Non-synonymous Sequence Variants Research Articles

NOTCH gene alterations in metastatic colorectal cancer in the Nationwide Cancer Genome Screening Project in Japan (SCRUM-Japan GI-SCREEN).

Activated Notch receptor signaling has been implicated in tumor growth and progression in colorectal cancer (CRC). However, the pathogenic relevance of NOTCH gene alterations remains unclear. The aim of this study was to clarify mutational landscapes and assess their clinical significance in patients with metastatic CRC. Pre-chemotherapy tumor tissues obtained from 1154 metastatic CRC patients in the Nationwide Cancer Genome Screening Project in Japan between April 2017 and March 2019 were studied using the Oncomine Comprehensive Assay. The frequencies of NOTCH1, NOTCH2, and NOTCH3 nonsynonymous sequence variants were 11.5%, 4.4%, and 10.4%, respectively. The majority of variants were missense of unknown significance that were distributed across all domains of all three NOTCH genes. The gain-of-function mutations in NOTCH reported in multiple malignancies were not identified. The NOTCH amplification rate was less than 1%. No NOTCH fusions were detected. In patients who were registered before, or within 1year of, first-line chemotherapy, overall survival for 51 patients with only NOTCH3 variants was significantly longer than for 540 patients with no NOTCH variants (median, 40.2months vs 27.7months; P = 0.04). Multivariate analysis revealed that variant NOTCH3 was an independent prognostic factor for increased survival (hazard ratio 0.61, 95% confidence interval, 0.39-0.94; P = 0.03) besides poor prognostic factors associated with mutant TP53, KRAS, and BRAF, as well as amplified MYC. NOTCH genes are unlikely to harbor driver mutations and amplifications in patients with metastatic CRC. NOTCH3 variant should be further investigated as a favorable prognostic marker.

Quantitative Alterations in Complement Alternative Pathway and Related Genetic Analysis in Severe Phenotype Preeclampsia.

Preeclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome share many clinical and biologic features with thrombotic microangiopathy syndromes caused by complement abnormalities. Our hypothesis was that similar functional and genetic alterations in the complement alternative pathway (CAP) are present in these disorders of pregnancy. We conducted quantitative analysis of proteins involved in CAP using ELISA and nephelometry on prospectively collected blood samples from patients with severe phenotype preeclampsia (defined as delivery ≤34 weeks due to preeclampsia), HELLP syndrome, or eclampsia, and matched normotensive controls (n=25 in each arm) between 2011 and 2016. Sequencing was performed to interrogate 14 genes encoding CAP components. Both groups were similar in age, gravidity, parity, marital status, and race. The study group had a higher BMI (mean±SD, 32±8 versus 25±4 kg/m2; P=0.002) and earlier gestational age at delivery (32.5±3.6 versus 40.3±1 weeks; P<0.001). Serologic studies demonstrated elevated Bb subunit (median [range], 1.2 [0.5-4.3] versus 0.6 [0.5-1] μg/ml; P<0.001), complement C5 concentration (28 [18-33] versus 24 [15-34] mg/dl; P=0.03), and sMAC (371 [167-761] versus 184 [112-249] ng/ml; P<0.001) concentrations in patients with preeclampsia. Two thirds of patients with preeclampsia had at least one nonsynonymous sequence variant in CAP genes. Patients with severe phenotype preeclampsia manifest functional alterations in CAP activation. Genetic variants in the CAP genes were detected in several patients, but a larger population study is necessary to fully evaluate genetic risk. Genetic screening and complement-targeted treatment may be useful in risk stratification and novel therapeutic approaches.

Non-Driver Mutations and Gene Sequence Variants Identified By Targeted Deep Sequencing in Polycythemia Vera and Essential Thrombocythemia

