Non-synonymous Polymorphisms Research Articles

The rs2304256, a Non-Synonymous Polymorphism in Tyrosine Kinase 2 Gene is Associated with the Risk of Endometriosis

The objective of the study was to investigate whether the TYK2 gene influences the risk of developing endometriosis in South Indian women. The non-synonymous SNP, rs2304256, in exon8 of the TYK2 gene was tested for association in a case–control study of 150 affected women and 150 women with no evidence of disease. The genotype frequencies of the polymorphism were compared using polymerase chain reaction and restriction fragment length polymorphism. Immunohistochemistry was used to analyze the distribution and expression of TYK2 in the endometrium of women with and without endometriosis. According to codominant, dominant, and recessive genotype models, statistically significant differences were observed in the genotype distribution and allele frequency (P=0.0432) between the cases and controls. The distribution and expression of TYK2 did not vary in the endometrium of cases and controls. In the present study, we could establish an association between the TYK2 rs2304256 non-synonymous polymorphism with the risk of endometriosis in South Indian women, indicating that this polymorphism may lead to significant disease susceptibility.

In Silico Analysis of nsSNPs of Human KRAS Gene and Protein Modeling Using Bioinformatic Tools.

The KRAS gene belongs to the RAS family and codes for 188 amino acid residues of KRAS protein, with a molecular mass of 21.6 kD. Non-synonymous single-nucleotide polymorphisms (nsSNPs) have been identified within the coding region in which some are associated with different diseases. However, structural changes are not well defined yet. In this study, we first categorized SNPs in the KRAS coding area and then used computational methods to determine their impact on the protein structure and stability. In addition, the three-dimensional model of KRAS was taken from the Protein Data Bank for structural modeling. Furthermore, genomic data were extracted from a variety of sources, including the 1000 Genome Project, dbSNPs, and ENSEMBLE, and assessed through in silico methods. Based on various tools used in this study, 10 out of 48 missense SNPs with rsIDs were found deleterious. The substitution of alanine for proline at position 146 pushed several residues toward the center of the protein. Arginine instead of leucine has a minor effect on protein structure and stability. In addition, the substitution of proline for leucine at the 34th position disrupted the structure and led to a bigger size than the wild-type protein, hence interrupting the protein interaction. Using the well-intended computational approach and applying several bioinformatic tools, we characterized and identified most damaging nsSNPs and further explored the structural dynamics and stability of KRAS protein.

Complete Genome Sequencing of an Embryonated Chicken Egg-Adapted Duck atadenovirus A.

Egg drop syndrome (EDS) is prevalent in industrial poultry globally. This disease is caused by Duck atadenovirus A or EDS virus (EDSV), a member of the genus Atadenovirus under the family Adenoviridae. The disease is attributed to significant economic losses in the poultry industry worldwide due to a drop in egg production, reduction in egg quality, and failure to reach maximum egg production. Oil-adjuvant inactivated vaccines, which are widely used in the poultry industry, provide good protection for immunized chickens against EDS. This study aimed to genetically and phylogenetically analyze the full-length genome of an embryonated chicken egg-adapted EDSV strain 127. After extraction of viral DNA from the allantoic fluid, overlapping fragments of the viral genome sequence were generated by polymerase chain reaction (PCR) using 25 pairs of primers. Purified PCR products were subjected to complete genome sequencing by the next-generation sequencing (NGS) approach. The nucleotide homology observed between genomes of the studied strain and that of the original strain 127 (NC_001813) of laying chickens was 99.9%. Its genome was 33,213 bp in length, with a G + C content of 43.01%. A comparison of the genome sequence of the egg-adapted virus with strain 127 revealed only three non-synonymous single-nucleotide polymorphisms (SNPs) between these viral genome sequences. Two mutations of S320G and I62K out of these SNPs were found within the coding regions of fiber and hypothetical proteins which may play a role in the adaptation of EDSV in the embryonated chicken eggs. The full genome sequencing of EDSV using NGS techniques provides insights into the discovery of genetic variants. Moreover, the genome sequence information of the EDSV provides valuable data for vaccine development in near future.

