Variants Of Uncertain Clinical Significance Research Articles

L-Type Calcium Channel: Predicting Pathogenic/Likely Pathogenic Status for Variants of Uncertain Clinical Significance.

(1) Background: Defects in gene CACNA1C, which encodes the pore-forming subunit of the human Cav1.2 channel (hCav1.2), are associated with cardiac disorders such as atrial fibrillation, long QT syndrome, conduction disorders, cardiomyopathies, and congenital heart defects. Clinical manifestations are known only for 12% of CACNA1C missense variants, which are listed in public databases. Bioinformatics approaches can be used to predict the pathogenic/likely pathogenic status for variants of uncertain clinical significance. Choosing a bioinformatics tool and pathogenicity threshold that are optimal for specific protein families increases the reliability of such predictions. (2) Methods and Results: We used databases ClinVar, Humsavar, gnomAD, and Ensembl to compose a dataset of pathogenic/likely pathogenic and benign variants of hCav1.2 and its 20 paralogues: voltage-gated sodium and calcium channels. We further tested the performance of sixteen in silico tools in predicting pathogenic variants. ClinPred demonstrated the best performance, followed by REVEL and MCap. In the subset of 309 uncharacterized variants of hCav1.2, ClinPred predicted the pathogenicity for 188 variants. Among these, 36 variants were also categorized as pathogenic/likely pathogenic in at least one paralogue of hCav1.2. (3) Conclusions: The bioinformatics tool ClinPred and the paralogue annotation method consensually predicted the pathogenic/likely pathogenic status for 36 uncharacterized variants of hCav1.2. An analogous approach can be used to classify missense variants of other calcium channels and novel variants of hCav1.2.

Clinicians' attitudes towards parental choice in the era of advanced genomic tests in pregnancy.

Israel is one of the first countries to incorporate chromosomal microarray analysis into routine prenatal care. We explored attitudes of Israeli healthcare professionals (HCPs) towards the disclosure of challenging findings: variants of uncertain clinical significance (VUS), susceptibility loci (SL) for neurodevelopmental disorders and variants associated with adult-onset (AO) conditions. Particularly, we sought their views on providing parental choice regarding the disclosure of these findings. Twenty-nine in-depth interviews were conducted with genetic counselors (n=19), medical geneticists (n=4), medical geneticists that are trained in and practice fetal medicine (n=3), and fetal medicine experts (n=3). Most participants (n=24) supported parental choice regarding uncertain genetic information. Engaging parents in disclosure decisions allows avoidance from potentially anxiety-provoking information, practicing parental autonomy, and better preparation in cases where uncertain findings are identified. HCPs believed that given appropriate preparation, parents can make informed decisions. Four participants believed that disclosure should be based on professional judgment and one supported full-disclosure. Unlike VUS or SL, all interviewees agreed that in cases of medically actionable AO conditions, the benefit of disclosure outweighs the damage. HCPs attitudes are largely in-line with the Israeli practice of involving parents in disclosure decisions regarding uncertain information. This may mitigate disclosure dilemmas and allow personalized disclosure based on parents' views.