Background:Driver mutations in myeloproliferative neoplasms involve mutations in the JAK2, MPL, and CALR genes. However, incorporation of next generation sequencing (NGS) testing into routine clinical practice may identify for mutations or variants in additional hematologic malignancy-associated genes, the roles of which have not been extensively elucidated in patients with polycythemia vera (PV) or essential thrombocythemia (ET). The purpose of this study is to characterize the clinical characteristics of ET/PV patients harboring additional non-driver mutations or gene variants.Methods:Eligible cases included patients with PV/ET diagnosed and treated at MD Anderson Cancer Center from January 2012 to January 2017. NGS sequencing of 28 genes known to be mutated in hematologic malignancies was performed on samples from 240 ET/PV patients. Non-driver mutations detected by the NGS platform were classified based on the allele frequency recorded in our database in conjunction with currently available and periodically updated public reference databases of somatic mutations or variants of unclear significance.Results:A total of 110 ET and 130 PV cases had NGS performed on samples collected at the time of initial referral. Among them, 21 (19%) ET cases and 28 (21.5%) PV cases had ≥1 non-driver somatic mutation detected. The distribution of non-driver somatic mutations in ET and PV are shown in Figure 1A. On grouping non-driver genes into functional pathways, the most frequent mutational targets were in genes associated with epigenetic regulation (PV, 22; ET, 25), mainly DNA methylation (PV, 18; ET, 20) followed by epigenetic modifier (PV, 4; ET, 5). In subjects with PV, TET2 (n = 12) was the most frequently mutated gene, followed by DNMT3A (n = 7), IDH1/2 (n = 5), and ASXL1 (n = 4). A similar pattern was noted in ET, with TET2 mutations (n = 8) being most frequent followed by DNMT3A (n = 7), and ASXL1 (n = 5). Older patients were more likely to display non-driver mutations in both ET (p < 0.001) and PV (p = 0.02). The combined prevalence of non-driver somatic mutations was 27% and 32% in PV and ET, respectively. Over a short median follow up of 3.9 (range, 1.0-37.1) months among PV patients with non-driver somatic mutations, no leukemic transformations occurred. Similarly, no leukemic transformations were observed in ET patients with additional non-driver somatic mutations over a median follow up of 3.8 (range, 1.1-36) months. Overall survival (OS) was significantly longer in patients without additional mutations in epigenetic modifiers (Figure 1C). The prognostic relevance of non-driver somatic mutations on OS was not determined due the small sample size and limited follow up duration. Of 13 (12%) triple-negative ET patients without non-driver somatic mutations, nine had non-synonymous gene sequence variants; the most common being in DNA methylation genes namely TET2 &IDH1/2 genes (Figure 1B).Conclusion:The spectrum of non-driver somatic mutations in our ET and PV study cohorts was dominated by mutations in the TET2, DNMT3A, ASXL1 genes, and were correlated with age (Buscarlet Blood, 2017). A larger sample size and longer follow-up is needed to determine the impact of non-driver somatic mutations on transformation-free and overall survival. Germline TET2 variants or their combinations with other variants/non-driver mutations may constitute low penetrance predisposition factors for myeloid neoplasms and their functional relevance warrants further exploration. [Display omitted] DisclosuresBose:Incyte Corporation: Honoraria. Kantarjian:Amgen: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding. Verstovsek:Astrazeneca: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; Blueprint Medicines Corp: Research Funding; Galena BioPharma: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Blueprint Medicines Corp: Research Funding; Lilly Oncology: Research Funding; Incyte: Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Research Funding; Lilly Oncology: Research Funding; CTI BioPharma Corp: Research Funding; Pfizer: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding; CTI BioPharma Corp: Research Funding; Bristol Myers Squibb: Research Funding; Astrazeneca: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding.

PNPLA3 gene polymorphism and severity of liver steatosis and fibrosis

IntroductionNon-alcoholic fatty liver disease (NAFLD) is one of the leading chronic liver diseases worldwide. Environmental and genetic factors have an impact on NAFLD development. Single nucleotide polymorphism in the PNPLA3 gene is strongly related to the increased fat content in hepatocytes. The aim of our study was to investigate the association between PNPLA3 variants and the intensity of liver steatosis and fibrosis assessed by liver biopsy in patients with NAFLD.Material and methodsThe study group comprised 127 patients with NAFLD confirmed by liver biopsy. The control group comprised 251 healthy volunteers. In every case genotyping of the PNPLA3 rs738409 C to G nonsynonymous sequence variant was performed. Distributions of genotypes and alleles were compared between NAFLD patients and controls, and analyzed in relation to different steatosis and fibrosis stages in the study group.ResultsFrequencies of PNPLA3 genotypes strongly differ between NAFLD patients and controls (p = 0.00001). Odds ratio (OR) for genotype GG in NAFLD was 2.94 (1.45–5.97, p = 0.0015). Also allele G was more frequent in NAFLD patients vs. controls (p = 0.00001); OR for G allele in NAFLD was 2.74 (1.77–4.27, p = 0.00000273). No significant differences in the genotype or allele distribution of PNLPA3 were observed in relation to steatosis intensity or between patients with or without cirrhosis. However, allele G was found is every hepatocellular carcinoma case.ConclusionsWe do not advocate universal genetic testing in every NAFLD case to select patients at risk of development of cirrhosis or steatohepatitis. Further investigation of the relationship between PNPLA3 variants and hepatocellular carcinoma is warranted.