Major-effect quantitative trait locus qLKR4.1 encodes a phospholipase Dδ protein associated with low-K+ stress tolerance by promoting root length

qLKR4.1, controlling low K+ resistance in tomato, was fine-mapped to an interval of 67.5kb on chromosome A04, and one gene encoding phospholipase Dδ was identified as a candidate gene. In plants, changes in root length are an important morphological response to low K+ (LK) stress; however, the underlying genetics in tomato remain unclear. Here, we combined bulked segregant analysis-based whole-genome sequencing, single-nucleotide polymorphism haplotyping, and fine genetic mapping to identify a candidate gene as a major-effect quantitative trait loci (QTL), i.e., qLKR4.1, which was associated with LK tolerance due to increased root elongation in the tomato line JZ34. Through multiple analyses, we found that Solyc04g082000 is the most likely candidate for qLKR4.1, which encodes phospholipase Dδ (PLDδ). Increased root elongation under LK in JZ34 may be attributed to a non-synonymous single-nucleotide polymorphism in the Ca2+-binding domain region of this gene. Solyc04g082000 increases root length through its PLDδ activity. Silencing of Solyc04g082000Arg in JZ34 led to a significant decrease in root length compared with silencing of Solyc04g082000His allele in JZ18 under LK conditions. Mutation of a Solyc04g082000 homologue in Arabidopsis, pldδ, resulted in decreased primary root lengths under LK conditions, compared to the wild type. Transgenic tomato expressing the qLKR4.1Arg allele from JZ34 exhibited a significant increase in root length compared with the wild type expressing the allele from JZ18 under LK conditions. Taken together, our results confirm that the PLDδ gene Solyc04g082000 exerts important functions in increasing tomato root length and LK tolerance.

Phylogenetic landscape of Monkeypox Virus (MPV) during the early outbreak in New York City, 2022

ABSTRACT Monkeypox (MPOX) is a zoonotic disease endemic to regions of Central/Western Africa. The geographic endemicity of MPV has expanded, broadening the human-monkeypox virus interface and its potential for spillover. Since May 2022, a large multi-country MPV outbreak with no proven links to endemic countries has originated in Europe and has rapidly expanded around the globe, setting off genomic surveillance efforts. Here, we conducted a genomic analysis of 23 MPV-infected patients from New York City during the early outbreak, assessing the phylogenetic relationship of these strains against publicly available MPV genomes. Additionally, we compared the genomic sequences of clinical isolates versus culture-passaged samples from a subset of samples. Phylogenetic analysis revealed that MPV genomes included in this study cluster within the B.1 lineage (Clade IIb), with some of the samples displaying further differentiation into five different sub-lineages of B.1. Mutational analysis revealed 55 non-synonymous polymorphisms throughout the genome, with some of these mutations located in critical regions required for viral multiplication, structural and assembly functions, as well as the target region for antiviral treatment. In addition, we identified a large majority of polymorphisms associated with GA > AA and TC > TT nucleotide replacements, suggesting the action of human APOBEC3 enzyme. A comparison between clinical isolates and cell culture-passaged samples failed to reveal any difference. Our results provide a first glance at the mutational landscape of early MPV-2022 (B.1) circulating strains in NYC.

Screening of candidate genes related to differences in growth and development between Chinese indigenous and Western pig breeds.

Neijiang (NJ) and Yacha (YC) are two indigenous pig breeds in the Sichuan basin of China, displaying higher resistance to diseases, lower lean ratio, and slower growth rate than the commercial Western pig breed Yorkshire (YS). The molecular mechanisms underlying the differences in growth and development between these pig breeds are still unknown. In the present study, five pigs from NJ, YC, and YS breeds were subjected to the whole genome resequencing, and then the differential single-nucleotide polymorphisms (SNPs) were screened using a 10-kb window sliding in 1-kb step using the Fst method. Finally, 48,924, 48,543, and 46,228 nonsynonymous single-nucleotide polymorphism loci (nsSNPs) were identified between NJ and YS, NJ and YC, and YC and YS, which highly or moderately affected 2,490, 800, and 444 genes, respectively. Moreover, three nsSNPs were detected in the genes of acetyl-CoA acetyltransferase 1 (ACAT1) insulin-like growth factor 2 receptor (IGF2R), insulin-like growth factor 2 and mRNA-binding protein 3 (IGF2BP3), which potentially affected the transformation of acetyl-CoA to acetoacetyl-CoA and the normal functions of the insulin signaling pathways. Moreover, serous determinations revealed significantly lower acetyl-CoA content in YC than in YS, supporting that ACAT1 might be a reason explaining the differences in growth and development between YC and YS breeds. Contents of phosphatidylcholine (PC) and phosphatidic acid (PA) significantly differed between the pig breeds, suggesting that glycerophospholipid metabolism might be another reason for the differences between Chinese and Western pig breeds. Overall, these results might contribute basic information to understand the genetic differences determining the phenotypical traits in pigs.

