Likely Pathogenic Research Articles

The diagnostic yield of exome sequencing in liver diseases from a curated gene panel

Exome sequencing (ES) has been used in a variety of clinical settings but there are limited data on its utility for diagnosis and/or prediction of monogenic liver diseases. We developed a curated list of 502 genes for monogenic disorders associated with liver phenotypes and analyzed ES data for these genes in 758 patients with chronic liver diseases (CLD). For comparison, we examined ES data in 7856 self-declared healthy controls (HC), and 2187 patients with chronic kidney disease (CKD). Candidate pathogenic (P) or likely pathogenic (LP) variants were initially identified in 19.9% of participants, most of which were attributable to previously reported pathogenic variants with implausibly high allele frequencies. After variant annotation and filtering based on population minor allele frequency (MAF ≤ 10–4 for dominant disorders and MAF ≤ 10–3 for recessive disorders), we detected a significant enrichment of P/LP variants in the CLD cohort compared to the HC cohort (X2 test OR 5.00, 95% CI 3.06–8.18, p value = 4.5e−12). A second-level manual annotation was necessary to capture true pathogenic variants that were removed by stringent allele frequency and quality filters. After these sequential steps, the diagnostic rate of monogenic disorders was 5.7% in the CLD cohort, attributable to P/LP variants in 25 genes. We also identified concordant liver disease phenotypes for 15/22 kidney disease patients with P/LP variants in liver genes, mostly associated with cystic liver disease phenotypes. Sequencing results had many implications for clinical management, including familial testing for early diagnosis and management, preventative screening for associated comorbidities, and in some cases for therapy. Exome sequencing provided a 5.7% diagnostic rate in CLD patients and required multiple rounds of review to reduce both false positive and false negative findings. The identification of concordant phenotypes in many patients with P/LP variants and no known liver disease also indicates a potential for predictive testing for selected monogenic liver disorders.

Correlation of TCR Repertoire Diversity with Myeloid Gene Mutations in Chronic and Advanced Phase Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are characterized by a chronic phase (CP), with indolent clinical behavior and, in 10-30% of cases, progression to advanced phases (AP), with aggressive behavior and compromised prognosis. Numerous factors have been associated with progression to AP, including the acquisition of mutations in recurrently mutated myeloid genes (1,2). On the other hand, decreased diversity in the T-cell receptor (TCR) repertoire has been associated with worse prognosis in solid tumors. The purpose of this work is to study the correlation between the acquisition of mutations in myeloid genes and the diversity of the TCR repertoire in chronic and advanced myeloproliferative neoplasms. Methods: Patients with chronic CP and AP myeloproliferative neoplasms were selected. The selected patients underwent an NGS panel of 48 myeloid genes. Only pathogenic (P) or likely pathogenic (LP) variants were considered. In parallel, the study of the TCR repertoire by NGS with the panel “AmpliSeq for Illumina Immune Repertoire Plus, TCR beta Panel” (Illumina™) was performed on the available samples. Finally, the obtained data were correlated. The study was approved by the local ethics committee. Results: A total of 22 patients were selected, 10 in CP and 12 in AP. Regarding diagnosis, 8 were polycythemia vera (PV), 4 essential thrombocythemia (ET), 6 secondary myelofibrosis (MFS) (1 to PV and 5 to ET), 2 systemic mastocytosis (MS) and 1 primary myelofibrosis (MFP). Eighty percent were male and the mean age was 68 years. The AP patients were significantly older (74 vs. 62 years of age, p<0,001). Regarding the myeloid NGS panel result, 41 mutations were found in 19 patients. Fourteen percent of the patients had no mutation, 36% had an isolated driver mutation and 50% had additional mutations. Of these, 9% were in CP and 81% were in AP. The most recurrently mutated genes were JAK2 (45.4%), CALR (18.2%), ASXL1, IDH2, SRSF2, IDH2 (all 13.6%), MPL and KIT (both 9.1%). Regarding the results of the TCR repertoire, it was possible to perform the study in 11 patients, 4 in CP and 7 in AP. Only 6/11 had results of sufficient quality to allow analysis. Of these 6 patients, 3 were in CP and 3 in AP. As can be seen in Figure 1, the diversity of the TCR between CP patients and PC patients was different. Thus, while the pattern of diversity in the CP phase shows the expected distribution, the diversity in AP shows a characteristic pattern. Conclusions: APs of MPNs show an abnormal TCR diversity pattern. This pattern may be related to the acquisition of myeloid gene mutations in AP. To our knowledge, this is the first study demonstrating an alteration in TCR diversity in AP and opens the door to future fields of research.

Del(20q) As a Somatic Gene Rescue in Adult Patients: Can Germline Mutations Other Than SBDS be Found in Affected Patients?

