Universal Germline Panel Testing for Individuals with Pheochromocytoma and Paraganglioma Produces High Diagnostic Yield

Background Practice guidelines to identify individuals with hereditary pheochromocytomas and paragangliomas (PPGLs) advocate for sequential gene testing strategy guided by specific clinical features and predate the routine use of multigene panel testing (MGPT). Objective To describe results of MGPT for hereditary PPGL in a clinically and ancestrally diverse cohort. Setting Commercial laboratory based in the United States. Methods Clinical data and test results were retrospectively reviewed in 1727 individuals who had targeted MGPT due to suspicion of hereditary PPGL from August 2013 through December 2019. Results Overall, 27.5% of individuals had a pathogenic or likely pathogenic variant (PV), 9.0% had a variant of uncertain significance, and 63.1% had a negative result. Most PVs were identified in SDHB (40.4%), followed by SDHD (21.1%), SDHA (10.1%), VHL (7.8%), SDHC (6.7%), RET (3.7%), and MAX (3.6%). PVs in FH, MEN1, NF1, SDHAF2, and TMEM127 collectively accounted for 6.5% of PVs. Clinical predictors of a PV included extra-adrenal location, early age of onset, multiple tumors, and positive family history of PPGL. Individuals with extra-adrenal PGL and a positive family history were the most likely to have a PV (85.9%). Restricting genetic testing to SDHB/C/D misses a third (32.8%) of individuals with PVs. Conclusion Our data demonstrate a high diagnostic yield in individuals with and without established risk factors, a low inconclusive result rate, and a substantial contribution to diagnostic yield from rare genes. These findings support universal testing of all individuals with PPGL and the use of concurrent MGPT as the ideal platform.

Revealing the Mutational Spectrum in Southern Africans With Amyotrophic Lateral Sclerosis

Background and ObjectivesTo perform the first screen of 44 amyotrophic lateral sclerosis (ALS) genes in a cohort of African genetic ancestry individuals with ALS using whole-genome sequencing (WGS) data.MethodsOne hundred three consecutive cases with probable/definite ALS (using the revised El Escorial criteria), and self-categorized as African genetic ancestry, underwent WGS using various Illumina platforms. As population controls, 238 samples from various African WGS data sets were included. Our analysis was restricted to 44 ALS genes, which were curated for rare sequence variants and classified according to the American College of Medical Genetics guidelines as likely benign, uncertain significance, likely pathogenic, or pathogenic variants.ResultsThirteen percent of 103 ALS cases harbored pathogenic variants; 5 different SOD1 variants (N87S, G94D, I114T, L145S, and L145F) in 5 individuals (5%, 1 familial case), pathogenic C9orf72 repeat expansions in 7 individuals (7%, 1 familial case) and a likely pathogenic ANXA11 (G38R) variant in 1 individual. Thirty individuals (29%) harbored ≥1 variant of uncertain significance; 10 of these variants had limited pathogenic evidence, although this was insufficient to permit confident classification as pathogenic.DiscussionOur findings show that known ALS genes can be expected to identify a genetic cause of disease in >11% of sporadic ALS cases of African genetic ancestry. Similar to European cohorts, the 2 most frequent genes harboring pathogenic variants in this population group are C9orf72 and SOD1.

Reviewing the Clinical Spectrum Related to Kmt2b Gene Mutations: Unusual Clinical Presentation and a Possible New Pathogenic Mutation

Introduction KMT2B related dystonia is a childhood onset generalized dystonia. Since its first description in 2016, different phenotypic spectrum have been reported. The aim of the case report is to provide data that may help to understand the spectrum of KMT2B-related disorders. We present two members of a family with a possible non-previously described pathogenic mutation and an unusual KMT2B related dystonia presentation: an adult onset and focal dystonia.Case Presentation The index patient is a 32 year-old woman with a generalized dystonia. Her maternal uncle presented a focal dystonia. Next-generation sequencing revealed a heterozygous missense mutation in KMT2B gene (19q13.12), described as a variant of uncertain significance (VUS). Although characteristic phenotype of KMT2B dystonia is a childhood onset generalized dystonia, different phenotypes have been related according to the kinds of mutations in this gene, also varying the age of symptom onset and the penetrance of the mutation. Asymptomatic or sub-clinical carriers and adult onset has been described. Due to the low prevalence of this variant in the general population and the low penetrance and high intrafamilial variability of this entity, we suggest that this mutation might be a pathogenic variant.ConclusionsKMT2B related dystonia is an emerging and prevalent monogenic dystonia whose incidence, genetic variability and clinical spectrum remain unknown. Despite the study of this gene is indicated in childhood onset dystonia, description of cases such as ours shows that its sequencing in patients with an adult-onset dystonia with family history can be useful for the diagnosis.

