Sequence Variants Of Unknown Significance Research Articles

Sequence variants of unknown significance in small fiber neuropathy : Characterization of a heterogeneous patient population

In almost half of patients suffering from small fiber neuropathies (SFN), the etiology remains elusive. For these patients with "idiopathic SFN", symptomatic analgesic therapy is the only option. Reports on apotential genetic background of neuropathic pain syndromes are increasing and particularly in SFN patients, several genetic variants were found mainly located in genes encoding voltage-gated sodium channels. Although up to 30% of SFN patients show genetic alterations, most of these remain of "unknown pathogenic significance" and little is known about "genetic SFN". The study aimed to determine clinical characteristics of SFN patients carrying arare genetic variant of unknown significance in pain-associated genes. From 2015 to 2020, 66patients with primarily idiopathic SFN were examined and rare gene variants of unknown significance detected in 13/66 (20%) of these. Adetailed medical history with focus on pain was recorded and patients filled in standardized questionnaires to assess physical and emotional burden due to pain. The authors found 13/66 (20%) patients with rare variants of unknown significance located in pain-associated genes who reported pain refractory to analgesic treatment, ahigher number of external factors influencing clinical symptoms, and ahigher level of physical impairment and emotional stress due to pain compared with patients without such genetic variants. Early genetic assessment is recommended to optimize the management of patients with potentially hereditary SFN. Early access to rehabilitation and mental support as well as aconsequent elimination of external triggering factors should be granted.

Expression Profiling in Ovarian Cancer Reveals Coordinated Regulation of BRCA1/2 and Homologous Recombination Genes.

Predictive biomarkers are crucial in clarifying the best strategy to use poly(ADP-ribose) polymerase inhibitors (PARPi) for the greatest benefit to ovarian cancer patients. PARPi are specifically lethal to cancer cells that cannot repair DNA damage by homologous recombination (HR), and HR deficiency is frequently associated with BRCA1/2 mutations. Genetic tests for BRCA1/2 mutations are currently used in the clinic, but results can be inconclusive due to the high prevalence of rare DNA sequence variants of unknown significance. Most tests also fail to detect epigenetic modifications and mutations located deep within introns that may alter the mRNA. The aim of this study was to investigate whether quantitation of BRCA1/2 mRNAs in ovarian cancer can provide information beyond the DNA tests. Using the nCounter assay from NanoString Technologies, we analyzed RNA isolated from 38 ovarian cancer specimens and 11 normal fallopian tube samples. We found that BRCA1/2 expression was highly variable among tumors. We further observed that tumors with lower levels of BRCA1/2 mRNA showed downregulated expression of 12 additional HR genes. Analysis of 299 ovarian cancer samples from The Cancer Genome Atlas (TCGA) confirmed the coordinated expression of BRCA1/2 and HR genes. To facilitate the routine analysis of BRCA1/2 mRNA in the clinical setting, we developed a targeted droplet digital PCR approach that can be used with FFPE samples. In conclusion, this study underscores the potential clinical benefit of measuring mRNA levels in tumors when BRCA1/2 DNA tests are negative or inconclusive.

Mechanisms of Congenital Heart Disease Caused by NAA15 Haploinsufficiency

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Characterizing variants of unknown significance in rhodopsin: A functional genomics approach.

Characterizing the pathogenicity of DNA sequence variants of unknown significance (VUS) is a major bottleneck in human genetics, and is increasingly important in determining which patients with inherited retinal diseases could benefit from gene therapy. A library of 210 rhodopsin (RHO) variants from literature and in‐house genetic diagnostic testing were created to efficiently detect pathogenic RHO variants that fail to express on the cell surface. This study, while focused on RHO, demonstrates a streamlined, generalizable method for detecting pathogenic VUS. A relatively simple next‐generation sequencing‐based readout was developed so that a flow cytometry‐based assay could be performed simultaneously on all variants in a pooled format, without the need for barcodes or viral transduction. The resulting dataset characterized the surface expression of every RHO library variant with a high degree of reproducibility (r 2 = 0.92–0.95), recategorizing 37 variants. For example, three retinitis pigmentosa pedigrees were solved by identifying VUS which showed low expression levels (p.G18D, p.G101V, and p.P180T). Results were validated across multiple assays and correlated with clinical disease severity. This study presents a parallelized, higher‐throughput cell‐based assay for the functional characterization of VUS in RHO, and can be applied more broadly to other inherited retinal disease genes and other disorders.

