Frequency Of Sequence Variants Research Articles

Comparative Clinicopathologic and Genomic Analysis of Hepatocellular Neoplasm, Not Otherwise Specified, and Hepatoblastoma

Accurate diagnosis and treatment of hepatocellular neoplasm, not otherwise specified (HCN-NOS), poses significant challenges. Our study aimed to investigate the clinicopathological and genomic similarities and differences between HCN-NOS and hepatoblastoma (HB) to guide diagnostic and treatment strategies. The clinicopathological characteristics of 16 patients with HCN-NOS and 23 patients with HB were compared. Molecular studies, including the OncoKids® DNA and RNA-based next-generation sequencing panel, chromosomal microarray, and targeted Sanger sequencing analyses of CTNNB1 and TERT promoter, were employed. We found that patients with HCN-NOS were older (p<0.001) and more frequently classified as high-risk (p<0.01), yet they showed no significant differences in alpha-fetoprotein (AFP) levels or survival outcomes compared to those with HB. HCN-NOS and HB had a comparable frequency of sequence variants, with CTNNB1 mutations being predominant in both groups. Notably, TERT promoter mutations (37.5%) and rare clinically significant variants (BRAF, NRAS, and KMT2D) were exclusive to HCN-NOS. HCN-NOS demonstrated a higher prevalence of gains in 1q, encompassing the MDM4 locus (17/17 vs. 11/24, p<0.001), as well as loss/loss of heterozygosity (LOH) of 1p (11/17 vs. 6/24, p<0.05) and chromosome 11 (7/17 vs. 1/24, p<0.01) when compared to HB. Furthermore, recurrent loss/LOH of chromosome 3, 4p, 9, 15q, and Y were only observed in HCN-NOS. However, no significant differences were noted in gains of chromosomes 2, 8, and 20, or loss/LOH of 4q and 11p between the two groups. Notably, no clinically significant gene fusions were detected in either group. In conclusion, our study reveals that HCN-NOS exhibits high-risk clinicopathological features and greater structural complexity compared to HB. However, patients with HCN-NOS exhibit comparable AFP levels at diagnosis, CTNNB1 mutation rates, and survival outcomes when subjected to aggressive treatment, as compared to those with HB. These findings have the potential to enhance diagnostic accuracy and inform more effective treatments for HCN-NOS.

Monogenik Obezite Ön Tanısı ile İncelenen Hastaların Klinik, Laboratuvar ve Genetik Sonuçlarının Değerlendirilmesi

Introduction:Monogenic forms of obesity have an important place in the genetic causes of severe and early-onset obesity. In this study, it was aimed to evaluate the clinical and molecular genetic analysis results of the cases followed up with a prediagnosis of monogenic obesity in our clinic. Materials and Methods: The demographic, clinical and biochemical data of the patients had molecular genetic analysis with a pre-diagnosis of monogenic obesity in our clinic between 2016 and 2018 were retrospectively analyzed and recorded. Results: 47 obese cases (20 girls, 39 pubertal, mean age 14.3±3.2 years) were included in the study. Pathogenic variant in MC4R was detected in three cases, and heterozygous variant in LEPR, was not accepted as a pathogen variant in the database in one case. The frequency of sequence variants in the MC4R gene was 6.4%, and the frequency of the sequence variants in the LEPR gene was 2.1%. Conclusions: In our study, 8.5% (n=4) sequence variant was found in children who were examined with suspicion of monogenic obesity. In these cases, obesity developed in the first year of life and at least one parent had obesity. Therefore, if early-onset obesity is accompanied by a family history of obesity, MC4R defect, one of the monogenic obesity forms, should be considered in differential diagnosis.

Is it time to report carrier state for recessive disorders in every microarray analysis?-A pilot model based on hearing loss genes deletions.

This study aimed to examine the implications of reporting heterozygous losses of recessive genes in Chromosomal Microarray Analysis (CMA), based on the incidence of microdeletions of three common hearing impairment genes in the local cohort and the prevalence of sequence variants in these genes in worldwide databases. Prevalence of heterozygous microdeletions in OTOA and STRC genes, as well as deletions in the DFNB1 locus encompassing GJB6 gene, was determined using electronic database of Rabin Medical Center. ClinVar archive and Deafness Variation Database were used to generate a list of clinically significant sequence variants in these three genes, as well as GJB2 gene, and estimation of the frequency of sequence variants was performed. Of the 19,189 CMA tests were performed in our laboratory, 107 STRC microdeletions were found (0.56%), followed in frequency by OTOA deletions (39, 0.2%), and DFNB1 locus deletions (10, 0.05%). The estimated risk for a hearing loss in the examined individual carrying the microdeletion was estimated as 0.11-0.67% for STRC, 0.016-0.13% for OTOA, and 1.9-7.5% in the DFNB1 locus (including double heterozygocity with GJB2 clinically significant sequence variants). The risks were higher in specific populations. In conclusion, we believe that that general decision whether to report or to disregard such incidental findings cannot be part of a uniform policy, but rather based on a detailed evaluation of origin-specific variants for each gene, with a careful consideration and discussion whether to include the microdeletion in the final report for each patient.

