Allele Frequencies Of Variants Research Articles

SUN-246 Low Frequency of Pathogenic Allelic Variants in the Disorders of Sex Development Related Genes in Small for Gestational Age Children with Hypospadias

Background: Small for gestational age (SGA) children are defined as the one with birth weight and/or length 2 or more standard deviations below the population mean for gestational age. The underlying causes of pre and postnatal growth failure are diverse1. SGA boys present a high frequency of hypospadias (15 to 30%) of unknown etiology2. Objective: To analyze the prevalence of SGA individuals with hypospadias in a large cohort of 46, XY disorders of sex development (DSD) patients (n=272) followed in a tertiary center and to search for a molecular etiology associated with this phenotype. Patients and methods: We evaluated 30 SGA children with medium or proximal hypospadias, four of them presented syndromic characteristics associated. DNA samples of 24 patients were studied by target massively parallel sequencing (TPMS) using an amplicon-based capture panel of 63 genes related to DSD, and one by whole exome sequencing (WES). TPMS and WES were performed in the Illumina MiSEQ/HiSEQ platforms, respectively. Paired-end reads were aligned to the hg19 assembly of the human genome with BWA-MEM. Variants were called and annotated with Platypus and ANNOVAR. Results: The prevalence of SGA individuals with hypospadias in the 46, XY DSD cohort was 11%. Two children had clinical features of Silver-Russell syndrome and their diagnosis was confirmed (11p15 LOM) by MLPA. The two other syndromic patients were suspected with three M syndrome and Mulibrey Nanism. The TPMS study identified three likely pathogenic variants in these patients: p.Trp1538* and p.Gln813fs variants in compound heterozygosis in CUL7 gene and the homozygous p.Gly340fs variant in TRIM37. Seven heterozygous variants with uncertain significance in 5 DSD related genes were identified: two in the DHX37, p.V717I and p.A737T, associated with GATA4 p.P407R variant and with WWOX p.Y85D, respectively; the WT1 p.C350R variant, the IGF1R p.R1337C variant, and the BMP8B p. Arg116Cys variant. The frequency of likely pathogenic variants in SGA children with hypospadias was 10%. Conclusion: The low frequency of pathogenic allelic variants identified in SGA children with hypospadias indicates that other mechanisms, as epigenetic changes, may be involved in the etiology of this condition. 1 CLAYTON, P. E. et al. Management of the Child Born Small for Gestational Age through to Adulthood: A Consensus Statement of the International Societies of Pediatric Endocrinology and the Growth Hormone Research Society. The Journal Of Clinical Endocrinology & Metabolism, [s.l.], v. 92, n. 3, p.804-810, mar. 2007. The Endocrine Society. http://dx.doi.org/10.1210/jc.2006-2017. 2 MOREL, Y et al. Aetiological diagnosis of male sex ambiguity: a collaborative study. European Journal Pediatric, [s. L.], v. 161, n. 1, p.49-59, jan. 2002.

Correlation of CYP2D6 allelic polymorphism to outcome of acute coronary syndrome in mid-Euphrates Iraqi patients on metoprolol therapy

This study aims to investigate the different clinically relevant allele variants (allele frequencies) of CYP2D6 gene and to determine whether a specific genotype of CYP2D6 gene (based on genetic polymorphism “allelic types” and combination) have impact on metoprolol effectiveness (clinical outcome) in patients who have acute coronary syndrome (ACS). The study included 250 patients with ACS who were classified into 2 study groups, 125 patients received metoprolol and served as a study group (Group1) and 125 who received no metoprolol therapy (due to contraindication to the medication) and served as a control group (Group 2). Venous blood samples were taken from all participants for DNA extraction. Urine samples were also collected to assess the metabolic ratio using High-performance liquid chromatography (HPLC) technique.There was significant variation in the distribution of Iraqi patients with respect to CYP2D6 allelic polymorphism as compared to similar patients in other countries. Besides, this significant difference existed in patients' outcome in terms of morbidity and mortality in respect to variable genotypes and phenotypes. We recommend a dose individualization of metoprolol in patients with ACS is essential to improve patients' outcome.

Utility of cfDNA Fragmentation Patterns in Designing the Liquid Biopsy Profiling Panels to Improve Their Sensitivity.