Highlights of This Issue

Cancer-associated fibroblasts (CAFs) promote cancer growth and metastasis. However, mechanistic understanding by which cancer cells promote activation of healthy fibroblasts into CAFs remains elusive. In the current issue, Goulet and colleagues demonstrate that exosomes released by bladder cancer cells are internalized by healthy fibroblasts (HFs) to promote CAF differentiation through the activation of the TGFβ/SMAD signaling pathway. In fact, exosomal TGFβ, shown to be localized inside the vesicle, contributed 53 to 86% of the total TGFβ present in the bladder cancer cell conditioned medium. In conclusion, this study highlights that TGFβ-derived cancer exosomes are important molecular modulators of tumor microenvironment and CAF differentiation.PARP inhibitors (PARPi) are synthetically lethal in tumors carrying BRCA1/2 mutations and with deficiencies in homologous recombination (HR)-mediated DNA repair. However, BRCA mutations are rare; thus, most tumors are resistant to PARPi. Sizemore and colleagues demonstrate that ionizing radiation (IR) induces BRCA1 sequestration to the cytoplasm and DNA repair deficiency, and subsequent PARPi synthetic lethality in multiple tumor model systems. Importantly, p53 status was discovered to be a significant determining factor of tumor cell sensitization to PARPi following IR. These results have the potential to significantly increase the number of patients who may clinically benefit from PARPi-based therapies.Cancer genomics has highlighted the prevalence of sequence variants of unknown significance. Singh and colleagues hypothesized that a fraction of these mutations reflect RNA-related selection processes, thereby impacting RNA metabolism and contributing to oncogenic processes. Analysis of whole-genome sequencing (WGS) data from multiple cancers uncovered frequent mutations in binding sites for RNA binding proteins and splicing regulators. Furthermore, RNA sequencing (RNA-seq) data from the same specimens validated a change in RNA processing in association to these mutations. The study describes new alterations in cancer that impact RNA processing and proposes a systematic method for the interpretation of non-coding variants in cancer genomes.Histone 2B monoubiquitination (H2Bub1) is commonly lost during carcinogenesis and is deubiquitinated by ubiquitin-specific peptidase 22 (USP22). Here, the impact of USP22 on stem cell characteristics and cisplatin resistance was investigated in cancer-initiating cells (CICs) derived from human lung adenocarcinomas. Elevated USP22 expression associated with stem-like characteristics and chemotherapy resistance. Furthermore, ALDH1A3 which has been implicated in cisplatin resistance in lung adenocarcinoma was down-regulated upon USP22 knockdown. These data demonstrate that USP22 plays a critical role in tumorgenicity and cisplatin resistance in lung adenocarcinoma and may be a useful target for therapy.

Identification of epigenetic signature associated with alpha thalassemia/mental retardation X-linked syndrome

