Likely Pathogenic Research Articles

Abstract 4134551: Outstanding Research Award in Pediatric Cardiology: Cardiomyopathy-Associated Pathogenic Variants in Pediatric Myocarditis: A Study from the Pediatric Cardiomyopathy Registry

Background: Recent studies have demonstrated that patients with myocarditis (MC) may have a higher burden of cardiomyopathy (CM)-associated gene variants than the general population. However, data in children is limited. The Pediatric Cardiomyopathy Registry (PCMR), a large multi-center registry of children with CM, includes patients with dilated CM (DCM) secondary to myocarditis (MC). Hypothesis: We hypothesized patients with DCM secondary to MC in the PCMR have a higher burden of CM-associated gene variants than healthy controls, and clinical genetic testing in this population is rare. Aims: We aimed to: (1) calculate the prevalence of pathogenic (P) or likely pathogenic (LP) CM-associated variants in patients with DCM secondary to MC and compare to the prevalence in heart-healthy controls and (2) report the rate of clinical genetic testing in this population. Methods: The PCMR was queried to identify patients with DCM secondary to MC who underwent exome sequencing as part of a prior prospective study at 14 sites in North America. Controls from the Indiana University Biobank were matched 4:1 with cases based on genomic similarity. A curated gene list was comprised of 46 genes associated with CM. A second gene list included 102 genes associated with CM or MC in the ClinVar database. The prevalence of variants was compared using Chi-Square or Fisher’s exact tests. Results: A total of 32 patients were identified with DCM secondary to MC. Median enrollment age was 1.5 years (IQR 0.4-10.3) and 53% were female. There was no family history in 88%. The prevalence of P/LP variants from the curated list was 34.4% in cases compared to 4.7% in controls (p<0.001). The prevalence of P/LP variants from the ClinVar list was 31.3% in cases compared to 6.3% in controls (p<0.001). Variants were identified in 8 genes: ABCC9, ANKRD1, MYH6, MYH7, MYPN, PLN, TPM1 and TTN. No P/LP variants were found in desmosomal genes. The prevalence of variants was higher but not significantly different in older children compared to infants (45.0% vs 16.6%, p=0.1). The prevalence of variants in MC was similar to previously reported rates in all causes of DCM (34.4% vs 28.8%, p=0.5). Only 22% of cases received clinical genetic testing. Conclusion: Approximately 1 in 3 children with DCM secondary to MC carry a CM-associated P/LP variant, which is significantly higher than the prevalence in healthy controls. Given the high prevalence, routine genetic testing should be considered in this population.

Abstract 4138455: Employing Bioinformatic Tools to Identify High-Risk Variants of Unknown Significance in Aortopathy Genes Associated with Aortic Dissection

Introduction: Pathogenic (P) or likely pathogenic (LP) aortopathy gene variants cause hereditary thoracic aortic aneurysm and dissection, but P/LP variants only explain approximately 20% of cases of thoracic aortic disease. Variants of unknown significance (VUS) are frequently identified among individuals undergoing genetic testing for thoracic aortic disease, however managing patients with VUSs remains clinically challenging. Hypothesis: Bioinformatic tools can be used to determine thresholds above which VUSs may be considered “high-risk.” Methods: Primary analyses were performed in the Penn Medicine Biobank (PMBB) which is composed of 43,731 participants who volunteered to have clinical information linked to biospecimen data including DNA which has undergone whole exome sequencing. PMBB participants were screened for VUSs in one of 11 aortopathy genes. VUS REVEL, AlphaMissense and minor allele frequency (MAF) high-risk thresholds were derived using cutpointR, a statistical package to optimize continuous variable thresholds based on binary outcomes. These thresholds were applied to VUS carriers in the PMBB and two independent validation cohorts. Logistic regression analysis was performed to test the association of high-risk VUSs with prevalent thoracic aortic disease. Results: There were 11,925 individuals in PMBB who carried at least one missense VUS. Carrying a VUS was associated with a modest increased risk of thoracic aortic aneurysm (TAA: OR=1.14, 95% confidence interval [CI] 1.01 to 1.29, P =0.034) but no increased risk of thoracic aortic dissection (OR=1.04, 95%CI 0.55 to 2.00, P =0.896). As VUS REVEL or AlphaMissense increased, or MAF decreased, the association between VUSs and thoracic aortic disease became statistically significant. Using cutpointR, we derived REVEL (>0.649), AlphaMissense (>0.2543), and MAF (<8.16x10 -6 ) thresholds that together identified 435 high-risk VUSs robustly associated with prevalent dissection (OR=7.85, 95%CI 4.73 to 13.03, P <0.001), though the association with TAA was attenuated (OR=2.35, 95%CI 1.62 to 3.42, P <0.001). Similar results were observed in the UK Biobank (UKB) and Early Onset Sporadic Thoracic Aortic Dissection (ESTAD) cohorts. Conclusions: VUSs that met high-risk bioinformatic thresholds were strongly associated with prevalent aortic dissection in the PMBB, UKB and ESTAD. Future investigation is warranted to determine if these thresholds can instruct clinical care of individuals carrying aortopathy gene VUSs.

