Andrey S. Glotov

Targeted next-generation sequencing (NGS) of nine candidate genes with custom AmpliSeq in patients and a cardiomyopathy risk group

BACKGROUND Hypertrophic cardiomyopathy is a common genetic cardiac disease. Prevention and early diagnosis of this disease are very important. Because of the large number of causative genes and the high rate of mutations involved in the pathogenesis of this disease, traditional methods of early diagnosis are ineffective. METHODS We developed a custom AmpliSeq panel for NGS sequencing of the coding sequences of ACTC1, MYBPC3, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPM1, and CASQ2. A genetic analysis of student cohorts (with and without cardiomyopathy risk in their medical histories) and patients with cardiomyopathies was performed. For the statistical and bioinformatics analysis, Polyphen2, SIFT, SnpSift and PLINK software were used. To select genetic markers in the patients with cardiomyopathy and in the students of the high risk group, four additive models were applied. RESULTS Our AmpliSeq custom panel allowed us to efficiently explore targeted sequences. Based on the score analysis, we detected three substitutions in the MYBPC3 and CASQ2 genes and six combinations between loci in the MYBPC3, MYH7 and CASQ2 genes that were responsible for cardiomyopathy risk in our cohorts. We also detected substitutions in the TNNT2 gene that can be considered as protective against cardiomyopathy. CONCLUSION We used NGS with AmpliSeq libraries and Ion PGM sequencing to develop improved predictive information for patients at risk of cardiomyopathy.

Placental microRNA expression in pregnancies complicated by superimposed pre‑eclampsia on chronic hypertension

Pre-eclampsia (PE) is a complication of pregnancy that affects 5–8% of women after 20 weeks of gestation. It is usually diagnosed based on the de novo onset of hypertension and proteinuria. Preexisting hypertension in women developing PE, also known as superimposed PE on chronic hypertension (SPE), leads to elevated risk of maternal and fetal mortality. PE is associated with an altered microRNA (miRNA) expression pattern in the placenta, suggesting that miRNA deregulation is involved in the pathogenesis of PE. Whether and how the miRNA expression pattern is changed in the SPE placenta remains unclear. The present study analyzed the placental miRNA expression profile in pregnancies complicated by SPE. miRNA expression profiles in SPE and normal placentas were investigated using an Ion Torrent sequencing system. Sequencing data were processed using a comprehensive analysis pipeline for deep miRNA sequencing (CAP-miRSeq). A total of 22 miRNAs were identified to be deregulated in placentas from patients with SPE. They included 16 miRNAs previously known to be associated with PE and 6 novel miRNAs. Among the 6 novel miRNAs, 4 were upregulated (miR-518a, miR-527, miR-518e and miR-4532) and 2 downregulated (miR-98 and miR-135b) in SPE placentas compared with controls. The present results suggest that SPE is associated with specific alterations in the placental miRNA expression pattern, which differ from alterations detected in PE placentas, and therefore, provide novel targets for further investigation of the molecular mechanisms underlying SPE pathogenesis.

Molecular association of pathogenetic contributors to pre-eclampsia (pre-eclampsia associome)

BackgroundPre-eclampsia is the most common complication occurring during pregnancy. In the majority of cases, it is concurrent with other pathologies in a comorbid manner (frequent co-occurrences in patients), such as diabetes mellitus, gestational diabetes and obesity. Providing bronchial asthma, pulmonary tuberculosis, certain neurodegenerative diseases and cancers as examples, we have shown previously that pairs of inversely comorbid pathologies (rare co-occurrences in patients) are more closely related to each other at the molecular genetic level compared with randomly generated pairs of diseases. Data in the literature concerning the causes of pre-eclampsia are abundant. However, the key mechanisms triggering this disease that are initiated by other pathological processes are thus far unknown. The aim of this work was to analyse the characteristic features of genetic networks that describe interactions between comorbid diseases, using pre-eclampsia as a case in point.ResultsThe use of ANDSystem, Pathway Studio and STRING computer tools based on text-mining and database-mining approaches allowed us to reconstruct associative networks, representing molecular genetic interactions between genes, associated concurrently with comorbid disease pairs, including pre-eclampsia, diabetes mellitus, gestational diabetes and obesity. It was found that these associative networks statistically differed in the number of genes and interactions between them from those built for randomly chosen pairs of diseases. The associative network connecting all four diseases was composed of 16 genes (PLAT, ADIPOQ, ADRB3, LEPR, HP, TGFB1, TNFA, INS, CRP, CSRP1, IGFBP1, MBL2, ACE, ESR1, SHBG, ADA). Such an analysis allowed us to reveal differential gene risk factors for these diseases, and to propose certain, most probable, theoretical mechanisms of pre-eclampsia development in pregnant women. The mechanisms may include the following pathways: [TGFB1 or TNFA]-[IL1B]-[pre-eclampsia]; [TNFA or INS]-[NOS3]-[pre-eclampsia]; [INS]-[HSPA4 or CLU]-[pre-eclampsia]; [ACE]-[MTHFR]-[pre-eclampsia].ConclusionsFor pre-eclampsia, diabetes mellitus, gestational diabetes and obesity, we showed that the size and connectivity of the associative molecular genetic networks, which describe interactions between comorbid diseases, statistically exceeded the size and connectivity of those built for randomly chosen pairs of diseases. Recently, we have shown a similar result for inversely comorbid diseases. This suggests that comorbid and inversely comorbid diseases have common features concerning structural organization of associative molecular genetic networks.