Exome Sequencing Highlights a Potential Role for Concealed Cardiomyopathies in Youthful Sudden Cardiac Death

Background: Sudden cardiac arrest (SCA) and sudden unexplained death (SUD) are feared sequelae of many genetic heart diseases. In rare circumstances, pathogenic variants in cardiomyopathy-susceptibility genes may result in electrical instability leading to SCA/SUD before any structural manifestations of underlying cardiomyopathy are evident. Methods: Collectively, 38 unexplained SCA survivors (21 males; mean age at SCA 26.4±13.1 years), 68 autopsy-inconclusive SUD cases (49 males; mean age at death 20.4±9.0 years) without disease-causative variants in the channelopathy genes, and 973 ostensibly healthy controls were included. Following exome sequencing, ultrarare (minor allele frequency ≤0.00005 in any ethnic group within Genome Aggregation Database [gnomAD, n=141 456 individuals]) nonsynonymous variants identified in 24 ClinGen adjudicated definitive/strong evidence cardiomyopathy-susceptibility genes were analyzed. Eligible variants were adjudicated as pathogenic, likely pathogenic, or variant of uncertain significance in accordance with current American College of Medical Genetics and Genomics guidelines. Results: Overall, 7 out of 38 (18.4%) SCA survivors and 14 out of 68 (20.5%) autopsy-inconclusive, channelopathic-negative SUD cases had at least one pathogenic/likely pathogenic or a variant of uncertain significance nonsynonymous variant within a strong evidence, cardiomyopathy-susceptibility gene. Following American College of Medical Genetics and Genomics criterion variant adjudication, a pathogenic or likely pathogenic variant was identified in 3 out of 38 (7.9%; P =0.05) SCA survivors and 8 out of 68 (11.8%; P =0.0002) autopsy-inconclusive SUD cases compared to 20 out of 973 (2.1%) European controls. Interestingly, the yield of pathogenic/likely pathogenic variants was significantly greater in autopsy-inconclusive SUD cases with documented interstitial fibrosis (4/11, 36%) compared with only 4 out of 57 (7%, P <0.02) SUD cases without ventricular fibrosis. Conclusion: Our data further supports the inclusion of strongevidence cardiomyopathy-susceptibility genes on the genetic testing panels used to evaluate unexplained SCA survivors and autopsy-inconclusive/negative SUD decedents. However, to avoid diagnostic miscues, the careful interpretation of genetic test results in patients without overt phenotypes is vital.

Prenatal Diagnosis and Genetic Analysis of 21q21.1–q21.2 Aberrations in Seven Chinese Pedigrees

Background: Chromosomal aberrations contribute to human phenotypic diversity and disease susceptibility, but it is difficult to assess their pathogenic effects in the clinic. Therefore, it is of great value to report new cases of chromosomal aberrations associated with normal phenotypes or clinical abnormalities.Methods: This was a retrospective analysis of seven pedigrees that carried 21q21.1–q21.2 aberrations. G-banding and single-nucleotide polymorphism array techniques were used to analyze chromosomal karyotypes and copy number variations in the fetuses and their family members.Results: All fetuses and their family members showed normal karyotypes in seven pedigrees. Here, it was revealed that six fetuses carried maternally inherited 21q21.1–q21.2 duplications, ranging from 1 to 2.7 Mb, but none of the mothers had an abnormal phenotype. In one fetus, an 8.7 Mb deletion of 21q21.1–q21.2 was found. An analysis of the pedigree showed that the deletion was also observed in the mother, brother, and maternal grandmother, but no abnormal phenotypes were found.Conclusion: This study identified 21q21.1–q21.2 aberrations in Chinese pedigrees. The carriers of 21q21.1–q21.2 duplications had no clinical consequences based on their phenotypes, and the 21q21.1–q21.2 deletion was transmitted through three generations of normal individuals. This provides benign clinical evidence for pathogenic assessment of 21q21.1–q21.2 duplication and deletion, which was considered a variant of uncertain significance and a likely pathogenic variant in previous reports.

Genetic epidemiology of autoinflammatory disease variants in Indian population from 1029 whole genomes

Background Autoinflammatory disorders are the group of inherited inflammatory disorders caused due to the genetic defect in the genes that regulates innate immune systems. These have been clinically characterized based on the duration and occurrence of unprovoked fever, skin rash, and patient’s ancestry. There are several autoinflammatory disorders that are found to be prevalent in a specific population and whose disease genetic epidemiology within the population has been well understood. However, India has a limited number of genetic studies reported for autoinflammatory disorders till date. The whole genome sequencing and analysis of 1029 Indian individuals performed under the IndiGen project persuaded us to perform the genetic epidemiology of the autoinflammatory disorders in India. Results We have systematically annotated the genetic variants of 56 genes implicated in autoinflammatory disorder. These genetic variants were reclassified into five categories (i.e., pathogenic, likely pathogenic, benign, likely benign, and variant of uncertain significance (VUS)) according to the American College of Medical Genetics and Association of Molecular pathology (ACMG-AMP) guidelines. Our analysis revealed 20 pathogenic and likely pathogenic variants with significant differences in the allele frequency compared with the global population. We also found six causal founder variants in the IndiGen dataset belonging to different ancestry. We have performed haplotype prediction analysis for founder mutations haplotype that reveals the admixture of the South Asian population with other populations. The cumulative carrier frequency of the autoinflammatory disorder in India was found to be 3.5% which is much higher than reported. Conclusion With such frequency in the Indian population, there is a great need for awareness among clinicians as well as the general public regarding the autoinflammatory disorder. To the best of our knowledge, this is the first and most comprehensive population scale genetic epidemiological study being reported from India.