Abstract 256: Sequence conservation of BRCA1 intron-exon junctions

Abstract Introduction: Clinical sequencing of BRCA1 and BRCA2 exons and intron-exon boundaries reveals sequence variants of uncertain clinical significance (VUSs). Many BRCA1/2 sequence variants near intron-exon boundaries are potentially pathogenic by disrupting normal splicing patterns. However, molecular analysis of these potential effects is complicated by naturally occurring alternative splicing products. It is not yet clear which of these are required for normal gene functions. We therefore aligned intron-exon boundary sequence strings for 21 BRCA1 exons in order to 1) determine whether VUSs near intron-exon boundaries disrupt conserved sequences, and 2) determine whether sequences flanking exons involved in alternative splicing events are sufficiently conserved. Methods: Sequence strings representing intron-exon junction regions were prepared for 21 BRCA1 exons. Each string consisted of 20 bases of 5' intron, the 5'-most 20 bases of each exon, the 3'-most 20 bases of each exon, and 20 bases of the 3' intron. Strings were similarly prepared for chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), orangutan (Pongo abelii), baboon (Papio anubis), and Rhesus macaque (Macaca mulatta). Each string was aligned using the MUSCLE multiple sequence alignment method. Results: Splice junction sequence conservation was from 68%-100% conserved (mean % conservation, 92.7%; median, 95%). In almost all cases, sequence differences were transitions or differing numbers of thymines in poly-T tracts. The two most divergent intron-exon junction regions flanked exons 17 and 18 (70% and 68% similar, respectively). The human exon 16-17 junction is not conserved in in gorilla, and the human exon 18-19 junction is not conserved in macaques. These differences account for the divergence among primates at these sites. Seven alternative splicing events in human BRCA1 are classified as “predominant,” and all are predicted to retain the translational reading frame. All of these alternative splicing events occur in nearly perfectly conserved splicing environments, suggesting they may occur in other species as well and possibly contribute to gene function. Seven clinically identified VUSs that fall within the human BRCA1 splice junction region are wild type base differences in other primates. c.231G>A from human exon 5 is seen in baboon, c.441+18C>T in human intron 6 is seen in orangutan, c.455T>C in exon 7 is seen in orangutan, c.547+7G>A in intron 7 is seen in macaque and baboon, and c.4097-10G>A, c.4097-20C>T, and c.4115G>A, all in or near exon 11, are seen in macaque, baboon, and orangutan. These observations suggest these VUSs are likely to be benign polymorphisms and not pathogenic variants. We note, however, that c.547+7G>A disrupts a candidate intron splicing enhancer as predicted by RegRNA 2.0 and believe this variant warrants further study. Citation Format: Talia Ishfaq, Maryam Salik, Uzma Jafry, Zaain Ahmad, James D. Fackenthal. Sequence conservation of BRCA1 intron-exon junctions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 256.

DNA-based screening and population health: a points to consider statement for programs and sponsoring organizations from the American College of Medical Genetics and Genomics (ACMG)

DNA-based screening and population health: a points to consider statement for programs and sponsoring organizations from the American College of Medical Genetics and Genomics (ACMG)

MO044TARGETED NEXT GENERATION SEQUENCING AND MULTIDISCIPLINARY APPROACH IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE, ALPORT DISEASE AND FAMILIAL HAEMATURIA: GENETIC SPECTRUM AND CLINICAL UTILITY IN A SPANISH COHORT

Abstract Background and Aims Autosomal Polycystic kidney Disease (ADPKD; ORPHA 730), Alport Syndrome (AS; ORPHA 63) and Familial Haematuria (FH) are the most frequent inherited kidney diseases. Next-generation Sequencing (NGS) has facilitated their molecular identification. A multidisciplinary team from four hospitals, with nephrologists, pediatricians, and clinical and molecular geneticists, has been formed in the Spanish region of Murcia (1.5 million inhabitants) with the lab implementation of NGS. Our aim is to evaluate the genetic spectrum in AS, FH and ADPKD and the clinical utility of this comprehensive approach. Method During 1-year activity, 114 individuals with diagnostic suspicion of ADPKD, AS or FH have been evaluated by a coordinated clinical protocol with periodic cases discussions. A customized Agilent panel was designed to capture 113 genes associated with several genetic diseases, including some related to PKD, AS or Familial Haematuria (FH): PKD1, PKD2, PKHD1, HNF1β, COL4A1, COL4A3, COL4A4 and COL4A5. Interpretation of sequence variants was performed according to the American College of Medical Genetics and Genomics (ACMG) Guidelines. Sanger sequencing was performed to confirm variants identified by NGS and to segregate them in the families. Exon 1 of PKD1 gene was also sequenced by Sanger method, due to the suboptimal capture of this region by NGS. Results We detected genetic variants in 63 patients (55.3%), pathogenic or probably pathogenic variants in 54 (47.8%). 31 patients had a variant in AS associated genes: 10 in COL4A3, 18 in COL4A4, 2 in COL4A1 and 1 in COL4A5. There were 13 pathogenic variants, 12 probably pathogenic variants and 6 variants of uncertain clinical significance (VUCS). Among them, 27 had an AD inheritance, 1 AR and 3 were sporadic. All the patients with any variant had microhaematuria, a 68% had also proteinuria, and mean eGFR at diagnostic was 63.79±21 ml/min/1.73m2. 61% had auditory disturbances and 11% ophthalmologic alterations. 4 of them had underwent kidney biopsy previously, but 3 were not adequately diagnosed, so they were reclassified after the molecular diagnosis. In total, 16 kidney biopsies were avoided by the genetic diagnosis. On the other hand, 29 patients had a variant in the ADPKD associated genes: 24 in PKD1 and 5 in PKD2. There were 20 pathogenic variants and 4 probably pathogenic variants, and their inheritance was confirmed AD in 27 patients, whereas new sporadic mutations were identified in 2 patients. 22 patients had big or enormous kidneys on sonography, although 7 had normal size. Hepatic cysts were present on 5%. 19 patients had hypertension, with a mean age of diagnosis of 47±14 years. Additionally, 1 patient was diagnosed on AR polycystic disease with homozygosity PKHD1 pathogenic variant. Among all the scope, familial history was clearly present in 43 patients, uncertain in 11, and not present in 9 patients. Conclusion The multidisciplinary approach to hereditary kidney diseases, with the active participation of nephrologists and clinical geneticists, has allowed a molecular diagnostic yield of 48% among patients with AS and ADPKD, employing NGS technology. This has led to a quicker diagnostic result in our region, the reclassification of some patient’s diagnosis, a decrease in invasive diagnostic procedures (such as kidney biopsy) and the correspondent adverse events and cost savings. Additionally, the AD inheritance pattern in AS has been confirmed as the most frequent in the region. The active participation of nephrologists in genomic medicine teams results in a better characterization of the hereditary kidney diseases, helping in the genetic variant interpretation and management of these patients and their families.