A novel homozygous variant in exon 10 of the GALNT3 gene causing hyperphosphatemic familial tumoral calcinosis in a family from North India.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is an extremely rare autosomal recessive disorder caused by variants in the GALNT3 (N-acetylgalactosaminyltransferase 3), FGF23 (Fibroblast Growth Factor-23) and αKL (α-Klotho) genes, which results in progressive calcification of soft tissues. We describe the case of a 9-year-old girl who presented with recurrent hard nodular swellings on her feet and knees which intermittently discharged chalky white material. Her younger brother also had a similar condition. Both siblings showed hyperphosphatemia, but the parentsbiochemical parameters were normal. The histological features of the material aspirated from a skin lesion were consistent with tumoral calcinosis. Sanger sequencing identified a novel homozygous non-synonymous sequence variant in exon 10 of the GALNT3 gene (NM_004482.3:c.[1681T>A];[1681T>A], NP_004473.2:p. [Cys561Ser];[Cys561Ser] in the proband and her affected brother. The parents were heterozygous carriers for the same sequence variant. In conclusion, we report a new variant in the GALNT3 gene that caused HFTC in a North Indian family.

Mutant GNLY is linked to Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare severe cutaneous adverse reactions to drugs. Granulysin (GNLY) plays a key role in keratinocyte apoptosis during SJS/TEN pathophysiology. To determine if GNLY-encoding mutations might be related to the protein's functional disturbances, contributing to SJS/TEN pathogenesis, we performed direct sequencing of GNLY's coding region in a group of 19 Colombian SJS/TEN patients. A GNLY genetic screening was implemented in a group of 249 healthy individuals. We identified the c.11G > A heterozygous sequence variant in a TEN case, which creates a premature termination codon (PTC) (p.Trp4Ter). We show that a mutant protein is synthesised, possibly due to a PTC-readthrough mechanism. Functional assays demonstrated that the mutant protein was abnormally located in the nuclear compartment, potentially leading to a toxic effect. Our results argue in favour of GNLY non-synonymous sequence variants contributing to SJS/TEN pathophysiology, thereby constituting a promising, clinically useful molecular biomarker.

Identification of c.1531C>T Pathogenic Variant in the CDH1 Gene as a Novel Germline Mutation of Hereditary Diffuse Gastric Cancer.

Germline pathogenic variants in the CDH1 gene are a well-established cause of hereditary diffuse gastric cancer (HDGC) syndrome. The aim of this study was to characterize CDH1 mutations associated with HDGC from Chile, a country with one of the highest incidence and mortality rates in the world for gastric cancer (GC). Here, we prospectively include probands with family history/early onset of diffuse-type of GC. The whole coding sequence of the CDH1 gene was sequenced from genomic DNA in all patients, and a multidisciplinary team managed each family member with a pathogenic sequence variant. Thirty-six cases were included (median age 44 years/male 50%). Twenty-seven (75%) patients had diffuse-type GC at ≤50 years of age and 19 (53%) had first or second-degree family members with a history of HDGC. Two cases (5.5%) carried a non-synonymous germline sequence variant in the CDH1 gene: (a) The c.88C>A missense variant was found in a family with three diffuse-type GC cases; and (b) c.1531C>T a nonsense pathogenic variant was identified in a 22-year-old proband with no previous family history of HDGC. Of note, six family members carry the same nonsense pathogenic variant. Prophylactic gastrectomy in the proband’s sister revealed stage I signet-ring cell carcinoma. The finding of 1531C>T pathogenic variant in the CDH1 in proband with no previous family history of HDGC warrants further study to uncover familial clustering of disease in CDH1 negative patients. This finding may be particularly relevant in high incidence countries, such as the case in this report.

FOXD1 mutations are related to repeated implantation failure, intra-uterine growth restriction and preeclampsia