Allele frequency analysis of variants reported to cause autosomal dominant inherited retinal diseases question the involvement of 19% of genes and 10% of reported pathogenic variants

BackgroundNext generation sequencing (NGS) generates a large amount of genetic data that can be used to better characterise disease-causing variants. Our aim was to examine allele frequencies of sequence variants...

Heterozygosity mapping for human dominant trait variants.

Homozygosity mapping is a well‐known technique to identify runs of homozygous variants that are likely to harbor genes responsible for autosomal recessive disease, but a comparable method for autosomal dominant traits has been lacking. We developed an approach to map dominant disease genes based on heterozygosity frequencies of sequence variants in the immediate vicinity of a dominant trait. We demonstrate through theoretical analysis that DNA variants surrounding an inherited dominant disease variant tend to have increased heterozygosity compared with variants elsewhere in the genome. We confirm existence of this phenomenon in sequence data with known dominant pathogenic variants obtained on family members and in unrelated population controls. A computer‐based approach to estimating empirical significance levels associated with our test statistics shows genome‐wide p‐values smaller than 0.05 for many but not all of the individuals carrying a pathogenic variant.

Accurate and Rapid Sequence Analysis of Adeno-Associated Virus Plasmids by Illumina Next-Generation Sequencing.

Sequence validation of plasmid DNA is a crucial quality control step that must occur prior to adeno-associated virus (AAV) vector packaging through plasmid transfection. AAV cis-plasmids present unique challenges to sequence analysis, as they contain inverted terminal repeats and are prone to sequence rearrangements. An accurate and rapid next-generation sequencing approach has been established to analyze full-length sequences of AAV cis-plasmids within 3.5 days. Here, a step-by-step protocol is described that can reliably detect and identify the location and frequency of sequence variants commonly observed in AAV cis-plasmids.

Identification of contemporary selection signatures using composite log likelihood and their associations with marbling score in Korean cattle.

Positive selection not only increases beneficial allele frequency but also causes augmentation of allele frequencies of sequence variants in close proximity. Signals for positive selection were detected by the statistical differences in subsequent allele frequencies. To identify selection signatures in Korean cattle, we applied a composite log-likelihood (CLL)-based method, which calculates a composite likelihood of the allelic frequencies observed across sliding windows of five adjacent loci and compares the value with the critical statistic estimated by 50,000 permutations. Data for a total of 11,799 nucleotide polymorphisms were used with 71 Korean cattle and 209 foreign beef cattle. As a result, 147 signals were identified for Korean cattle based on CLL estimates (P < 0.01). The signals might be candidate genetic factors for meat quality by which the Korean cattle have been selected. Further genetic association analysis with 41 intragenic variants in the selection signatures with the greatest CLL for each chromosome revealed that marbling score was associated with five variants. Intensive association studies with all the selection signatures identified in this study are required to exclude signals associated with other phenotypes or signals falsely detected and thus to identify genetic markers for meat quality.

Exome sequencing pilot study in children with carbamazepine‐induced serious skin reactions