Genotyping of cell-free DNA (cfDNA) in plasma samples has the potential to allow for a noninvasive assessment of tumor biology, avoiding the inherent shortcomings of tissue biopsy. Next generation sequencing (NGS), a leading technology for liquid biopsy analysis, continues to be hurdled with several major issues with cfDNA samples, including low cfDNA concentration and high fragmentation. In this study, by employing Ion Torrent PGM semiconductor technology, we performed a comparison between two multi-biomarker amplicon-based NGS panels characterized by a substantial difference in average amplicon length. In course of the analysis of the peripheral blood from 13 diagnostic non-small cell lung cancer patients, equivalence of two panels, in terms of overall diagnostic sensitivity and specificity was shown. A pairwise comparison of the allele frequencies for the same somatic variants obtained from the pairs of panel-specific amplicons, demonstrated an identical analytical sensitivity in range of 140 to 170 bp amplicons in size. Further regression analysis between amplicon length and its coverage, illustrated that NGS sequencing of plasma cfDNA equally tolerates amplicons with lengths in the range of 120 to 170 bp. To increase the sensitivity of mutation detection in cfDNA, we performed a computational analysis of the features associated with genome-wide nucleosome maps, evident from the data on the prevalence of cfDNA fragments of certain sizes and their fragmentation patterns. By leveraging the support vector machine-based machine learning approach, we showed that a combination of nucleosome map associated features with GC content, results in the increased accuracy of prediction of high inter-sample sequencing coverage variation (areas under the receiver operating curve: 0.75, 95% CI: 0.750–0.752 vs. 0.65, 95% CI: 0.63–0.67). Thus, nucleosome-guided fragmentation should be utilized as a guide to design amplicon-based NGS panels for the genotyping of cfDNA samples.

Relationship of four vitamin D receptor gene polymorphisms with type 1 diabetes mellitus susceptibility in Kuwaiti children

BackgroundThe incidence of type 1 diabetes mellitus (T1DM) in Kuwait is amongst the highest in the world. Vitamin D is considered to be involved in immune modulation and its deficiency contribute to autoimmune destruction of insulin producing beta cells in T1DM patients. Vitamin D has been shown to exert its effects via a nuclear vitamin D receptor (VDR) and therefore, VDR gene may be considered a candidate for T1DM susceptibility.MethodsThe genotypes of four VDR gene polymorphisms were determined in 253 Kuwaiti Arab T1DM patients and 214 healthy controls by PCR-RFLP analysis. Serum concentrations of three autoantibodies i.e. ICA (Islet cell autoantibody), GADA (Glutamic acid decarboxylase) and INS (Insulin autoantibody) were determined by radio-immunoassays.ResultsStatistically significant differences were detected between the genotypes of two VDR gene polymorphisms (FokI, C > T, rs10735810 and TaqI, C > T, rs731236) between T1DM patients and controls (P < 0.0001). In both, the frequency of variant alleles was considerably high in T1DM than in the controls. In contrast, the VDR gene ApaI (G > T, rs7975232) and BsmI (A > G, rs1544410) polymorphisms did not show association with T1DM. The homozygous variant genotypes of FokI, ApaI and TaqI polymorphisms show significant differences between various age-of-onset subgroups while no such association was detected in the case of BsmI polymorphism. Significant differences were also noted between heterozygous genotypes of all four polymorphisms especially between 4-6y and > 6y age-of-onset subgroups of T1DM patients. Three autoantibodies, ICA (Islet cell), GADA (glutamate decarboxylase) and INS (insulin) were positively associated to, varying degrees, with T1DM in Kuwaiti Arabs harboring different VDR gene polymorphism genotypes.ConclusionsOur results demonstrate a significant effect of two VDR gene polymorphisms (FokI and TaqI) and three autoantibodies on genetic susceptibility of T1DM in Kuwaiti Arabs along with other factors.

Retrospective Data Analysis of the Influence of Age and Sex on TPMT Activity and Its Phenotype-Genotype Correlation.

Therapeutic efficacy and toxicity of thiopurine drugs (used as anticancer and immunosuppressant agents) are affected by thiopurine S-methyltransferase (TPMT) enzyme activity. TPMT genotype and/or phenotype is used to predict the risk for adverse effects before drug administration. Inosine triphosphate pyrophosphatase (ITPA) is another enzyme involved in thiopurine metabolism. In this study, we aimed to evaluate (a) frequency of various TPMT phenotypes and genotypes, (b) correlations between them, (c) influence of age and sex on TPMT activity, and (d) distribution of ITPA variants among various TPMT subgroups. TPMT enzyme activity was determined by LC-MS/MS. TPMT (*2,*3A-C) and ITPA (rs1127354, rs7270101) genotypes were determined using a customized TaqMan® OpenArray®. TPMT enzyme activity varied largely (6.3-90 U/mL). The frequency of low, intermediate, normal, and high activity was 0.5% (n = 230), 13.1% (n = 5998), 86.1% (n = 39448), and 0.28% (n = 126), respectively. No significant difference in TPMT activity in relation to age and sex was found. Genotype analysis revealed the frequency of variant TPMT alleles was 6.73% (*3A, n = 344), 0.05% (*3B, n = 2), 2.22% (*3C, n = 95), and 0.42% (*2, n = 19). Analysis of paired phenotype and genotype showed that TPMT activity in samples with variant allele(s) was significantly lower than those without variant alleles. Lastly, an equal distribution of ITPA variants was found among normal and abnormal TPMT activity. This retrospective data analysis demonstrated a clustering of variant TPMT genotypes with phenotypes, no significant influence of age and sex on TPMT activity, and an equal distribution of ITPA variants among various TPMT subgroups.

