Sequence variant analysis of RNA sequences in severe equine asthma

Background. Severe equine asthma is a chronic inflammatory disease of the lung in horses similar to low-Th2 late-onset asthma in humans. This study aimed to determine the utility of RNA-Seq to call gene sequence variants, and to identify sequence variants or potential relevance to the pathogenesis of asthma. Methods. RNA-Seq data were generated from endobronchial biopsies collected from 6 asthmatic and 7 non-asthmatic horses before and after challenge (26 samples total). Sequences were aligned to the equine genome with Spliced Transcripts Alignment to Reference software. Read preparation for sequence variant calling was performed with Picard tools and Genome Analysis Toolkit (GATK). Sequence variants were called and filtered using GATK and Ensembl Variant Effect Predictor (VEP) tools, and two RNA-Seq predicted sequence variants were investigated with both PCR and Sanger sequencing. Supplementary analysis of novel sequence variant selection with VEP was based on a score of <0.01 predicted with Sorting Intolerant From Tolerant (SIFT) software, missense nature, location within the protein coding sequence and presence in all asthmatic individuals. For select variants, effect on protein function was assessed with Polymorphism Phenotyping (PolyPhen) 2 and Screening for Non-Acceptable Polymorphism (SNAP) 2 software. Sequences were aligned and 3D protein structures predicted with Geneious software. Difference in allele frequency between the groups was assessed using a Pearson's Chi-squared test with Yates' continuity correction, and difference in genotype frequency was calculated using the Fisher's exact test for count data. Results. RNA-Seq variant calling and filtering correctly identified substitution variants in PACRG and RTTN. Sanger sequencing confirmed that the PACRG substitution was appropriately identified in all 26 samples while the RTTN substitution was identified correctly in 24 of 26 samples. These variants of uncertain significance had substitutions that were predicted to result in loss of function and to be non-neutral. Amino acid substitutions projected no change of hydrophobicity and isoelectric point in PACRG, and a change in both for RTTN. For PACRG, no difference in allele frequency between the two groups was detected but a higher proportion of asthmatic horses had the altered RTTN allele compared to non-asthmatic animals. Discussion. RNA-Seq was sensitive and specific for calling gene sequence variants in this disease model. Even moderate coverage (<10-20 cpm) yielded correct identification in 92% of samples, suggesting RNA-Seq may be suitable to detect sequence variants in low coverage samples. The impact of amino acid alterations in PACRG and RTTN proteins, and possible association of the sequence variants with asthma, is of uncertain significance, but their role in ciliary function may be of future interest.

Characterization of Germline Variants in Multiple Myeloma

AB0002 Analysis of nlrp3 and nod2 genes in adult onset still’s disease

This paper reports a molecular genetics analysis of the loci of two genes in the catecholamine degradation system (COMT rs4680 and MAOA-uVNTR) in samples from the Hadza (n = 353) and Datoga (n = 465) African ethnic populations. The results of pairwise comparisons of the COMT rs4680 locus allele and genotype frequency distribution revealed a statistically significant difference between men from the Hadza and Datoga populations (p < 0.001), while the differences observed between women from these two tribes remained below the significance threshold (p = 0.064; p = 0.076). In the Hadza population, we have also observed a statistically significant increase in the portion of G/A heterozygotes and a decrease in the portion of A/A homozygotes among women (0.576 and 0.127, respectively) compared with men (0.482 and 0.231, respectively). In the case of the MAOA-uVNTR locus, no statistical differences in the allele frequency distribution were found between the men from the two populations (p = 0.993). Women also showed no statistical differences in either allele (p = 0.229) or genotype (p = 0.057) frequencies. Moreover, in each tribe, we observed no differences in allele frequencies in the MAOA-uVNTR locus between the men and women of the same tribe. The obtained data may be further used to detect connections between gene variants and different forms of aggressive behavior in human beings.