AGTR2 gene polymorphism is associated with muscle fibre composition, athletic status and aerobic performance

What is the central question of this study? Variations in genes are considered to be molecular determinants maintaining the expression of the slow or fast myosin heavy chains of adult skeletal muscle. The role of polymorphisms of candidate genes involved in skeletal muscle development, energy homeostasis and thyroid and calcium metabolism in the determination of muscle fibre type has not previously been reported. What is the main finding and its importance? We show that the AGTR2 rs11091046 C allele is associated with an increased proportion of slow‐twitch muscle fibres, endurance athlete status and aerobic performance. Such findings have important implications for our understanding of muscle function in both health and disease.

Catching hidden variation: systematic correction of reference minor allele annotation in clinical variant calling

PurposeWe comprehensively assessed the influence of reference minor alleles (RMAs), one of the inherent problems of the human reference genome sequence.MethodsThe variant call format (VCF) files provided by the 1000 Genomes and Exome Aggregation Consortium (ExAC) consortia were used to identify RMA sites. All coding RMA sites were checked for concordance with UniProt and the presence of same codon variants. RMA-corrected predictions of functional effect were obtained with SIFT, PolyPhen-2, and PROVEAN standalone tools and compared with dbNSFP v2.9 for consistency.ResultsWe systematically characterized the problem of RMAs and identified several possible ways in which RMA could interfere with accurate variant discovery and annotation. We have discovered a systematic bias in the automated variant effect prediction at the RMA loci, as well as widespread switching of functional consequences for variants located in the same codon as the RMA. As a convenient way to address the problem of RMAs we have developed a simple bioinformatic tool that identifies variation at RMA sites and provides correct annotations for all such substitutions. The tool is available free of charge at http://rmahunter.bioinf.me.ConclusionCorrection of RMA annotation enhances the accuracy of next-generation sequencing–based methods in clinical practice.

Genetic determination of the vascular reactions in humans in response to the diving reflex

The purpose of this study was to investigate the genetic mechanisms of the defense vascular reactions in response to the diving reflex in humans with polymorphisms in the genes ADBR2, ACE, AGTR1, BDKRB2, and REN We hypothesized that protective vascular reactions, in response to the diving reflex, are genetically determined and are distinguished in humans with gene polymorphisms of the renin-angiotensin and kinin-bradykinin system. A total of 80 subjects (19 ± 1.4 yr) participated in the study. The intensity of the vascular response was estimated using photoplethysmogram. The I/D polymorphism (rs4340) of ACE was analyzed by PCR. REN (G/A, rs2368564), AGTR1 (A/C, rs5186), BDKRB2 (T/C, rs1799722), and ADBR2 (A/G, rs1042713) polymorphisms were examined using the two-step multiplex PCR followed by carrying allele hybridization on the biochip. Subjects with the BDKRB2 (C/C), ACE (D/D), and ADBR2 (G/G, G/A) genotypes exhibited the strongest peripheral vasoconstriction in response to diving. In subjects with a combination of the BDKRB2 (C/C) plus ACE (D/D) genotypes, we observed the lowest pulse wave amplitude and pulse transit time values and the highest arterial blood pressure during face immersion compared with the heterozygous individuals, suggesting that these subjects are more susceptible to diving hypoxia. This study observed that humans with gene polymorphisms of the renin-angiotensin and kinin-bradykinin systems demonstrate various expressions of protective vascular reactions in response to the diving reflex. The obtained results might be used in estimation of resistance to hypoxia of any origin in human beings or in a medical practice.NEW & NOTEWORTHY Our study demonstrates that the vascular reactions in response to the diving reflex are genetically determined and depend on gene polymorphisms of the kinin-bradykinin and the renin-angiotensin systems.

Systematic dissection of biases in whole-exome and whole-genome sequencing reveals major determinants of coding sequence coverage

Next generation DNA sequencing technologies are rapidly transforming the world of human genomics. Advantages and diagnostic effectiveness of the two most widely used resequencing approaches, whole exome (WES) and whole genome (WGS) sequencing, are still frequently debated. In our study we developed a set of statistical tools to systematically assess coverage of CDS regions provided by several modern WES platforms, as well as PCR-free WGS. Using several novel metrics to characterize exon coverage in WES and WGS, we showed that some of the WES platforms achieve substantially less biased CDS coverage than others, with lower within- and between-interval variation and virtually absent GC-content bias. We discovered that, contrary to a common view, most of the coverage bias in WES stems from mappability limitations of short reads, as well as exome probe design. We identified the ~ 500 kb region of human exome that could not be effectively characterized using short read technology. We also showed that the overall power for SNP and indel discovery in CDS region is virtually indistinguishable for WGS and best WES platforms. Our results indicate that deep WES (100x) using least biased technologies provides similar effective coverage (97% of 10x q10+ bases) and CDS variant discovery to the standard 30x WGS, suggesting that WES remains an efficient alternative to WGS in many applications. Our work could serve as a guide for selection of an up-to-date resequencing approach in human genomic studies.