Association of Foxp3 and TGF-β1 Polymorphisms with Pre-Eclampsia Risk in Chinese Women.

The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the genes that encode forkhead box p3 (Foxp3) (rs3761549 C>T, rs2280883T>C, rs2232365 A>G and rs3761548 C>A) and transforming growth factor (TGF)-β1 (rs11466359 C>T, rs11466345 A>G and rs1800469 T>C) are associated with pre-eclampsia (PE) risk in Chinese women. SNPs were identified by polymerase chain reaction and ligase detection reaction. Allelic variant and genotype frequencies for Foxp3 and TGF-β1 were compared between PE women (n = 203) and healthy pregnant (HP) controls (n = 243). The TGF-β1 rs1800469 TT genotype was found more frequently in PE patients than in HP controls [CC vs. CT+TT: odds ratio (OR) = 1.71; 95% confidence interval (CI): 1.04-2.81; p = 0.033], indicating that the T allele of rs1800469 confers a risk for PE [OR = 1.46; 95% CI: 1.12-1.92; p = 0.006]. The Foxp3 rs2232365 A allele was associated with severe PE specifically [OR = 1.70; 95% CI: 1.12-2.58; p = 0.01], compared with mild PE. There were no haplotype associations with PE. These findings indicate that allelic variants of TGF-β1 rs1800469 T influence PE risk in Chinese women. Pregnant Han Chinese women carrying the rs1800469 TT genotype were at increased risk of PE.

10-year follow-up of the Super-Seniors Study: compression of morbidity and genetic factors

BackgroundSuper-Seniors are healthy, long-lived individuals who were recruited at age 85 years or older with no history of cancer, cardiovascular disease, diabetes, dementia, or major pulmonary disease. In a 10-year follow-up, we aimed to determine whether surviving Super-Seniors showed compression of morbidity, and to test whether the allele frequencies of longevity-associated variants in APOE and FOXO3 were more extreme in such long-term survivors.MethodsSuper-Seniors who survived and were contactable were re-interviewed 10 years after initial characterization. Health and lifestyle were characterized via questionnaire. Geriatric tests including the Timed Up and Go (TUG), Geriatric Depression Scale (GDS), Instrumental Activities of Daily Living (IADL) and the Mini-Mental State Exam (MMSE) were administered, and data were compared to results from on average 10 years earlier. As well, genotype and allele frequencies for SNPs rs7412 and rs429358 in APOE, and rs2802292 in FOXO3 were compared to the frequencies in the original collection of Super-Seniors and mid-life controls.ResultsOf the 480 Super-Seniors recruited from 2004 to 2007, 13 were alive, contactable, and consented to re-interview (mean age = 100.1 ± 3.3). Eight of these 13 participants (62%) still met Super-Senior health criteria. Diseases that occurred in late life were cardiovascular (5 of 13; 38%) and lung disease (1 of 13; 8%). MMSE and IADL scores declined in the decade between interviews, and GDS and TUG scores increased. The surviving group of centenarians had a higher frequency of APOE and FOXO3 longevity-associated variants even when compared to the original long-lived Super-Senior cohort.ConclusionsAlthough physical and mental decline occurred in the decade between interviews, the majority of Super-Seniors re-interviewed still met the original health criteria. These observations are consistent with reports of compression of morbidity at extreme ages, particularly in centenarians. The increased frequency of longevity- associated variants in this small group of survivors is consistent with studies that reported genetics as a larger contributor to longevity in older age groups.

Haplotype Analysis on the Relationship of the DNAJC6 Gene with Early-Onset Parkinson's Disease Risk in a Chinese Population.

