clarifying variant pathogenicity. METHODS: Five families with disparate clinical presentations and different genetic diseases evaluated and treated in multiple diagnostic settings are summarized. RESULTS: Extended evaluations indicated possible genetic diagnoses and assigned candidate causal variants, but the cumulative clinical, biochemical, and molecular information in each instance was not completely consistent with the identified disease. Initiation of treatment specific to the suspected diagnoses in the affected individuals led to clinical improvement in all five families. CONCLUSION: We propose that the effect of therapies that are specific and targeted to treatable genetic diseases embodies an in vivo physiological response and could be considered as additional criteria within the 2015 ACMG/AMP guidelines in determining genomic variant pathogenicity.

Laboratory analysis of acylcarnitines, 2020 update: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Acylcarnitine analysis is a useful test for identifying patients with inborn errors of mitochondrial fatty acid ß-oxidation and certain organic acidemias. Plasma is routinely used in the diagnostic workup of symptomatic patients. Urine analysis of targeted acylcarnitine species may be helpful in the diagnosis of glutaric acidemia type I and other disorders in which polar acylcarnitine species accumulate. For newborn screening applications, dried blood spot acylcarnitine analysis can be performed as a multiplex assay with other analytes, including amino acids, succinylacetone, guanidinoacetate, creatine, and lysophosphatidylcholines. Tandem mass spectrometric methodology, established more than 30 years ago, remains a valid approach for acylcarnitine analysis. The method involves flow-injection analysis of esterified or underivatized acylcarnitines species and detection using a precursor-ion scan. Alternative methods utilize liquid chromatographic separation of isomeric and isobaric species and/or detection by selected reaction monitoring. These technical standards were developed as a resource for diagnostic laboratory practices in acylcarnitine analysis, interpretation, and reporting.

Experiences with offering pro bono medical genetics services in the West Indies: Benefits to patients, physicians, and the community.

We describe our experiences with organizing pro bono medical genetics and neurology outreach programs on several different resource-limited islands in the West Indies. Due to geographic isolation, small population sizes, and socioeconomic disparities, most Caribbean islands lack medical services for managing, diagnosing, and counseling individuals with genetic disorders. From 2015 to 2019, we organized 2-3 clinics per year on various islands in the Caribbean. We also organized a week-long clinic to provide evaluations for children suspected of having autism spectrum disorder. Consultations for over 100 different individuals with suspected genetic disorders were performed in clinics or during home visits following referral by locally registered physicians. When possible, follow-up visits were attempted. When available and appropriate, clinical samples were shipped to collaborating laboratories for molecular analysis. Laboratory tests included karyotyping, cytogenomic microarray analysis, exome sequencing, triplet repeat expansion testing, blood amino acid level determination, biochemical assaying, and metabolomic profiling. We believe that significant contributions to healthcare by genetics professionals can be made even if availability is limited. Visiting geneticists may help by providing continuing medical education seminars. Clinical teaching rounds help to inform local physicians regarding the management of genetic disorders with the aim of generating awareness of genetic conditions. Even when only periodically available, a visiting geneticist may benefit affected individuals, their families, their local physicians, and the community at large.

Evaluation of pathogenetic mutations in breast cancer predisposition genes in population-based studies conducted among Chinese women.

PURPOSE: Limited studies have been conducted to evaluate pathogenetic mutations in breast cancer predisposition genes among Chinese women. To fully characterize germline mutations of these genes in this population, we used the whole-exome sequencing data in a population-based case-control study conducted in Shanghai, China. METHODS: We evaluated exonic, splicing, and copy number variants in 11 established and 14 candidate breast cancer predisposition genes in 831 invasive breast cancer cases and 839 controls. We identified 55 pathogenic variants, including 15 newly identified in this study. RESULTS: Approximately 8% of the cases and 0.6% of the cancer-free controls carried these pathogenetic variants (P = 3.05 × 10-15). Among cases, 3.7% had a BRCA2 pathogenic variant and 1.6% had a BRCA1 pathogenic variant, while 2.5% had a pathogenic variant in other genes including ATM, CHEK2, NBN, NF1, CDH1, PALB2, PTEN, TP53 as well as BARD1, BRIP, and RAD51D. Patients with BRCA1/2 pathogenic variants were more likely to have a family history of breast cancer and hormone receptor negative tumors compared with patients without pathogenic variants. CONCLUSIONS: This study highlighted the importance of hereditary breast cancer genes in the breast cancer etiology in this understudied population. Together with previous studies in East Asian women, this study suggested a relatively more prominent role of BRCA2 compared to BRCA1. This study also provides additional evidence to design cost-efficient genetic testing among Chinese women for risk assessment and early detection of breast cancer.