430 Diagnosis and management of a case of biventricular arrhythmogenic cardiomyopathy: a case report

Arrhythmogenic cardiomyopathy (ACM) is a genetically determined heart muscle disorder characterized by fibro-fatty replacement of myocardium which may affect the right ventricle (‘dominant right’ variant), the left ventricle (‘dominant left’ variant) or both (‘biventricular disease’ variant). Despite of ACM is one of the main causes of sudden cardiac death (SCD) in young people and athletes as well, the diagnosis of this complex clinical entity still remains a challenge. We report on a case of a 29 years old non-athletic woman with family history of SCD and a long-standing personal history of palpitations leading to frequent accesses to the Accident & Emergency Department. Her echocardiogram was normal while twelve leads ECG revealed negative T waves in the right precordial leads (V1–V3) and post-excitation epsilon waves. ECG Holter monitoring recorded frequent (up to 17 000 in 24 h) polymorphic premature ventricular contractions (PVCs) seldom organized in couplets or triplets. She was followed-up in our clinic and started on Propanolol 40 mg bid with slight relief of symptoms and reduction of PVCs. Cardiac magnetic resonance (CMR) showed regional dyskinesia of both ventricles and RV dilatation. Moreover, tissue characterization findings revealed the presence of diffuse subepicardial late gadolinium enhancement (LGE) of the LV. Genetic analysis was performed and a variant of uncertain significance (VUS) in Desmoplakin was identified. Non sustained monomorphic ventricular tachycardia was induced during electrophysiologic study by programmed ventricular stimulation. In light of these results, we diagnosed biventricular ACM and, after carefully discussion with the patient, a subcutaneous implantable cardioverter defibrillator (ICD) was implanted. The present case should be a persisting reminder that must be considered more than just a single diagnostic tool when dealing with arrhythmic presentations especially in young patients and the importance of contrast CMR along with genetic testing in order to aid clinicians in the demanding selection of the best candidates for ICD implantation.

Comprehensive molecular-genetic analysis of mid-frequency sensorineural hearing loss

The genetic heterogeneity of sensorineural hearing loss (SNHL) is a major hurdle to the detection of disease-causing variants. We aimed to identify underlying causal genes associated with mid-frequency hearing loss (HL), which contributes to less than about 1% of SNHL cases, by whole exome sequencing (WES). Thirty families segregating mid-frequency SNHL, in whom biallelic GJB2 mutations had been previously excluded, were selected from among 851 families in our DNA repository of SNHL. DNA samples from the probands were subjected to WES analysis and searched for candidate variants associated with SNHL. We were able to identify the genetic aetiology in six probands (20%). In total, we found three pathogenic and three likely pathogenic variants in four genes (COL4A5, OTOGL, TECTA, TMPRSS3). One more proband was a compound heterozygote for a pathogenic variant and a variant of uncertain significance (VUS) in MYO15A gene. To date, MYO15A and TMPRSS3 have not yet been described in association with mid-frequency SNHL. In eight additional probands, eight candidate VUS variants were detected in five genes (DIAPH1, MYO7A, TECTA, TMC1, TSPEAR). Seven of these 16 variants have not yet been published or mentioned in the available databases. The most prevalent gene was TECTA, identified in 23% of all tested families. Furthermore, we confirmed the hypothesis that a substantive portion of cases with this conspicuous audiogram shape is a consequence of a genetic disorder.

Classification of the canonical splice alteration MUTYH c.934-2A>G is likely benign based on RNA and clinical data

MUTYH-associated polyposis (MAP) is an autosomal recessive disorder characterized by the development of multiple adenomatous colonic polyps and an increased lifetime risk of colorectal cancer. Germline biallelic pathogenic variants in MUTYH are responsible for MAP. The MUTYH c.934-2A>G (NM_001128425.1) variant, which is also known as c.850-2A>G for NM_001048174.2, has been identified in our laboratory in more than 800 patients, including homozygous and compound heterozygote carriers. The variant was initially classified as a variant of uncertain significance (VUS) due to lack of a MAP phenotype in biallelic carriers. In two unrelated female patients who were heterozygous carriers of this variant, further testing by RNA sequencing identified an aberrant transcript with a deletion of 9 nucleotides at the start of exon 11 (MUTYH r.934_942del9). This event is predicted to lead to an in-frame loss of 3 amino acids in a non-critical domain of the protein. This was the only splice defect identified in these patients which was not present in the controls and the aberrant transcript is derived exclusively from the variant allele, strongly supporting the cause of this splice defect as being the intronic variant, MUTYH c.934-2A>G. The splicing analysis demonstrating a small in-frame skipping of 3 amino acids in a non-critical domain along with the absence of a MAP phenotype in our internal cohort of biallelic carriers provides evidence that the variant is likely benign and not of clinical significance.