DNAJC6 gene is one of the Parkinson's disease (PD) related genes, but relationship between DNAJC6 polymorphisms and PD remains unclear. We aims to examine the association between genetic variations in DNAJC6 gene and idiopathic early-onset PD (EOPD) in the Chinese population. Exons and intron/exon boundaries of DNAJC6 gene was amplified and sequenced in 135 EOPD patients and 212 healthy controls. Single nucleotide polymorphisms (SNP)-based and haplotype-based association study between EOPD and DNAJC6 was conducted. SNP-SNP interactions were investigated using the generalized multifactor dimensionality reduction (GMDR) method. We further evaluated the effect of variants on gene function and expression using online in silico algorithms and databases. We found fourteen previously reported SNPs in the DNAJC6 gene. The frequencies of variant alleles in rs11208644, rs4582839 and rs4915691 were observed significantly higher in EOPD group compared to healthy controls, while in rs6588144 was significantly lower. Additionally, haplotype analysis indicated that the CTCACTCGGC, CTTACTCGGC and TTTGTTCGAC haplotypes were associated with higher EOPD risk in EOPD patients. SNP-SNP interaction analysis showed that rs12077111-rs4592284 SNP combination was the best model with higher EOPD risk. Based on the in silico analysis results, these SNPs were predicted to be no harm to the protein function, but might lead to possible changes in splice site and alter the expression level of DNAJC6. Our study indicated that EOPD was associated with several SNPs and haplotypes of DNAJC6 gene.

Circulating leptin concentration, LEP gene variants and haplotypes, and polycystic ovary syndrome in Bahraini and Tunisian Arab women

Background and aimEpidemiological studies suggested that ethnic/racial background influences the associations of altered leptin secretion and leptin gene (LEP) polymorphisms with polycystic ovary syndrome (PCOS). We investigated the association between LEP variants and plasma leptin levels with PCOS in Tunisian and Bahraini Arab women. Subjects and methodsRetrospective case-control study, involving 255 PCOS cases and 253 control subjects from Bahrain, and 320 women PCOS cases and 447 controls from Tunisia. LEP genotyping was done by allele exclusion on real-time PCR. ResultsMinor allele frequencies of rs10487506, rs7799039, rs2167270, rs12706832, and rs10954173 LEP variants were not significantly different between PCOS cases and control women among Bahraini and Tunisians, even before applying the Bonferroni correction. Similarly, the genotype distribution of the tested LEP variants was comparable between women with PCOS and control women among Bahraini and Tunisian subjects. None of the tested LEP variants was linked with altered leptin serum concentrations. However, five-locus haplotype analysis identified GGGGG and GAGGG haplotypes to be positively, and haplotype AAGGG to be negatively associated with PCOS in Bahraini women, after adjusting for HOMA-IR. No LEP haplotype associated with PCOS was identified in Tunisians. ConclusionThis is the first study to document differential contribution of LEP gene variants with PCOS according to ethnic/racial background of study subjects, highlighting the need for controlling for ethnicity in genetic association studies.

Can we predict long-term survival in resectable pancreatic ductal adenocarcinoma?

ObjectivePancreatic ductal adenocarcinoma (PDAC) is an aggressive tumour associated with poor 5-year survival. We aimed to determine factors which differentiate short and long-term survivors and identify a prognostic biomarker.MethodsOver a ten-year period, patients with resected PDAC who developed disease recurrence within 12 months (Group I) and those who had no disease recurrence for 24 months (Group II) were identified. Clinicopathological data was analysed. Ion Torrent high-throughput sequencing on DNA extracted from FFPE tumour samples was used to identify mutations. Additionally, peripheral blood samples were analysed for variants in cell-free DNA, circulating tumour cells (CTCs), and microRNAs.ResultsMultivariable analysis of clinicopathological factors showed that a positive medial resection margin was significantly associated with short disease-free survival (p = 0.007). Group I patients (n = 21) had a higher frequency of the KRAS mutant mean variant allele (16.93% ± 11.04) compared to those in Group II (n = 13; 7.55% ± 5.76, p = 0.0078). Group I patients also trended towards having a KRAS c.35G>A p.Gly12Asp mutation in addition to variants in other genes, such as TP53, CDKN2A, and SMAD4. Mutational status of cell-free DNA, and number of CTCs, was not found to be useful in this study. A circulating miRNA (hsa-miR-548ah-5p) was found to be significantly differentially expressed.ConclusionsMedial resection margin status and the frequency of KRAS mutation in the tumour tissue are independent prognostic indicators for resectable PDAC. Circulating miRNA hsa-miR-548ah-5p has the potential to be used as a prognostic biomarker.