Clinical Interpretation of Sequence Variants.

Clinical interpretation of DNA sequence variants is a critical step in reporting clinical genetic testing results. Application of next-generation sequencing technology in molecular genetic testing has facilitated diagnoses of genetic disorders in clinical practice. However, the large number of DNA sequence variants detected in clinical specimens, many of which have never been seen before, make clinical interpretation challenging. Recommendations by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) have been widely adopted by clinical laboratories around the world to guide clinical interpretation of sequence variants. The ClinGen Sequence Variant Interpretation Working Group and various disease-specific variant curation expert panels have also developed specifications for the ACMG/AMP recommendations. Despite these efforts to standardize variant interpretation in clinical practice, different laboratories may subjectively use professional judgment to determine which criteria are applicable when classifying a variant. In addition, clinicians and researchers who are not familiar with the variant interpretation process may have difficulty understanding clinical genetic reports and communicating the clinical significance of genetic testing results. Here we provide a step-by-step protocol for clinical interpretation of sequence variants, including practical examples. By following this protocol, clinical laboratory geneticists can interpret the clinical significance of sequence variants according to the ACMG/AMP recommendations and ClinGen framework. Furthermore, this article will help clinicians and researchers to understand variant classification in clinical genetic testing reports and evaluate the quality of the reports. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Interpreting the clinical significance of sequence variants Support Protocol: Reevaluating the clinical significance of sequence variants.

Advantages and Perils of Clinical Whole-Exome and Whole-Genome Sequencing in Cardiomyopathy.

As the price of next-generation sequencing keeps decreasing, cost is becoming a less important discriminator for diagnostic laboratories in choosing the preferred type of approach to genetic testing. Genome-wide sequencing strategies will plausibly become the standard first-tier tools for genetic testing, with the potential for deeper understanding of the genetic architecture of cardiomyopathies and discovery of the underlying aetiology in the many patients in whom the genetic cause remains elusive. Routine usage of extended sequencing assays will also enable "genetic-first diagnostics", particularly for those patients affected with syndromic conditions of unclear genetic origin, often resulting in costly and distressing diagnostic odysseys before reaching a diagnosis. However, access to genome-wide data for all patients will need to be managed with rigour and caution by (cardiovascular) genetic professionals to avoid erroneous variant pathogenicity assertions and over-reporting uncertain findings, both damaging scenarios to patients and their family members. Researchers will also be required to adopt robust methods to demonstrate novel genetic associations with disease, given the high "narrative potential" of such large datasets and the dangers of generating further false positive associations (that have previously blighted the field of cardiac genetics). Here, we discuss advantages and dangers associated with the routine adoption of whole-exome (and whole-genome) sequencing in diagnostic facilities and in the research setting in the context of cardiomyopathies but relevant to several other conditions.

Laboratory diagnosis of disorders of peroxisomal biogenesis and function: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Peroxisomal disorders are a clinically and genetically heterogeneous group of diseases caused by defects in peroxisomal biogenesis or function, usually impairing several metabolic pathways. Peroxisomal disorders are rare; however, the incidence may be underestimated due to the broad spectrum of clinical presentations. The inclusion of X-linked adrenoleukodystrophy to the Recommended Uniform Screening Panel for newborn screening programs in the United States may increase detection of this and other peroxisomal disorders. The current diagnostic approach relies heavily on biochemical genetic tests measuring peroxisomal metabolites, including very long-chain and branched-chain fatty acids in plasma and plasmalogens in red blood cells. Molecular testing can confirm biochemical findings and identify the specific genetic defect, usually utilizing a multiple-gene panel or exome/genome approach. When next-generation sequencing is used as a first-tier test, evaluation of peroxisome metabolism is often necessary to assess the significance of unknown variants and establish the extent of peroxisome dysfunction. This document provides a resource for laboratories developing and implementing clinical biochemical genetic testing for peroxisomal disorders, emphasizing technical considerations for sample collection, test performance, and result interpretation. Additionally, considerations on confirmatory molecular testing are discussed.