Abstract 4136581: Shared Genetic Burden of Ischemic and Non-Ischemic Dilated Cardiomyopathy

Introduction: Ischemic dilated cardiomyopathy (IDCM) is defined as DCM caused by severe stenosis of one or more coronary arteries. Patients with non-ischemic dilated cardiomyopathy (NIDCM) have normal coronary arteries and may possess rare disease-causing variants in cardiac proteins such as TTN . However, IDCM and NIDCM share echocardiographic and pathologic features, possibly suggesting a shared etiology. Hypothesis: There is a similar prevalence of pathogenic (P)/likely pathogenic (LP) variants in patients with IDCM compared to patients with NIDCM. Aims: We compared the prevalence of P/LP genetic variants in DCM-associated genes in a cohort of patients with NIDCM or IDCM who received a heart transplant. Methods: We conducted a retrospective cohort study of patients who received a heart transplant between May 2017 and October 2023 at Cedars Sinai Medical Center in Los Angeles, CA. All subjects had LVEF ≤ 50% and LVEDD ≥ 95 th percentile for sex and height. Whole exome sequencing was performed on explanted heart tissue. We assessed for rare variants (minor allele frequency <0.1%) in genes with a definite, strong, or moderate rating in the ClinGen DCM Gene Curation Expert Panel. Variant pathogenicity was interpreted using the American College of Genetics and Genomics criteria for DCM. Ischemic cardiomyopathy was defined by the Felker criteria (Felker et al., JACC 2002). Results: We included 60 subjects: 16 with IDCM and 44 with NIDCM. There was no difference in LVEF or LVEDD between groups (TABLE). 2/16 (12.5%) patients with IDCM carried a P/LP variant, both of which were in TTN . 11/44 (25.0%) patients with NIDCM carried a P/LP variant: 6 in TTN and 1 each in FLNC, ILK, LMNA, MYH7 , and RBM20 . The prevalence of TTN variants in each group was similar (12.5% in IDCM vs. 13.6% in NIDCM) and higher than the expected frequency in the general population (0.2%). There was no significant difference in the prevalence of P/LP variants between the groups (chi-squared p-value 0.493, Bayes factor 0.45). Conclusion: Patients with IDCM exhibited a similar prevalence of P/LP variants to NIDCM patients, which suggests a shared genetic burden. If further studies confirm our findings, genetic testing for individuals with IDCM may be warranted.

CASRdb: A Publicly Accessible Comprehensive Database for Disease-associated Calcium-sensing Receptor Variants.

Genetic testing of the calcium-sensing receptor (CASR) gene is crucial for confirming diagnoses of familial hypocalciuric hypercalcemia type I (FHH1) and autosomal dominant hypocalcemia type I (ADH1). Therefore, we created a publicly accessible comprehensive database of the disease-causing variants of the CASR gene. We used two sources for variant reports: (1) we conducted a systematic review in the Embase and Pubmed databases from inception to March 2023, using search strategies associated with CASR. We identified all articles reporting CASR variants associated with disorders of calcium metabolism. (2) Additionally, data associated with pathogenic (P) or likely pathogenic (LP) variants in the ClinVar and LOVD databases were retrieved. Benign or likely benign variants were excluded. Variants of uncertain significance (VUS) were included only if they were reported in the literature. We generated a library of CASR variants associated with phenotypes, which has been made available on a website. We identified a total of 498 variants, of which 121 (24.3%) were associated with ADH1 and 377 (75.7%) with FHH1. Most included variants were identified from the literature (117 activating and 352 inactivating variants), and the majority of these were not documented in ClinVar/LOVD (73/117, 62.4% activating variants; 207/352, 58.8% inactivating variants). We developed CASRdb, a database that compiles information on all CASR variants associated with disorders of calcium metabolism from existing literature and genomic databases. Our database stands out due to the substantially higher number of disease-associated variants it contains, highlighting its comprehensive nature. The website is available at http://casrdb.mgh.harvard.edu.

