Likely Pathogenic Research Articles

Genetic and clinical screening for hereditary primary hyperparathyroidism in a large Chinese cohort: a single-center study.

Primary hyperparathyroidism (PHPT) includes sporadic PHPT and hereditary PHPT. However, until now, there have been no exact data on the proportion and composition of hereditary PHPT in the Chinese PHPT population. This study aimed to clarify the proportion and composition of hereditary PHPT in patients at a large academic center in Beijing, China, and to analyze genotype-phenotype characteristics. A total of 394 newly diagnosed Han PHPT patients who consented to genetic screening were enrolled. Targeted next-generation sequencing (T-NGS) (including for MEN1, RET, CDKN1B, CaSR, HRPT2/CDC73, GNA11, AP2S1, GCM2), combined with MEN1-multiplex ligation-dependent probe amplification (MLPA) and CDC73-MLPA, was used for genetic screening. Diagnosis of hereditary PHPT was based on clinical manifestations, family history, and genetic screening. Thirty-seven pathogenic (P)/likely pathogenic (LP) variants were detected in 41 patients via T-NGS, and three patients carried long-range deletions of MEN1 or CDC73 detected by MLPA, with a variant detection rate of 11.2% (44/394). In total, 30 patients were clinically diagnosed with MEN1. Combined with genetic and clinical screening, the rate of hereditary PHPT in this study was 18.8% (74/394). For purposes of comparison, the rate of unequivocal nonhereditary PHPT was 66.5% (262/394); 14.7% (58/394) did not exhibit the clinical features of hereditary PHPT but carried variants of uncertain clinical significance and so could not be clearly categorized. Both the age at hospital visit (43.6 ± 14.0 versus 53.7 ± 14.9 years) and age at onset (35.4 ± 13.8 versus 50.6 ± 14.8 years) in the hereditary group (n = 74) were significantly lower than those in the nonhereditary group (n = 262). Higher levels of ionized calcium and serum β-CTX were observed in the hereditary group; proportions of parathyroid hyperplasia and multigland involvement were also higher. In addition to multigland disease and positive family history, it is recommended that patients with an age of onset less than 38 should be screened for hereditary forms. © 2023 American Society for Bone and Mineral Research (ASBMR).

Prenatal Chromosomal Microarray Analysis: Does Increased Resolution Equal Increased Yield?

Chromosomal microarray analysis (CMA) is considered a first-tier test for patients with developmental disabilities and congenital anomalies and is also routinely applied in prenatal diagnosis. The current consensus size cut-off for reporting copy number variants (CNVs) in the prenatal setting ranges from 200 Kb to 400 Kb, with the intention of minimizing the impact of variants of uncertain significance (VUS). Very limited data are currently available on the application of higher resolution platforms prenatally. The aim of this study is to investigate the feasibility and impact of applying high-resolution CMA in the prenatal setting. To that end, we report on the outcomes of applying CMA with a size cut-off of 20 Kb in 250 prenatal samples and discuss the findings and diagnostic yield and also provide follow-up for cases with variants of uncertain significance. Overall, 19.6% (49) showed one or more chromosomal abnormalities, with the findings classified as Pathogenic (P) or Likely Pathogenic (LP) in 15.6% and as VUS in 4%. When excluding the cases with known familial aberrations, the diagnostic yield was 12%. The smallest aberration detected was a 32 Kb duplication of the 16p11.2 region. In conclusion, this study demonstrates that prenatal diagnosis with a high-resolution aCGH platform can reliably detect smaller CNVs that are often associated with neurodevelopmental phenotypes while providing an increased diagnostic yield, regardless of the indication for testing, with only a marginal increase in the VUS incidence. Thus, it can be an important tool in the prenatal setting.

Genetic characterization of dilated cardiomyopathy patients undergoing heart transplantation in the Chinese population by whole-exome sequencing

BackgroundDilated cardiomyopathy (DCM) is one of the most frequent causes of heart failure and heart transplantation (HTx). The genetic basis of DCM among patients undergoing HTx remains to be further studied. This study aimed to characterize the genetic basis of DCM HTx in the Chinese population.MethodsIn total, 208 unrelated DCM patients who underwent HTx at Fuwai Hospital between June 2004 and June 2017 were included in this study. Whole-exome sequencing (WES) was performed for all patients. Gene burden analysis, variant classification, and genotype–phenotype correlation analysis were subsequently performed.ResultsAfter completing the bioinformatics analysis, gene burden analysis suggested that titin (TTN), filamin C (FLNC) and lamin A/C (LMNA) were significantly enriched with rare protein-altering variants. The frequencies of TTN and FLNC truncating variants in our cohort were 18.8% and 8.7%, respectively. Among the 165 rare variants in high evidence DCM-related genes, 27 (16.4%) and 59 (35.8%) were interpreted as pathogenic (P) and likely pathogenic (LP), respectively. In addition, 41 (47.7%) and 16 (18.6%) of these 86 P/LP variants are located in TTN and FLNC, respectively. The FLNC group contained more patients with NYHA class IV than the P/LP-negative group (FLNC, 16/18 vs. P/LP-negative, 81/123, P = 0.049).ConclusionsBased on WES, we provided a primary genetic spectrum of DCM patients undergoing HTx in the Chinese population. TTN and FLNC harbour the most P/LP variants. FLNC truncation may lead to severe clinical symptoms in DCM patients.

