Causal and Putative Pathogenic Mutations Identified in 38.6% of Children with Primary SRNS in South Africa

The aim was to identify causal mutations in genes implicated in steroid resistant nephrotic syndrome (SRNS) within a South African population. We enrolled 119 children with primary NS; 71 SRNS and 48 steroid-sensitive NS. All children with SRNS underwent kidney biopsy. We first genotyped the NPHS2 gene for the p.V260E variant in all NS cases (n= 119) and controls (n= 219). To further identify additional variants, we performed whole-exome sequencing and interrogated ten genes (NPHS1, NPHS2, WT1, LAMB2, ACTN4, TRPC6, INF2, CD2AP, PLCE1, MYO1E) implicated in SRNS/FSGS in 56 SRNS cases and 29 controls; we also performed exome sequencing on two patients carrying the NPHS2 p.V260E mutation as positive controls. The overall detection rate of pathogenic mutations in children with SRNS was 27/70(38.57%): 15(21.43%) carried the NPHS2 p.V260E mutation and 12(17.14%) carried a pathogenic mutation in the heterozygous state in INF2 (n=8), CD2AP (n=3) or TRPC6 (n=1) genes. NPHS2 p.V260E homozygosity was specifically associated with biopsy-proven FSGS, accounting for 23.81% of Black children (15 of 63) with SR-FSGS. No causal mutations were identified in NPHS1, WT1, LAMB2, PLCE1, MYO1E and ACTN4. We report four novel variants in INF2, PLCE1, ACTN4 and TRPC6.Conclusion: The NPHS2 p.V260E mutation is a prevalent cause of SR-FSGS among Black South African children occurring in 23.81% of children with SRNS. Screening all Black African children presenting with NS for NPHS2 p.V260E will provide a precision diagnosis of SR-FSGS and inform clinical management.

Prevalence of germline TP53 mutation among early onset middle eastern breast cancer patients

Background The data on prevalence and clinical relevance of TP53 germline mutations in early onset Middle-Eastern breast cancer (BC) is limited. Methods We determined TP53 germline mutations in a cohort of 464 early onset BC patients from Saudi Arabia using capture sequencing based next generation sequencing. Results Germline TP53 pathogenic mutations were found in 1.5% (7/464) of early onset Saudi BC patients. A total of six pathogenic missense mutations, one stop gain mutation and two variants of uncertain significance (VUS) were detected in our cohort. No TP53 pathogenic mutations were detected among 463 healthy controls. TP53 mutations carriers were significantly more likely to have bilateral breast cancer (p = 0.0008). At median follow-up of 41 months, TP53 mutations were an unfavorable factor for overall survival in univariate analysis. All the patients carrying TP53 mutations were negative for BRCA1 and BRCA2 mutations. Majority of patients (85.7%; 6/7) carrying TP53 mutation had no family history suggestive of Li-Fraumeni Syndrome (LFS) or personal history of multiple LFS related tumors. Only one patient had a positive family history suggestive of LFS. Conclusions TP53 germline mutation screening detects a clinically meaningful risk of early onset BC from this ethnicity and should be considered in all early onset BC regardless of the family history of cancer, especially in young patients that are negative for BRCA mutations.

Genetic Insight into Birt-Hogg-Dubé syndrome in Indian patients reveals novel mutations in FLCN

Background: Birt-Hogg-Dub&eacute syndrome (BHDS) is a rare monogenic condition mostly associated with germline mutations at FLCN. It is characterized by either one or more manifestations of primary spontaneous pneumothorax (PSP), skin fibrofolliculomas and renal carcinoma. Here, we comprehensively studied germline mutations in BHDS patients and asymptomatic members from 15 Indian families. Methods: Targeted amplicon NGS and Sanger sequencing was performed to detect germline mutations at FLCN in 31 clinically diagnosed patients and 74 asymptomatic family members. Functional effects and protein-protein interaction of FLCN variants were evaluated in-silico and molecular docking method. Family-based association study between pathogenic mutations and BHDS was also performed. Germline mutations at genes associated with phenotypically similar diseases were also addressed in few families. Results: Six different types of pathogenic FLCN mutations were observed in the patients. Two of them: 11-nucleotide deletion (c.1150_1160delGTCCAGTCAGC) and splice acceptor mutation (c.1301-1G>A), were novel mutations. Two unreported Clinvar pathogenic mutations: stop-gain (c.634C>T) and 4-nucleotide duplication (c.1329_1332dupAGCC), and known mutations: hotspot mutation (c.1285delC) and splice donor mutations (c.1300+1G>A) were also detected. All these mutations greatly affected the protein stability and FLCN-FNIP2 protein interaction. Family-based association studies suggested pathogenic FLCN mutations are significantly associated with BHDS. Two pathogenic SNPs, rs1801133 and rs138189536, at MTHFR, associated with Homocystinuria, were found in one family. Conclusion: Pathogenic mutations at FLCN may play key roles in deregulating metabolic pathways leading to disease pathogenesis. Instead of FLCN mutations, MTHFR pathogenic SNPs were also detected in clinically diagnosed BHDS patients, therefore, genetic evaluation is necessary to avoid confounding diagnosis.