BackgroundAlpha thalassemia/mental retardation X-linked syndrome (ATR-X) is caused by a mutation at the chromatin regulator gene ATRX. The mechanisms involved in the ATR-X pathology are not completely understood, but may involve epigenetic modifications. ATRX has been linked to the regulation of histone H3 and DNA methylation, while mutations in the ATRX gene may lead to the downstream epigenetic and transcriptional effects. Elucidating the underlying epigenetic mechanisms altered in ATR-X will provide a better understanding about the pathobiology of this disease, as well as provide novel diagnostic biomarkers.ResultsWe performed genome-wide DNA methylation assessment of the peripheral blood samples from 18 patients with ATR-X and compared it to 210 controls. We demonstrated the evidence of a unique and highly specific DNA methylation “epi-signature” in the peripheral blood of ATRX patients, which was corroborated by targeted bisulfite sequencing experiments. Although genomically represented, differentially methylated regions showed evidence of preferential clustering in pericentromeric and telometric chromosomal regions, areas where ATRX has multiple functions related to maintenance of heterochromatin and genomic integrity.ConclusionMost significant methylation changes in the 14 genomic loci provide a unique epigenetic signature for this syndrome that may be used as a highly sensitive and specific diagnostic biomarker to support the diagnosis of ATR-X, particularly in patients with phenotypic complexity and in patients with ATRX gene sequence variants of unknown significance.

Clinical Experience in the Screening and Management of a Large Kindred With Familial Isolated Pituitary Adenoma Due to an Aryl Hydrocarbon Receptor Interacting Protein (AIP) Mutation

Germline AIP mutations usually cause young-onset acromegaly with low penetrance in a subset of familial isolated pituitary adenoma families. We describe our experience with a large family with R304* AIP mutation and discuss some of the diagnostic dilemmas and management issues. The aim of the study was to identify and screen mutation carriers in the family. Forty-three family members participated in the study. The study was performed in university hospitals. We conducted genetic and endocrine screening of family members. We identified 18 carriers of the R304* mutation, three family members with an AIP-variant A299V, and two family members who harbored both changes. One of the two index cases presented with gigantism and pituitary apoplexy, the other presented with young-onset acromegaly, and both had surgery and radiotherapy. After genetic and clinical screening of the family, two R304* carriers were diagnosed with acromegaly. They underwent transsphenoidal surgery after a short period of somatostatin analog treatment. One of these two patients is in remission; the other achieved successful pregnancy despite suboptimal control of acromegaly. One of the A299V carrier family members was previously diagnosed with a microprolactinoma; we consider this case to be a phenocopy. Height of the unaffected R304* carrier family members is not different compared to noncarrier relatives. Families with AIP mutations present particular problems such as the occurrence of large invasive tumors, poor response to medical treatment, difficulties with fertility and management of pregnancy, and the finding of AIP sequence variants of unknown significance. Because disease mostly develops at a younger age and penetrance is low, the timing and duration of the follow-up of carriers without overt disease requires further study. The psychological and financial impact of prolonged clinical screening must be considered. Excellent relationships between the family, endocrinologists, and geneticists are essential, and ideally these families should be managed in centers with specialist expertise.

Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes

A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUSs of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using five splicing prediction programs, and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. A total of 10 VUSs were predicted by two or more programs to induce a significant reduction of splice site strength or activation of cryptic splice sites or generation of new splice sites. Minigene-based splicing assays confirmed four of these predictions. Five additional VUSs, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A>G and BRCA1 c.5194-12G>A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977-7C>G induced in frame inclusion of 6 nt from the 3' end of intron 17. The novel variants BRCA2 c.520C>T and BRCA2 c.7992T>A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions.

BRCA1 and BRCA2 germline mutations in Uruguayan breast and breast–ovarian cancer families. Identification of novel mutations and unclassified variants