Deletions involving chromosome 20 (20q-) are present in up to 5-8% of myeloid neoplasms (MN) and have been associated with either myelodysplastic (MDS) or myeloproliferative (MPN) features, chiefly myelofibrosis. Theoretically, any chromosomal deletion may result in haploinsufficient expression of tumor suppressor genes (TSGs) or in loss of a protective allele in heterozygous carriers of germline (GL) variants or heterozygous somatic (SM) mutations. However, deletions have also been implicated as a maladaptive somatic gene rescue (SGR) event in carriers of GL bone marrow failure (BMF) genes such as SAMD9 and SBDS. The latter is found in Shwachman-Diamond syndrome whereby the upregulated EIF6 gene, responsible for hematopoietic dysfunction, is clonally removed by SM 20q deletion event. We identified an interesting 35-years-old patient with mild cytopenia and 20q- in which a cryptic homozygous SBDS c.A148T GL mutation was found. This prompted us to search in other adult patients with 20q deletion for: i) other cases with SBDS gene alteration (both hetero and homozygous), ii) other GL variants which could be clonally relieved by compensatory 20q- or, iii) disease-prone GL variants on 20q, whereby either disease-prone suppressive allele is included in the deleted region. Within a cohort of 5,308 cases with various MN, 215 patients with 20q- were identified. WES results were available for 350 patients. We applied a stringent bioanalytic algorithm to identify unique GL alterations (not deposited in SM databases) in genes expressed in hematopoietic tissues and involved in BMF and cancer, and a population allelic frequency of <1% which were classified as pathogenic (P), likely pathogenic (LP), and VUS with particular level of clinical suspicion (sVUS). In 76 of 215 patients with 20q-, 346 GL mutations in genes of potential interest were identified and further analyzed. No P/LP variants were detected on genes within commonly deleted region on chr.20q, and no additional carriers of SBDS variants were found. However, we identified 9 pathogenic GL variants in potentially pertinent genes including GL alterations in DDX41, STAG2 and TP53 which have been described in the context of MN. However, these gene variants were also present in patients with diploid 20q. The remaining 6 variants included heterozygous P/LP GL alterations in 3 different Fanconi anemia genes ( FANCA, FANCM, FANCD2), SUZ12, and TINF2. The latter, TINF2, a telosome gene, known to be mutated in dyskeratosis congenita (DC), carried an LOF c.605-1G>A in a patient with MDS/MPN with a family history of cancer who later developed 20q-. The patient did not show any symptoms of DC-like features, which usually present at an early age. Since FANCA LOF with SGR via overepression of EIF6 has previously been described 1, we investigated and did not identify any cases in our cohort of EIF6 SGR resultant overexpression. While occasional patients with no previously diagnosed SBDS can be found among MDS with 20q-, screening for SBDS genes demonstrates that such a genetic configuration among adult patients with 20q is very rare. In addition, only 9 out of 215 patients with 20q- carried other pathogenic GL variants in non-recurrent genes which appear to be likely coincidental. This suggests that del20q is not a SGR in response to a GL mutation, but rather a SGR due to other aforementioned mechanisms, which are currently being explored in our cohort of patients.

The VUS Challenge in Cystic Kidney Disease: A Case-Based Review.

Genetic testing in nephrology is becoming increasingly important to diagnose patients and to provide appropriate care. This is especially true for autosomal dominant polycystic kidney disease (ADPKD) because this is a common cause of kidney failure and genetically complex. In addition to the major genes, PKD1 and PKD2 , there are at least six minor loci, and phenotypic, and in some cases, genetic overlap with other cystic disorders. Targeted next-generation sequencing, a low-cost, high-throughput technique, has made routine genetic testing viable in nephrology clinics. Appropriate pre- and post-testing genetic counseling is essential to the testing process. Carefully assessing variants is also critical, with the genetic report classifying variants in accordance with American College of Medical Genetics and Genomics guidelines. However, variant of uncertain significance (VUSs) may pose a significant challenge for the ordering clinician. In ADPKD, and particularly within PKD1 , there is high allelic heterogeneity; no single variant is present in more than 2% of families. The Mayo/Polycystic Kidney Disease Foundation variant database, a research tool, is the best current database of PKD1 and PKD2 variants containing over 2300 variants identified in individuals with polycystic kidney disease, but novel variants are often identified. In patients with a high pretest probability of ADPKD on the basis of clinical criteria, but no finding of a pathogenic (P) or likely pathogenic (LP) variant in a cystic kidney gene, additional evaluation of cystic gene VUS can be helpful. In this case-based review, we propose an algorithm for the assessment of such variants in a clinical setting and show how some can be reassigned to a diagnostic grouping. When assessing the relevance of a VUS, we consider both patient/family-specific and allele-related factors using population and variant databases and available prediction tools, as well as genetic expertise. This analysis plus further family studies can aid in making a genetic diagnosis.