Analytical “Bake-Off” of Whole Genome Sequencing Quality for the Genome Russia Project Using a Small Cohort for Autoimmune Hepatitis

A comparative analysis of whole genome sequencing (WGS) and genotype calling was initiated for ten human genome samples sequenced by St. Petersburg State University Peterhof Sequencing Center and by three commercial sequencing centers outside of Russia. The sequence quality, efficiency of DNA variant and genotype calling were compared with each other and with DNA microarrays for each of ten study subjects. We assessed calling of SNPs, indels, copy number variation, and the speed of WGS throughput promised. Twenty separate QC analyses showed high similarities among the sequence quality and called genotypes. The ten genomes tested by the centers included eight American patients afflicted with autoimmune hepatitis (AIH), plus one case’s unaffected parents, in a prelude to discovering genetic influences in this rare disease of unknown etiology. The detailed internal replication and parallel analyses allowed the observation of two of eight AIH cases carrying a rare allele genotype for a previously described AIH-associated gene (FTCD), plus multiple occurrences of known HLA-DRB1 alleles associated with AIH (HLA-DRB1-03:01:01, 13:01:01 and 7:01:01). We also list putative SNVs in other genes as suggestive in AIH influence.

Targeted sequencing analysis of ACVR2A gene identifies novel risk variants associated with preeclampsia

Abstract Background: Preeclampsia (PE) is the most common complication of pregnancy that remains to be a major cause of maternal and fetal mortality. Prediction and early diagnosis of PE would allow for timely initiation of preventive therapy. According to recent studies of ACVR2A gene polymorphism is associated with PE, but it is still unclear whether these findings reflect specific pathogenetic mechanisms of this disease. Methods: We performed targeted next-generation sequencing (NGS) sequencing of ACVR2A gene by means of Ion Torrent Personal Genome machine (PGM) Sequencer. A genetic analysis of patients with PE and control group was performed. Bioinformatics analysis using Polyphen2 (Boston, MA), SIFT (La Jolla, CA), and SnpSift software were used. To select genetic markers in PE patients two additive models and score analysis were applied. Results: Based on the score analysis, we detected two substitutions (rs145399059 and rs17692648) and one insertion insAA at position 148642724 that were associated with PE in our cohorts. We also detected a variant rs17742573 that can be considered as protective against preeclampsia. Conclusions: Our data suggest that some variants in ACVR2A gene are associated with PE. But more studies are required to reveal the role of ACVR2A gene in the pathogenesis of this disease during pregnancy.

Identification of Novel Candidate Markers of Type 2 Diabetes and Obesity in Russia by Exome Sequencing with a Limited Sample Size

Type 2 diabetes (T2D) and obesity are common chronic disorders with multifactorial etiology. In our study, we performed an exome sequencing analysis of 110 patients of Russian ethnicity together with a multi-perspective approach based on biologically meaningful filtering criteria to detect novel candidate variants and loci for T2D and obesity. We have identified several known single nucleotide polymorphisms (SNPs) as markers for obesity (rs11960429), T2D (rs9379084, rs1126930), and body mass index (BMI) (rs11553746, rs1956549 and rs7195386) (p < 0.05). We show that a method based on scoring of case-specific variants together with selection of protein-altering variants can allow for the interrogation of novel and known candidate markers of T2D and obesity in small samples. Using this method, we identified rs328 in LPL (p = 0.023), rs11863726 in HBQ1 (p = 8 × 10−5), rs112984085 in VAV3 (p = 4.8 × 10−4) for T2D and obesity, rs6271 in DBH (p = 0.043), rs62618693 in QSER1 (p = 0.021), rs61758785 in RAD51B (p = 1.7 × 10−4), rs34042554 in PCDHA1 (p = 1 × 10−4), and rs144183813 in PLEKHA5 (p = 1.7 × 10−4) for obesity; and rs9379084 in RREB1 (p = 0.042), rs2233984 in C6orf15 (p = 0.030), rs61737764 in ITGB6 (p = 0.035), rs17801742 in COL2A1 (p = 8.5 × 10−5), and rs685523 in ADAMTS13 (p = 1 × 10−6) for T2D as important susceptibility loci in Russian population. Our results demonstrate the effectiveness of whole exome sequencing (WES) technologies for searching for novel markers of multifactorial diseases in cohorts of limited size in poorly studied populations.