The aim of the present study was to analyze BRCA1 and BRCA2 mutations in Uruguayan families with breast and breast/ovarian cancer. Probands from 42 families with at least three cases of female breast cancer (BC) or two cases and subcriteria (paternal transmission, ovarian cancer, bilateral BC, male BC, Ashkenazi Jewish ancestry) in the same lineage, at least one diagnosed before age 50, were screened for germline mutations. PCR amplification of all exons and intron-exon boundaries were performed, followed by protein truncation test, heteroduplex analysis, and direct sequencing. We identified seven different truncating mutations in seven families, five in BRCA2 (three in site-specific BC families and two in breast-ovarian cancer families) and two in BRCA1 (one in a site-specific BC family and the other in a breast-ovarian cancer family). Both BRCA1 mutations (5583insT and 2687T>G) and one of the five BRCA2 mutations (3829insTdel35) were not previously reported. We also detected ten sequence variants of unknown significance, five of them not described before. The low frequency of BRCA1/2 mutations (0.17) is in agreement with that reported in studies which included families with similar selection criteria. However, the observed predominance of BRCA2 (0.12) over BRCA1 mutations (0.05) is in contrast with the higher proportion of BRCA1 mutations communicated for most previous studies, even those with a predominance of site-specific BC families. Meanwhile, it has been described in one Chilean and some Spanish and Italian reports, highlighting the strong dependence between the mutational spectra and the ethnicity of the population analyzed.

CDH1 gene mutations do not contribute in hereditary diffuse gastric cancer in Poland

Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome characterized by a high risk of diffuse stomach cancer and lobular breast cancer. HDGC is caused by germline mutations in the CDH1 gene encoding the E-cadherin which is a member of the transmembrane glycoprotein family responsible for calcium-dependent, cell-to-cell adhesion and plays a fundamental role in the maintenance of cell differentiation and the normal architecture of epithelial tissues. Mutations in the CDH1 gene are detected in 30–46% of families that fulfil strong clinical criteria for HDGC and in about 11% of families fulfilling the modified criteria. In the present study, we investigated germline mutations in the CDH1 gene in Polish patients with HDGC. The entire coding sequence of CDH1 gene was analyzed by sequencing in 86 Polish cancer patients from families fulfilling the modified criteria of HDGC. We found several silent mutations including one common variant (c.2076T>C) present in 56 patients, and three rare variants (c.2253C>T, c.1896C>T, c.2634C>T) detected in 2 patients. In addition, we found four rare sequence variants of unknown significance localized in introns. We did not detect any deleterious mutations of the CDH1 gene. CDH1 gene mutations are not present in Polish families with HDGC defined by the modified clinical criteria. Further studies of families with HDGC matching the restrictive criteria for HDGC are needed.

Prediction of BRCA2-association in hereditary breast carcinomas using array-CGH

Germline mutations in BRCA1/2 increase the lifetime risk for breast and ovarian cancer dramatically. Identification of such mutations is important for optimal treatment decisions and pre-symptomatic mutation screening in family members. Although current DNA diagnostics is able to identify many different mutations, it remains unclear, how many BRCA2-associated breast cancer cases remain unidentified as such. In addition, mutation scanning detects many unclassified variants (UV) for which the clinical relevance is uncertain. Therefore, our aim was to develop a test to identify BRCA2-association in breast tumors based on the genomic signature. A BRCA2-classifier was built using array-CGH profiles of 28 BRCA2-mutated and 28 sporadic breast tumors. The classifier was validated on an independent group of 19 BRCA2-mutated and 19 sporadic breast tumors. Subsequently, we tested 89 breast tumors from suspected hereditary breast (and ovarian) cancer (HBOC) families, in which either no BRCA1/2 mutation or an UV had been found by routine diagnostics. The classifier showed a sensitivity of 89% and specificity of 84% on the validation set of known BRCA2-mutation carriers and sporadic tumor cases. Of the 89 HBOC cases, 17 presented a BRCA2-like profile. In three of these cases additional indications for BRCA2-deficiency were found. Chromosomal aberrations that were specific for BRCA2-mutated tumors included loss on chromosome arm 13q and 14q, and gain on 17q. Since we could separate BRCA1-like, BRCA2-like, and sporadic-like tumors, using our current BRCA2- and previous BRCA1-classifier, this method of breast tumor classification could be applied as additional test for current diagnostics to help clinicians in decision making and classifying sequence variants of unknown significance.