METB-09. GERMLINE PATHOGENIC VARIANTS IN 838 PEDIATRIC BRAIN TUMOR PATIENTS

Abstract The genetic contribution of rare pathogenic germline variation in cancer patients without a family history remains unclear. We sought to characterize the prevalence, spectrum, and clinical significance of pathogenic germline variation in cancer predisposition genes (CPGs) in pediatric brain tumor patients. Paired tumor and normal whole genome or exome sequencing was performed for 838 patients in the Pediatric Brain Tumor Atlas (PBTA). Rare variants in 196 CPGs were annotated as pathogenic (P) or likely pathogenic (LP) in an automated manner consistent with American College of Medical Genetics criteria and reviewed by an interdisciplinary panel. To assess enrichment, the frequency of CPG P-LP variants in cases was compared to the gnomAD v3.1 cancer-free control cohort (n=74,023). Second hit, mutational signature, and gene set enrichment analyses (GSEA) were performed on matched tumor sequencing to characterize differences in patients with and without germline P-LP variants in DNA repair genes. We observed 178 germline CPG P-LP variants in 163 PBTA patients (19.5%). CPG P-LP variants were most prevalent among Neurofibroma plexiform patients (64.3%), and NF1 and TSC2 harbored the most significant P-LP variant burden compared to controls (OR=69.5, p=2.4E-23, CI=34.6-137 and OR=357, p=1.1E-14, CI=71-3669, respectively). DNA repair gene P-LP variants were enriched in cases compared to controls (OR=4.4, p=8.3E-30). Patients harboring mismatch repair (MMR) gene P-LP variants exhibited significantly higher tumor-associated MMR-deficiency mutational signature exposures (p<0.05) and DNA repair GSEA scores relative to other patients (p=0.04). Second hit SNVs in tumors were identified in four patients with P-LP germline variants in NF1 (n=2), APC (n=1), and SUFU (n=1). Germline P-LP variants are enriched in PBTA patients, and DNA repair variants are predictive of repair-deficiency signatures in tumor samples. Our work emphasizes the need for germline sequencing to inform patient care.

METB-06. HIGH FREQUENCY OF CLINICALLY RELEVANT ALTERATIONS DETECTED BY INTEGRATED TUMOR AND GERMLINE GENE PANEL TESTING IN CHILDREN WITH CNS TUMORS – RESULTS FROM THE TEXAS KIDSCANSEQ STUDY

Abstract BACKGROUND Tumor and germline alterations detected by sequencing have the potential to inform management of children with CNS tumors. Comparative clinical utility data for available molecular sequencing modalities are needed to optimize testing strategies. METHODS Cancer patients less than 18 years of age were eligible to enroll in the Texas KidsCanSeq (KCS) clinical genomics study at six Texas institutions. Blood testing was performed on all patients using a pediatric solid tumor targeted DNA panel (124 genes), which included 35 genes associated with known cancer predisposition syndromes. Tumor testing was conducted on high-risk or recurrent tumors using the same DNA panel as well as an 81-gene RNA fusion panel. Tumor variants were classified as being of strong (Tier 1) or potential (Tier 2) clinical significance following AMP/ASCO/CAP guidelines. Germline pathogenic (P) or likely pathogenic (LP) variants per ACMG/AMP guidelines were considered clinically relevant. RESULTS A total of 224 children with CNS tumors were enrolled, including 146 patients with high-risk or recurrent tumors. Tissues were sufficient for analysis from 132 tumors and 211 germline samples. Clinically relevant alterations were identified in 90 of 132 (68%) tumors tested. Of 50 with therapeutic implications, 21 (42%) were not known from prior clinical testing. Therapeutically relevant tumor variants most frequently involved BRAF (n=20), CDKN2A/B (n=16), SMARCB1 (n=9), PIK3CA (n=8), and NF1 (n=7). One high grade glioma (in a patient with Lynch syndrome) was found to be hypermutated (tumor mutation burden >190 mut/Mb). Germline analysis detected P/LP variants in cancer predisposition genes in 25 of 211 (12%) patients tested, including 12 of 135 (9%) with high-risk/recurrent tumors. CONCLUSION Combined tumor and germline panel testing revealed alterations of clinical relevance according to consensus guidelines in approximately 70% of children with high-risk or recurrent CNS tumors.

Diagnostic yield and predictive value on left ventricular remodelling of genetic testing in dilated cardiomyopathy.