Interleukin (IL)-8 polymorphisms and risk of prostate disorders

Prostate cancer (PCa) and benign prostate hyperplasia (BPH) as two prevalent age-related disorders in male have some shared genetic and immunological underlying mechanisms. However, researchers have aimed at identification of specific biomarkers with the ability to differentiate between these disorders. In the present study, we genotyped the rs4073, rs2227306 and rs1126647 single nucleotide polymorphisms within IL-8 gene in 530 individuals including 130 PCa patients, 200 BPH patients and 200 male controls. The rs2227306 alleles and genotypes were distributed equally in the three study groups. The frequency of the A allele of the rs4073 was significantly lower in PCa group compared with BPH group (OR (95% CI) = 0.62 (0.46–0.84), adjusted P value = 0.006). This allele was negatively associated with PCa risk in dominant model (OR (95% CI) = 0.53 (0.34–0.83), adjusted P value = 0.02). When comparing PCa and BPH groups, the rs1126647 was associated with PCa risk in recessive model (OR (95% CI) = 2.14 (1.23–3.72), adjusted P value = 0.02). The A T A haplotype (rs4073, rs2227306 and rs1126647 respectively) was less frequent in PCa group compared with BPH group (OR (95% CI) = 0.4 (0.22–0.75), adjusted P value = 0.03). Consequently, our data demonstrated significant differences in allele, genotype and haplotype frequencies of IL-8 variants between BPH and PCa patients which might imply distinct role for this chemokine in the pathogenesis of these disorders. Future studies are needed to elaborate the underlying mechanism of these observations.

Familial Mediterranean Fever Gene Mutations and Gout as an Auto-Inflammatory Arthropathy.

Introduction:The auto-inflammatory diseases (AID) are a heterogeneous group of multi-system disorders of innate immunity dysregulation. MEFV gene has major role in AID.Aim:The aim of this study is to investigate the frequency of MEFV variant alleles in gout patients as an AID and their genotype-phenotype relationship.Methods:Total amount of 224 of the healthy people as a control group (113 male and 111 female) and 24 gouty arthritis patients (20 male and 4 female) entered this study. Blood samples screened for the 12 common pathogenic mutations according to manufacturer’s instructions.Results:The mean age of patients was 54 years. MTP joint was the most involved joint (91.66%). Mutations were shown in 5 patients (20.83%) that were not different from healthy population (25%). Five patients carry one mutated MEFV allele, E148Q in 4 patients and V726A in 1 patient. Control group showed 25% mutations as E148Q (18.3%), P369S (3.1%), V726A (2.2%), A744S (1.3%) respectively. The most common mutation detected in patients was E148Q (16.66%) and all of them were males. No significant and meaningful associations were detected between the MEFV gene mutations and gouty arthritis patients.Conclusion:There was not any correlation between MEFV gene mutations carriage with age, sex, the number of joint involvement and the course of disease in gouty arthritis. MEFV gene mutations were more frequent in men than women, but this is not statistically significant.

Assessment of Correlation of Growth Hormone Receptor Gene with Tooth Dimensions: A CBCT and Genotyping Study.

Aim:Assessment of root morphology, size, and amount of bone around tooth is essential before starting the orthodontic treatment. The present study aimed to assess the relationship between tooth dimensions with two gene variants of growth hormone (GH), namely rs6184 and rs6180.Materials and Methods:This study was conducted on 218 subjects (males: 104, females: 114) requiring orthodontic treatment. All underwent cone beam computed tomography (CBCT) scan for orthodontic treatment planning with Kodak CBCT machine. In all teeth, crown height (CH), root length (RL), and crown–root ratio were evaluated. Two growth hormone receptor (GHR) variants (rs6184 and rs6180) were genotyped using the TaqMan genotyping assay.Results:The mean CH and RL of all teeth, that is, maxillary and mandibular central incisors, lateral incisor, canine, first premolar, second premolar, first molar, and second molar, were measured. There was no significant difference in males and females (P > 0.05). Allele frequencies of GHR variants for rs6180 and rs6184 were 48.1% and 8.92%, respectively. Multiple regression analysis showed GHR rs6184 association with maxillary central incisor CH, maxillary canine RL, mandibular canine CH, and mandibular first premolar RL (P < 0.05).Conclusion:There was correlation of CH of maxillary and mandibular canine and RL of maxillary canine and mandibular first premolar with GHR rs6184.