Theme 2 Genetics and genomics.

Background: A genetic basis is found in ∼70% of familial and ∼15% of sporadic ALS, in research cohorts. Clinical trials of gene-targeted therapies are underway, heralding a new era of personalized medicine in ALS treatment. However, ALS management guidelines do not include recommendations for the offer of genetic testing. Many persons with ALS who desire genetic testing are not currently offered it, and the yield of genetic testing in clinic-based ALS populations is unknown. The ALS GAP program, sponsored by the Northeast ALS (NEALS) Consortium, provides free genetic testing for patients with ALS who have a family history of ALS or dementia. We report genetic testing outcomes in the first 142 patients tested in the program.Objectives: 1) To create a pilot ALS genetic testing program for NEALS clinics, 2) To study the rate of ALS gene identification in a US clinic-based populationMethods: Persons with ALS and a family history of ALS (fALS) or dementia (dALS) who receive care at a US NEALS clinic are eligible for testing. Patients classified as fALS (having a positive family history of ALS in a 1st, 2nd, or 3rd degree relative) are eligible for C9orf72 testing, with the option to reflex to a 5 gene (SOD1, FUS, TARDBP, TBK1, VCP) panel. Patients classified as dALS (having a positive family history of dementia of any type in a 1st or 2nd degree relative) are eligible for C9orf72 testing only.Results: Currently, 29.5% (34/115) of US NEALS clinics have participated in the program. Of 142 patients who have completed testing to date, 78 (54.9%) were classified as fALS and 64 (45.1%) as dALS. Among fALS cases, 42/78 (53.9%) tested positive, including 32/78 (41%) with a C9orf72 repeat expansion, and 10/78 (12.8%) with other pathogenic or likely pathogenic variants in SOD, FUS, TARDP or VCP. Variants of uncertain significance (VUS) in FUS were identified in 2/78 (2.6%). Among dALS cases, 12/60 (20%) tested positive for C9orf72.Discussion and conclusions: Participation in ALS-GAP indicates significant clinician and patient interest in ALS genetic testing. This program addresses several current barriers to testing access, including cost, identifying appropriate candidates for testing, and appropriate test selection. Although 38% of patients who participated in the program have thus far received a genetic diagnosis, our testing outcome data suggests that the gene identification rate in fALS cases may be lower in clinic-based patients than in research cohorts, particularly for genes other than C9orf72. This program may serve as a model for the practice of ALS genetic testing in the clinic setting. Consistent, equitable testing policies, as well as an accurate understanding of the genetic profile of clinic-based ALS populations, are needed as gene-targeted therapies reach patient care.

Multimodal Analysis of SCN1A Missense Variants Improves Interpretation of Clinically Relevant Variants in Dravet Syndrome.

Objective: We aimed to improve the classification of SCN1A missense variants in patients with Dravet syndrome (DS) by combining and modifying the current variants classification criteria to minimize inconclusive test results. Methods: We established a score classification workflow based on evidence of pathogenicity to adapt the classification of DS-related SCN1A missense variants. In addition, we compiled the variants reported in the literature and our cohort and assessed the proposed pathogenic classification criteria. We combined information regarding previously established pathogenic amino acid changes, mode of inheritance, population-specific allele frequencies, localization within protein domains, and deleterious effect prediction analysis. Results: Our meta-analysis showed that 46% (506/1,101) of DS-associated SCN1A variants are missense. We applied the score classification workflow and 56.5% (286/506) of the variants had their classification changed from VUS: 17.8% (90/506) into "pathogenic" and 38.7% (196/506) as "likely pathogenic." Conclusion: Our results indicate that using multimodal analysis seems to be the best approach to interpret the pathogenic impact of SCN1A missense changes for the molecular diagnosis of patients with DS. By applying the proposed workflow, most DS related SCN1A variants had their classification improved.

Recommendations for the clinical interpretation of genetic variants and presentation of results to patients with inherited bleeding disorders. A UK Haemophilia Centre Doctors' Organisation Good Practice Paper.

This paper sets out good practice for clinicians involved in interpreting variant reports for patients with inherited bleeding disorders. It is aimed primarily at doctors, nurses and allied healthcare professionals who may not have had specific training in genetic testing methodology or reporting. It deals with uncertainty in classification of variant pathogenicity and the handling of incidental findings.