We assessed the diagnostic yield of genetic testing and the relationship of left ventricular (LV) reverse remodelling (LVRR) with the presence of DNA pathogenic (P) or likely pathogenic (LP) variants in patients with dilated cardiomyopathy (DCM). From 680 outpatients followed at the Heart Failure Outpatient Clinic of our institution, we selected subjects with a diagnosis of DCM as defined by LV ejection fraction (LVEF) ≤40% and LV dilatation not explained by coronary artery disease or other causes. All patients were offered genetic investigation of 42 disease-associated DCM genes with next-generation sequencing. Seventy patients fulfilled the definition of DCM and 66 underwent genetic investigation. We identified 18 P/LP variants in 16 patients, with a diagnostic yield of 24%. The most common variants were truncating TTN variants (n=7), followed by LMNA (n=3), cytoskeleton Z-disc (n=3), ion channel (n=2), motor sarcomeric (n=2), and desmosomal (n=1) genes. After a median follow-up of 53months (inter-quartile range 20-111), patients without P/LP variants exhibited higher systolic and diastolic blood pressure, lower plasma brain natriuretic peptide levels, and a larger extent of LVRR, as reflected by the increase in LVEF (+14% vs. +1%, P=0.0008) and decrease in indexed LV end-diastolic diameter (-6.5 vs. -2mm/m2 , P=0.03) compared with patients with P/LP variants. Our results confirm the high diagnostic yield of genetic testing in selected DCM patients and suggest that identification of P/LP variants in DCM portends poorer LVRR in response to guideline-directed medical therapy.

Clinical and genomic characteristics between DDR germline and somatic mutations in pan-cancer.

e15139 Background: The defects DNA-damage repair (DDR) genes would drive tumor formation and are associated with increased genomic instability and tumor mutational burden (TMB) in cancer. Although, alterations of DDR genes are common in tumor, the differences between the germline and somatic alterations are poorly characterized. Methods: The DNA sequencing data of 540 genes from 6174 pan-cancer patients were retrospectively collected and 276 DDR genes were analyzed. The variations were annotated as pathogenic (P), likely pathogenic (LP), and nonpathogenic (NP) according to ACMG (American College of Medical Genetics) guideline. The patients were divided into 3 groups: DDR-germline (P&LP germline variants, N = 766), DDR-somatic group (only P&LP somatic variants, N = 2923) and non-DDR group (NP variants, N = 2485). Results: In pan-cancer, 12.41% patients have germline DDR gene mutations, of which tumors with high germline mutation frequency include ovarian cancer (27/102, 26.47%), glioma (22/136, 16.18%), breast cancer (25/159, 15.72%), genitourinary cancer (17/114, 14.91%), biliary tract cancer (42/284, 14.79%), pancreatic cancer (28/193, 14.51%), colorectal cancer (95/727, 13.07%), head-neck tumors (11/85, 12.94%) and esophagus cancer (16/124, 12.90%). Meanwhile, 47.34% patients have somatic DDR gene mutations, of which tumors with high somatic mutation frequency include esophagus cancer (89/124, 71.77%), colorectal cancer (488/727, 67.13%), endometrial cancer (49/73, 67.12%), head-neck tumors (50/85, 58.82%), pancreatic cancer (111/193, 57.51%), gastric carcinoma (233/415, 56.14%), ovarian cancer (53/102, 51.96%) and biliary tract cancer (138/284, 48.59%). The most commonly germline alterations were found in TP53 (33%), PTEN (14%) and BRCA2 (14%), while in the somatic mutations, TP53 (52%) showed the highest frequency, PTEN (9%) and MSH3 (8%) followed. In pairwise comparisons of the three groups’ actionable mutations, DDR-somatic group was more likely to have alterations in TP53 (OR = 5.68), AKT3 (OR = 5.81) and APC (OR = 1.42) compared with DDR-germline group. Taken non-DDR group as reference, mutations in TP53 (OR = 5.09), PTEN (OR = 7.65), BRCA2 (OR = 5.32) were more common in DDR-germline group, but TP53 (OR = 28.90), PTEN (OR = 7.73), APC (OR = 2.68) in DDR-somatic group. The tumor mutational burden (TMB) was statistically different among the three groups (p < 2.22e−16). The DDR-somatic group exhibits the highest median TMB (5.68) compared with 1.42 in DDR-germline and 2.13 in non-DDR group. Conclusions: The differences in the mutation profile between the DDR-germline and DDR-somatic groups and the distinct actionable genes may indicate the different target-therapy choices. Besides, DDR-somatic group exhibited the highest TMB, which might indicate patients with somatic DDR alterations may better benefit from the immunotherapy.

Redefining early-onset cancer and risk of hereditary cancer predisposition.