Family screening in hypertrophic cardiomyopathy - clinical and genetic yield at baseline

Abstract Background Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disorder, often with monogenic inheritance and accordingly, familial aggregation of HCM is common. International guidelines recommend family screening of relatives of patients with HCM to allow pre-symptomatic identification and early interventions. The clinical and genetic yield of implemented screening programs have been scarcely described and might improve by adjustments. We hypothesized that screening can be improved. Purpose To assess the yield of clinical and genetic family screening in HCM at first screening visit. Methods The study was a retrospective study of clinical and genetic cascade screening of adult relatives (age ≥18 years) of HCM probands, screened for familial occurrence of HCM from 2006 to 2022 at a regional assembly of clinics for inherited cardiac diseases (population 2.6 million). We collected demographic, clinical, symptomatic, and genetic characteristics from electronic health records, and a national pedigree database. Diagnostic work-up included a 12-lead ECG, and echocardiography. Relatives of probands carrying a likely pathogenic (LP) or pathogenic (P) variant were also offered cascade genetic testing. Patients and relatives were diagnosed according to the 2014 ESC guidelines. Results We included 603 adult relatives (55% female, mean age 44±15 years) (table 1). At baseline, 118 relatives (20%) fulfilled diagnostic criteria for HCM, and 135 (22%) were carriers of LP/P variants; 78 (13%) of these did not fulfill diagnostic criteria for HCM. Thus, the combined clinical and genetic yield of baseline screening was 33%. A total of 108 (18%) clinically unaffected relatives did not carry the family’s LP/P variant and were discharged from follow-up. This indicates that 50% of the relatives at baseline could be categorized as affected, genetically predisposed or clinically and genetically not at risk. Relatives fulfilling criteria for HCM at baseline were older (51 vs. 42 years, p<0.001), more likely to report cardiopulmonary symptoms (46% vs 16%, p<0.001), be male (59% vs 41%, p<0.001), and have abnormal ECG features (40% vs. 12%, p<0.001), compared to relatives not fulfilling criteria for HCM at baseline. On echocardiography, relatives fulfilling criteria for HCM had a mean maximal wall thickness of 17±4 mm and 14% had left ventricular outflow tract obstruction (gradient at rest/Valsalva≥30 mmHg). Conclusion At baseline screening, one in three relatives of HCM patients were diagnosed with HCM or found to be genetically predisposed to the disease. One in five clinically unaffected relatives not carrying the proband’s disease-causing variant could be discharged from screening. Considering the pattern of inheritance in HCM, the theoretical risk of developing HCM in the remaining 50% of the relatives is low. The non-diagnostic findings may be useful for identification of the remaining truly at-risk relatives.Table 1:Baseline characteristics

Contribution of copy number variants to the genetic etiology of dilated cardiomyopathy

Abstract Background/Introduction Dilated cardiomyopathy (DCM) is one of the most common cardiomyopathies, with a prevalence of 1 in 250. Single nucleotide variants (missense, nonsense, or frameshift) in definitive DCM genes are the most frequently identified in positive genetic studies. Despite major advances in genetic diagnostic technologies in recent decades, and more than 100 genes potentially associated with DCM identified to date, the diagnostic yield is still lower than 40%. Copy number variants (CNVs) have been identified as cause of the disease in several studies, especially in genes intolerant to haploinsufficiency. However, the contribution of CNVs to the etiology of DCM has not been evaluated in large cohorts of DCM patients and remains unknown. Purpose To determine the percentage of cases in which CNVs have been identified as the cause of disease in a large cohort of DCM patients evaluated genetically. Methods Retrospective study in which the presence of CNVs was evaluated in a cohort of patients diagnosed with DCM and studied by next-generation-sequencing (NGS). CNVs were detected using a read depth approach and confirmed by an orthogonal molecular technique (Sanger sequencing, MLPA, or dPCR). Results 6,512 DCM patients referred for genetic testing were sequenced in our center by NGS, and the median age at the time of genetic study was 51.1 years (range 0-93). A pathogenic (P) or likely-pathogenic (LP) disease-causing variant was identified in 1,495 of the patients (diagnostic yield of 22.9%). In 67 (1.02%) of the cases, a CNV classified as P/LP was identified. DMD was the gene with the highest number of CNVs identified (n=40), followed by TTN (n=7) and LMNA (n=5). Of the 67 cases, 45 (67%) corresponded to deletions, with a lesser extent to duplications (n=9; 13%), complex alterations (n=7; 11%), and deletion/insertion (n=6; 9%). Only two of the patients harboring disease-causing CNVs had other variants potentially associated with DCM: a likely-pathogenic variant in MYH7 and a homozygous pathogenic variant in the ALMS1 gene. Conclusion In our cohort of DCM patients, 1.02% harbored CNVs considered P/LP, being DMD the most frequently identified gene. These results show that systematic CNVs analysis in NGS studies improves the yield of genetic testing in DCM and highlights the importance of a complete genetic study that includes structural variant detection in the diagnosis of the disease.