Contribution of Pathogenic CNVs and Noonan Syndrome in Fetuses with Increased Nuchal Translucency and Persistently Increased Nuchal Fold

Prenatal genetic diagnosis in euploid fetuses with increased Nuchal Translucency (NT) and persistently increased Nuchal Fold (NF) is challenging. The aims of this study is to evaluate the prevalence of pathogenic copy number variants and Noonan Syndrome (NS) in fetuses with increased NT and persistently increased Nuchal Fold (NF) to provide recommendations for pre-natal diagnostic strategies. This is a prospective study from 118 prenatal samples from fetuses with increased NT (≥3.5mm) in first trimester. Multiplex Ligation Dependent Probe Amplification (MLPA), Chromosomal Microarray Analysis (CMA) and karyotyping were conducted. For fetuses with increased NF (≥6mm) in the second trimester with normal karyotype and CMA, targeted Next-Generation Sequencing (NGS) tests for NS were carried out. A total of 118 fetuses had an NT measurement of ≥3.5mm performed MLPA and karyotyping, 89 euploid fetuses were further investigated with CMA, which yielded eight pathogenic CNVs (size ranged from 0.85Mb to 14.5Mb). Twenty fetuses had persistently increased NF at the second trimester. NS testing revealed 3/20 (15%) fetuses had pathogenic variants, and one (5%) with a novel variant of uncertain clinical significance inherited from the father. Our study suggested that NS targeted sequencing facilitates additional genetic diagnosis in fetuses with high NT and persistently increased NF.

12. Analysis of the clinical utility of mate pair sequencing to further characterize congenital chromosome abnormalities

The field of clinical cytogenetic testing relies on chromosomal microarray (CMA) and karyotype to detect copy number imbalance and structural variation. Karyotype can both detect balanced and unbalanced rearrangements but cannot define breakpoint boundaries or gene content. CMA is less subjective and can more accurately define gene content but can only detect unbalanced rearrangements without structure or orientation information. Both methodologies can lead to variants of uncertain clinical significance for which no further clinical testing has historically been available.