Exome sequencing data reanalysis of 200 hypertrophic cardiomyopathy patients: the HYPERGEN French cohort 5 years after the initial analysis

BackgroundApproximately half of hypertrophic cardiomyopathy (HCM) patients lack a precise genetic diagnosis. The likelihood of identifying clinically relevant variants increased over time.MethodsIn this study, we conducted a gene-centric reanalysis of exome data of 200 HCM cases 5 years after the initial analysis. This reanalysis prioritized genes with a matched HCM entry in the OMIM database and recently emerging HCM-associated genes gathered using a text mining-based literature review. Further classification of the identified genes and variants was performed using the Clinical Genome Resource (ClinGen) resource and American College of Medical Genetics and Genomics (ACMG) guidelines to assess the robustness of gene–disease association and the clinical actionability of the prioritized variants.ResultsAs expected, the majority of patients carried variants in MYBPC3 and MYH7 genes, 26% (n = 51) and 8% (n = 16), respectively, in accordance with the initial analysis. The vast majority of pathogenic (P) and likely pathogenic (LP) variants were found in MYBPC3 (22 out of 40 variants) and MYH7 (8 out of 16 variants) genes. Three genes—not included in the initial analysis—were identified: SVIL, FHOD3, and TRIM63. Considering only patients with unique variants in the last three genes, there was a 9% enhancement in variant identification. Importantly, SVIL variant carriers presented apical and septal HCM, aortopathies, and severe scoliosis for one patient. Ten patients (5%) carried variants in the FHOD3 gene, six in hotspot regions (exons 12 and 15). We identified seven variants within the TRIM63 gene in 12 patients (6%). Homozygous variants were detected in 2.5% of the cohort in MYBPC3 (n = 1), MYL3 (n = 1), and TRIM63 (n = 3) genes.ConclusionOur study revealed that no variants were found in the ACTC1, TPM1, and TNNI3 genes in the HYPERGEN cohort. However, we identified variants in five out of the eight HCM core genes, with a high prevalence in young patients. We identified variants in three recent HCM-associated genes (SVIL, FHOD3, and TRIM63) in 35 patients, with 18 patients carrying unique variants (9%). Our results further emphasize the usefulness of exome data reanalysis, particularly in genotype-negative patients.

Updating probability of pathogenicity for RYR1 and CACNA1S exon variants in individuals without malignant hyperthermia after exposure to triggering anesthetics

Objectives We aimed to classify genetic variants in RYR1 and CACNA1S associated with malignant hyperthermia using biobank genotyping data in patients exposed to triggering anesthetics without malignant hyperthermia phenotype. Methods We identified individuals who underwent surgery and were exposed to triggering anesthetics without malignant hyperthermia phenotype and who had RYR1 or CACNA1S genotyping data available in our biobank. We classified all variants in the cohort using a Bayesian framework of the American College of Medical Genetics and Genomics and the Association of Molecular Pathologists guidelines for variant classification and updated the posterior probabilities from this model with the new information from our biobank cohort. Results We identified 253 patients with 95 RYR1 variants and 12 CACNA1S variants. After applying a Bayesian framework, we classified 17 variants as benign (B), 31 as likely benign (LB), 57 as uncertain (VUS), and 2 as likely pathogenic (LP). When we incorporated evidence about unique exposures to malignant hyperthermia triggering anesthetic agents, 48 of 107 (45%) variants were downgraded (9 to B, 37 to LB, and 2 to VUS). Notably, 41 (72%) of 57 VUSs were downgraded to B or LB. When repeat anesthetics in the same individual were counted as one exposure, 42 of 107 (39%) of variants were downgraded (5 to B, 35 to LB, and 2 to VUS). Specifically, 37 (65%) of 57 VUSs were downgraded to LB. Conclusion Deidentified biorepositories linked with anesthetic data offer a new method of integrating clinical evidence into the assessment of variant probability of pathogenicity.

Genotype-phenotype correlations in patients with titin variants and cardiomyopathy from a Swiss cohort