Abstract 16991: Rare Variants in BMP10 Confer Risk of Pulmonary Arterial Hypertension

Background: Genetic mutations in the genes of BMPR2 pathway are the most crucial cause of pulmonary arterial hypertension. BMP10 is the major ligand of BMPR2, but it remains elusive that whether BMP10 mutation are associated with pulmonary arterial hypertension (PAH). Methods: Whole exome sequencing was applied to screen the BMP10 mutations in 378 patients with congenital heart disease associated with pulmonary arterial hypertension (CHD-PAH). The pathogenicity of variants was assessed by ACMG guideline. Pedigree structures and familial transmission of variants were analyzed for the case who carried the BMP10 mutation. The deleterious function of BMP10 mutations were analyzed via in vitro experiments. Results: Five rare and pathogenic BMP10 variants were identified in five patients with CHD-PAH, including three missense mutations (p.Q74R, p.R96Q, p.I335S) and two nonsense mutations (p.Q116Ter, p.R313Ter). These five cases did not carry mutation in other known PAH risk genes. Five BMP10 mutations were classified as uncertain significance(UVS) or likely pathogenic(LP) variants according to ACMG guideline. It was notworthy that the high impact likely loss-of-function variants in BMP10 were found in 0.53% (2 out of 378) of cases, which was significiantly hihger than those of 0.002% (4 out 251106) in the general population from the GnomAD database (P<10 -4 ). Familial analysis revealed that a case had inherited the heterozygous BMP10 p.I335S missense variant from her affected mother, strongly indicating the co-segregation of BMP10 mutaiton and PAH phenotype. Functional experiments implied that the missense mutation p.I335S and the nonsense mutation p.Q116Ter both resulted in reduced BMP10 protein expression in 293T cells. Conclusion: We identified strong association between rare damaging variants in BMP10 and PAH. The novel BMP10 mutations found in this study will provide new insights into the genetic mechanism of PAH.

Abstract 14779: Whole Exome Sequencing Study in Pediatric Postural Orthostatic Tachycardia Syndrome Unveils a Path Forward From Genes to Drugs

Introduction: Postural orthostatic tachycardia syndrome (POTS) is more common in Caucasian individuals with a positive family history, while the risk in females is ~4-times of that in males. To date, the etiology of POTS is poorly understood. Hypothesis: Integrative sequencing and translational approaches to identify its genetic underpinning may better understand its pathogenesis and molecular mechanisms for the development of novel precision-based therapies. Methods: We adopted exome sequencing to evaluate the etiology and molecular mechanisms of POTS. We examined a unique collection of 87 unrelated pediatric POTS cases (61 females and 26 males, 6~21 years old), using exome sequencing. Non-relationship between individuals were validated by genome wide genotyping and identity by descent (IBD) analysis. Results: All the individuals were classified as European by principal component analysis (Figure 1). We identified 184 pathogenic (P) or likely pathogenic (LP) variants (in 162 genes). Sixty-eight (78%) cases have at least one P/LP variant. Each P/LP mutation is supported by a minimum of two databases, including ClinVar, InterVar or HGMD_Pro_2023.1. Over-representation analysis (ORA) by the DisGeNET approach showed the enriched gene sets of muscle hypotonia and epilepsy/seizures with statistical significance (FDR<E-10). Clinically, among the 87 WES patients, 41 (47%) suffered from chronic fatigue, and 13 (15%) patients have the symptoms of epilepsy, seizure or convulsion. A number of drug targets in these gene sets are highlighted, including vitamin D receptor, carbamoyl-phosphate synthase 1, methylmalonyl-CoA mutase, androgen receptor, and phenylalanine hydroxylase. Conclusions: Our results suggests that chronic fatigue and epilepsy/seizure symptoms are comorbid symptoms in POTS patients, resulting from common genetic defects. Future precision-based therapies targeting these specific genetic defects may lead to improved outcome in POTS.

Abstract 14922: Cardiovascular Genetic Testing: Insights Into Patient Demographics, Provider Characteristics, and Indications

Introduction: Genetic testing offers the opportunity for early diagnosis of cardiovascular disease (CVD). Despite guideline recommendations, genetic testing is vastly underutilized in CVD. Methods: This single-center, retrospective study included CVD patients who underwent outpatient genetic testing, using Invitae kits, at UCSD Sulpizio Cardiovascular Center from September 2018 to February 2023. Patient demographics, provider characteristics, indications for testing, genetic testing data, and genetic counselor referrals were collected. Results: A total of 374 patients were included with a median age of 57 years. Patients were predominantly male (58.6%) and Caucasian (64.7%). Prevalence of genetic testing increased yearly from 2019 (n = 51) to 2022 (n = 142). The most common indication for genetic testing was cardiomyopathy (76.7%) followed by aortopathy (13.6%) and hyperlipidemia (5.3%). Heart failure (71.3%) providers were most likely to order genetic testing followed by adult congenital (14.4%) providers (Figure 1). There were 79 (21.1%) patients who had either a pathogenic (P) or likely pathogenic (LP) mutation, 166 (44.4%) patients who had a variant of uncertain significance, and 44 (55.7%) patients who had more than one gene mutation present. The most common genes implicated were those that cause cardiomyopathy, MYBC3 (n = 9) and TTR (n= 9). For patients who had either a P or LP mutation, 59.5% of them were referred to a genetic counselor and only 36.7% were actually seen by one. Conclusion: In this study, the prevalence of outpatient genetic testing for CVD is increasing with a significant proportion ordered by heart failure physicians for cardiomyopathy. Of the patients who tested positive, less than half received genetic counseling. These data suggest an opportunity for research focused on improving access to and patient acceptance of genetic counseling. As genetic testing becomes more prevalent, genetic counseling should increase in parallel.