The BRCA1 c.5434C→G (p.Pro1812Ala) variant induces a deleterious exon 23 skipping by affecting exonic splicing regulatory elements

BackgroundA large fraction of the sequence variants of unknown significance or unclassified variants (UVs) could be pathogenic by affecting mRNA splicing. The breast and ovarian cancer susceptibility gene BRCA1 exhibits...

The contribution of CHEK2 to the TP53-negative Li-Fraumeni phenotype

BackgroundCHEK2 has previously been excluded as a major cause of Li-Fraumeni syndrome (LFS). One particular CHEK2 germline mutation, c.1100delC, has been shown to be associated with elevated breast cancer risk. The prevalence of CHEK2*1100delC differs between populations and has been found to be relatively high in the Netherlands. The question remains nevertheless whether CHEK2 germline mutations contribute to the Li-Fraumeni phenotype.MethodsWe have screened 65 Dutch TP53-negative LFS/LFL candidate patients for CHEK2 germline mutations to determine their contribution to the LFS/LFL phenotype.ResultsWe identified six index patients with a CHEK2 sequence variant, four with the c.1100delC variant and two sequence variants of unknown significance, p.Phe328Ser and c.1096-?_1629+?del.ConclusionOur data show that CHEK2 is not a major LFS susceptibility gene in the Dutch population. However, CHEK2 might be a factor contributing to individual tumour development in TP53-negative cancer-prone families.

A Systematic Genetic Assessment of 1,433 Sequence Variants of Unknown Clinical Significance in the BRCA1 and BRCA2 Breast Cancer–Predisposition Genes

Mutation screening of the breast and ovarian cancer-predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare nontruncating sequence variants in these genes is problematic, because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. Using data from the Myriad Genetic Laboratories database of nearly 70,000 full-sequence tests, we assessed the clinical significance of 1,433 sequence variants of unknown significance (VUSs) in the BRCA genes. Three independent measures were employed in the assessment: co-occurrence in trans of a VUS with known deleterious mutations; detailed analysis, by logistic regression, of personal and family history of cancer in VUS-carrying probands; and, in a subset of probands, an analysis of cosegregation with disease in pedigrees. For each of these factors, a likelihood ratio was computed under the hypothesis that the VUSs were equivalent to an "average" deleterious mutation, compared with neutral, with respect to risk. The likelihood ratios derived from each component were combined to provide an overall assessment for each VUS. A total of 133 VUSs had odds of at least 100 : 1 in favor of neutrality with respect to risk, whereas 43 had odds of at least 20 : 1 in favor of being deleterious. VUSs with evidence in favor of causality were those that were predicted to affect splicing, fell at positions that are highly conserved among BRCA orthologs, and were more likely to be located in specific domains of the proteins. In addition to their utility for improved genetics counseling of patients and their families, the global assessment reported here will be invaluable for validation of functional assays, structural models, and in silico analyses.

Functional characterization of missense variants in the creatine transporter gene (SLC6A8): improved diagnostic application

Creatine transporter deficiency is an X-linked mental retardation disorder caused by mutations in the creatine transporter gene (SLC6A8). So far, 20 mutations in the SLC6A8 gene have been described. We have developed a diagnostic assay to test creatine uptake in fibroblasts. Additionally, we expanded the assay to characterize novel SLC6A8 missense variants. A total of 13 variants were introduced in the SLC6A8 cDNA by site-directed mutagenesis. All variants were transiently transfected in SLC6A8-deficient fibroblasts and tested for restoration of creatine uptake in deficient primary fibroblasts. Thus, we proved that nine variants (p.Gly87Arg, p.Phe107del, p.Tyr317X, p.Asn336del, p.Cys337Trp, p.Ile347del, p.Pro390Leu, p.Arg391Trp, and p.Pro554Leu) are pathogenic mutations and four variants (p.Lys4Arg, p.Gly26Arg, p.Met560Val, and p.Val629Ile) are nonpathogenic. The present study provides an improved diagnostic tool to classify sequence variants of unknown significance.