Abstract Background Alterations in titin (TTN) are mostly described with dilated cardiomyopathy (DCM). Few studies identified TTN variants in arrhythmogenic cardiomyopathy (ACM), but the overlap of dilated and arrhythmogenic phenotypes in TTN variants is scarcely studied. Most pathogenic TTN variants are located on the A-band, but there is few knowledge about the phenotypic presentation of I-band and Z-disc (non-A-band) variants. Purpose The aim of this study is to characterize and investigate the outcome of patients with cardiomyopathy and pathogenic (P) or likely pathogenic (LP) TTN variants with a particular focus on the cardiomyopathy phenotype (DCM vs. ACM) at initial presentation and follow-up. Further, we studied the association of the TTN variant (A-band vs. non-A-band regions) and a unique phenotype. Methods This is a retrospective multicenter study from a Swiss cohort including 34 patients with P/LP TTN variants and cardiomyopathy. The variables of the secured anonymized database include patient characteristics, genetic, ECG, echocardiographic, and cardiac magnetic resonance (CMR) data. Ventricular arrhythmia (VA) events were divided into non-sustained ventricular tachycardia (nsVT), sustained ventricular tachycardia (sVT) and sudden cardiac arrest (SCA +/- ventricular fibrillation). Results Of 34 included patients, 68% were male. Median age at presentation was 43.5 years. 29 (85%) patients presented with DCM, 7 (21%) left dominant ACM (2020 Padua criteria) and none ARVC (2010 ARVC Task Force criteria). At median follow-up of 48.5 months, 17 (50%) patients had VA events (15 nsVT, 5 sVT and 3 SCA events). VA events significantly increased over time (p=0.008) and were not associated with gender or left ventricular ejection fraction (LVEF) <35%. Under optimal guideline-directed medical therapy (GDMT) mean LVEF and right ventricular function increased significantly (LVEF: 28.3±14.4% vs. 41.9±9.8% and right ventricular fractional area change (RV FAC): 24.7±7.5% vs. 35.8±4.8%, p<0.05). Late gadolinium enhancement (LGE) on CMR was commonest in the basal septum, but no patient had higher degree atrioventricular block (AVB) at baseline and follow-up. At last follow-up, 8 (24%) patients fulfilled ACM and 13 (38%) DCM criteria (Figure 1). Most TTN variants were located on the A-band (21, 62%), 10 (29%) on the non-A-band regions. Latter had higher rates of sVT (log-rank p=0.041) during follow-up (Figure 2). 20 (59%) had LP and 11 (32%) P variants. P variant carriers were younger at disease presentation compared to LP variant carriers. Conclusion Patients with TTN variants frequently present as DCM, whereas presentation as left dominant ACM is less common. TTN does not seem to be a classical ARVC causing gene. Despite favourable biventricular remodelling under GDMT, ventricular arrhythmic events increase during follow-up. I-band and Z-disk variants are associated with a higher risk of sVT, which may help in the risk stratification process.

High incidence of malignant arrhythmias and heart failure in patients with RBM20-associated cardiomyopathy: a multicenter cohort study and review of the literature

Abstract Background Patients with RBM20 cardiomyopathy commonly experience malignant arrhythmias, sudden cardiac death (SCD), and progressive heart failure (HF). Objective The aim of this study is to investigate genotype-phenotype correlations, clinical outcomes and causes of death in patients with RBM20-associated cardiomyopathy in a multicenter cohort, combined with an overview of the current literature. Methods This international, multicenter cohort included all patients with cardiomyopathy who had a pathogenic (P) or likely pathogenic (LP) RBM20 variant. For survival and regression analysis, we matched a control group based on sex, age, and presence of left ventricular dysfunction. Additionally, we conducted a literature search on studies investigating RBM20-associated cardiomyopathy. Results Sixty-two patients (45% male) were included in the study, with a mean age of 42 ± 15 years at presentation. Of the LP/P RBM20 variants identified, 9 were novel. Eleven variants were truncating, while the remaining variants consisted of missense and splice-site variants. Patients with a truncating variant who developed HF were on average older at the time of diagnosis compared to patients with missense variants (mean age 62 ± 9 vs. 45 ± 14; p=0.01). After a median follow-up duration of 5.0 [1.0 – 10.5] years, 21 (34%) patients reached the composite endpoint of ventricular arrhythmias (VA), implantable cardioverter-defibrillator therapy, heart transplantation (HTx), left ventricular assist device (LVAD) implantation, or cardiovascular death. Of these, 19 (31%) patients experienced malignant VA (mean age 45 ± 15 years, 63% males). Males were at higher risk for the composite endpoint (log-rank p= 0.02), particularly for VA (log-rank p=0.007). The literature review analyzed 34 studies with a total of 678 patients, of whom 53% were male. In these studies, 123 (24%) patients experienced a VA, 58 (12%) underwent HTx or were treated with LVAD, and 52 (11%) died. Conclusion In this multicenter study the patients carrying a LP/P variant in RBM20 variants had a severe phenotype, with a high incidence of VA, particularly in males. Additionally, this study presents 9 novel DNA variants, of which 8 were truncating, and were mainly observed in older individuals. Finally, we report a lower mortality rate compared to the literature, possibly due to a higher number of heart transplantations performed in our patient cohort.Survival composite endpoint for sex

Variant reclassification over time decreases the level of diagnostic uncertainty in monogenic obesity: Experience from two centres.