Integration of functional assay data results provides strong evidence for classification of hundreds of BRCA1 variants of uncertain significance

PurposeBRCA1 pathogenic variant heterozygotes are at a substantially increased risk for breast and ovarian cancer. The widespread uptake of testing has led to a significant increase in the detection of missense variants in BRCA1, the vast majority of which are variants of uncertain clinical significance (VUS), posing a challenge to genetic counseling. Here, we harness a wealth of functional data for thousands of variants to aid in variant classification. MethodsWe have collected, curated, and harmonized functional data for 2701 missense variants representing 24.5% of possible missense variants in BRCA1. Results were harmonized across studies by converting data into binary categorical variables (functional impact versus no functional impact). Using a panel of reference variants we identified a subset of assays with high sensitivity and specificity (≥80%) and apply the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant interpretation guidelines to assign evidence criteria for classification. ResultsIntegration of data from validated assays provided ACMG/AMP evidence criteria in favor of pathogenicity for 297 variants or against pathogenicity for 2058 representing 96.2% of current VUS functionally assessed. We also explore discordant results and identify limitations in the approach. ConclusionHigh quality functional data are available for BRCA1 missense variants and provide evidence for classification of 2355 VUS according to their pathogenicity.

Epidemiological and ES cell-based functional evaluation of BRCA2 variants identified in families with breast cancer.

The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.

Diagnostic Utility of Genome-Wide DNA Methylation Analysis in Mendelian Neurodevelopmental Disorders.

Mendelian neurodevelopmental disorders customarily present with complex and overlapping symptoms, complicating the clinical diagnosis. Individuals with a growing number of the so-called rare disorders exhibit unique, disorder-specific DNA methylation patterns, consequent to the underlying gene defects. Besides providing insights to the pathophysiology and molecular biology of these disorders, we can use these epigenetic patterns as functional biomarkers for the screening and diagnosis of these conditions. This review summarizes our current understanding of DNA methylation episignatures in rare disorders and describes the underlying technology and analytical approaches. We discuss the computational parameters, including statistical and machine learning methods, used for the screening and classification of genetic variants of uncertain clinical significance. Describing the rationale and principles applied to the specific computational models that are used to develop and adapt the DNA methylation episignatures for the diagnosis of rare disorders, we highlight the opportunities and challenges in this emerging branch of diagnostic medicine.

Maturity-Onset Diabetes of the Young Identified Among Algerian Probands with Early-Onset Diabetes

AimTo investigate the prevalence of variants within selected maturity-onset diabetes of the young (MODY)-genes among Algerian patients initially diagnosed with type 1 diabetes (T1D) or type 2 diabetes (T2D), yet presenting with a MODY-like phenotype.MethodsEight unrelated patients with early-onset diabetes (before 30 years) and six relatives with diabetes were examined by targeted re-sequencing for variants in genes known to be involved in MODY (HNF1A, GCK, HNF4A, HNF1B, INS, ABCC8, KCNJ1). Clinical data for probands were retrieved from hospital records.ResultsA total of 12 variants were identified, of which three were classified as pathogenic and one as a variant of uncertain clinical significance (VUS). Two of the pathogenic variants were found in GCK (p.Gly261Arg and p.Met210Lys, respectively) in one proband each and the remaining pathogenic variant was found in HNF1B (p.Gly76Cys) in a proband also carrying the VUS in HNF1A (p.Thr156Met).ConclusionVariants in known MODY-genes can be the cause of early-onset diabetes in Algerians diagnosed with T1D or T2D among patients presenting with a MODY-like phenotype; thus, genetic screening should be considered.

A 300-kb microduplication of 7q36.3 in a patient with triphalangeal thumb-polysyndactyly syndrome combined with congenital heart disease and optic disc coloboma: a case report