Genomic Basis of Adaptation to a Novel Precipitation Regime.

Energy production and metabolism are intimately linked to ecological and environmental constraints across the tree of life. In plants, which depend on sunlight to produce energy, the link between primary metabolism and the environment is especially strong. By governing CO2 uptake for photosynthesis and transpiration, leaf pores, or stomata, couple energy metabolism to the environment and determine productivity and water-use efficiency (WUE). Although evolution is known to tune physiological traits to the local environment, we lack knowledge of the specific links between molecular and evolutionary mechanisms that shape this process in nature. Here, we investigate the evolution of stomatal conductance and WUE in an Arabidopsis population that colonized an island with a montane cloud scrubland ecosystem characterized by seasonal drought and fog-based precipitation. We find that stomatal conductance increases and WUE decreases in the colonizing population relative to its closest outgroup population from temperate North Africa. Genome-wide association mapping reveals a polygenic basis of trait variation, with a substantial contribution from a nonsynonymous single-nucleotide polymorphism in MAP KINASE 12 (MPK12 G53R), which explains 35% of the phenotypic variance in WUE in the island population. We reconstruct the spatially explicit evolutionary history of MPK12 53R on the island and find that this allele increased in frequency in the population due to positive selection as Arabidopsis expanded into the harsher regions of the island. Overall, these findings show how adaptation shaped quantitative eco-physiological traits in a new precipitation regime defined by low rainfall and high humidity.

Association of cytochrome P450 2D15 (CYP2D15) nonsynonymous polymorphisms and exon 3 deleted RNA splice variant with CYP2D15 protein content and enzyme function in dog liver microsomes.

CYP2D15 is a major drug metabolizing P450 in canine liver. Like the human orthologue (CYP2D6), this enzyme is highly polymorphic with at least five common nonsynonymous variants reported that result in amino acid changes, including p.Ile109Val, p.Leu115Phe, p.Gly186Ser, p.Ile250Phe and p.Ile307Val. Furthermore, a mRNA splice variant of CYP2D15 has been found in canine liver that lacks the exon 3 gene region resulting in an inactive enzyme. The objective of this study was to evaluate whether any of these amino acid variants or the exon 3 deletion mRNA variant (exon3-delta) was associated with differences in CYP2D15-selective activities or protein content in a bank of canine livers. Livers were obtained from 25 Beagles and 34 dogs of various other breeds. CYP2D15-selective activities measured included dextromethorphan o-demethylation and tramadol o-demethylation. Reverse transcription PCR showed that 76% of livers (44/58) expressed both exon3-delta and normally spliced CYP2D15 RNA, while the remaining 24% (14/58) expressed only normally spliced RNA. The presence of exon3-delta was not correlated with CYP2D15 activities or protein content. Compared with wild-type livers, Beagle dog livers heterozygous for the p.Ile109Val and p.Gly186Ser variants showed from 40 to 50% reductions in median enzyme activities, while heterozygous p.Gly186Ser livers were associated with a 41% reduction in median CYP2D15 protein content (p< .05; Dunn's test). In the entire liver bank, livers homozygous for p.Ile109Val were also associated with a 40% reduction in median dextromethorphan O-demethylation activities versus wild-type livers (p< .05). These results identify several nonsynonymous CYP2D15 gene variants associated with variable CYP2D15 metabolism in canine liver.

Evaluation of Resistance and Susceptibility to Prion Diseases in Pelibuey Sheep from Mexico

Abstract Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases with a chronic and fatal course, which are caused by a misfolded form of the cellular prion protein that is encoded by the host. The purpose of this work was to evaluate the resistance and genetic susceptibility to prion diseases in Pelibuey sheep from Mexico. The sequences of 99 Pelibuey sheep from the central and southwestern regions of Mexico were analyzed to determine the polymorphisms related to resistance and genetic susceptibility to scrapie, the Hardy-Weinberg equilibrium test and the D-Tajima test was used to identify the effect of evolutionary forces on the PRNP gene. Twelve non-synonymous polymorphisms Q101R, M112T, A116P, G127A, A136V, M137T, L141F, H143R, R154H, Q171R, Q171H, N176K were detected, in addition two synonymous substitutions 231R (agg/cgg) and 237L (ctc/ctg) were found. As a result of the sequence analysis, the ARR allele was not under the Hardy-Weinberg equilibrium, indicating that there is an evolutionary force at work, and the D-Tajima suggest the existence of purifying genetic selection. In conclusion, Pelibuey sheep exhibit genotypes for the PRNP gene that make them resistant to developing classic scrapie; at least 98% of the population is a carrier of an allele related to scrapie resistance; while for atypical scrapie there is a very high probability that an outbreak occurred in the herds, since the ARQ allele in combination with the L substitution at codon 141 confers susceptibility to carrier sheep.