BackgroundHuman reproductive disorders consist of frequently occurring dysfunctions including a broad range of phenotypes affecting fertility and women’s health during pregnancy. Several female-related diseases have been associated with hypofertility/infertility phenotypes, such as recurrent pregnancy loss (RPL). Other occurring diseases may be life-threatening for the mother and foetus, such as preeclampsia (PE) and intra-uterine growth restriction (IUGR). FOXD1 was defined as a major molecule involved in embryo implantation in mice and humans by regulating endometrial/placental genes. FOXD1 mutations in human species have been functionally linked to RPL’s origin.MethodsFOXD1 gene mutation screening, in 158 patients affected by PE, IUGR, RPL and repeated implantation failure (RIF), by direct sequencing and bioinformatics analysis. Plasmid constructs including FOXD1 mutations were used to perform in vitro gene reporter assays.ResultsNine non-synonymous sequence variants were identified. Functional experiments revealed that p.His267Tyr and p.Arg57del led to disturbances of promoter transcriptional activity (C3 and PlGF genes). The FOXD1 p.Ala356Gly and p.Ile364Met deleterious mutations (previously found in RPL patients) have been identified in the present work in women suffering PE and IUGR.ConclusionsOur results argue in favour of FOXD1 mutations’ central role in RPL, RIF, IUGR and PE pathogenesis via C3 and PlGF regulation and they describe, for the first time, a functional link between FOXD1 and implantation/placental diseases. FOXD1 could therefore be used in clinical environments as a molecular biomarker for these diseases in the near future.KeywordsRecurrent pregnancy loss, Preeclampsia, Intra-uterine growth restriction, FOXD1

Distinct Roles for Two Chromosome 1 Loci in Ethanol Withdrawal, Consumption, and Conditioned Place Preference.

We previously identified a region on chromosome 1 that harbor quantitative trait loci (QTLs) with large effects on alcohol withdrawal risk using both chronic and acute models in mice. Here, using newly created and existing QTL interval-specific congenic (ISC) models, we report the first evidence that this region harbors two distinct alcohol withdrawal QTLs (Alcw11and Alcw12), which underlie 13% and 3–6%, respectively, of the genetic variance in alcohol withdrawal severity measured using the handling-induced convulsion. Our results also precisely localize Alcw11 and Alcw12 to discreet chromosome regions (syntenic with human 1q23.1–23.3) that encompass a limited number of genes with validated genotype-dependent transcript expression and/or non-synonymous sequence variation that may underlie QTL phenotypic effects. ISC analyses also implicate Alcw11and Alcw12 in withdrawal-induced anxiety-like behavior, representing the first evidence for their broader roles in alcohol withdrawal beyond convulsions; but detect no evidence for Alcw12 involvement in ethanol conditioned place preference (CPP) or consumption. Our data point to high-quality candidates for Alcw12, including genes involved in mitochondrial respiration, spatial buffering, and neural plasticity, and to Kcnj9 as a high-quality candidate for Alcw11. Our studies are the first to show, using two null mutant models on different genetic backgrounds, that Kcnj9 −/− mice demonstrate significantly less severe alcohol withdrawal than wildtype littermates using acute and repeated exposure paradigms. We also demonstrate that Kcnj9 −/− voluntarily consume significantly more alcohol (20%, two-bottle choice) than wildtype littermates. Taken together with evidence implicating Kcnj9 in ethanol CPP, our results support a broad role for this locus in ethanol reward and withdrawal phenotypes. In summary, our results demonstrate two distinct chromosome 1 QTLs that significantly affect risk for ethanol withdrawal, and point to their distinct unique roles in alcohol reward phenotypes.

Further dissection of QTLs for salt-induced stroke and identification of candidate genes in the stroke-prone spontaneously hypertensive rat

We previously revealed that two major quantitative trait loci (QTLs) for stroke latency of the stroke-prone spontaneously hypertensive rat (SHRSP) under salt-loading were located on chromosome (Chr) 1 and 18. Here, we attempted further dissection of the stroke-QTLs using multiple congenic strains between SHRSP and a stroke-resistant hypertensive rat (SHR). Cox hazard model among subcongenic strains harboring a chromosomal fragment of Chr-1 QTL region showed that the most promising region was a 2.1 Mbp fragment between D1Rat177 and D1Rat97. The QTL region on Chr 18 could not be narrowed down by the analysis, which may be due to multiple QTLs in this region. Nonsynonymous sequence variations were found in four genes (Cblc, Cxcl17, Cic, and Ceacam 19) on the 2.1 Mbp fragment of Chr-1 QTL by whole-genome sequence analysis of SHRSP/Izm and SHR/Izm. Significant changes in protein structure were predicted in CBL-C and CXCL17 using I-TASSER. Comprehensive gene expression analysis in the kidney with a cDNA microarray identified three candidate genes (LOC102548695 (Zinc finger protein 45-like, Zfp45L), Ethe1, and Cxcl17). In conclusion, we successfully narrowed down the QTL region on Chr 1, and identified six candidate genes in this region.

Brain region-specific expression of genes mapped within quantitative trait loci for behavioral responsiveness to acute stress in Fisher 344 and Wistar Kyoto male rats.