10510 Background: For most solid tumors, early age of onset, a marker for potential increased inherited risk, has been arbitrarily defined as a diagnosis of cancer at age < 50. However, this disregards the variation in mean age of diagnoses across solid tumors resulting in the potential misclassification of patients at increased risk of germline alterations. Methods: 25,305 prospectively ascertained cancer patients, representing 32 solid tumor types, underwent sequencing analysis of up to 90 cancer predisposition genes using next-generation genetic analysis with reporting of germline likely pathogenic (LP) and pathogenic variants (P). Mean age of diagnosis by cancer type, with standard deviations (STD), were calculated according to Surveillance, Epidemiology, and End Results Program (SEER) data. Early-onset (EO-CA), average-onset (AO-CA), and late-onset (LO-CA) cancers were defined as cancers diagnosed < -1 STD, -1 < STD > 1, and > 1 STD around the mean age at diagnosis for the specific cancer type, respectively. Prevalence of LP/P variants was classified according to age of onset. Statistical analyses were performed using STATA. Results: Among 25,305 patients with 32 solid tumor types classified as per SEER data, 31% (n = 7,854) of patients were categorized as having EO-CA. Overall prevalence of germline alterations varied as a function of age at diagnosis with 19.2%(n = 1509), 15.9% (n = 2588/16,299), and 12.9% (149/1152) of EO-CA, AO-CA, and LO-CA patients harboring a germline LP/P variant (p < 0.0001). The enrichment of variants in EO-CAs was driven by high- and moderate-penetrance genes with 13.2%, 9.2% and 5.1% of EO-CA, AO-CA, and LO-CA patients having an LP/P variant (p < 0.0001); low-penetrance germline alterations were static across the age groups (6%, 6.7%, 7.8%). An age cutoff of 50 for early-onset cancer was > 1 STD below the mean age at diagnosis in 20 cancer types resulting in 2,226 patients who would not have been classified as having EO-CA inclusive of 394 patients who tested positive for a germline LP/P, representing 26.1% of all germline EO-CA positives. Conclusions: Early-onset cancer should be defined taking into consideration the mean age at diagnosis for a specific cancer type as an arbitrary cutoff of age < 50 misses over 25% of patients with an inherited cancer predisposition syndrome. Due to the high prevalence of inherited predisposition syndromes, genetic testing of all patients with EO-CA, irrespective of tumor type, is warranted.

Whole genome sequencing in the 100,000 Genomes project identifies a RYR2 variant associating with dilated cardiomyopathy and sudden unexpected death in the young

Abstract Funding Acknowledgements Type of funding sources: None. Background Previous studies have shown genetic testing in sudden unexpected death in the young (SUDY) cases identify a pathogenic variant in 13-27% of cases. This diagnostic yield increased up to 39% when genetic testing was combined with family evaluation. The 100,000 Genomes Project was launched to investigate the role of whole genome sequencing (WGS) as a diagnostic tool for cancers and rare diseases in a national health care system. Young sudden death victims with no finding on postmortem testing were enrolled to the SUDY cohort. Purpose We aimed to analyse WGS data in the SUDY cohort of 100,000 Genomes Project with panel analysis and trio studies in order to examine the yield of WGS in this population. Methods Sudden death victims, aged 40 or younger, without pre-existing condition that would explain the event and with no diagnosis established on postmortem examination were enrolled between 2017 and 2019 at different Genomic Medicine Centres (GMCs) across the UK. Parents were recruited in all paediatric cases. DNA sequencing was performed by Illumina. The platform's automated pipeline was used for variant calling. Panel based and hypothesis free approaches were used for data analysis. Variant analysis was performed with focus on variants with combined annotation-dependent depletion (CADD) score >15 and American College of Medical Genetics and Genomics (ACMG) classification of pathogenic (P), likely pathogenic (LP), or variant of unknown significance (VUS). Results A total of 15 participants, including 6 trios, were included in the study. Participants had a median age of 18 [IQR 1-29.5] and two thirds were male. Following assessment of all protein coding and intronic variants identified, only the missense variant RYR2:c.10046C>T (p.Ser3349Leu) was thought to be suitable for investigation for upgrade to likely pathogenic, giving a diagnostic yield of 6.67% for WGS in SUDY cases. This variant was detected in a 24-year-old sudden death victim. Family evaluation led to the diagnosis of dilated cardiomyopathy (DCM) in four family members and segregation testing confirmed affected family members harboured the variant. Further investigation in 100,000 Genomes Project, laboratory data, and our inherited cardiac conditions database as well as previous publications, identified additional cases of this variant in association with DCM and sudden cardiac death (see table and figure). Conclusion WGS with targeted gene analysis can be an effective diagnostic tool for SUDY. The study’s findings show the importance of large population databases and collaboration in the research community, allowing reclassification of a RYR2 VUS as a LP variant, the strongest genetic data so far to associate a missense variant with familial DCM and the risk for SCD. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project.

Update on rare variants in inherited arrhythmogenic syndromes: when to reclassify?

Abstract Funding Acknowledgements Type of funding sources: None. Background Discerning a definite pathogenic role of rare variants in genes associated with inherited arrhythmogenic syndromes (IAS) is critical because of their potential implications for clinical decision-making. The discovery of new gene-disease associations, the publication of functional studies and update on population genetic data may lead to changes in the interpretation of genetic variants classified in previous studies. Aim To update the classification of rare variants previously identified in genetic testing of IAS cases. Methods This is a retrospective descriptive analysis that enrolled rare variants previously identified in genetic tests from 145 families with a clinical diagnosis of any IAS between 2015-2020. These variants were re-evaluated and reclassified following the American College of Medical Genetics and Genomics guidelines, including newly available data. Results In their previous study, 78% of all cases harboured a rare variant classified as a variant of uncertain significance (VUS). Only 22% had a definitive diagnosis with a variant classified as pathogenic (P) or likely pathogenic (LP). In our comprehensive reanalysis following current available data, the classification of 26.7% of these variants changed, mainly due to updated population frequencies, but also to the addition of clinical data and segregation studies. As showed in 2015, the majority of variants were now classified as VUS (57.5%). The percentage of rare variants classified as potentially deleterious increased from 22% to 30%. In addition, 76.5% of reclassified variants gained certainty. Conclusions The large number of variants remaining as VUS is one of the main limitations in current diagnosis of IAS. Clarifying the role of rare variants can help to obtain a definitive genetic diagnosis, allowing the identification of those carriers at risk of developing the disease and ruling out those who are not. We propose that a periodic review of rare variants no later than 5 tears may help to improve clinical decision-making in the diagnosis, follow-up, treatment and risk stratification of families with IAS.