Stevens-Johnson syndrome (SJS) and drug-induced hypersensitivity syndrome (HSS, also known as DRESS) are life-threatening drug hypersensitivity reactions. Previous studies on genetic predictors for SJS and HSS were focused on immune-related genes and on known, frequent polymorphisms in the genome. For the anticonvulsant carbamazepine (CBZ), genetic markers for CBZ-induced SJS and HSS have been identified in the HLA region (HLA-B*15:02, HLA-A*31:01). However, many patients carriyng HLA-B*15:02 or HLA-A*31:01 tolerate CBZ, resulting in a low positive predictive value of a predictive test based on these genetic markers alone. In this pilot study, we aimed to identify rare genetic variants potentially associated with CBZ-induced SJS and HSS using whole exome sequencing in seven children with CBZ-induced SJS and five children with CBZ-induced HSS. Whole exome sequencing was performed using Illumina TruSeq exome capture and sequencing technology. Paired-end sequence reads were aligned and variants within 150 bp of the 64 Mb target region were identified using Bowtie, BWA and GATK. DNA samples from 95 CBZ-tolerant Canadian children were pooled and sequenced to a median depth of 300-fold to estimate frequencies of sequence variants identified in the hypersensitivity cases. All study participants were recruited through the Canadian Pharmacogenomics Network for Drug Safety. Children with CBZ-induced SJS or HSS were of European, Asian and First Nations ancestry with three SJS cases of Asian origin carrying HLA-B*15:02 and two HSS patients of European and First Nations origin carrying HLA-A*31:01. No individual rare single nucleotide variant (SNV) was strongly overrepresented in the SJS or HSS cases either in combined or in hypersensitivity reaction-specific analyses. Gene-based analyses revealed several candidate genes with rare nonsynonymous SNVs observed in a majority of SJS or HSS cases, respectively, which require follow-up in additional patients. With a hypothesis-free, comprehensive genetic screening approach, we identified new candidate genes and variants potentially involved in the development of CBZ-induced SJS or HSS for further investigation. The identified genes and pathways may lead to novel hypotheses for the pathomechanisms of these serious dermatologic reactions, potentially opening up new possibilities for prevention and treatment strategies.

Altered frequency of sequence variants in growth-related genes in children with short stature

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

BRCA1 and BRCA2 Gene Mutations Screening In Sporadic Breast Cancer Patients In Kazakhstan.

BackgroundA large number of distinct mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known regarding the role of these inherited susceptibility genes in breast cancer risk among Kazakhstan women.AimTo evaluate the role of BRCA1/2 mutations in Kazakhstan women presenting with sporadic breast cancer.MethodsWe investigated the distribution and nature of polymorphisms in BRCA1 and BRCA2 entire coding regions in 156 Kazakhstan sporadic breast cancer cases and 112 age-matched controls using automatic direct sequencing.ResultsWe identified 22 distinct variants, including 16 missense mutations and 6 polymorphisms in BRCA1/2 genes. In BRCA1, 9 missense mutations and 3 synonymous polymorphisms were observed. In BRCA2, 7 missense mutations and 3 polymorphisms were detected. There was a higher prevalence of observed mutations in Caucasian breast cancer cases compared to Asian cases (p<0.05); higher frequencies of sequence variants were observed in Asian controls. No recurrent or founder mutations were observed in BRCA1/2 genes. There were no statistically significant differences in age at diagnosis, tumor histology, size of tumor, and lymph node involvement between women with breast cancer with or without the BRCA sequence alterations.ConclusionsConsidering the majority of breast cancer cases are sporadic, the present study will be helpful in the evaluation of the need for the genetic screening of BRCA1/2 mutations and reliable genetic counseling for Kazakhstan sporadic breast cancer patients. Evaluation of common polymorphisms and mutations and breast cancer risk in families with genetic predisposition to breast cancer is ongoing in another current investigation.

Chromogranin B P413L variant as risk factor and modifier of disease onset for amyotrophic lateral sclerosis

Recently, chromogranins were reported to interact specifically with mutant forms of superoxide dismutase that are linked to amyotrophic lateral sclerosis (ALS). This interaction led us to analyze the frequencies of sequence variants of the CHGB gene in ALS patients and matched controls from three different countries. Of particular interest was the finding of the P413L CHGB variant present in 10% of ALS patients (n = 705) as compared to 4.5% in controls (n = 751), conferring a 2.2-fold greater relative risk to develop the disease (P < 0.0001). This effect was mainly contributed by the samples of French origin that yielded a frequency of the P413L variation at 17% in ALS (n = 289) and 5% in controls (n = 448), conferring a 3.3-fold greater risk to develop ALS. Furthermore, the P413L CHGB variant is associated with an earlier age of onset by almost a decade in both sporadic ALS and familial ALS cases. Genetic variation influencing age of onset in ALS had not previously been reported. Expression of fusion CHGB-EGFP constructs in SHSY-5Y cells revealed that the P413L variation can cause defective sorting of CHGB into secretory granules. The finding that CHGB may act as a susceptibility gene and modifier of onset in ALS is consistent with the emerging view that dysfunction of the secretory pathway may contribute to increased vulnerability of motor neurons.