DNA Mismatch Repair Gene Variant Classification: Evaluating the Utility of Somatic Mutations and Mismatch Repair Deficient Colonic Crypts and Endometrial Glands.

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. Lynch syndrome specific tumor features were evaluated for their ability to support the ACMG/InSiGHT framework in classifying variants of uncertain clinical significance (VUS) in the MMR genes. Twenty-eight CRC or EC tumors from 25 VUS carriers (6xMLH1, 9xMSH2, 6xMSH6, 4xPMS2), underwent targeted tumor sequencing for the presence of microsatellite instability/MMR-deficiency (MSI-H/dMMR) status and identification of a somatic MMR mutation (second hit). Immunohistochemical testing for the presence of dMMR crypts/glands in normal tissue was also performed. The ACMG/InSiGHT framework reclassified 7/25 (28%) VUS to likely pathogenic (LP), three (12%) to benign/likely benign, and 15 (60%) VUS remained unchanged. For the seven re-classified LP variants comprising nine tumors, tumor sequencing confirmed MSI-H/dMMR (8/9, 88.9%) and a second hit (7/9, 77.8%). Of these LP reclassified variants where normal tissue was available, the presence of a dMMR crypt/gland was found in 2/4 (50%). Furthermore, a dMMR endometrial gland in a carrier of an MSH2 exon 1-6 duplication provides further support for an upgrade of this VUS to LP. Our study confirmed that identifying these Lynch syndrome features can improve MMR variant classification, enabling optimal clinical care.

THU616 Analysis Of Germline Mutations In Patients With Sporadic Adrenocortical Carcinoma

Abstract Disclosure: M. Terzolo: None. S. Grisanti: None. M. Scatolini: None. P. Tomaiuolo: None. E. Grosso: None. V. Basile: None. D. Cosentini: None. S. Puglisi: None. M. Laganà: None. P. Perotti: None. L. Saba: None. E. Rossini: None. S. Sigala: None. M. Volante: None. A. Berruti: None. Background: Adrenocortical carcinoma (ACC) is a rare cancer associated with hereditary syndromes in 10% of cases. Germline variants (GVs) in patients with ACC have been identified mainly in pediatric patients and in family cases, while data on GVs in adult patients with sporadic ACC are limited. Methods: We analyzed germline DNA from 150 adult patients with sporadic ACC sequentially referred to our centers between 1998 and 2019. We designed a custom panel of 17 genes potentially involved in the pathogenesis of ACC: AIP, APC, ARMC5, ARNT, BRCA1, BRCA2, CTNNB1, IGF2, MEN1, MSH2, MSH6, PDE8B, PDE11A, PRKACA, PRKACB, PRKAR1A, and TP53. NGS data were analyzed by a semi-automated bioinformatic pipeline. All GVs were studied using effect predictor tools (PolyPhen and SIFT). Specific databases (ClinVar, Varsome, gnomAD, IARC TP53, HGMD) were used for variant classification according to ACMG criteria. Variants interpreted as pathogenic (P) or likely pathogenic (LP) were considered as positive. Clinical, pathological and genomic data were analyzed in different Cox models to study prognostic impact of covariates for disease-free survival (DFS), progression-free survival (PFS) and overall survival (OS). Results: We identified 21 unique GVs in 24/150 patients (16%). GVs were found in 9 genes including APC (n=3), ARMC5 (n=3), MSH2 (n=3), PDE11A (n=3), TP53 (n=3), MSH6 (n=2), PDE8B (n=2), AIP (n=1) and CTNNB1 (n=1). Eleven positive GVs including 3 GVs with deleterious potential were found in 14/150 patients (9.3%). Some variants were particularly enriched, with a frequency of 0.02% compared to an overall frequency of 0.001-0.004% in the gnomAD database. We found a new GV in TP53 (G105D) in a patient who was later found to have a sister with ACC and, for the first time, 3 GVs in ARMC5 (P731R). Patients with ARMC5 GVs had large cortisol-secreting tumors and one case displayed pathologic features of ACC and combined macronodular cortical disease. Positive GVs were associated with a shorter OS (50 vs 142 months, HR 1.81; 95%CI, 0.86-3.82, p=0.118) and PFS (8 vs 30 months, HR 3.11; 95%CI, 1.57-6.16, p=0.001) but not DFS (27 vs 32 months, HR 1.07; 95%CI, 0.52-2.22, p=0.845). At multivariate analysis, known clinical factors (age, surgery of primary ACC, ENSAT stage, hypercortisolism) were found to have prognostic impact on OS. However, GVs remained independent predictors of PFS and OS in metastatic patients. Conclusions: In a series of 150 patients with ACC, we found that 9.3% of them had positive GVs, thus confirming findings from the TCGA analysis. We describe for the first time the presence of ARMC5 GVs in patients with ACC and we found a novel pathogenic variant of TP53. Pathologic features of one ARMC5 case suggest a possible progression from macronodular hyperplasia to ACC. Finally, the present findings suggest that GVs can affect ACC progression and survival of affected patients. Presentation: Thursday, June 15, 2023

Frequency of actionable secondary findings in 7472 Korean genomes derived from the National Project of Bio Big Data pilot study.