Genotype-phenotype correlation of rare variants in the SCN5A gene in children population

Abstract Funding Acknowledgements Type of funding sources: None. Background Rare variants in the SCN5A gene have been associated with Brugada syndrome (BS), long QT syndrome type 3 (LQTS3), and other conduction disorders that can lead to life-threatening arrhythmias. Variable expressivity, incomplete penetrance and other modifying factors make it difficult to establish a genotype-phenotype correlation, especially in paediatric patients. This, together with the finding of variants of uncertain significance (VUS), makes the genetic diagnosis of arrhythmogenic syndromes challenging. Purpose To describe the phenotypes associated with rare variants in the SCN5A gene in children. Methods This is a retrospective, single-centre descriptive analysis of the phenotypes found in patients (≤18 years of age at diagnosis) carriers of rare variants in the SCN5A gene. Sixty-four children (75% male, median age 8 years – IQR: 3-13 years) from 41 families were included. The variants were classified according to the recommendations of the American College of Medical Genetics. Results 82.8% of patients harboured pathogenic (P) or likely pathogenic (LP) variants and 17.2% VUS. 89.1% was diagnosed with BS, 6.3% with LQTS3, and one family presented atrial fibrillation. Two patients died before the first year of life. Of the patients, 41.9% had symptoms: 21.9% cardiac events (cardiac syncope, resuscitated sudden death, ventricular fibrillation and ventricular tachycardia), 7.9% conduction disorders (sinus node dysfunction, atrioventricular block, bifascicular block) and 4.7% supraventricular arrhythmias (supraventricular tachycardia, atrial fibrillation, flutter). Non-cardiac phenotypes included: epilepsy (10.9%) and febrile seizures (7.8%). 58.1% of patients remained asymptomatic during follow-up (median follow-up 5 years – IQR: 2.25-6). In total, 39 variants were identified, 87.2 were classified as P/LP. Segregation studies allowed the reclassification of 28.2% of VUS as P/LP, 12.8% of the variants remained as VUS. 53.8% of the variants were located in the cytosolic region (Figure 1). Conclusions The presence of rare variants in the SCN5A gene is associated with arrhythmic phenotypes that can trigger cardiac events and lead to sudden death. The most common cardiac symptom in children is syncope. Patients may present with neurological symptoms such as epilepsy and febrile seizures. Identified VUS should be considered and not discarded until a definitive classification is made. VUS segregation can help to clarify the meaning of these variants, but should not be used to identify individuals at risk in the family, nor in clinical decisions. The high number of asymptomatic patients may be explained by the fact that these are diseases in which the onset of symptoms usually occurs in adolescence and later. Most of the variants were found in mutational hot spots, mainly located in the cytosolic and extracellular region of the protein.

The genetic spectrum of a Chinese series of patients with 46, XY disorders of the sex development.

The etiology of 46, XY disorders of sex development (46, XY DSD) is complex, and studies have shown that different series of patients with 46, XY DSD has different genetic spectrum. In this study, we aimed to investigate the underlying genetic etiology in a Chinese series of patients with 46, XY DSD by whole exome sequencing (WES). Seventy patients with 46, XY DSD were enrolled from the Peking Union Medical College Hospital (Beijing, China). The detailed clinical characteristics were evaluated, and peripheral blood was collected for WES to find the patients' rare variants (RVs) of genes related to 46, XY DSD. The clinical significance of the RVs was annotated according to American College of Medical Genetics and Genomics (ACMG) guidelines. A total of 57 RVs from nine genes were identified in 56 patients with 46, XY DSD, which include 21 novel RVs and 36 recurrent RVs. Based on the American ACMG guidelines, 43 variants were classified as pathogenic(P) or likely pathogenic (LP) variants and 14 variants were defined as variants of uncertain significance (VUS). P or LP variants were identified in 64.3% (45/70) patients of the series. Thirty-nine, 14, and 4 RVs were involved in the process of androgen synthesis and action, testicular determination and developmental process, and syndromic 46, XY DSD, respectively. The top three genes most frequently affected to cause 46, XY DSD were AR, SRD5A2, and NR5A1. Seven patients were found harboring RVs of the 46, XY DSD pathogenic genes identified in recent years, namely DHX37 in four patients, MYRF in two patients, and PPP2R3C in one patient. We identified 21 novel RVs of nine genes, which extended the genetic spectrum of 46, XY DSD pathogenic variants. Our study showed that 60% of the patients were caused by AR, SRD5A2 or NR5A1 P/LP variants. Therefore, polymerase chain reaction (PCR) amplification and Sanger sequencing of these three genes could be performed first to identify the pathogeny of the patients. For those patients whose pathogenic variants had not been found, whole-exome sequencing could be helpful in determining the etiology.

The Impact of Germline Alterations in Appendiceal Adenocarcinoma.