Sequence analysis of P21-activated kinase 3 (PAK3) in chronic schizophrenia with cognitive impairment

The P21-activated kinase PAK3 is critical for cognitive development and truncating mutations cause non-syndromic mental retardation (MR). Missense mutations are also associated with psychotic disorders, most commonly with schizophrenia involving premorbid MR, namely “pfropfschizophrenie”. We set out to measure the frequency of sequence variants in PAK3 in schizophrenia without premorbid MR. We conducted complete gene reseqeuncing of all coding exons and exon–intron boundaries in patients with schizophrenia with cognitive impairment but without premorbid MR. Deleterious variants in schizophrenia alone were rare (< 1/159 or 0.6%). Thereby, while PAK3 remains a strong biological candidate in psychosis, evidence from human genetics provides strongest support for a link to pfropfschizophrenie and not to schizophrenia without premorbid intellectual disability.

Novel mutations in epithelial sodium channel (ENaC) subunit genes and phenotypic expression of multisystem pseudohypoaldosteronism

Multisystem pseudohypoaldosteronism (PHA) is a rare autosomal recessive aldosterone unresponsiveness syndrome that results from mutations in the genes encoding epithelial sodium channel (ENaC) subunits alpha, beta and gamma. In this study we examined three PHA patients to identify mutations responsible for PHA with different clinical presentations. All three patients presented uniformly with symptoms of severe salt-loss during the first week of life and were hospitalized for up to a year. Beyond infancy, one of the patients showed mild renal salt loss and had no lower respiratory tract infections until 8 years of age, while the other patients continue with a severe course. We sequenced the complete coding regions and intron-exon junctions of the genes encoding alpha, beta and gamma subunits of ENaC for all patients. The results revealed that the mild case represents a novel compound heterozygote including a missense (Gly327Cys) mutation in the alphaENaC gene. Sequences of relatives over three generations confirmed that the missense mutation co-segregates with PHA. This mutation was not found in 60 control subjects. The other patients with severe PHA had two homozygous mutations, a novel deletion mutation in exon 8 of the alphaENaC gene and a splice site mutation in intron 12 of the betaENaC gene. Most of the PHA-causing mutations appear in the alphaENaC gene located on chromosome 12 rather than in the beta and gammaENaC genes located tandemly on chromosome 16. However, the frequency of sequence variants in patients and control subjects showed no difference between genes. Severe PHA cases are associated with mutations leading to absence of normal-length alpha, beta or gammaENaC, while a mild case has been found to be associated with a missense mutation in alphaENaC. The predominance of PHA-causing mutations in the alphaENaC gene may be related to the function of this subunit.

Effect of the codon 72 polymorphism (c.215G>C, p.Arg72Pro) in combination with somatic sequence variants in the TP53 gene on survival in patients with advanced ovarian carcinoma.

This study was conducted to evaluate the frequency and prognostic impact of TP53 alterations stratified for the TP53 codon 72 polymorphism (c.215G>C, p.Arg72Pro) in a cohort of 109 patients with advanced ovarian carcinomas. TP53 sequence variants were observed in 80 of the 109 (73.4%) tumors and were significantly associated with grade of differentiation (P=0.001). A tendency towards higher frequency of sequence variants in tumors with higher FIGO stages was seen (P=0.05). The type of TP53 sequence variant (transition A:T>G:C vs. G:C>A:T at CpG dinucleotides, and transversion G:C>T:A) had significant correlation with patients' age (P=0.04) with more A:T>G:C in patients over 60 years old. No significant associations were found between frequency of sequence variants and age at diagnosis, histological type, size of residual tumor after primary surgery, or long-term survival. Analyses of the codon 72 polymorphism in tumor DNA gave a higher frequency of homozygosity/hemizygosity than expected from the population frequency, particularly for the Pro allele. Tumors homozygous or hemizygous for the Pro allele had significantly higher frequency of TP53 sequence variants, particularly of the nonmissense type (P=0.002), and patients with these types of alterations had significantly shorter survival (P=0.04). TP53 protein accumulation, determined by immunohistochemistry (IHC), was found in 67.9% (74 out of 109) of the tumors, was significantly more common among serous than nonserous ovarian carcinomas (P=0.008), and had a significant effect on progression-free survival (P=0.03). p63 (TP73L; formerly TP63) and p73 (TP73) protein accumulation detected by IHC was seen in 67.9 and 0% of the tumors, respectively. A significantly higher frequency of p63-positive cases was seen among serous tumors (P=0.008) and tended to increase with increasing FIGO stages (P=0.05), but had no significant effect on survival. No association between p63 protein accumulation and TP53 protein accumulation was seen.

Involvement of ATM missense variants and mutations in a series of unselected breast cancer cases.