CYP19A1 facilitates the bioconversion of estrogens from androgens. CYP19A1 intron single nucleotide polymorphisms (SNPs) may alter mRNA splicing, resulting in altered CYP19A1 activity, and potentially influencing disease susceptibility. Genetic studies of CYP19A1 SNPs have been well documented in populations of European ancestry; however, studies in populations of African ancestry are limited. In the present study, ten ‘candidate’ intronic SNPs in CYP19A1 from 125 African Americans (AA) and 277 European Americans (EA) were genotyped and their frequencies compared. Allele frequencies were also compared with HapMap and ASW 1000 Genomes populations. We observed significant differences in the minor allele frequencies between AA and EA in six of the ten SNPs including rs10459592 (p<0.0001), rs12908960 (p<0.0001), rs1902584 (p = 0.016), rs2470144 (p<0.0001), rs1961177 (p<0.0001), and rs6493497 (p = 0.003). While there were no significant differences in allele frequencies between EA and CEU in the HapMap population, a 1.2- to 19-fold difference in allele frequency for rs10459592 (p = 0.004), rs12908960 (p = 0.0006), rs1902584 (p<0.0001), rs2470144 (p = 0.0006), rs1961177 (p<0.0001), and rs6493497 (p = 0.0092) was observed between AA and the Yoruba (YRI) population. Linkage disequilibrium (LD) blocks and haplotype clusters that is unique to the EA population but not AA was also observed. In summary, we demonstrate that differences in the allele frequencies of CYP19A1 intron SNPs are not consistent between populations of African and European ancestry. Thus, investigations into whether CYP19A1 intron SNPs contribute to variations in cancer incidence, outcomes and pharmacological response seen in populations of different ancestry may prove beneficial.

Objective To investigate the relationship between the brain-derived neurotrophic factor（BDNF) gene Tag SNPs(rs11030101 and rs6265) and early-onset schizophrenia in the Chinese Han population. Methods The tag single nucleotide polymorphisms（tag SNPs) rs11030101 and rs6265 in the BDNF gene were genotyped in 360 early-onset schizophrenics and 399 controls subjects.Single nucleotide polymorphism association and haplotype analysis were performed. Results (1) There were significantly difference in allele and genotype frequencies between patients and control subjects for rs11030101 (χ2=5.13, df=1, P=0.024; χ2=6.12, df=2, P=0.047) and haplotypes A-Val and T-Val (χ2=7.96, P=0.005；χ2=8.53, P=0.004）,and the difference in allele frequencies was still statistically significant after Bonferroni correction（P=0.048）.(2) Stratification of the study by gender of the samples yielded significant evidence for an association with the polymorphisms rs11030101 in female population (genotype-wise: χ2=7.76, df=2, P=0.021), and the difference still existed after Bonferroni correction（P=0.042）. The haplotype analysis revealed that there was significant differences between patients and control subjects for haplotypes T-Val and T-Met（χ2=6.90, P=0.009；χ2=4.60, P=0.032）in the female population. Conclusions The BDNF gene may play important roles in the susceptibility to early-onset and female schizophrenia in the Chinese population. Key words: Schizophrenia; Brain-derived neurotrophic factor; Polymorphism, single nucleotide

Genetic variants causing underlying pharmacogenetic and disease phenotypes have been used as the basis for clinical decision-making. However, due to the lack of standards for next-generation sequencing (NGS) pipelines, reproducing genetic variants among institutions is still difficult. The aim of this study is to show how many important variants for clinical decisions can be individually detected using different pipelines. Genetic variants were derived from 105 breast cancer patient target DNA sequences via three different variant-calling pipelines. HaplotypeCaller, Mutect2 tumor-only mode in the Genome Analysis ToolKit (GATK), and VarScan were used in variant calling from the sequence read data processed by the same NGS preprocessing tools using Variant Effect Predictor. GATK HaplotypeCaller, VarScan, and MuTect2 found 25,130, 16,972, and 4232 variants, comprising 1491, 1400, and 321 annotated variants with ClinVar significance, respectively. The average number of ClinVar significant variants in the patients was 769.43, 16.50% of the variants were detected by only one variant caller. Despite variants with significant impact on clinical decision-making, the detected variants are different for each algorithm. To utilize genetic variants in the clinical field, a strict standard for NGS pipelines is essential.