More than 10% of assessed patients with appendiceal adenocarcinoma have a pathogenic (P) or likely pathogenic (LP) germline variant, including genes implicated in heritable gastrointestinal cancer syndromes, such as Lynch syndrome. We defined the clinical and molecular impact of heritable alterations in appendiceal adenocarcinoma to evaluate the need for dedicated appendiceal screening and prevention strategies in patients with LP/P germline variants. We performed an integrated germline and somatic molecular analysis for patients with confirmed appendiceal adenocarcinoma. Patients underwent paired tumor-normal sequencing for up to 90 hereditary cancer risk genes and 505 genes for somatic mutation profiling. We defined the cooccurrence of LP/P germline variants and second-hit pathogenic somatic alterations. The associations between germline variants and patient clinicopathologic features were also evaluated. Twenty-five of 237 patients (10.5%) carried pathogenic or likely pathogenic germline variants in cancer susceptibility genes. Clinicopathologic characteristics and appendiceal adenocarcinoma-specific survival were similar in patients with or without germline variants. Most (92%, N = 23/25) patients with germline variants demonstrated no second-hit somatic alterations, including loss of heterozygosity. Two patients with a germline APC I1307K low-penetrance founder variant exhibited secondary somatic pathogenic alterations in APC. However, only one patient tumor exhibited APC-mediated WNT signaling dysregulation: a plausible consequence of multiple somatic APC mutations with no germline variant contribution. Four patients had germline variants in PMS2 or MSH2 associated with Lynch syndrome, yet their cancers were microsatellite-stable. Germline variants are likely incidental without a contributory driver role in appendiceal adenocarcinoma. Appendiceal adenocarcinoma screening in patients with germline variants is not clearly merited.

PO-04-125 SPECTRUM, PREVALENCE, AND IMPLICATIONS OF INCIDENTAL VARIANT FINDINGS IN CARDIAC DISEASE-ASSOCIATED GENES DURING PREVENTATIVE GENETIC TESTING

Classically, genetic testing has been reserved for individuals with a reasonable pre-test probability of disease. However, the use of commercial preventative genetic testing (PGT) to identify asymptomatic individuals at risk for various heritable disorders, most commonly those linked to cancer and cardiovascular disease, has increased significantly. To describe our experience regarding incidentally discovered pathogenic (P)/likely pathogenic (LP) variants in otherwise healthy patients undergoing PGT. An institutionally developed natural language processing algorithm was used to identify all patients who underwent PGT at Mayo Clinic. The clinical characteristics, demographics, rationale for PGT, family history, variant adjudication, and cardiac work-up results for those whose PGT result included a channelopathy- or cardiomyopathy-inclusive cardiac panel were abstracted from the electronic medical record. Overall, 666/1269 (52%) patients that underwent PGT had ACMG reportable genetic heart disease (GHD)-susceptibility genes included. Of those, 10/666 (2%, 3 females, and mean age at time of PGT 59±10 years) had an incidentally discovered P/LP variant in one of 7 Clinical Genome Resource-adjudicated strong/definitive evidence GHD-susceptibility genes (DSG2, DSP, FLNC, KCNE1, MYBPC3 [n=2], MYH7 [n=2], and PKP2). Comprehensive cardiac work-up was recommended for all P/LP variant-positive patients and pursued in 8 (80%). Whereas no evidence of an underlying cardiac channelopathy or genetic cardiomyopathy was identified, 1 patient did have a positive test for ischemia. Interestingly, none of the patients with an incidentally discovered P/LP variant in a strong evidence cardiomyopathy (n=8) or channelopathy (n=1) gene that underwent further clinical evaluation had overt electrocardiographic or imaging evidence of an associated disease phenotype. Furthermore, family history was reviewed, and no patients had a family history consistent with the GHD in question. Overall, in 10 patients who had an incidental P/LP variant upon PGT, there was limited evidence of an underlying genetic channelopathy or cardiomyopathy phenotype suggesting the penetrance/expressivity of these incidentally discovered variants is exceedingly low. Patients considering preventative genetic testing should be counselled on the rate of incidentally identified findings as well as the potential clinical and psychosocial ramifications.