Acute stress responsiveness is a quantitative trait that varies in severity from one individual to another; however, the genetic component underlying the individual variation is largely unknown. Fischer 344 (F344) and Wistar Kyoto (WKY) rat strains show large differences in behavioral responsiveness to acute stress, such as freezing behavior in response to footshock during the conditioning phase of contextual fear conditioning (CFC). Quantitative trait loci (QTL) have been identified for behavioral responsiveness to acute stress in the defensive burying (DB) and open field test (OFT) from a reciprocal F2 cross of F344 and WKY rat strains. These included a significant QTL on chromosome 6 (Stresp10). Here, we hypothesized that the Stresp10 region harbors genes with sequence variation(s) that contribute to differences in multiple behavioral response phenotypes between the F344 and WKY rat strains. To test this hypothesis, first we identified differentially expressed genes within the Stresp10 QTL in the hippocampus, amygdala, and frontal cortex of F344 and WKY male rats using genome-wide microarray analyses. Genes with both expression differences and non-synonymous sequence variations in their coding regions were considered candidate quantitative trait genes (QTGs). As a proof-of-concept, the F344.WKY-Stresp10 congenic strain was generated with the Stresp10 WKY donor region into the F344 recipient strain. This congenic strain showed behavioral phenotypes similar to those of WKYs. Expression patterns of Gpatch11 (G-patch domain containing 11), Cdkl4 (Cyclin dependent kinase like 4), and Drc1 (Dynein regulatory complex subunit 1) paralleled that of WKY in the F344.WKY-Stresp10 strain matching the behavioral profiles of WKY as opposed to F344 parental strains. We propose that these genes are candidate QTGs for behavioral responsiveness to acute stress.

Rare Risk Variants Identification by Identity-by-Descent Mapping and Whole-Exome Sequencing Implicates Neuronal Development Pathways in Schizophrenia and Bipolar Disorder.

Schizophrenia (SCZ) and bipolar disorder (BPD) are highly heritable disorders with an estimated co-heritability of 68%. Hundreds of common alleles have been implicated, but recently a role for rare, high-penetrant variants has been also suggested in both disorders. This study investigated a familial cohort of SCZ and BPD patients from a closed population sample, where the high recurrence of the disorders and the homogenous genetic background indicate a possible enrichment in rare risk alleles. A total of 230 subjects (161 cases, 22 unaffected relatives, and 47 controls) were genetically investigated through an innovative strategy that integrates identity-by-descent (IBD) mapping and whole-exome sequencing (WES). IBD analysis allowed to track high-risk haplotypes (IBDrisk) shared exclusively by multiple patients from different families and possibly carrying the most penetrant alleles. A total of 444 non-synonymous sequence variants, of which 137 disruptive, were identified in IBDrisk haplotypes by WES. Interestingly, gene sets previously implicated in SCZ (i.e., post-synaptic density (PSD) proteins, voltage-gated calcium channels (VGCCs), and fragile X mental retardation protein (FMRP) targets) were found significantly enriched in genes carrying IBDrisk variants. Further, IBDrisk variants were preferentially affecting genes involved in the extracellular matrix (ECM) biology and axon guidance processes which appeared to be functionally connected in the pathway-derived meta-network analysis. Results thus confirm rare risk variants as key factors in SCZ and BPD pathogenesis and highlight a role for the development of neuronal connectivity in the etiology of both disorders.

313: Quantitative functional alterations in complement alternative pathway and related genetic analysis in severe phenotype preeclampsia and HELLP syndrome