BackgroundTriphalangeal thumb-polysyndactyly syndrome (TPT-PS) is a rare well-defined autosomal dominant disorder characterized by long thumbs with three phalanges combined with pre- and postaxial polydactyly/syndactyly of limbs.By now, the syndrome has been reported in several large families from different ethnic backgrounds, with a high degree of inter- and intrafamilial variability. The genome locus responsible for TPT-PS has been mapped to the 7q36.3 region harboring a long-range sonic hedgehog (SHH) regulatory sequence (ZRS). Both single-nucleotide variants and complete duplications of ZRS were shown to cause TPT-PS and similar limb phenotypes. TPT-PS usually forms as isolated limb pathology not associated with additional malformations, in particular, with cardiovascular abnormalities.Case presentationHere we report on a rare Russian neonatal case of TPT-PS combined with severe congenital heart disease, namely double outlet right ventricle, and microphthalmia with optic disc coloboma. Pedigree analysis revealed TPT-PS of various expressivity in 10 family members throughout five generations, while the cardiac defect and the eye pathology were detected only in the proband. To extend the knowledge on genotype–phenotype spectrum of TPT-PS, the careful clinical and genomic analysis of the family was performed. High-resolution array-based comparative genomic hybridization (array-CGH) revealed a ~ 300 kb microduplication of 7q36.3 locus (arr[GRCh37] 7q36.3(156385810_156684811) × 3) that co-segregated with TPT-PS in the proband and her mother. The duplication encompassed three genes including LMBR1, the intron 5 of which is known to harbor ZRS. Based on whole-exome sequencing data, no additional pathogenic mutations or variants of uncertain clinical significance were found in morbid cardiac genes or genes associated with a microphthalmia/anophthalmia/coloboma spectrum of ocular malformations.ConclusionsThe results support the previous data, indicating that complete ZRS duplication underlies TPT-PS, and suggest a broader phenotypic impact of the 7q36.3 microduplication. Potential involvement of the 7q36.3 microduplication in the patient’s cardiac and eye malformations is discussed. However, the contribution of some additional genetic/epigenetic factors to the complex patient`s phenotype cannot be excluded entirely. Further comprehensive functional studies are needed to prove the possible involvement of the 7q36.3 locus in congenital heart disease and eye pathology.

Personalized prenatal genomic testing: Couples' experience with choice regarding uncertain and adult-onset findings from chromosomal-microarray-analysis.

Chromosomal-microarray-analysis (CMA) can identify variants of uncertain clinical significance, susceptibility-loci for neurodevelopmental conditions, and risk for adult-onset conditions. We explored choices made by couples undergoing prenatal CMA, their understanding of these findings, reasons for and against receiving them, and whether they believe parents or professionals should decide which are disclosed. Semi-structured interviews were conducted with women (n = 27) or their partners (n = 15) during the week following prenatal CMA testing and analyzed using grounded theory. Over half the interviewees (55%) recalled at least two of the three types of CMA results they chose whether to receive. Sixty-four percent found the choice simple, whereas 36% found it difficult. All participants could clearly explain their choices, which were based on the perceived actionability and psychological impact of the information. Sixty percent viewed their choice favorably, whereas ~21% would have preferred clinicians to decide for them. More women than men, and more decisive than indecisive participants supported parental choice. Overall, expectant parents can make informed choices about which uncertain findings about their fetuses they wish to receive, and value the opportunity to tailor results to their values and wishes. Arguments presented provide the basis for a decision-aid tool for expecting parents.

Results and Clinical Interpretation of Germline RET Analysis in a Series of Patients with Medullary Thyroid Carcinoma: The Challenge of the Variants of Uncertain Significance.

Simple SummaryAbout 25% of Thyroid Carcinomas of Medullary type occur in carriers of hereditary alterations in the RET gene. Different alterations are associated with different risks (highest, high, and moderate) and management depends on risk category. We explored prevalence, clinical presentation and management of inherited RET variants in patients tested at our center. We found inherited RET variants in 31.9% of tested individuals: the vast majority of patients with Medullary Thyroid Carcinoma who had a family history positive for the disease was found to carry a RET change, but also 14.3% of those with no family history tested positive, supporting the recommendation to perform genetic testing in all cases of Medullary Thyroid Carcinoma. For known variants, findings in our patients were consistent with available risk classification. Besides, we obtained evidence supporting the classification of two rare variants of uncertain clinical significance (p.Ser904Phe and p.Asp631_Leu633delinsGlu), which may help future management of carriers.Germline RET variants are responsible for approximately 25% of medullary thyroid carcinoma (MTC) cases. Identification of RET variant carriers allows for the adoption of preventative measures which are dependent on the risk associated with the specific alteration. From 2002 to 2020, at our cancer genetics clinic, RET genetic testing was performed in 163 subjects (102 complete gene analyses and 61 targeted analyses), 72 of whom presented with MTC. A germline RET variant was identified in 31.9% of patients affected by MTC (93.8% of those having positive family history and 14.3% of clinically sporadic cases). Subsequent target testing in relatives allowed us to identify 22 asymptomatic carriers, who could undertake appropriate screening. Overall, patients with germline RET variants differed significantly from those who tested negative by family history (p < 0.001) and mean age at MTC diagnosis (44.45 vs. 56.42 years; p = 0.010), but the difference was not significant when only carriers of moderate risk variants were considered (51.78 vs. 56.42 years; p = 0.281). Out of 12 different variants detected in 49 patients, five (41.7%) were of uncertain significance (VUS). For two of these, p.Ser904Phe and p.Asp631_Leu633delinsGlu, co-segregation and genotype/phenotype analysis, matched with data from the literature, provided evidence supporting their classification in the moderate and the highest/high risk class (with a MEN2B phenotype), respectively.