Exome and genome sequencing (ES/GS) in genetic medicine and research leads to discovering genomic secondary findings (SFs) unrelated to the purpose of the primary test. There is a lack of agreement to return the SF results for individuals undergoing the test. The aim of this study is to investigate the frequency of actionable secondary findings using GS data obtained from the rare disease study and the Korean Genome and Epidemiology Study (KoGES) in the National Project of Bio Big Data pilot study. Pathogenic (P) or likely pathogenic (LP) variants of 78 SF genes recommended by the American College of Medical Genetics and Genomics (ACMG) were screened in the rare disease study and KoGES. The pathogenicity of SF gene variants was determined according to the ACMG interpretation. The overall SF rate was 3.75% for 280 individuals with 298 P/LP variants of 41 ACMG SF genes which were identified among 7472 study participants. The frequencies of genes associated with cardiovascular, cancer, and miscellaneous phenotypes were 2.17%, 1.22%, and 0.58%, respectively. The most frequent SF gene was TTN followed by BRCA2. The frequency of actionable SFs among participants with rare disease and general population participants in the Korean population presented here will assist in reporting results of medically actionable SFs in genomic medicine.

Diagnostic yield of genetic testing in a multinational heterogeneous cohort of 2088 DCM patients.

Familial dilated cardiomyopathy (DCM) causes heart failure and may lead to heart transplantation. DCM is typically a monogenic disorder with autosomal dominant inheritance. Currently disease-causing variants have been reported in over 60 genes that encode proteins in sarcomeres, nuclear lamina, desmosomes, cytoskeleton, and mitochondria. Over half of the patients undergoing comprehensive genetic testing are left without a molecular diagnosis even when patient selection follows strict DCM criteria. This study was a retrospective review of patients referred for genetic testing at Blueprint Genetics due to suspected inherited DCM. Next generation sequencing panels included 23-316 genes associated with cardiomyopathies and other monogenic cardiac diseases. Variants were considered diagnostic if classified as pathogenic (P) or likely pathogenic (LP). Of the 2,088 patients 514 (24.6%) obtained a molecular diagnosis; 534 LP/P variants were observed across 45 genes, 2.7% (14/514) had two diagnostic variants in dominant genes. Nine copy number variants were identified: two multigene and seven intragenic. Diagnostic variants were observed most often in TTN (45.3%), DSP (6.7%), LMNA (6.7%), and MYH7 (5.2%). Clinical characteristics independently associated with molecular diagnosis were: a lower age at diagnosis, family history of DCM, paroxysmal atrial fibrillation, absence of left bundle branch block, and the presence of an implantable cardioverter-defibrillator. Panel testing provides good diagnostic yield in patients with clinically suspected DCM. Causative variants were identified in 45 genes. In minority, two diagnostic variants were observed in dominant genes. Our results support the use of genetic panels in clinical settings in DCM patients with suspected genetic etiology.

BARD1 germline variants induce haploinsufficiency and DNA repair defects in neuroblastoma.

High-risk neuroblastoma is a complex genetic disease that is lethal in more than 50% of patients despite intense multimodal therapy. Through genome-wide association studies (GWAS) and next-generation sequencing, we have identified common single nucleotide polymorphisms and rare, pathogenic or likely pathogenic germline loss-of-function variants in BARD1 enriched in neuroblastoma patients. The functional implications of these findings remain poorly understood. We correlated BARD1 genotype with expression in normal tissues and neuroblastomas, along with the burden of DNA damage in tumors. To validate the functional consequences of germline pathogenic or likely pathogenic BARD1 variants, we used CRISPR-Cas9 to generate isogenic neuroblastoma (IMR-5) and control (RPE1) cellular models harboring heterozygous BARD1 loss-of-function variants (R112*, R150*, E287fs, and Q564*) and quantified genomic instability in these cells via next-generation sequencing and with functional assays measuring the efficiency of DNA repair. Both common and rare neuroblastoma-associated BARD1 germline variants were associated with lower levels of BARD1 mRNA and an increased burden of DNA damage. Using isogenic heterozygous BARD1 loss-of-function variant cellular models, we functionally validated this association with inefficient DNA repair. BARD1 loss-of-function variant isogenic cells exhibited reduced efficiency in repairing Cas9-induced DNA damage, ineffective RAD51 focus formation at DNA double-strand break sites, and enhanced sensitivity to cisplatin and poly (ADP-ribose) polymerase (PARP) inhibition both in vitro and in vivo. Taken together, we demonstrate that germline BARD1 variants disrupt DNA repair fidelity. This is a fundamental molecular mechanism contributing to neuroblastoma initiation that may have important therapeutic implications.

A curated census of pathogenic and likely pathogenic UTR variants and evaluation of deep learning models for variant effect prediction.