The diagnosis of monogenic obesity is burdened by frequent variants of uncertain significance (VUS). We describe our real-life approach of variant reassessment over time and we assess whether inconclusive variants are decreasing in monogenic obesity. We tested for monogenic obesity (genes: LEPR, POMC, ADCY3, PCSK1, CARTPT, SIM1, MRAP2, LEP, NTRK2, BDNF, KSR2, MAGEL2, SH2B1, MC4R, MC3R) in 101 children/adolescents (11.7 [7.3-13.7] years, 3.6 [3.3-4.0] z-BMI) in Verona and 183 (11.3 [8.4-12.2] years, 3.2 [2.7-3.9] z-BMI) in Naples from January 2020 to February 2023. In March-July 2024 we reassessed the baseline variants by updated software interpretation and literature renavigation. We initially found 20 VUS, 4 Likely Pathogenic (LP), 5 Likely Benign (LB) and 1 benign variant in 33 individuals. At follow-up, 6 VUS were reclassified as benign/LB, one LP as pathogenic and 3 LB as benign. Overall, 10/30 variants (6/18 in Verona, 3/11 in Naples and a variant found in both centres) were reclassified, leading to a less uncertain report for 13 of 33 variant-carrying patients. Monogenic obesity was diagnosed in 3 probands in Verona and 4 in Naples, carrying variants at MC4R or NTRK2. Our variant reassessment was effective to improve classification certainty for the 39% of patients and suggested that the molecular diagnosis of monogenic obesity is becoming more accurate over time.

Chromosomal Abnormalities Detected by Chromosomal Microarray Analysis and Karyotype in Fetuses with Ultrasound Abnormalities.

Chromosomal microarray analysis (CMA) is a first-line test to assess the genetic etiology of fetal ultrasound abnormalities. The aim of this study was to evaluate the effectiveness of CMA in detecting chromosomal abnormalities in fetuses with ultrasound abnormalities, including structural abnormalities and non-structural abnormalities. A retrospective study was conducted on 368 fetuses with abnormal ultrasound who received interventional prenatal diagnosis at Meizhou People's Hospital from October 2022 to December 2023. Samples of villi, amniotic fluid, and umbilical cord blood were collected according to different gestational weeks, and karyotype and CMA analyses were performed. The detection rate of chromosomal abnormalities in different ultrasonic abnormalities was analyzed. There were 368 fetuses with abnormal ultrasound, including 114 (31.0%) with structural abnormalities, 225 (61.1%) with non-structural abnormalities, and 29 (7.9%) with structural combined with non-structural abnormalities. The detection rate of aneuploidy and pathogenic (P)/likely pathogenic (LP) copy number variations (CNVs) of CMA in fetuses with structural abnormalities was 5.26% (6/114), the detection rate of karyotype was 2.63% (3/114), and the additional diagnosis rate of CMA was 2.63%. In the fetuses with ultrasonic non-structural abnormalities, the detection rate of karyotype was 6.22% (14/225), the detection rate of aneuploidy and P/LP CNVs in fetuses with ultrasonic structural abnormalities was 9.33% (21/225), and the additional diagnosis rate of CMA was 3.11%. There was no significant difference in chromosome abnormality detection rate of CMA among structural abnormality, non-structural abnormality, and structural abnormality combined with non-structural abnormality groups (5.3%, 9.3%, and 13.8%, p = 0.241), also among multiple ultrasonic abnormality and single ultrasonic abnormality groups (14.8%, and 7.3%, p = 0.105). CMA can significantly improve the detection rate of genetic abnormalities in prenatal diagnosis of ultrasonic abnormal fetuses compared with karyotype analysis. CMA is a more effective tool than karyotyping alone in detecting chromosomal abnormalities in fetuses with ultrasound abnormalities.

Homologous recombination deficiency gene panel analysis results in synchronous endometrial and ovarian cancers.

The objective of this study was to analyze the genetic alterations of tumors within the scope of the homologous recombination deficiency gene panel in patients diagnosed with synchronous endometrial ovarian cancer who have been followed for over 5 years using next-generation sequencing. DNA was isolated from the patient's formalin-fixed, paraffin-embedded tissue blocks. Next-generation sequencing was performed using the Illumina capture-based sequencing method. Samples were sequenced using the Sophia HR Solution DNA Kit. Seven patients were included in this study. The ratios of likely pathogenic (LP)/pathogenic (P) somatic mutations in ATM (serine/threonine kinase or Ataxia-telangiectasia mutated gene), BRCA2 (breast cancer type 2 susceptibility gene), BARD1 (BRCA1 associated RING domain 1), TP53 (tumor protein p53), BIRP1 (BRCA1-interacting helicase 1 gene), PALB2 (partner and localizer of BRCA2), and CHECK2 were 21 (48.8%), 8 (18.6%), 5 (11.6%), 3 (6.9%), 2 (4.6%), 2 (4.6%), and 2 (4.6%), respectively, in endometrium, and the ratios of somatic mutations in ATM, BRCA2, TP53, BARD1, RAD54L (DNA repair/recombination protein like), BIRP1, and RAD51D (RAD51 recombinase paralog D) were 24 (60%), 6 (15%), 5 (12.5%), 2 (5%), 2 (5%), 1 (2.5%), and 1 (2.5%), respectively, in ovary. In endometrioid-synchronous endometrial ovarian cancer cases, P/LP mutations were observed in ATM and CHECK2 genes in endometrium and ATM, BRCA2, and TP53 genes in ovary. In two non-endometrioid-synchronous endometrial ovarian cancer cases, CHEK2 (checkpoint kinase 2) mutations were observed in endometrium and ATM and TP53 mutations in ovary, whereas in one case, P/LP mutations in ATM and TP53 genes were common in both tissues. Pathogenic variations confirming the diagnosis of synchronous endometrial ovarian cancer with genetic alterations were identified in all but one case. ATM gene mutation emerged as the most common alteration and has a potential association with a favorable prognosis.