Folliculin variants linked to Birt-Hogg-Dubé syndrome are targeted for proteasomal degradation.

Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dubé (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present an integrated computational and experimental study that reveals that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant (ΔE510) forms perinuclear protein aggregates. A yeast-based stability screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature led to a stabilization of two FLCN missense proteins, and for one (R362C), function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization and resulting restoration of function may hold therapeutic potential of certain disease-linked variants. Our computational saturation scan encompassing both missense variants and single site deletions in FLCN may allow classification of rare FLCN variants of uncertain clinical significance.

NGS Panel Testing of Triple-Negative Breast Cancer Patients in Cyprus: A Study of BRCA-Negative Cases.

Simple SummaryIn Cyprus, approximately 9% of triple-negative (negative in common breast cancer receptors—estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2) receptors) breast cancer (TNBC) patients carry inherited mutations in the BRCA1/2 breast cancer (BC) susceptibility genes. These mutations increase the risk of BC. However, the contribution of other BC susceptibility genes has not yet been determined. To this end, we aimed to investigate the prevalence of mutations in BRCA1/2-negative TNBC patients in Cyprus. Ninety-five cancer susceptibility genes were sequenced for 163 TNBC patients. The frequency of non-BRCA mutations and especially PALB2 in TNBC patients in Cyprus appears to be higher compared to other populations, and half of the mutation-positive patients were diagnosed over the age of 60 years. Based on these results, we believe that PALB2 and TP53 along with BRCA1/2 genetic testing could be beneficial for a large proportion of TNBC patients in Cyprus, irrespective of their age of diagnosis.In Cyprus, approximately 9% of triple-negative (estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative) breast cancer (TNBC) patients are positive for germline pathogenic variants (PVs) in BRCA1/2. However, the contribution of other genes has not yet been determined. To this end, we aimed to investigate the prevalence of germline PVs in BRCA1/2-negative TNBC patients in Cyprus, unselected for family history of cancer or age of diagnosis. A comprehensive 94-cancer-gene panel was implemented for 163 germline DNA samples, extracted from the peripheral blood of TNBC patients. Identified variants of uncertain clinical significance were evaluated, using extensive in silico investigation. Eight PVs (4.9%) were identified in two high-penetrance TNBC susceptibility genes. Of these, seven occurred in PALB2 (87.5%) and one occurred in TP53 (12.5%). Interestingly, 50% of the patients carrying PVs were diagnosed over the age of 60 years. The frequency of non-BRCA PVs (4.9%) and especially PALB2 PVs (4.3%) in TNBC patients in Cyprus appears to be higher compared to other populations. Based on these results, we believe that PALB2 and TP53 along with BRCA1/2 genetic testing could be beneficial for a large proportion of TNBC patients in Cyprus, irrespective of their age of diagnosis.