The formation of sporadic abdominal aortic aneurysm (AAA) is explained by a remodelling of the extracellular matrix (ECM) and breakdown of structural components of the vascular wall. Matrix metalloproteinases are the principle matrix-degrading proteases and are known to play a major role in the remodelling of the extracellular matrix in arterial vessels. Their activity is controlled by tissue inhibitors of metalloproteinases (TIMPs). Decreased TIMP-1 and TIMP-2 expression in the extracellular matrix of the walls of AAAs has been demonstrated in several studies. This case-control study was designed to investigate the possible impact of genetic variants of the TIMP-2 gene in the aetiology of AAA and to reproduce a recently described significant difference in allele frequency of the SNP 303G>A in a German population. TIMP-2 single nucleotide polymorphisms (SNPs) were analysed in a study sample of 50 patients with AAA and 41 controls. Differences in genotype and allele frequencies of the identified polymorphisms were determined after sequencing the entire coding region and selected parts of the promoter using the automated laser fluorescence technique. Six polymorphisms were identified, one of which is described for the first time, located in the intron, (231+23C>T). An association of the SNP 303G>A with the phenotype was not confirmed in our study (p=0.648). However, the CT genotype of the SNP -479C>T was more frequent in patients with AAA than in the control group (p=0.054). In our analysis of the TIMP-2 gene, we identified one new SNP. A previously published association of the SNP 303G>A with the phenotype could not be validated in our population. However, we detected an association for the CT genotype of one polymorphism in the promoter region (g-479C>T) and AAA. This result has to be proved in a second study sample.

The uncertainty of copy number variants: pregnancy decisions and clinical follow-up

The aim of this study was to estimate the allele frequencies in polymorphic site of exon six of POU1F1 gene in three Iranian native and Holstein cattle. Genomic DNA was extracted from 3 Iranian native cattle breeds, including 97 Mazandarani, 87 Sarabi, 112 Golpaygani and also 110 Holstein cattle. A 451 bp fragment of intron 5 and exon 6 were amplified and digested with HinfI restriction enzyme. Frequencies of allele A were 0.37, 0.27, 0.34 and 0.21 for Mazandarani, Sarabi, Golpaygani and Holstein cattle, respectively. Significant differences in genotype frequencies were found between Mazandarani or Golpaygani and Holstein cattle. No significant differences in genotype frequencies were found between Sarabi and Holstein cattle. Transition A to G in nucleotide 1256 is responsible for HinfI(-) allele. No significant association was observed between POU1F1 polymorphism and milk production. Differences in allelic frequency between native Bos indicus breeds (Mazandarani, Golpaygani) and Holstein at the present study might be due to differences in origin breeds, low number of samples and/or as the effect of natural selection in native breeds.

SLC1A1, which encodes the neuronal and epithelial glutamate transporter, is a promising candidate gene for obsessive-compulsive disorder (OCD). In this study, we conducted capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) screen for all 12 identified exons, including all coding regions and approximately 50 bp of flanking introns of the human SLC1A1 in 378 OCD-affected individuals. Full sequencing was completed on samples that showed an aberrant SSCP tracing for identification of the underlying sequence variants. Our aim was to determine if there are differences in the frequencies of relatively common alleles, or rare functional alleles, in 378 OCD cases and 281 ethnically matched controls. We identified one nonsynonymous coding SNP (c.490A > G, T164A) and three synonymous coding SNP (c.81G > C, A27A; c.414A > G, T138T; c.1110T > C, T370T) in case samples. We found no statistical differences in genotype and allele frequencies of common cSNPs in SLC1A1 between the OCD cases and controls. The rare variant T164A was found only in one family. Further investigation of this variant is necessary to determine whether and how it is related to OCD. There was no other evidence of significant accumulation of deleterious coding mutations in SLC1A1 in the OCD cases.