Polygenic Adaptation Underlies Evolution of Brain Structures and Behavioral Traits

Many of the characteristics of psychiatric disorders, including their early age of onset, moderate to high prevalence (i.e., 1% for schizophrenia, >15% for major depression), reduced fecundity, and high heritability have led many to speculate on how risk alleles have persisted throughout evolutionary history. While several potential mechanisms for maintaining high allele frequency of risk variants have been hypothesized (e.g., weak positive selection, balancing selection), few have been empirically tested. Given the recent analytic advancements in detecting polygenic adaptation, we systematically evaluated evidence for selection across a total of twenty-five complex traits including ten neuropsychiatric disorders, three personality traits, total intracranial volume, seven subcortical brain structure volume traits, and four complex traits with no known neuropsychiatric associations. We tested each set of trait-associated variants for evidence of classical hard sweeps (i.e., extreme integrated haplotype scores, iHS), partial sweeps (i.e., extreme population differentiation, Fst), rapid evolution since divergence from Neanderthal (i.e., Neanderthal depletion score, NDS), ancient polygenic selection (i.e., Qx scores), and very recent polygenic selection within the past 2,000 years (i.e., trait singleton density scores, tSDS). Variants associated with schizophrenia (Qx = 208.36, p < 0.001), extraversion (Qx = 88.04, p = 0.001), subjective well-being (Qx = 113.90, p < 0.001), hippocampus volume (Qx = 108.98, p < 0.001), and putamen volume (Qx = 115.76, p < 0.001) were enriched for signatures of weak polygenic adaptation after accounting for genetic drift. However, a limitation of this approach is that the null model of neutral drift is computed with respect to ancestral and derived allelic annotations, therefore it is not possible to determine whether polygenic adaptation has favored risk/trait-increasing alleles or protective/trait-decreasing alleles. Thus, we also employed a directional test of selection (tSDS) which overcomes this limitation. Remarkably, tSDS is powered to detect very recent selection that has been active only in the past ~2,000 years. Here, we find that signatures of recent polygenic adaptation are enriched among SCZ-protective alleles (Z=-3.06, p=0.002) total intracranial volume-increasing alleles (Z=3.09, p = 0.002), but putamen (Z=-4.93, p < 0.001), amygdala (Z=-3.49, p<0.001) and pallidum (Z=-3.39, p<0.001) volume-decreasing alleles. These results provide the first genome-wide genetic evidence in support of the mosaic theory of brain evolution in humans, suggesting that individual brain regions can evolve and vary in size independent of one another. No trait studied displayed significant consistent evidence of classical hard sweeps or partial sweeps. To characterize the biological processes and functional elements potentially targeted by polygenic adaptation, we conducted expression Quantitative Trait Locus (eQTL) and gene set enrichment analyses. SNPs associated with schizophrenia were enriched for eQTLs in brain (p = 0.007) while immune eQTLs were overrepresented among putamen (p = 0.001) and intracranial volume (p = 0.001) associated variants. Our results suggest that alleles associated with behavioral and brain structure volume traits have experienced polygenic adaptation acting through neurodevelopmental and immune-mediated pathways.

Ethnic differences in distributions of allelic and genotypic frequencies of NAT2 polymorphic variants in patients with pulmonary tuberculosis

Ethnic differences in distributions of allelic and genotypic frequencies of NAT2 polymorphic variants in patients with pulmonary tuberculosis