Loss-of-function, gain-of-function and dominant-negative mutations have profoundly different effects on protein structure: implications for variant effect prediction

Most known pathogenic mutations occur in protein-coding regions of DNA and change the way proteins are made. Taking protein structure into account has therefore provided great insight into the molecular mechanisms underlying human genetic disease. While there has been much focus on how mutations can disrupt protein structure and thus cause a loss of function (LOF), alternative mechanisms, specifically dominant-negative (DN) and gain-of-function (GOF) effects, are less understood. Here, we have investigated the protein-level effects of pathogenic missense mutations associated with different molecular mechanisms. We observe striking differences between recessive vs dominant, and LOF vs non-LOF mutations, with dominant, non-LOF disease mutations having much milder effects on protein structure, and DN mutations being highly enriched at protein interfaces. We also find that nearly all computational variant effect predictors underperform on non-LOF mutations, even those based solely on sequence conservation. However, we do find that non-LOF mutations could potentially be identified by their tendency to cluster in space. Overall, our work suggests that many pathogenic mutations that act via DN and GOF mutations are likely being missed by current variant prioritisation strategies, but that there is considerable scope to improve computational predictions through consideration of molecular disease mechanisms.

Spectrum of Mutations of Familial Mediterranean Fever Gene by Whole Sequencing Method in Iranian Patients

Background: Familial Mediterranean fever (FMF) is the most common type of periodic fever syndrome. The disease is most prevalent in the western Mediterranean population, but today it is widespread in the world due to the large ethnic migrations of Turks, Jews, Arabs and Armenians. The MEFV gene is the only gene known to be associated with the disease. Objectives: The aim of this study was to characterize pathogenic mutations in patients with typical FMF symptoms by sequencing the entire MEFV gene. Methods: This is a descriptive-analytical study that was performed during ten years from 2009 to 2019. On 252 patients after clinical diagnosis based on existing criteria to determine mutations referred to Tehran Medical Genetics Laboratory and the whole sequencing method for MEFV gene was used to determine mutations. Results: Out of 252 patients, 143 (56.7%) had pathogenic variants, and 109 (43.3%) had no variants reported as pathogenic mutations. Variants were identified as fallow: (1) 8.7% as homozygous; (2) 22.2% as compound heterozygous; (3) 25.7% as heterozygous. The most common variants were M694V (c.2080A > G) and E148Q (c.442G > C). Conclusions: This study showed that the age of onset of the disease was in the first and second decades of life amongst our patients and the most common complaints of patients were periodic fever and abdominal pain. The most frequent allele was M694V (c.2080A > G) followed by E148Q (c.442G > C) allele.

Implementation of molecular genetic tests in the diagnostics of thyroid cancer: own research

Aim — to investigate the significance of BRAFV600E, NRAS, KRAS, HRAS, RET/PTC (RET/PTC1 and RER/PTC3), PAX8/PPARg mutations for the development and course of thyroid cancer with the determination of a significant predictor mutation. Materials and methods. The analysis included 63 selected case histories of patients who, at the preoperative stage, underwent molecular genetic testing (MHT) of the thyroid gland (TG) masses and subsequently underwent surgical treatment in the surgical department of the Ukrainian Scientific and Practical Center for Endocrine Surgery, Transplantation of Endocrine Organs and Tissues. The average age of the patients was 41.0 ± 1.8 years; from them 7men and56women. All patients underwent fine-needle aspiration puncture biopsy(FNAB) of thyroid nodules according to the standard method followed by a cytological conclusion in accordance with the Bethesda system.The Bethesda III category was revealed in 3 patients (4.8 %), Bethesda IV — 13 patients (20.6 %), Bethesda V — 12 patients (19.0 %), Bethesda VI in 35 (55.6 %) patients. The pathogenic mutations were detected in 47 (74.6 %) patients (group 1), among them two mutations were simultaneously found in two subjects. In 16 cases (25.4 %), no pathogenic mutation was found at all (group 2). Results. The genes that occurred most often were BRAFV600E — in 35 patients (55.6 %), NRAS — in 11 patients (17.5 %), KRAS — in 3 patients (4.8 %). In case of thyroid cancerdiagnosis, pathogenic mutations were found in 38 (79.1 %) subjects. The BRAFV600E gene mutation was observed when establishing a cytological conclusion classified as Bethesda III—V in 28.9 %, and in Bethesda V and in 77.1 %. Accordingly, the sensitivity of the test was low — 0.646, specificity — 0.733. The high prognostic significance of a positive result(PPV) value (from 0.784 to 0.943) indicated the likelihood of detecting thyroid cancer. This assumption confirms the calculation of the c-square criterion with the Yates correction, which is 5.207 (p = 0.023). The use of this test for the presence of the NRAS gene to detect thyroid cancer was ineffective, the value of the c -square criterion with Yates correction = 0.009 (p = 0.927). The incidence of thyroid cancer in group 1 in the cytological class Bethesda III—V was higher than in group 2, but it did not differ significantly between the groups. The aggressiveness of thyroid cancer in patients of group 1 with a positive MHT result did not have significant differences compared with the results obtained in group 2 (39.5 % and 33.3 %, respectively). Conclusions. The use of the kit for the determination of MHTmade it possible to identify pathogenic mutations in the genes BRAFV600E, NRAS, KRAS in 79.1 % of cases. The presence of these genes in combination with the analysis of cytomorphological findings classified according to the Bethesda III—V system did not increase the detection of thyroid cancer in the studied patients. The BRAFV600E gene, which was observed in 64.6 % of cases (PPV from 0.784 to 0.943), was a significant predictor among the studied candidate genes for establishing the diagnosis of thyroid cancer. Detection of a pathogenic mutation in patients with thyroid cancer did not indicate in favour of its aggressive course.