PO-04-143 CARDIOVASCULAR OUTCOMES IN LMNA-MEDIATED CARDIAC LAMINOPATHY: THE MAYO CLINIC EXPERIENCE

Pathogenic loss-of-function variants in LMNA-encoding lamin A/C cause a range of phenotypes, including lipodystrophy, premature aging, muscular dystrophy, and cardiomyopathy. LMNA-mediated cardiac laminopathy is viewed as a particularly malignant form of arrhythmogenic dilated cardiomyopathy (DCM). However, a paucity of data exists comparing the cardiovascular outcomes in cardiac laminopathy to other DCM genotypes. Therefore, we sought to ascertain whether the prevalence of advanced heart failure (AHF), ventricular arrhythmia (VA), and stroke events differ between patients with either LMNA-mediated DCM or TTN-mediated DCM who were evaluated in a single tertiary medical center. All patients with either a pathogenic (P) or likely pathogenic (LP) variant in either LMNA or TTN in the Mayo Clinic Genetic ACM Registry (n=389) were included. A VA event was defined as sudden cardiac death (SCD), sudden cardiac arrest (SCA), sustained VA, or an appropriate ICD shock while an AHF event was defined as ventricular assist device implantation or heart transplantation. Overall, 88 patients with a P/LP variant in LMNA (61% female) and 72 patients with a P/LP variant in TTN (42% female) were identified. LMNA-positive patients were more likely to have a family history of SCA/SCD (56% vs 35%; p=0.008) and more likely to receive an ICD (60% versus 33%; p=0.001). Interestingly, the prevalence of atrial fibrillation (51% vs. 40%; p=0.2), stroke (8% vs. 8%; p=0.9), and AHF events (12% vs 8%; p=0.4) was similar between LMNA- and TTN-positive patients. However, LMNA-positive patients trended towards more VA events (32% vs 20%; p=0.08). Independent of sex, time-to-event analysis showed LMNA patients had an earlier time (p=0.0009) to a composite endpoint (AHF event or VA event) and remained significant when separating the endpoints: AHF event (p=0.008) and VA event (p=0.0009). LMNA-mediated cardiac laminopathy is a highly arrhythmogenic form of DCM with nearly one-third experiencing a SCA and/or appropriate ICD therapy and half with a history of atrial fibrillation. Although the overall rate of ventricular arrhythmic and AHF events was similar between LMNA and TTN P/LP variant-positive patients, cardiac laminopathy results in earlier end stage heart failure and potentially lethal ventricular arrhythmias.

PO-01-015 YIELD OF GENETIC TESTING AND RESULT UTILITY IN A COHORT OF 2100 DCM PATIENTS

Familial dilated cardiomyopathy (DCM) is typically an autosomal dominant (AD), monogenic disorder. Advances in gene-specific precision medicine approaches increasingly highlight the importance of genetic testing for patients with this diagnosis. The yield of genetic testing in DCM has largely been documented by single center studies and have not evaluated the utility of results for gene-specific management purposes. We report the yield of genetic testing in a large cohort of patients with a clinical suspicion of DCM from multiple centers and describe the potential clinical utility of results. This was a retrospective review of consecutive, deidentified patients who underwent genetic testing with a multigene, cardiomyopathy-focused next generation sequencing (NGS) panel. Patients had a confirmed or suspected diagnosis of DCM based on the test requisition received. Panels included sequence and copy number variant (CNV) analysis by NGS of 23 to 316 genes. An informative result was defined as the identification of pathogenic (P) or likely pathogenic (LP) variant(s) using a modified ACMG/AMP classification scheme. A total of 2,100 index patients underwent testing; 56.4% were tested with large panels (101-316 genes; n=1,184). Sequencing and CNV analysis was performed in 73.0% (n=1,533) of patients and panels that included mtDNA were performed for 38.3% (n=804). In this cohort, 24.2% (n=508) of patients received an informative result. AD inheritance was seen in 94.1% (n=478), followed by autosomal recessive (AR) in 2.6% (n=13), X-linked in 2.0%; (n=10) and mitochondrial in 1.4%; (n=7). Thirteen patients (2.6%) had two P/LP variants in AD genes. The most frequent genes were TTN (46.3%), DSP (6.9%), LMNA (6.9%), RBM20 (5.6%) and MYH7 (5.4%). Among patients with informative results, 12.5% (n=64) had a result associated with neuromuscular or metabolic conditions, 17.9% (n=91) had a result associated with ICD recommendations, and 61.8% (n=314) had a result associated with a clinical trial (clinicaltrials.gov). In this cohort, almost 1 in 4 patients received an informative genetic test result. A total of 5.9% of informative results were in genes associated with an inheritance other than AD. In this cohort, 22.3% received an informative result requiring extra-cardiac management, applicable gene-specific management or potential clinical trial eligibility. This work further supports the utility of genetic testing using large multigene panels for DCM.

Genetic Findings from a Multigene Panel for Autoinflammatory Disease

Systemic autoinflammatory diseases (AIDs) are disorders resulting from inappropriate activation of the innate immune system. While 4 genes (MEFV, NLRPS, TNFRSF1A, MVK) explain most clinical diagnoses, recent use of expanded genetic testing panels has identified numerous monogenic AIDs, as well as somatic VEXAS syndrome, an adult-onset inflammatory condition primarily affecting males. To further investigate the utility of genetic testing for patients with AIDs, we performed a retrospective analysis of results from a next-generation sequencing (NGS) multigene panel test at Blueprint Genetics. Testing included sequence and copy number variants (CNVs) analyses of up to 47 genes associated with AIDs by NGS data from a clinically validated assay. The patient sex, age, and clinical history were collected from the test requisition. Variant classification was performed applying a modified variant classification scheme of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). A positive result was defined as the identification of a pathogenic (P) or likely pathogenic (LP) variant(s) consistent with the patient’s reported phenotype and with associated disease inheritance. In total, deidentified test results from 912 consecutive patients were included for analysis. The median age at testing was 20 years (range 3 months to 84 years) and 57% (n = 517) of patients were female. A positive result was identified in 7% (n = 61) of patients, including 71 (P/LP) variants (28 unique) from 9 different genes. The 4 most common genes (MEFV n = 37, NLRP3 n = 9, MVK n = 5, and TNFRSF1A n = 3) accounted for 88.5% of positive results. Six positive results (11.5%) were in less commonly reported genes (TNFAIP3 n = 2, NOD2, PSTPIP1, TMEM173, and IL36RN), all of which may inform treatment decisions. A mosaic variant (allele ratio of 4%) in UBA1 was also identified and consistent with somatic VEXAS syndrome. This data supports the use of a multiple-gene panel for patients with suspected AIDs.