BackgroundThis study is to explore the relationship between the ZBRK1/ZNF350 (Zinc finger and BRCA1-interacting protein with KRAB domain-1; also known as zinc-finger protein 350) gene polymorphism and early-onset breast cancer.MethodsThe ZBRK1/ZNF350 gene exon detection analysis was performed with the direct sequencing and Snapshot methods in 80 cases of breast cancer (aged ≤ 40 years old) and 240 healthy subjects (aged ≤ 40 years old).ResultsTotally 9 sequence variants were detected, including 5 missense mutations and 4 synonymous mutations, located at EXON3, EXON4 and EXON5, respectively. The rs4987241 and rs3764538 variants were published for the first time, while the remaining variants had been reported before. There were significant differences in the frequency distribution of family history between the breast cancer and control groups. Moreover, there were significant differences in the CT genotype frequency at the rs138898320 locus between the breast cancer and healthy control groups. Compared with the carriers of CC wild genotype at rs138898320, the risk of breast cancer was reduced by 88.3% in the CT mutant genotype carriers, with significant difference. In the stratification with no family history, compared with the carriers of CC wild genotype at rs138898320, significant differences were observed for the CT mutant genotype carriers. In the stratification with family history, there was no significant difference in the variation of rs138898320.ConclusionThe rs138898320 CT mutation genotype of ZBRK1/ZNF350 may reduce the risk of breast cancer, and the protecting effect would be increased in the stratification with no family history.Trial registration Not applicable.

Ensembl (https://www.ensembl.org) is unique in its flexible infrastructure for access to genomic data and annotation. It has been designed to efficiently deliver annotation at scale for all eukaryotic life, and it also provides deep comprehensive annotation for key species. Genomes representing a greater diversity of species are increasingly being sequenced. In response, we have focussed our recent efforts on expediting the annotation of new assemblies. Here, we report the release of the greatest annual number of newly annotated genomes in the history of Ensembl via our dedicated Ensembl Rapid Release platform (http://rapid.ensembl.org). We have also developed a new method to generate comparative analyses at scale for these assemblies and, for the first time, we have annotated non-vertebrate eukaryotes. Meanwhile, we continually improve, extend and update the annotation for our high-value reference vertebrate genomes and report the details here. We have a range of specific software tools for specific tasks, such as the Ensembl Variant Effect Predictor (VEP) and the newly developed interface for the Variant Recoder. All Ensembl data, software and tools are freely available for download and are accessible programmatically.

BackgroundTo date, mutations in the MAPT (i.e., tau) gene within Alzheimer’s disease (AD) patients are either benign or have an unclear effect on pathogenicity. Significant prior literature and evidence from other tauopathies support the hypothesis that mutations can alter tau’s structure and function. Approximately one‐third of neuropathological autopsies identify individuals with comparable AD neuropathology (i.e., extracellular amyloid‐beta (Aβ) plaques and intracellular tau‐containing neurofibrillary tangles (NFTs)) without similar clinical manifestations or cognitive deficits (herein referred to as non‐demented with Alzheimer’s neuropathology or NDAN). The comparable neuropathological burden with divergent clinical manifestations between AD patients and NDAN individuals led us to hypothesize that a mutation in the NDAN MAPT gene could explain, at least in part, this dichotomous presentation.MethodRNA was isolated from the frontal cortices of seven NDAN individuals (4M, 3F) using a commercial RNA kit. Primers to amplify the tau coding sequence were found in the literature and confirmed to amplify the tau coding sequence. With assistance from UTMB’s Next Generation Sequencing core, the RNA quality was first assessed, and subsequently, library generation and sequencing were performed.ResultTwo novel somatic mutations were identified in all seven individuals with frequencies consistent with mosaic mutant frequencies. The first, an aspartic acid to alanine mutation, occurred at a position where no prior mutations have been documented. The second occurs at the serine immediately preceding tau’s amyloid motif, VQIVYK; in this mutation, the phosphoresidue is replaced by a charged and bulky arginine. Analysis using the Ensembl Variant Effect Predictor (VEP) predicted both mutations to be, at a minimum, moderately damaging to tau’s function, which is logical considering the amino acid substitutions occurring. The severity of each mutation’s effect on the tau structure and function was isoform specific.ConclusionWe identified, to the best of our knowledge, two completely novel tau mutations uniquely associated with individuals that remain cognitively intact despite abundant AD neuropathology. These mutations may help contextualize the unique phenotype observed in NDAN individuals, such as cognitive resilience and spreading with minimal toxicity.Funding Support: R01422460 and R0142690 to GT. Attendance at this conference was supported by an AAIC 2024 Conference Fellowship.

Genetic Susceptibility in Sinusoidal Obstruction Syndrome/Veno-Occlusive Disease