Introduction: Variants in 5' and 3' untranslated regions (UTR) contribute to rare disease. While predictive algorithms to assist in classifying pathogenicity can potentially be highly valuable, the utility of these tools is often unclear, as it depends on carefully selected training and validation conditions. To address this, we developed a high confidence set of pathogenic (P) and likely pathogenic (LP) variants and assessed deep learning (DL) models for predicting their molecular effects. Methods: 3' and 5' UTR variants documented as P or LP (P/LP) were obtained from ClinVar and refined by reviewing the annotated variant effect and reassessing evidence of pathogenicity following published guidelines. Prediction scores from sequence-based DL models were compared between three groups: P/LP variants acting though the mechanism for which the model was designed (model-matched), those operating through other mechanisms (model-mismatched), and putative benign variants. PhyloP was used to compare conservation scores between P/LP and putative benign variants. Results: 295 3' and 188 5' UTR variants were obtained from ClinVar, of which 26 3' and 68 5' UTR variants were classified as P/LP. Predictions by DL models achieved statistically significant differences when comparing modelmatched P/LP variants to both putative benign variants and modelmismatched P/LP variants, as well as when comparing all P/LP variants to putative benign variants. PhyloP conservation scores were significantly higher among P/LP compared to putative benign variants for both the 3' and 5' UTR. Discussion: In conclusion, we present a high-confidence set of P/LP 3' and 5' UTR variants spanning a range of mechanisms and supported by detailed pathogenicity and molecular mechanism evidence curation. Predictions from DL models further substantiate these classifications. These datasets will support further development and validation of DL algorithms designed to predict the functional impact of variants that may be implicated in rare disease.

Identification of a second genetic alteration in patients with SHOX deficiency individuals: a potential explanation for phenotype variability.

Our study aimed to assess the impact of genetic modifiers on the significant variation in phenotype that is observed in individuals with SHOX deficiency, which is the most prevalent monogenic cause of short stature. We performed a genetic analysis in 98 individuals from 48 families with SHOX deficiency with a target panel designed to capture the entire SHOX genomic region and 114 other genes that modulate growth and/or SHOX action. We prioritized rare potentially deleterious variants. We did not identify potential deleterious variants in the promoter or intronic regions of the SHOX genomic locus. In contrast, we found eight heterozygous variants in 11 individuals from nine families in genes with a potential role as genetic modifiers. In addition to a previously described Likely Pathogenic (LP) variant in CYP26C1 observed in two families, we identified LP variants in PTHLH and ACAN, and variants of uncertain significance in NPR2, RUNX2 and TP53 in more affected individuals from families with SHOX deficiency. Families with a SHOX alteration restricted to the regulatory region had a higher prevalence of a second likely pathogenic variant (27%) than families with an alteration compromising the SHOX coding region (2.9%, p= 0.04). In conclusion, variants in genes related to the growth plate have a potential role as genetic modifiers of the phenotype in individuals with SHOX deficiency. In individuals with SHOX alterations restricted to the regulatory region, a second alteration could be critical to determine the penetrance and expression of the phenotype.

Defining the scope of extended NIPS in Western China: evidence from a large cohort of fetuses with normal ultrasound scans

BackgroundStandard noninvasive prenatal screening(NIPS) is an accurate and reliable method to screen for common chromosome aneuploidies, such as trisomy 21, 18 and 13. Extended NIPS has been used in clinic for not only aneuploidies but also copy number variants(CNVs). Here we aim to define the range of chromosomal abnormalities that should be able to identify by NIPS in order to be an efficient extended screening test for chromosomal abnormalities.MethodsA prospective study was conducted, involving pregnant women without fetal sonographic structural abnormalities who underwent amniocentesis. Prenatal samples were analyzed using copy number variation sequencing(CNV-seq) to identify fetal chromosomal abnormalities.ResultsOf 28,469 pregnancies included 1,022 (3.59%) were identified with clinically significant fetal chromosome abnormalities, including 587 aneuploidies (2.06%) and 435 (1.53%) pathogenic (P) / likely pathogenic (LP) CNVs. P/LP CNVs were found in all chromosomes, but the distribution was not uniform. Among them, P/LP CNVs in chromosomes 16, 22, and X exhibited the highest frequencies. In addition, P/LP CNVs were most common on distal ends of the chromosomes and in low copy repeat regions. Recurrent microdeletion/microduplication syndromes (MMS) accounted for 40.69% of total P/LP CNVs. The size of most P/LP CNVs (77.47%) was < 3 Mb.ConclusionsIn addition to aneuploidies, the scope of extended NIPS should include the currently known P/LP CNVs, especially the regions with recurrent MMS loci, distal ends of the chromosomes, and low copy repeat regions. To be effective detection should include CNVs of < 3 Mb. Meanwhile, sufficient preclinical validation is still needed to ensure the clinical effect of extended NIPS.

Identifying patients at risk for hereditary myeloid malignancy syndromes incorporating a novel, self-administered questionnaire to an initial screening platform.