Frequency of Known Genetic Variants for Parkinson’s Disease in the PD GENEration Study Cohort (S42.002)

Frequency of Known Genetic Variants for Parkinson’s Disease in the PD GENEration Study Cohort (S42.002)

Abstract 2041: Research strategies for rare germline variants and cancer study

Abstract Exploration of rare germline variants in cancer is the fields of ongoing research. So, we would like to suggest several considerations for the rare pathogenic germline variants studies. We confirmed the pathogenicity of each variant using ClinVar (https://www.ncbi.nlm.nih.gov/clinvar). In addition, we utilized in silico prediction tools such as Polyphen and SIFT. Even for the same variant, the report of the variant may vary depending on the database used. We conducted rare germline variants screening using ICGC (International Cancer Genome Consortium) PCAWG (Pan-Cancer Analysis of Whole Genomes) data. we filtered allele frequencies using the gnomAD (v2.1.1) exome database. We utilized several ClinVar versions to check the clinical significance of germline variants. Next, we have investigated some in silico tools that predict ClinVar pathogenic and benign variants well. We observed inconsistent variant reports across versions of ClinVar. Although the information on variants may change over time, there have been inconsistent changes in the clinical significance of variants reported as P (Pathogenic) or LP (Likely pathogenic). The clinical significance of the variants reported in the previous version was P or LP, but those in the updated version were changed to “Conflicting interpretations of pathogenicity”. It was confirmed that the variant report changes when at least one “Uncertain Significance” review is added. Among various prediction tools, we found that the predictive power of a tool called “MetaRNN” was useful. Compared with several tools, we confirmed that MetaRNN’s AUC (Area Under the ROC Curve) value was 0.985, which is the best performance. But, it is not enough to judge the pathogenicity of a variant only by the prediction result of the tool. And, prediction tools are also known to be more efficient in predicting missense variants than others. Therefore, it is necessary to examine the pathogenicity of germline variants using both the prediction tool and the clinical significance of ClinVar. In summary, for rare germline variants in cancer studies, it is necessary to observe variants compared to the previous versions of ClinVar. Moreover, if prediction tools are appropriately used, researchers will increase the possibility of finding a variant or gene with more crucial clinical significance to reveal the association between the rare germline variants and cancer development. Citation Format: Seokhyeon Kim, Youngil Koh, Sung-Soo Yoon. Research strategies for rare germline variants and cancer study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2041.

SPECTRUM OF GERMLINE CANCER SUSCEPTIBILITY GENE MUTATIONS IN BREAST AND OVARIAN CANCER PATIENTS IN THE BLACK SEA REGION OF TURKEY: SINGLE CENTER EXPERIENCE

Introduction: Breast cancer consists huge amount of the cancerrelated death in population. Ovarian cancer is the second most frequent seen type of gynecological cancer and has the highest mortality among gynecological cancers since most cases are detected late. The current study intended to determine the prevalence of oncogene mutations, especially BRCA1 and BRCA2, in high-risk patients diagnosed with ovarian and breast cancer in the Black Sea region of our country. Material and method: Between August 2017 and January 2022, a total of 223 individuals who applied to our center and met the genetic test criteria were included in the study. Next-generation sequencing (NGS) was used to detect germ-line deleterious variants in genes included in the oncogenetic panel of patients (34 genes). Results: Among the 223 patients analyzed within the scope of the study, 195 had breast cancer, and 28 had ovarian cancer, resulting in the detection of 15 different pathogenic variants of BRCA1 (%4,9) and BRCA2 (%6,7) genes in 26 (11.6%) patients. In the analysis of 32 oncogenes other than BRCA1 and BRCA2 genes, 26 different pathogenic (P) or likely pathogenic (LP) variants were detected in a total of 35 patients (15.7%). Based on the analysis of 223 breast/ ovarian cancer patients together, 41 different pathogenic (P) or likely pathogenic (LP) variants were found in 61 patients (27.3%). Furthermore, 65 different VUSs (Variant of Uncertain Significance) were detected in 73 patients (32.7%). Conclusion: This is the first study to be conducted in our region in a single center located in the Black Sea region. The study was conducted in a single center within the Black Sea region and, to our knowledge, provides the first data in this region in terms of cancer genes other than BRCAs. To appreciate of the genetic susceptibility spectrum of hereditary breast and/or ovarian cancer better, it is imperative to clarify the risks associated with genes other than BRCAs, which carry a high risk for other breast and ovarian cancers, as well as BRCA1 and BRCA2. Therefore, patients in the risk group must undergo multigene panel testing in addition to routine BRCA1 and BRCA2 gene testing. We detected two novel variants in the BRCA2 gene and five novel variants other than BRCA oncogenes. Furthermore, the results of this study contributed to the development of our country's specific variant pool.