Preeclampsia and HELLP syndrome share many clinical and biologic features with thrombotic microangiopathy syndromes caused by abnormalities in complement pathways. It is unclear whether functional and genetic alterations in the complement alternative pathway (CAP) are associated with preeclampsia and HELLP syndrome. We used prospectively collected blood samples (2012 to 2016) from cases of severe phenotype preeclampsia, defined as delivery <34 weeks, HELLP syndrome and eclampsia, and matched normotensive controls (n=25 in each arm). Quantitative and functional analysis of 15 CAP components and activation fragments was conducted using clinically available immunoassays. Whole exome sequencing was performed on study group to interrogate 36 genes encoding CAP factors for coding non-synonymous sequence variants with a population allele frequency of <5%. Study and control groups were similar in age, gravidity, parity, marital status and race. However, the study group had a higher BMI (mean±SD; 32±8 vs 25±4 kg/m2; p=0.002) and earlier gestational age at delivery (32.5±3.6 vs 40.3±1 weeks; p<0.001). Serological studies in cases compared with controls demonstrated elevated Bb subunit (median [range]; 1.2 [0.5-4.3] vs 0.6 [0.5-1] mcg/mL; p<0.001), C5 concentrations (28 [18-33] vs 24 [15-34] mg/dL; p=0.03) and sMAC (371 [167-761] vs 184 [112-249] ng/mL; p<0.001) levels. Of 25 cases, 17 (68%) had at least 1 sequence variant in CAP genes (5 x CFB, 2 x CFI, 3 x CFH, 5 x CFHR, 1 x MCP, 5 x C2, 3 x C3, 1 x C3R, 1x C5, 5 x C7, 5 x C8, 1x C9). Specific listings are found in Figure. Patients with severe phenotype preeclampsia manifest functional alterations in complement alternative pathway activation, as evident by elevated levels of Bb, C5 and sMAC. Associated genetic sequence variants in complement pathway genes are also more common than expected, some of which are potentially pathogenic, suggesting shared genetic pathogenesis. Genetic screening and complement-targeted treatment may be useful in risk stratification and novel therapeutic approaches.

Elevation of fumarase attenuates hypertension and can result from a nonsynonymous sequence variation or increased expression depending on rat strain.

The activity of fumarase, an enzyme in the tricarboxylic acid cycle, is lower in Dahl salt-sensitive SS rats compared with SS.13BN rats. SS.13BN rats have a Brown Norway (BN) allele of fumarase and exhibit attenuated hypertension. The SS allele of fumarase differs from the BN allele by a K481E sequence variation. It remains unknown whether higher fumarase activities can attenuate hypertension and whether the mechanism is relevant without the K481E variation. We developed SS-TgFh1 transgenic rats overexpressing fumarase on the background of the SS rat. Hypertension was attenuated in SS-TgFh1 rats. Mean arterial pressure in SS-TgFh1 rats was 20 mmHg lower than transgene-negative SS littermates after 12 days on a 4% NaCl diet. Fumarase overexpression decreased H2O2, while fumarase knockdown increased H2O2 Ectopically expressed BN form of fumarase had higher specific activity than the SS form. However, sequencing of more than a dozen rat strains indicated most rat strains including salt-insensitive Sprague-Dawley (SD) rats had the SS allele of fumarase. Despite that, total fumarase enzyme activity in the renal medulla was still higher in SD rats than in SS rats, which was associated with higher expression of fumarase in SD. H2O2 can suppress the expression of fumarase. Renal medullary interstitial administration of fumarase siRNA in SD rats resulted in higher blood pressure on the high-salt diet. These findings indicate elevation of total fumarase activity attenuates the development of hypertension and can result from a nonsynonymous sequence variation in some rat strains and higher expression in other rat strains.

Molecular Characterization of Somatic Alterations in Dukes' B and C Colorectal Cancers by Targeted Sequencing.

Despite global progress in research, improved screening and refined treatment strategies, colorectal cancer (CRC) remains as the third most common malignancy. As each type of cancer is different and exhibits unique alteration patterns, identifying and characterizing gene alterations in CRC that may serve as biomarkers might help to improve diagnosis, prognosis and predict potential response to therapy. With the emergence of next generation sequencing technologies (NGS), it is now possible to extensively and rapidly identify the gene profile of individual tumors. In this study, we aimed to identify actionable somatic alterations in Dukes’ B and C in CRC via NGS. Targeted sequencing of 409 cancer-related genes using the Ion AmpliseqTM Comprehensive Cancer Panel was performed on genomic DNA obtained from paired fresh frozen tissues, cancer and normal, of Dukes’ B (n = 10) and Dukes’ C (n = 9) CRC. The sequencing results were analyzed using Torrent Suite, annotated using ANNOVAR and validated using Sanger sequencing. A total of 141 somatic non-synonymous sequence variations were identified in 86 genes. Among these, 64 variants (45%) were predicted to be deleterious, 38 variants (27%) possibly deleterious while the other 39 variants (28%) have low or neutral protein impact. Seventeen genes have alterations with frequencies of ≥10% in the patient cohort and with 14 overlapped genes in both Dukes’ B and C. The adenomatous polyposis coli gene (APC) was the most frequently altered gene in both groups (n = 6 in Dukes’ B and C). In addition, TP53 was more frequently altered in Dukes’ C (n = 7) compared to Dukes’ B (n = 4). Ten variants in APC, namely p.R283∗, p.N778fs, p.R805∗, p.Y935fs, p.E941fs, p.E1057∗, p.I1401fs, p.Q1378∗, p.E1379∗, and p.A1485fs were predicted to be driver variants. APC remains as the most frequently altered gene in the intermediate stages of CRC. Wnt signaling pathway is the major affected pathway followed by P53, RAS, TGF-β, and PI3K signaling. We reported the alteration profiles in each of the patient which has the potential to affect the clinical decision. We believe that this study will add further to the understanding of CRC molecular landscape.