Genetic analysis of pregnancy loss and fetal structural anomalies by whole exome sequencing

BackgroundWhole exome sequencing (WES) has been recommended to investigate the genetic cause of fetal structural anomalies. In this retrospective study, we aimed to evaluate the diagnostic yield of WES in our cohort of families with pregnancy loss or termination of pregnancy due to structural anomalies.MethodsAs aneuploidy, triploidy and copy number variations (CNVs) could be detected by exome-based CNV analysis, only WES is performed in this study. And the results of 375 cases assessed by WES were analyzed.ResultsThe overall detection rate was 32.3% (121/375), including aneuploidy and triploidy (7.5%, 28/375), CNVs (5.1%, 19/375) and single-nucleotide variants (SNVs) /insertions or deletions (Indels) (19.7%, 74/375). Among these, the diagnostic yield for likely pathogenic (LP) or pathogenic (P) CNVs is 4.8% (18/375), and the diagnostic yield for LP or P SNVs/Indels is 15.2% (57/375). And an additional 4.8% (18/375) of cases had CNVs or SNVs/Indels classified as variants of uncertain significance (VUS) with potential clinical significance.ConclusionsOur findings expand the known mutation spectrum of genetic variants related to fetal abnormalities, increase our understanding of prenatal phenotypes, and enable more accurate counseling of recurrence risk for future pregnancies.

Single Nucleotide Polymorphism array analysis for fetuses from balanced translocation carriers at the second trimester

Prenatal diagnosis is crucial for pregnancies from couples with a carrier of a balanced translocation. We retrospectively reviewed 195 pregnancies from 189 couples with a balanced translocation carrier. Of these, 126 were from natural conception, while 69 were conceived through assisted reproductive technology (ART) with preimplantation genetic diagnosis (PGD). Both single nucleotide polymorphism (SNP) array analysis and conventional karyotyping were conducted on all pregnancies, and karyotype-visible imbalances and pathogenic/likely pathogenic copy number variations (CNVs) were categorized as clinically significant abnormalities. In natural conception group, couples with a female carrier experiencing more than two miscarriages accounted for 30.2%, significantly higher than the 14.0% in male carrier couples (p<0.05). In the PGD group, similar difference was observed between female and male carrier couples (p<0.05). In the natural pregnancies, SNP array analysis yielded additional 12 cases of CNVs, including two cases of pathogenic (P)/likely pathogenic (LP) aberrations, four variants with uncertain significance (VUS), and six likely benign variants. Only two CNVs were found to be associated with translocation breakpoints, which were finally confirmed to be of parental inheritance. In the PGD pregnancies, two cases of VUS unrelated to the translocation breakpoints were revealed. Taken together, repeated miscarriage was more frequently observed in couples where the carrier was female than male. Conventional SNP array analysis in prenatal diagnosis indicated insufficient evidence to support the notion that balanced translocations increase the likelihood of fetuses having clinically significant CNVs.

Germline AIP variants in sporadic young acromegaly and pituitary gigantism: clinical and genetic insights from a Han Chinese cohort.