SU94 - EXPLORING THE SOMATIC VARIATION IN SCHIZOPHRENIA

Background The presence of somatic variation in the human brain has been elucidated over the past several decades. Most recently, single-cell whole-genome sequencing studies have provided unequivocal evidence that individual human brain cells harbor unique genetic variants. The functional significance of this phenomenon is unclear, but some studies have suggested that it contributes to risk of neuropsychiatric disease. Currently, cost and labor requirement render difficult the sequencing of individual genomes at the scale that will likely be necessary to fully uncover the extent and role of somatic variation in the brain. A scalable alternative, however, has been developed in the field of cancer genomics wherein somatic variants are identified by comparing the frequencies of variant alleles in tumor and normal bulk tissue specimens from the same individual. Here, we apply this approach to investigate the role of somatic variation in schizophrenia in a pilot study of 9 individuals (5 schizophrenia cases and 4 unaffected controls). Methods Specimens from the prefrontal cortex and temporal muscle of 5 schizophrenia cases and 4 controls were obtained from the Mount Sinai NIH Brain and Tissue Repository. Deep whole-exome sequencing (250X coverage) was performed using DNA extracted from neuronal and non-neuronal nuclei, which were isolated from cortical specimens by fluorescent-activated nuclear sorting. We followed standard protocols for mapping and filtering reads, then called somatic Single Nucleotide Variants (sSNVs) using a suite of 6 calling algorithms. For each individual, the calling algorithms were utilized to call variants in the exomes of both the neuronal and non-neuronal cells of the brain by comparing them to a matched “normal” sample from the same individual (i.e., the temporal muscle). Variants were then filtered using an in-house quality control pipeline, and a subset of the remaining calls were validated with digital PCR (dPCR) and parallel sequencing of multiple clones (clone-seq). Results We identified 26 sSNVs in neuronal and non-neuronal cells from the brains of 9 individuals. We chose 10 of 26 for experimental validation using dPCR and clone-seq, observing a validation rate of 80%. We noted that 13 of the 26 sSNVs were found in a single schizophrenia case; as such, 4 of the 10 sSNVs included in validation experiments were from this individual, 3 of which were successfully validated. For 5 sSNVs, only the neuronal exome of the individual had the variant, for 17 only the non-neuronal exome and for 4 both the neuronal and non-neuronal exomes. At least 1 sSNV was observed in all 5 of the cases and in 2 of the 4 controls. We noted that 2 of the 23 somatic SNVs in cases fell within one of the 16 copy number variants implicated in schizophrenia by the largest study of copy number variation in schizophrenia to date. Several of the sSNVs observed in our cohort fall within genes known to function in determining cellular fate and development, including WNT10B, SMAD1 and FGF1. Discussion Here, we present a pipeline suitable for the large-scale investigation of somatic variation in the human brain. We show evidence that somatic variation occurs in a cell-type specific manner in the human brain. Genes affected by sSNVs in the brain have known roles in determining cell fate. Greater number of sSNVs in schizophrenia cases compared to controls was observed, and multiple case sSNVs fall within known schizophrenia CNVs. Larger sample sizes are needed so as to further elucidate the extent and role of somatic variation in the human brain.

Correlation of CYP2C19 genotype with plasma voriconazole exposure in South-western Chinese Han patients with invasive fungal infections.

The aim of this study was to investigate the correlation between CYP2C19 genotype and dose-adjusted voriconazole (VCZ) trough concentrations (C0/dose).We analyzed the correlation between CYP2C192(681G>A), CYP2C193(636G>A), and CYP2C1917(-806C>T) genetic polymorphisms and the dose-corrected pre-dose concentration (C0/dose) in 106 South-western Chinese Han patients.The frequencies of variant alleles of CYP2C192, 3, and 17 were 29.7%, 4.25%, and 0.92%. For 49.3% of the VCZ samples, the therapeutic window between 1.5 and 5.5 μg/ml was reached. Following the first dose VCZ measurement, in subsequent samples the proportion of VCZ C0 within the therapeutic window increased, suggesting effective therapeutic drug monitoring (TDM) (P = .001). The VCZ C0 was significantly different (P = .010) between patients with normal metabolism (NMs), intermediate metabolism (IMs), and poor metabolism (PMs). The VZC C0/dose was 12.2 (interquartile range (IQR), 8.33-18.2 μg·ml/kg·day), and 7.68 (IQR, 4.07-16.3 μg·ml/kg·day) in PMs and IMs patients, respectively, which was significantly higher than in NMs phenotype patients (4.68; IQR, 2.51-8.87 μg·ml/kg·day, P = .008 and P = .014).This study demonstrated that the VCZ C0/dose was significantly influenced by the CYP2C19 genotype in South-western Chinese Han patients. In this patient population, more over-exposure was observed in patients with a CYP2C19 genotype associated with poor or intermediate metabolism. CYP2C19 genotype-based dosing combined with TDM will support individualization of VCZ dosing, and potentially will minimize toxicity and maximize therapeutic efficacy.

Modification of Cytotoxic Lymphocytes with T Cell Receptor Specific for Minor Histocompatibility Antigen ACC-1Y