It has been proposed that women carrying heterozygous mutations of the ATM gene could be at increased risk of developing breast cancer. However, data in the literature are contrasting and no firm conclusion has been reached. Our aim was to verify whether ATM inactivation could play a role in breast tumor development. Following the classical tumor suppressor inactivation scheme, tumors showing loss of heterozygosity (LOH) at the ATM locus should present an increased proportion of mutated ATM forms. We screened a cohort of 173 nonselected primary breast tumors for LOH in a 4 cM region at 11q23 spanning the ATM gene. We analyzed 25 tumors presenting LOH within the ATM locus for mutations in the ATM coding sequence using an RT-PCR-SSCP approach. Five patients were found to bear a coding missense variant, out of which four corresponded to a frequent polymorphism in exon 39. One patient presented a previously unreported variant in exon 19 (2614C>T) resulting in a nonconservative change (Pro>Ser) at aa 872. This variant was not found in any of the other 172 patients nor in 63 healthy controls tested, indicating that it is a rare ATM variant. LOH involved the ATM wild-type allele in the tumor presenting variant 2614. However, because the ATM gene presents a relatively large number of rare coding polymorphism it is difficult, in the absence of familial data, to be conclusive on the significance of this variant. Searching for further variants in exons 19 and 39 in the whole set of 173 breast tumors, we found one tumor showing an acquired deletion of four bases in the ATM gene. Somatic mutations affecting the ATM gene thus seem rare in breast cancer. In our cohort of breast cancer patients, tumors presenting LOH at the ATM locus did not show an increased frequency of sequence variants. Furthermore, allelic imbalance profiles in a 4-cM region of chromosome arm 11q spanning the ATM locus revealed that hot spots of LOH were more likely to correspond to a region localized telomeric to the gene. Therefore, these data suggest that other target genes for genetic inactivation exist in the 11q23 region.

Mutation analysis reveals novel sequence variants in NTRK1 in sporadic human medullary thyroid carcinoma.

Tyrosine kinase NTRK1 is expressed in neural and nonneuronal tissues. Like RET, NTRK1 is often activated by rearrangements that involve one of at least five other genes in papillary thyroid carcinoma (PTC). Because of similarities in involvement of the two tyrosine kinases RET (rearranged during transfection) and NTRK1 in the pathogenesis of PTC, the obvious parallels between RET and NTRK1 and between PTC and medullary thyroid carcinoma (MTC), NTRK1 seemed to be an excellent candidate gene to play a role in the genesis of MTC. Single-strand conformational polymorphism analysis of 16 exons of NTRK1, from 31 sporadic MTC, revealed variants in five exons (exons 4 and 14-17). Sequence analysis demonstrated one sequence variant each in exons 4, 14, 16, and 17, and four different variants in exon 15. Differential restriction enzyme digestion specific for each variant confirmed the sequencing results. All variants were also present in the corresponding germline DNA. Interestingly, the sequence variants at codon 604 (c1810C>T) and codon 613 (c1838G>T) ofexon 15 always occurred together and might represent linkage disequilibrium. The frequencies of the sequence variants in germline DNA from patients with sporadic MTC did not differ significantly from those in a race-matched control group. Although we did not find any somatic mutations of NTRK1 in sporadic MTC, the single-strand conformational polymorphism conditions reported here, together with the knowledge of the frequency of various sequence variants, may help in future mutation analyses of DNA from other neural and nonneural tissues.

Nucleotide sequence of RNA of a Polish isolate of potato leafroll luteovirus.

The nucleotide sequence of the genomic RNA of a Polish isolate of a potato leafroll virus was determined. Some variations between the determined sequences were observed. A comparison of the frequency of sequence variants in particular regions of the genome is presented.

Discontinuous sequence change of human immunodeficiency virus (HIV) type 1 env sequences in plasma viral and lymphocyte-associated proviral populations in vivo: implications for models of HIV pathogenesis

Sequence change in different hypervariable regions of the external membrane glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1) was studied. Viral RNA associated with cell-free virus particles circulating in plasma and proviral DNA present in HIV-infected peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of two currently asymptomatic hemophiliac patients over a 5-year period. HIV sequences were amplified by polymerase chain reaction to allow analysis in the V3, V4, and V5 hypervariable regions of gp120. Rapid sequence change, consisting of regular replacements by a succession of distinct viral populations, was found in both plasma virus and PBMC provirus populations. Significant differences between the frequencies of sequence variants in DNA and RNA populations within the same sample were observed, indicating that at any one time point, the predominant plasma virus variants were antigenically distinct from viruses encoded by HIV DNA sequences in PBMCs. How these findings contribute to current models of HIV pathogenesis is discussed.