Screening of targeted panel genes in Brazilian patients with primary ovarian insufficiency

Primary ovarian insufficiency (POI) is a heterogeneous disorder associated with several genes. The majority of cases are still unsolved. Our aim was to identify the molecular diagnosis of a Brazilian cohort with POI. Genetic analysis was performed using a customized panel of targeted massively parallel sequencing (TMPS) and the candidate variants were confirmed by Sanger sequencing. Additional copy number variation (CNV) analysis of TMPS samples was performed by CONTRA. Fifty women with POI (29 primary amenorrhea and 21 secondary amenorrhea) of unknown molecular diagnosis were included in this study, which was conducted in a tertiary referral center of clinical endocrinology. A genetic defect was obtained in 70% women with POI using the customized TMPS panel. Twenty-four pathogenic variants and two CNVs were found in 48% of POI women. Of these variants, 16 genes were identified as BMP8B, CPEB1, INSL3, MCM9, GDF9, UBR2, ATM, STAG3, BMP15, BMPR2, DAZL, PRDM1, FSHR, EIF4ENIF1, NOBOX, and GATA4. Moreover, a microdeletion and microduplication in the CPEB1 and SYCE1 genes, respectively, were also identified in two distinct patients. The genetic analysis of eleven patients was classified as variants of uncertain clinical significance whereas this group of patients harbored at least two variants in different genes. Thirteen patients had benign or no rare variants, and therefore the genetic etiology remained unclear. In conclusion, next-generation sequencing (NGS) is a highly effective approach to identify the genetic diagnoses of heterogenous disorders, such as POI. A molecular etiology allowed us to improve the disease knowledge, guide decisions about prevention or treatment, and allow familial counseling avoiding future comorbidities.

Copy number variations in ultrasonically abnormal late pregnancy fetuses with normal karyotypes

Many fetuses are found to have ultrasonic abnormalities in the late pregnancy. The association of fetal ultrasound abnormalities in late pregnancy with copy number variations (CNVs) is unclear. We attempted to explore the relationship between types of ultrasonically abnormal late pregnancy fetuses and CNVs. Fetuses (n = 713) with ultrasound-detected abnormalities in late pregnancy and normal karyotypes were analyzed. Of these, 237 showed fetal sonographic structural malformations and 476 showed fetal non-structural abnormalities. Single nucleotide polymorphism (SNP)-based chromosomal microarray (CMA) was performed on the Affymetrix CytoScan HD platform. Using the SNP array, abnormal CNVs were detected in 8.0% (57/713) of the cases, with pathogenic CNVs in 32 cases and variants of uncertain clinical significance (VUS) in 25 cases. The detection rate of abnormal CNVs in fetuses with sonographic structural malformations (12.7%, 30/237) was significantly higher (P = 0.001) than that in the fetuses with non-structural abnormalities (5.7%, 27/476). Overall, we observed that when fetal sonographic structural malformations or non-structural abnormalities occurred in the third trimester of pregnancy, the use of SNP analysis could improve the accuracy of prenatal diagnosis and reduce the rate of pregnancy termination.

Integrated analysis of metabolomic profiling and exome data supplements sequence variant interpretation, classification, and diagnosis

PurposeA primary barrier to improving exome sequencing diagnostic rates is the interpretation of variants of uncertain clinical significance. We aimed to determine the contribution of integrated untargeted metabolomics in the analysis of exome sequencing data by retrospective analysis of patients evaluated by both exome sequencing and untargeted metabolomics within the same clinical laboratory. MethodsExome sequencing and untargeted metabolomic data were collected and analyzed for 170 patients. Pathogenic variants, likely pathogenic variants, and variants of uncertain significance in genes associated with a biochemical phenotype were extracted. Metabolomic data were evaluated to determine if these variants resulted in biochemical abnormalities that could be used to support their interpretation using current American College of Genetics and Genomics (ACMG) guidelines. ResultsMetabolomic data contributed to the interpretation of variants in 74 individuals (43.5%) over 73 different genes. The data allowed for the reclassification of 9 variants as likely benign, 15 variants as likely pathogenic, and 3 variants as pathogenic. Metabolomic data confirmed a clinical diagnosis in 21 cases, for a diagnostic rate of 12.3% in this population. ConclusionUntargeted metabolomics can serve as a useful adjunct to exome sequencing by providing valuable functional data that may not otherwise be clinically available, resulting in improved variant classification.

Functional Categorization of BRCA1 Variants of Uncertain Clinical Significance in Homologous Recombination Repair Complementation Assays.

Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway. Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1-deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families. All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants. We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.