Genetic reanalysis of patients with a difference of sex development carrying the NR5A1/SF-1 variant p.Gly146Ala has discovered other likely disease-causing variations.

NR5A1/SF-1 (Steroidogenic factor-1) variants may cause mild to severe differences of sex development (DSD) or may be found in healthy carriers. The NR5A1/SF-1 c.437G>C/p.Gly146Ala variant is common in individuals with a DSD and has been suggested to act as a susceptibility factor for adrenal disease or cryptorchidism. Since the allele frequency is high in the general population, and the functional testing of the p.Gly146Ala variant revealed inconclusive results, the disease-causing effect of this variant has been questioned. However, a role as a disease modifier is still possible given that oligogenic inheritance has been described in patients with NR5A1/SF-1 variants. Therefore, we performed next generation sequencing (NGS) in 13 DSD individuals harboring the NR5A1/SF-1 p.Gly146Ala variant to search for other DSD-causing variants and clarify the function of this variant for the phenotype of the carriers. Panel and whole-exome sequencing was performed, and data were analyzed with a filtering algorithm for detecting variants in NR5A1- and DSD-related genes. The phenotype of the studied individuals ranged from scrotal hypospadias and ambiguous genitalia in 46,XY DSD to opposite sex in both 46,XY and 46,XX. In nine subjects we identified either a clearly pathogenic DSD gene variant (e.g. in AR) or one to four potentially deleterious variants that likely explain the observed phenotype alone (e.g. in FGFR3, CHD7). Our study shows that most individuals carrying the NR5A1/SF-1 p.Gly146Ala variant, harbor at least one other deleterious gene variant which can explain the DSD phenotype. This finding confirms that the NR5A1/SF-1 p.Gly146Ala variant may not contribute to the pathogenesis of DSD and qualifies as a benign polymorphism. Thus, individuals, in whom the NR5A1/SF-1 p.Gly146Ala gene variant has been identified as the underlying genetic cause for their DSD in the past, should be re-evaluated with a NGS method to reveal the real genetic diagnosis.

DNA Methylation Description of Hippocampus, Cortex, Amygdala, and Blood of Drug-Resistant Temporal Lobe Epilepsy.

Epigenetic changes such as DNA methylation were observed in drug-resistant temporal lobe epilepsy (DR-TLE), a disease that affects 25-30% of epilepsy patients. The main objective is to simultaneously describe DNA methylation patterns associated with DR-TLE in hippocampus, amygdala, surrounding cortex to the epileptogenic zone (SCEZ), and peripheral blood. An Illumina Infinium MethylationEPIC BeadChip array was performed in 19 DR-TLE patients and 10 postmortem non-epileptic controls. Overall, 32, 59, and 3210 differentially methylated probes (DMPs) were associated with DR-TLE in the hippocampus, amygdala, and SCEZ, respectively. These DMP-affected genes were involved in neurotrophic and calcium signaling in the hippocampus and voltage-gated channels in SCEZ, among others. One of the hippocampus DMPs (cg26834418 (CHORDC1)) showed a strong blood-brain correlation with BECon and IMAGE-CpG, suggesting that it could be a potential surrogate peripheral biomarker of DR-TLE. Moreover, in three of the top SCEZ's DMPs (SHANK3, SBF1, and MCF2L), methylation status was verified with methylation-specific qPCR. The differentially methylated CpGs were classified in DMRs: 2 in the hippocampus, 12 in the amygdala, and 531 in the SCEZ. We identified genes that had not been associated to DR-TLE so far such as TBX5, EXOC7, and WRHN. The area with more DMPs associated with DR-TLE was the SCEZ, some of them related to voltage-gated channels. The DMPs found in the amygdala were involved in inflammatory processes. We also found a potential surrogate peripheral biomarker of DR-TLE. Thus, these results provide new insights into epigenetic modifications involved in DR-TLE.

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.

Characterization of spliceogenic variants located in regions linked to high levels of alternative splicing: BRCA2 c.7976+5G > T as a case study.

Many BRCA1 and BRCA2 (BRCA1/2) genetic variants have been studied at mRNA level and linked to hereditary breast and ovarian cancer due to splicing alteration. In silico tools are reliable when assessing variants located in consensus splice sites, but we may identify variants in complex genomic contexts for which bioinformatics is not precise enough. In this study, we characterize BRCA2 c.7976 + 5G > T variant located in intron 17 which has an atypical donor site (GC). This variant was identified in three unrelated Spanish families and we have detected exon 17 skipping as the predominant transcript occurring in carriers. We have also detected several isoforms (Δ16-18, Δ17,18, Δ18, and ▼17q224 ) at different expression levels among carriers and controls. This study remarks the challenge of interpreting genetic variants when multiple alternative isoforms are present, and that caution must be taken when using in silico tools to identify potential spliceogenic variants located in GC-AG introns.

Recommendations regarding the genetic and immunological study of reproductive dysfunction. / Recomendaciones para el estudio genético e inmunológico en la disfunción reproductiva.

In this article several members of diverse scientific associations and reproduction experts from Spain have updated different genetic and immunological procedure recommendations in couples affected by reproductive dysfunction with the goal of providing a set of useful guidelines for the clinic. The laboratory test has been considered as highly recommendable for making clinical decisions when the result of the diagnostic test is relevant, moderately recommendable when the results are of limited evidence because they are inconsistent, and low when the benefit of the test is uncertain. It is expected that these recommendations will provide some useful guidelines for the diagnosis, prognosis and treatment of couples presenting reproductive dysfunction.

Appropriate utilization of clinical laboratory tests.

BACKGROUND: The use of laboratory tests has been increasing in recent years due to various factors affecting laboratories, physicians, legal aspects, or patients themselves. METHODS: The efficacy of laboratory tests must be evaluated on the basis of the clinical benefits that they provide in terms of prevention, diagnosis, follow-up, or treatment; with the aim of optimizing health results in general. RESULTS: There are techniques that can be used to determine the clinical validity of the efficacy, effectiveness, and safety of treatment or preventive measures performed on individuals with abnormal tests, as well as providing an economic evaluation of the process. Once the test is incorporated into clinical service, it must be evaluated by retrospective audits in test utilization. To improve the use of laboratory tests, many strategies have been devised that incorporate clinical practice guidelines (CPGs), the conduct of professionals and the patients, and organization of the process. We discuss the importance of the involvement of health professionals. CONCLUSIONS: Strategies in relation to CPGs, conduct of professionals, conduct of patients, or organization of health care processes improve the use of tests in relation to clinical processes. Laboratory professionals have the appropriate knowledge and can improve the quality and efficacy of health care.