New mutations in non-syndromic primary ovarian insufficiency patients identified via whole-exome sequencing.

Is it possible to identify new mutations potentially associated with non-syndromic primary ovarian insufficiency (POI) via whole-exome sequencing (WES)? WES is an efficient tool to study genetic causes of POI as we have identified new mutations, some of which lead to protein destablization potentially contributing to the disease etiology. POI is a frequently occurring complex pathology leading to infertility. Mutations in only few candidate genes, mainly identified by Sanger sequencing, have been definitively related to the pathogenesis of the disease. This is a retrospective cohort study performed on 69 women affected by POI. WES and an innovative bioinformatics analysis were used on non-synonymous sequence variants in a subset of 420 selected POI candidate genes. Mutations in BMPR1B and GREM1 were modeled by using fragment molecular orbital analysis. Fifty-five coding variants in 49 genes potentially related to POI were identified in 33 out of 69 patients (48%). These genes participate in key biological processes in the ovary, such as meiosis, follicular development, granulosa cell differentiation/proliferation and ovulation. The presence of at least two mutations in distinct genes in 42% of the patients argued in favor of a polygenic nature of POI. It is possible that regulatory regions, not analyzed in the present study, carry further variants related to POI. WES and the in silico analyses presented here represent an efficient approach for mapping variants associated with POI etiology. Sequence variants presented here represents potential future genetic biomarkers. This study was supported by the Universidad del Rosario and Colciencias (Grants CS/CIGGUR-ABN062-2016 and 672-2014). Colciencias supported Liliana Catherine Patiño´s work (Fellowship: 617, 2013). The authors declare no conflict of interest.

Mitochondrial DNA Mutations in Grade II and III Glioma Cell Lines Are Associated with Significant Mitochondrial Dysfunction and Higher Oxidative Stress.

The role of mitochondria in tumorigenesis has regained much attention as it could dysregulate cellular energetics, oxidative stress and apoptosis. However, the role of mitochondria in different grade gliomasis still unknown. This study aimed to identify mitochondrial DNA (mtDNA) sequence variations that could possibly affect the mitochondrial functions and also the oxidative stress status. Three different grades of human glioma cell lines and a normal human astrocyte cell line were cultured in-vitro and tested for oxidative stress biomarkers. Relative oxidative stress level, mitochondria activity, and mitochondrial mass were determined by live cell imaging with confocal laser scanning microscope using CM-H2DCFDA, MitoTracker Green, and MitoTracker Orange stains. The entire mitochondrial genome was sequenced using the AffymetrixGeneChip Human Mitochondrial Resequencing Array 2.0. The mitochondrial sequence variations were subjected to phylogenetic haplogroup assessment and pathogenicity of the mutations were predicted using pMUT and PolyPhen2. The Grade II astrocytoma cells showed increased oxidative stress wherea high level of 8-OHdG and oxidative stress indicator were observed. Simultaneously, Grade II and III glioma cells showed relatively poor mitochondria functions and increased number of mutations in the coding region of the mtDNA which could be due to high levels of oxidative stress in these cells. These non-synonymous mtDNA sequence variations were predicted to be pathogenic and could possibly lead to protein dysfunction, leading to oxidative phosphorylation (OXPHOS) impairment, mitochondria dysfunction and could create a vicious cycle of oxidative stress. The Grade IV cells had no missense mutation but preserved intact mitochondria and excellent antioxidant defense mechanisms thus ensuring better survival. In conclusion, Grade II and III glioma cells demonstrated coding region mtDNA mutations, leading to mitochondrial dysfunction and higher oxidative stress.