Variants in the Aryl hydrocarbon receptor-interacting protein (AIP) gene have been identified in sporadic acromegaly and pituitary gigantism, especially in young patients, with a predisposition to aggressive clinical phenotype and poor treatment efficacy. The clinical characteristics of patients with sporadic acromegaly and pituitary gigantism as well as AIP variants in Han Chinese have been rarely reported. We aimed to identify AIP gene variants and analyze the clinical characteristics of patients with sporadic acromegaly and pituitary gigantism in Han Chinese. The study included 181 sporadic acromegaly (N = 163) and pituitary gigantism (N = 18) patients with an onset age of no more than 45 years old, who were diagnosed, treated, and followed up in Huashan Hospital. All 6 exons and their flanking regions of the AIP gene were analyzed with Sanger sequencing or NGS. The clinical characteristics were compared between groups with and without AIP variants. Germline AIP variants were found in 15/181 (8.29%) cases. In patients with an onset age ≤30 years old, AIP variants were identified in 12/133 (9.02%). Overall, 13 variants were detected. The pathogenic (P) variants p.R304X and p.R81X were identified in four cases, with two instances of each variant. Six exon variants (p.C254R, p.K103fs, p.Q228fs, p.Y38X, p.Q213*, and p.1115 fs) have not been reported before, which were likely pathogenic (LP). Patients with P/LP variants had younger onset ages, a higher prevalence of pituitary gigantism, larger tumor volumes, and a higher percentage of Ki-67-positive cells in tumors. In addition, the group with P/LP variants showed a less significant reduction of GH levels in an acute octreotide suppression test (OST) [17.7% (0, 65.0%) vs. 80.5% (63.9%, 90.2%), P = 0.001], and a trend of less GH decrease after the 3-month treatment with long-acting somatostatin analogs (SSAs). Germline AIP variants existed in sporadic Chinese Han acromegaly and pituitary gigantism patients and were more likely to be detected in young patients. AIP variants were associated with more aggressive tumor phenotypes and less response to SSA treatment.

Rare forms of monogenic diabetes in non-European individuals. First reports of CEL and RFX6 mutations from the Indian subcontinent.

Monogenic diabetes is one of the few examples in metabolic diseases in which a real precision medicine approach can be implemented in daily clinical work. Unfortunately, most of what is known today comes from studies in Whites, thus leaving much uncertainty about the genetics and the clinical presentation of monogenic diabetes in non-Europeans. To fill this gap, we report here two pedigrees from Bangladesh with CEL- and RFX6- diabetes, two rare types of monogenic diabetes which have never been described so far in individuals of the Indian subcontinent. Next generation, Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) were performed. Variants' interpretation was according to the American College of Medical Genetics and Genomics guidelines. In the pedigree with CEL-diabetes, a large and never described deletion of exon 2-11 of CEL (confirmed by MLPA) affecting the entire catalytic domain and being likely pathogenic (LP) was observed in both the proband (who had diabetes at 16) and his mother (diabetes at 31), but not in relatives with normoglycemia. In the pedigree with RFX6-diabetes, a LP protein truncation variant (PTV, p.Tyr192*) in RFX6 was found in both the proband (diabetes at 9) and his mother (diabetes at 30), thus suggesting high heterogeneity in disease onset. Normoglycemic relatives were not available for genetic testing. We report genetic features and clinical presentation of the first two cases of CEL- and RFX6-diabetes from the Indian subcontinent, thus contributing to fill the gap of knowledge on monogenic diabetes in non-Europeans.

Genotype-first analysis in an unselected health system-based population reveals variable phenotypic severity of COL4A5 variants.

Our knowledge of X-linked Alport Syndrome [AS] comes mostly from selected cohorts with more severe disease. We examined the phenotypic spectrum of X-linked AS in males and females with a genotype-based approach using data from the Geisinger MyCode DiscovEHR study, an unselected health system-based cohort with exome sequencing and electronic health records. Patients with COL4A5 variants reported as pathogenic (P) or likely pathogenic (LP) in ClinVar, or protein-truncating variants (PTVs), were each matched with up to 5 controls without COL4A3/4/5 variants by sociodemographics, diabetes diagnosis, and year of first outpatient encounter. AS-related phenotypes included dipstick hematuria, bilateral sensorineural hearing loss (BSHL), proteinuria, decreased eGFR, and ESKD. Out of 170,856 patients, there were 29 hemizygous males (mean age 52.0 y [SD 20.0]) and 55 heterozygous females (mean age 59.3 y [SD 18.8]) with a COL4A5 P/LP variant, including 48 with the hypomorphic variant p.Gly624Asp. Overall, penetrance (having any AS phenotypic feature) was highest for non-p.Gly624Asp P/LP variants (males: 94%, females: 85%), intermediate for p.Gly624Asp (males: 77%, females: 69%), compared to controls (males: 32%; females: 50%). The proportion with ESKD was highest for males with P/LP variants (44%), intermediate for males with p.Gly624Asp (15%) and females with P/LP variants (10%), compared to controls (males: 3%, females 2%). Only 47% of individuals with COL4A5 had completed albuminuria screening, and a minority were taking renin-angiotensin aldosterone system (RAAS) inhibitors. Only 38% of males and 16% of females had a known diagnosis of Alport syndrome or thin basement membrane disease. In an unselected cohort, we show increased risks of AS-related phenotypes in men and women compared to matched controls, while showing a wider spectrum of severity than has been described previously and variability by genotype. Future studies are needed to determine whether early genetic diagnosis can improve outcomes in Alport Syndrome.