Four to 10% of cases of myeloid malignancies are inherited. We report our experience on hereditary myeloid malignancy syndromes (HMMS) incorporating a novel questionnaire in the screening platform for patients with myeloid malignancies and aplastic anemia. The questionnaire was sent via electronic patient portal prior to clinic visits. Patients screened positive based on responses to questionnaire items, presence of suspicion disease characteristics (young age, family history, monosomy 7 etc.) and/or presence of signs of HMMS. Those deemed at-risk based on questionnaire responses, clinical features and/or somatic mutation profile were offered germline testing. A total of 408 patients were screened, 141 (35%) were deemed at-risk. Fifty-four (38%) of at-risk patients were seen in the genetics clinic. Forty-one (76%) of the patients seen agreed to germline testing and 13 declined due to cost or personal decision. Twenty pathogenic (P)/likely-pathogenic (LP) germline mutations were identified in 16 (39%) of the tested patients. Five patients also had a variant of uncertain significance (VUS) and an additional 13 had at least 1 VUS without P/LP mutations (total 29 VUS's were found in 18 (44%) of tested patients). The median age of diagnosis for patients with P/LP mutations was 56 years versus 66 years in the entire cohort. Incorporating an electronic questionnaire is an effective screening method for HMMS. Many patients declined testing due to cost. These results highlight the importance of germline testing in patients with myeloid malignancies, further research in HMMS, and coverage by healthcare plans.

Genetic Characteristics of Latvian Patients with Familial Hypercholesterolemia: The First Analysis from Genome-Wide Sequencing.

There is limited data on the genetic characteristics of patients with familial hypercholesterolemia (FH) in Latvia. We aim to describe monogenic variants in patients from the Latvian Registry of FH (LRFH). Whole genome sequencing with 30× coverage was performed in unrelated index cases from the LRFH and the Genome Database of Latvian Population. LDLR, APOB, PCSK9, LDLRAP1, ABCG5, ABCG8, LIPA, LPA, CYP27A1, and APOE genes were analyzed. Only variants annotated as pathogenic (P) or likely pathogenic (LP) using the FH Variant Curation Expert Panel guidelines for LDLR and adaptations for APOB and PCSK9 were reported. Among 163 patients, the mean highest documented LDL-cholesterol level was 7.47 ± 1.60 mmol/L, and 79.1% of patients had LDL-cholesterol ≥6.50 mmol/L. A total of 15 P/LP variants were found in 34 patients (diagnostic yield: 20.9%): 14 in the LDLR gene and 1 in the APOB gene. Additionally, 24, 54, and 13 VUS were detected in LDLR, APOB, and PCSK9, respectively. No P/LP variants were identified in the other tested genes. Despite the high clinical likelihood of FH, confirmed P/LP variants were detected in only 20.9% of patients in the Latvian cohort when assessed with genome-wide next generation sequencing.

DNA damage response (DDR) variants and effects on the tumor transcriptome and microenvironment in east Asian (EA) prostate cancers (PCa).

75 Background: Aberrations of the DDR pathway have been shown to be prognostic of survival and predictive of PARP inhibition in PCa. We have previously reported on the prevalence of germline DDR mutations based on a 32-gene set in localized PCa in EA men, and observed comparable rates with that of White men. Here, we performed additional analyses investigating for associations between DDR variants to the somatic transcriptome of localized PCa. Methods: We utilized a prospectively recruited cohort of 172 EA patients, with centrally assessed Gleason’s score (GS) by an expert pathologist. Germline profiling was performed by whole exome sequencing (100X) using the Illumina Novaseq (CA). Joint genotyping was performed to annotate variants that were deemed to be pathogenic (P)/likely pathogenic (LP)/conflict of interest pathogenic (CIP) based on the CADD and REVEL scores. Tumor transcriptome was profiled using the Decipher Genomic Classifier (GC, Veracyte, CA) based on the Affymetrix ST array (ThermoFisher, CA). Results: Among the subset of 172 EA men, we observed DDR variants in 26 (15%) patients, which was comparable to the larger cohort of 890 men from the same institution. Among the DDR mutations, 2 (7.7%) were classified as P/LP, while 24 (92.3%) were CIP. Decipher GC scores were comparable between patients with and without DDR mutations (median GC score 0.87 vs 0.53, Mann-Whitney U P=0.10). For luminal-basal subtyping by either the PAM50 and Prostate specific subtyping models, we did not observe a difference for the basal (P=0.48) and luminal subtypes (P=0.21). Interestingly, DNA repair gene-set expression were comparable between patients with DDR mutations versus those without (median score -0.2 vs -0.17). However, we observed microenvironment differences for immune-related genes, where patients harboring DDR mutations manifested a less immune-suppressive environment versus those without (PDL2: -0.15 vs 0.2; Treg: 0.09 vs 0.13; MDSC: 0.03 vs 0.07, respectively). Conclusions: Herein, we report an extension of our previous work (Lua, et al. ASCO GU, 2023), where we show potential linkages between the germline DDR variant status and the tumor transcriptome. Our findings suggest the influence of germline DDR variants that extends beyond tumorigenesis.