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for hematopoietic malignancies. The graft-derived donor lymphocytes are capable of eliminating the residual recipient malignant cells in the course of allogeneic immune response, thus decreasing the chances of a relapse of the disease. Foreign peptides of the recipient presented by the MHC molecules are able to elicit the immune response immunologically. These polymorphic peptides are known as minor histocompatibility antigens (MiHAs). MiHAs occur due to the nonsynonymous single nucleotide polymorphisms in human genome. Transfusion of T cells specific to MiHAs presented predominantly in the cells of hematopoietic origin will allow the targeted elimination of residual malignant clones avoiding undesirable damage to healthy tissues. To induce the immune response, the donor must be homozygous by the MiHA allele and the recipient must either be homozygous or heterozygous by the alternative MiHA allele. The therapeutic mismatch occurs in 25% of cases under the optimal frequency of allelic variants. Minor antigen ACC-1Y originates from polymorphism in the BCL-2A1 gene; its immunogenic mismatch occurrence approaches the theoretical maximum. In addition, BCL2A1 is overexpressed in cells of various lymphomas. ACC-1Y is presented on allele HLA-A*24:02, which is relatively frequent in the Russian population. Combination of these factors makes the minor antigen ACC-1Y a promising target for immunotherapy. Transfusion of donor CD8^(+) lymphocytes modified with transgenic MiHA-specific TCR is one of the promising methods of posttransplant leukemia therapy and relapse prophylaxis. We obtained a sequence of high-affinity ACC-1Y-specific TCR after the antigen-specific expansion of T cells derived from a healthy ACC-IY^(-/-) donor. We cloned this sequence into the lentiviral vector and obtained the assembled viral particles. Further, we transduced the CD8^(+) lymphocyte culture and demonstrated its antigen-specific cytotoxic activity. It is suggested that CD8^(+) lymphocytes modified by the described method could be potentially transferred to recipients as a therapy against relapse after allo-HSCT.

VarGenius executes cohort-level DNA-seq variant calling and annotation and allows to manage the resulting data through a PostgreSQL database

BackgroundTargeted resequencing has become the most used and cost-effective approach for identifying causative mutations of Mendelian diseases both for diagnostics and research purposes. Due to very rapid technological progress, NGS laboratories are expanding their capabilities to address the increasing number of analyses. Several open source tools are available to build a generic variant calling pipeline, but a tool able to simultaneously execute multiple analyses, organize, and categorize the samples is still missing.ResultsHere we describe VarGenius, a Linux based command line software able to execute customizable pipelines for the analysis of multiple targeted resequencing data using parallel computing. VarGenius provides a database to store the output of the analysis (calling quality statistics, variant annotations, internal allelic variant frequencies) and sample information (personal data, genotypes, phenotypes). VarGenius can also perform the “joint analysis” of hundreds of samples with a single command, drastically reducing the time for the configuration and execution of the analysis.VarGenius executes the standard pipeline of the Genome Analysis Tool-Kit (GATK) best practices (GBP) for germinal variant calling, annotates the variants using Annovar, and generates a user-friendly output displaying the results through a web page.VarGenius has been tested on a parallel computing cluster with 52 machines with 120GB of RAM each. Under this configuration, a 50 M whole exome sequencing (WES) analysis for a family was executed in about 7 h (trio or quartet); a joint analysis of 30 WES in about 24 h and the parallel analysis of 34 single samples from a 1 M panel in about 2 h.ConclusionsWe developed VarGenius, a “master” tool that faces the increasing demand of heterogeneous NGS analyses and allows maximum flexibility for downstream analyses. It paves the way to a different kind of analysis, centered on cohorts rather than on singleton. Patient and variant information are stored into the database and any output file can be accessed programmatically. VarGenius can be used for routine analyses by biomedical researchers with basic Linux skills providing additional flexibility for computational biologists to develop their own algorithms for the comparison and analysis of data.The software is freely available at: https://github.com/frankMusacchia/VarGenius

Frequency of allelic variants of the TMPRSS2 gene in a prostate cancer-free Southwestern Colombian population

OBJECTIVE: To describe the frequency of the TMPRSS2 gene and its variants in a prostate cancer-free Southwestern Colombian population.MATERIALS AND METHODS: An observational study was conducted that included cancer-free persons, regardless of age, from Southwestern Colombia. Blood samples were drawn from the patients for DNA extraction. Blood drops were collected and dried on filters and immersed in phosphate buffer, utilizing the DNeasy kit. The preparation process was carried out using the TruSeq Exome Library Prep® kit and the resulting libraries were normalized with the TruSeq Rapid Exome® kit. The commercial kits were provided by Illumina®. We sequenced the full exome and identified the variants associated with the TMPRSS2 gene. Descriptive statistics were employed for the data analysis.RESULTS: The study population was made up of 162 persons from whom 7,315,466 sequence data were obtained. The TMPRSS2 gene was found in 414 data (4.3%). The most common SNP was rs140530035 (32.1%) and the most relevant SNP sequenced was rs12329760 (10.6%).CONCLUSION: TMPRSS2 was not frequent in the population studied. The most important polymorphism associated with the TMPRSS2 gene was rs12329760.KEYWORDS: Gene; Prostate cancer; TMPRSS2; Polymorphism.