Systematic analysis of SCN5A variants associated with inherited cardiac diseases

BackgroundSCN5A variants are associated with a spectrum of cardiac electrical disorders with clear phenotypes. However, they may also be associated with complex phenotypic traits like overlap syndromes or pleiotropy, which have not been systematically described. In addition, the involvement of SCN5A in dilated cardiomyopathies (DCMs) remains controversial. ObjectiveWe aimed to evaluate the different phenotypes associated with pathogenic (P)/likely pathogenic (LP) SCN5A variants and to determine the prevalence of pleiotropy in a large multicentric cohort of P/LP SCN5A variant carriers. MethodsThe DNA of 13,510 consecutive probands (9960 with cardiomyopathies) was sequenced with a custom panel of genes. Individuals carrying a heterozygous single P/LP SCN5A variant were selected and phenotyped. ResultsThe study included 170 P/LP variants found in 495 patients. Of them, 119 (70%) were exclusively associated with a single well-established phenotype: 91 with Brugada syndrome, 15 with type 3 long QT syndrome, 6 with progressive cardiac conduction disease, 4 with multifocal ectopic Purkinje-related premature contractions, and 3 with sick sinus syndrome. Thirty-two variants (19%) were associated with overlap syndromes or pleiotropy. The 19 remaining variants (11%) were associated with atypical or unclear phenotypes. Of those, 8 were carried by 8 patients presenting with DCM with a debatable causative genotype/phenotype link. ConclusionMost P/LP SCN5A variants were found in patients with primary electrical disorders, mainly Brugada syndrome. Nearly 20% were associated with overlap syndromes or pleiotropy, underscoring the need for comprehensive phenotypic evaluation. The concept of SCN5A variants causing DCM is extremely rare (8/9960) if not questionable.

Implementation of long-read sequencing for routine molecular diagnosis of familial mediterranean fever

BackgroundLong-read sequencing technology, widely used in research, is proving useful in clinical diagnosis, especially for infectious diseases. Despite recent advances, it hasn't been routinely applied to constitutional human diseases. Long-read sequencing detects intronic variants and phases variants, crucial for identifying recessive diseases. MethodsWe integrated long-read sequencing into the clinical diagnostic workflow for the MEFV gene, responsible for familial Mediterranean fever (FMF), using a Nanopore-based workflow. This involved long-range PCR amplification, native barcoding kit library preparation, GridION sequencing, and in-house bioinformatics. We compared this new workflow against our validated method using 39 patient samples and 3 samples from an external quality assessment scheme to ensure compliance with ISO15189 standards. ResultsOur evaluation demonstrated excellent performance, meeting ISO15189 requirements for reproducibility, repeatability, sensitivity, and specificity. Since October 2022, 150 patient samples were successfully analyzed with no failures. Among these samples, we identified 13 heterozygous carriers of likely pathogenic (LP) or pathogenic (P) variants, 1 patient with a homozygous LP/P variant in MEFV, and 4 patients with compound heterozygous variants. ConclusionThis study represents the first integration of long-read sequencing for FMF clinical diagnosis, achieving 100 % sensitivity and specificity. Our findings highlight its potential to identify pathogenic variants without parental segregation analysis, offering faster, cost-effective, and accurate clinical diagnosis. This successful implementation lays the groundwork for future applications in other constitutional human diseases, advancing precision medicine.

"Re-evaluation of variants of uncertain significance in patients with hereditary arrhythmogenic disorders"

BackgroundGenetic diagnostics support the diagnosis of hereditary arrhythmogenic diseases, but variants of uncertain significance (VUS) complicate matters, emphasising the need for regular reassessment. Our study aims to reanalyse rare variants in different genes in order to decrease VUS diagnoses and thus improve risk stratification and personalized treatment for patients with arrhythmogenic disorders.MethodsGenomic DNA was analysed using Sanger sequencing and next-generation sequencing (NGS). The Data was evaluated using various databases and in silico prediction tools and classified according to current ACMG standards by two independent experts.ResultsWe identified 53 VUS in 30 genes, of which 17 variants (32%) were reclassified. 13% each were downgraded to likely benign (LB) and benign (B) and 6% were upgraded to likely pathogenic (LP). Reclassifications mainly occurred among variants initially classified in 2017–2019, with rates ranging from 50 to 60%.ConclusionThe results support the assumption that regular reclassification of VUS is important, as it provides new insights for genetic diagnostics, that benefit patients and guide therapeutic approach.

Variant classification changes over time in the clinical molecular diagnostic laboratory setting

BackgroundVariant classification in the setting of germline genetic testing is necessary for patients and their families to receive proper care. Variants are classified as pathogenic (P), likely pathogenic (LP), uncertain...