Genome-Wide Association Screening Determines Peripheral Players in Male Fertility Maintenance

Deciphering the functional relationships of genes resulting from genome-wide screens for polymorphisms that are associated with phenotypic variations can be challenging. However, given the common association with certain phenotypes, a functional link should exist. We have tested this prediction in newly sequenced exomes of altogether 100 men representing different states of fertility. Fertile subjects presented with normal semen parameters and had naturally fathered offspring. In contrast, infertile probands were involuntarily childless and had reduced sperm quantity and quality. Genome-wide association study (GWAS) linked twelve non-synonymous single-nucleotide polymorphisms (SNPs) to fertility variation between both cohorts. The SNPs localized to nine genes for which previous evidence is in line with a role in male fertility maintenance: ANAPC1, CES1, FAM131C, HLA-DRB1, KMT2C, NOMO1, SAA1, SRGAP2, and SUSD2. Most of the SNPs residing in these genes imply amino acid exchanges that should only moderately affect protein functionality. In addition, proteins encoded by genes from present GWAS occupied peripheral positions in a protein-protein interaction network, the backbone of which consisted of genes listed in the Online Mendelian Inheritance in Man (OMIM) database for their implication in male infertility. Suggestive of an indirect impact on male fertility, the genes focused were indeed linked to each other, albeit mediated by other interactants. Thus, the chances of identifying a central player in male infertility by GWAS could be limited in general. Furthermore, the SNPs determined and the genes containing these might prove to have potential as biomarkers in the diagnosis of male fertility.

Race, ethnicity, F8 variants, and inhibitor risk: analysis of the “My Life Our Future” hemophilia A database

BackgroundSeveral studies have suggested Black and Hispanic hemophilia A (HA) patients in the United States suffer higher incidences of neutralizing anti-FVIII antibodies (inhibitors) than their White counterparts. The possible influence of nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in the F8 gene sequence has been proposed as a possible race-associated contributing factor. Some earlier studies indicated that intron-22 inversion mutations carry a lower inhibitor risk than other mutations resulting in large F8 gene disruptions. ObjectivesThe objectives of the study were to test the following hypotheses: (1) The risk of developing an inhibitor differs among racial/ethnic groups in the United States, (2) specific non-HA-causing ns-SNPs in the F8 gene are correlated with inhibitor risk, and (3) inhibitor risk associated with intron-22 inversions mutations is similar to that associated with other large structural changes in the F8 gene. MethodsAdjusted logistic regression analysis of the “My Life Our Future” database containing demographic, clinical, and F8 sequence data from >6000 mild, moderate, and severe HA participants. ResultsBlack and Hispanic severe HA subjects had a higher inhibitor risk than non-Hispanic Whites (adjusted odds ratio = 1.65, 95% CI: 1.22-2.21 and adjusted odds ratio = 1.88, 95% CI: 1.43-2.48), confirming this racial/ethnic/medical disparity; however, F8 ns-SNPs were not associated with inhibitor development. There was no difference in inhibitor risk among severe HA subjects with an intron-22 inversion vs other large structural changes in the F8 gene. ConclusionsNonpathogenic ns-SNPs in the F8 gene are not correlated with inhibitor risk. Inhibitor risk associated with intron-22 inversion mutations is similar to that of other large structural changes in F8 that preclude intact FVIII expression.

A next generation targeted amplicon sequencing method to screen for insecticide resistance mutations in Aedes aegypti populations reveals a rdl mutation in mosquitoes from Cabo Verde.