Genetic variability in E6, E7 and L1 genes of Human Papillomavirus 62 and its prevalence in Mexico

BackgroundHuman papillomavirus (HPV) is the main etiological agent of cervical cancer, the third most common cancer among women globally and the second most frequent in Mexico. Persistent infection with high-risk HPV genotypes is associated with premalignant lesions and cervical cancer development. HPVs considered as low risk or not yet classified, are often found in coinfection with different HPV genotypes. Indeed, HPV62 is one of the most prevalent HPV detected in some countries, but there is limited information about its prevalence in other regions and there are no HPV62 variants currently described. The aim of this study was to determine the prevalence of HPV62 in cervical samples from Mexican women and to identify mutations in the L1, E6 and E7 genes, which have never been reported in our population.MethodsHPV screening was performed by Cobas HPV Test in women who attended prevention health programs and dysplasia clinics. All HPV positive samples (n = 491) and 87 additional cervical cancer samples were then genotyped with Linear Array HPV Genotyping test. Some samples were selected to corroborate genotyping by Next-Generation sequencing. On the other hand, nucleotide changes in L1, E6 and E7 genes were determined using PCR, Sanger sequencing and analysis with the CLC-MainWorkbench 7.6.1 software. L1 protein structure was predicted with the I-TASSER server.ResultsUsing Linear Array, HPV62 prevalence was 7.6% in general population, 8% in Cervical Intraepithelial Neoplasia grade 1 (CIN1) samples and 4.6% in cervical samples. The presence of HPV62 was confirmed with Next-Generation sequencing. Regarding L1 gene, novel sequence variations were detected, but they did not alter the tertiary structure of the protein. Moreover, several nucleotide substitutions were found in E6 and E7 genes compared to reference HPV62 genomic sequence. Specifically, three non-synonymous sequence variations were detected, two in E6 and one in E7.ConclusionsHPV62 is a frequent HPV genotype found mainly in general population and in women with CIN1, and in 90.5% of the cases it was found in coinfection with other HPVs. Novel nucleotide changes in its L1, E6 and E7 genes were detected, some of them lead to changes in the protein sequence.

A novel mutation in exon 1 of GATA4 in Egyptian patients with congenital heart disease.

Congenital heart disease (CHD) is a common birth defect. Many studies have reported GATA4 mutations in patients with CHD, mainly septal defects. This study aimed to investigate the GATA4 exon 1 mutation in Egyptian patients with isolated congenital heart defects as a possible causative mutation. Screening for mutations or any sequence variations in exon 1 of the GATA4 gene was carried out by PCR amplification followed by direct sequencings in 165 Egyptian patients with different nonsyndromic congenital heart diseases and 93 controls who were matched in terms of age and sex. Thorough clinical assessments were done for all subjects, along with X-ray, 2D echocardiography, and Doppler examinations. The most common CHD among our cases was isolated ventricular septal defect (VSD) in 47.3% (78/165), followed by isolated atrial septal defect. A novel nonsynonymous sequence variation in fragment 2 (P193H) of exon 1 of GATA4 was detected in 15 (9.1%) of the subjects with septal defects. This mutation was not seen in any of the control group subjects. There is a high prevalence of exon 1 GATA4 mutation (9.1%) in our study compared to other studies in different populations, which may correlate with different ethnic populations.

Sequence analysis of four vitamin D family genes (VDR, CYP24A1, CYP27B1 and CYP2R1) in Vogt-Koyanagi-Harada (VKH) patients: identification of a potentially pathogenic variant in CYP2R1.

BackgroundVKH is a rare autoimmune disease. Decreased level of vitamin D has recently been found to be involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. This study was designed to screen the vitamin D pathway genes for pathogenic mutations, if any, in VKH patients.MethodsGenomic DNA was extracted from blood samples collected from patients with VKH disease and healthy controls. Entire coding region, exon-intron junctions of four genes were sequenced in DNA from 39 Saudi VKH patients and 50 ethnically matched healthy individuals. All patients and controls were unrelated.ResultsVitamin D levels in VKH patients were found either insufficient (21–29 ng/mL) or deficient (<20 ng/mL). Sequencing analysis of the VDR, CYP24A1, CYP27B1 and CYP2R1 detected twelve nucleotide changes in these genes in our cohort of 39 patients; 4 of which were non-coding, 6 were synonymous coding and 2 were non-synonymous coding sequence changes. All synonymous coding variants were benign polymorphisms with no apparent clinical significance. A non-synonymous coding sequence variant (c.2 T > C; p.1Met?) found in VDR is an initiation coding change and was detected in control individuals as well, while another variant (c.852G > A; p.284 M > I) found in CYP2R1 is predicted to be disease causing by mutationtaster software. This potentially pathogenic variant was found in 17 out of 39 VKH patients.ConclusionsScreening of four Vitamin D pathway genes in 39 VKH patients shows that a potentially pathogenic sequence variant in CYP2R1 may cause VKH in a subset of patients. These findings support the previous observation that low vitamin D levels might play a role in VKH pathogenesis and mutations in genes involved in vitamin D anabolism and catabolism might be of importance in VKH pathobiology.