Aedes mosquito vectors transmit many viruses of global health concern, including dengue, chikungunya and Zika. These vector-borne viral diseases have a limited number of treatment options, and vaccines vary in their effectiveness. Consequently, integrated vector management is a primary strategy for disease control. However, the increasing emergence and spread of insecticide resistance is threatening the efficacy of vector control methods. Identifying mutations associated with resistance in vector populations is important to monitor the occurrence and evolution of insecticide resistance and inform control strategies. Rapid and cost-effective genome sequencing approaches are urgently needed. Here we present an adaptable targeted amplicon approach for cost-effective implementation within next generation sequencing platforms. This approach can identify single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) in genes involved in insecticide resistance in Aedes aegypti mosquitoes. We designed and tested eleven amplicons, which included segments of the ace-1 (carbamate target), the Voltage-Gated Sodium Channel (vgsc; pyrethroids, DDT and organochlorines), and rdl (dieldrin) genes; thereby covering established knockdown resistance (kdr) mutations (e.g., S989P, I1011M/V, V1016G/I and F1534C), with the potential to identify novel ones. The amplicon assays were designed with internal barcodes, to facilitate multiplexing of large numbers of mosquitoes at low cost, and were sequenced using an Illumina platform. Our approach was evaluated on 152 Ae. aegypti mosquitoes collected in Cabo Verde, an archipelago with a history of arbovirus outbreaks. The amplicon sequence data revealed 146 SNPs, including four non-synonymous polymorphisms in the vgsc gene, one in ace-1 and the 296S rdl mutation previously associated with resistance to organochlorines. The 296S rdl mutation was identified in 98% of mosquitoes screened, consistent with the past use of an organochlorine compound (e.g., DDT). Overall, our work shows that targeted amplicon sequencing is a rapid, robust, and cost-effective tool that can be used to perform high throughput monitoring of insecticide resistance.

Polygamy and purifying selection in birds

Good genes theories of sexual selection predict that polygamy will be associated with more efficient removal of deleterious alleles (purifying selection), due to the alignment of sexual selection with natural selection. On the other hand, runaway selection theories expect no such alignment of natural and sexual selection, and may instead predict less efficient purifying selection in polygamous species due to higher reproductive variance. In an analysis of polymorphism data extracted from 150-bird genome assemblies, we show that polygamous species carry significantly fewer nonsynonymous polymorphisms, relative to synonymous polymorphisms, than monogamous bird species (p = .0005). We also show that this effect is independent of effective population size, consistent with the alignment of natural selection with sexual selection and "good genes" theories of sexual selection. Further analyses found no impact of polygamy on genetic diversity, while polygamy in females (polyandry) had a marginally significant impact (p = .045). We also recapitulate previous findings that smaller body mass and greater geographic range size are associated with more efficient purifying selection, more intense GC-biased gene conversion, and greater genetic diversity.

Molecular surveillance of artemisinin resistance-related Pfk13 and pfcrt polymorphisms in imported Plasmodium falciparum isolates reported in eastern China from 2015 to 2019

BackgroundArtemisinin-based combination therapy (ACT) has been recommended as the first-line treatment by the World Health Organization to treat uncomplicated Plasmodium falciparum malaria. However, the emergence and spread of P. falciparum resistant to artemisinins and their partner drugs is a significant risk for the global effort to reduce disease burden facing the world. Currently, dihydroartemisinin-piperaquine (DHA-PPQ) is the most common drug used to treat P. falciparum, but little evidence about the resistance status targeting DHA (ACT drug) and its partner drug (PPQ) has been reported in Shandong Province, China.MethodsA retrospective study was conducted to explore the prevalence and spatial distribution of Pfk13 and Pfcrt polymorphisms (sites of 72–76, and 93–356) among imported P. falciparum isolates between years 2015–2019 in Shandong Province in eastern China. Individual epidemiological information was collected from a web-based reporting system were reviewed and analysed.ResultsA total of 425 P. falciparum blood samples in 2015–2019 were included and 7.3% (31/425) carried Pfk13 mutations. Out of the isolates that carried Pfk13 mutations, 54.8% (17/31) were nonsynonymous polymorphisms. The mutant alleles A578S, Q613H, C469C, and S549S in Pfk13 were the more frequently detected allele, the mutation rate was the same as 9.7% (3/31). Another allele Pfk13 C580Y, closely associated with artemisinin (ART) resistance, was found as 3.2% (2/31), which was found in Cambodia. A total of 14 mutant isolates were identified in Western Africa countries (45.2%, 14/31). For the Pfcrt gene, the mutation rate was 18.1% (77/425). T76T356 and T76 were more frequent in all 13 different haplotypes with 26.0% (20/77) and 23.4% (18/77). The CVIET and CVIKT mutant at loci 72–76 have exhibited a prevalence of 19.5% (15/77) and 3.9% (3/77), respectively. The CVIET was mainly observed in samples from Congo (26.7%, 4/15) and Mozambique (26.7%, 4/15). No mutations were found at loci 97, 101 and 145. For polymorphisms at locus 356, a total of 24 isolates were identified and mainly from Congo (29.2%, 7/24).ConclusionThese findings indicate a low prevalence of Pfk13 in the African isolates. However, the emergence and increase in the new alleles Pfcrt I356T, reveals a potential risk of drug pressure in PPQ among migrant workers returned from Africa. Therefore, continuous molecular surveillance of Pfcrt mutations and in vitro susceptibility tests related to PPQ are necessary.

Natural variation in CRABS CLAW contributes to fruit length divergence in cucumber.

Fruit length is a key domestication trait that affects crop yield and appearance. Cucumber (Cucumis sativus) fruits vary from 5 to 60 cm in length. Despite the identification of several regulators and multiple quantitative trait loci (QTLs) underlying fruit length, the natural variation, and molecular mechanisms underlying differences in fruit length are poorly understood. Through map-based cloning, we identified a nonsynonymous polymorphism (G to A) in CRABS CLAW (CsCRC) as underlying the major-effect fruit size/shape QTL FS5.2 in cucumber. The short-fruit allele CsCRCA is a rare allele that has only been found in round-fruited semi-wild Xishuangbanna cucumbers. A near-isogenic line (NIL) homozygous for CsCRCA exhibited a 34∼39% reduction in fruit length. Introducing CsCRCG into this NIL rescued the short-fruit phenotype, and knockdown of CsCRCG resulted in shorter fruit and smaller cells. In natural cucumber populations, CsCRCG expression was positively correlated with fruit length. Further, CsCRCG, but not CsCRCA, targets the downstream auxin-responsive protein gene CsARP1 to regulate its expression. Knockout of CsARP1 produced shorter fruit with smaller cells. Hence, our work suggests that CsCRCG positively regulates fruit elongation through transcriptional activation of CsARP1 and thus enhances cell expansion. Using different CsCRC alleles provides a strategy to manipulate fruit length in cucumber breeding.

Origin, distribution, and function of three frequent coding polymorphisms in the gene for the human P2X7 ion channel.

P2X7, an ion channel gated by extracellular ATP, is widely expressed on the plasma membrane of immune cells and plays important roles in inflammation and apoptosis. Several single nucleotide polymorphisms have been identified in the human P2RX7 gene. In contrast to other members of the P2X family, non-synonymous polymorphisms in P2X7 are common. Three of these occur at overall frequencies of more than 25% and affect residues in the extracellular "head"-domain of P2X7 (155 Y/H), its "lower body" (270R/H), and its "tail" in the second transmembrane domain (348T/A). Comparison of the P2X7 orthologues of human and other great apes indicates that the ancestral allele is Y-R-T (at 155-270-348). Interestingly, each single amino acid variant displays lower ATP-sensitivity than the ancestral allele. The originally published reference sequence of human P2X7, often referred to as "wildtype," differs from the ancestral allele at all three positions, i.e. H-H-A. The 1,000 Genome Project determined the sequences of both alleles of 2,500 human individuals, including roughly 500 persons from each of the five major continental regions. This rich resource shows that the ancestral alleles Y155, R270, and T348 occur in all analyzed human populations, albeit at strikingly different frequencies in various subpopulations (e.g., 25%-59% for Y155, 59%-77% for R270, and 13%-47% for T348). BLAST analyses of ancient human genome sequences uncovered several homozygous carriers of variant P2X7 alleles, possibly reflecting a high degree of inbreeding, e.g., H-R-T for a 50.000year old Neanderthal, H-R-A for a 24.000year old Siberian, and Y-R-A for a 7,000 year old mesolithic European. In contrast, most present-day individuals co-express two copies of P2X7 that differ in one or more amino acids at positions 155, 270, and 348. Our results improve the understanding of how P2X7 structure affects its function and suggest the importance of considering P2X7 variants of participants when designing clinical trials targeting P2X7.

Protective and pathogenic role of collagen subtypes genes COL4A3 and COL4A4 polymorphisms in the onset of keratoconus in South-Asian Pakistani cohort

Collagen sub-types have an important role in corneal structure and are reported to be an important genetic predictor for keratoconus (KC) development, therefore we assessed the association of collagen subtypes by screening non-synonymous polymorphisms of COL4A3 and COL4A4 in South-Asian (Pakistani) patients. MethodsA total of 257 KC sporadic cases, gender and ethnicity matched 253 control individuals were screened for three non-synonymous single nucleotide polymorphisms (SNPs) rs55703767and rs10178458 in COL4A3 and rs2229814 and one synonymous SNP rs2228555 in COL4A4. The genotyping was done by Competitive Allele specific polymerase chain reaction (PCR) and the data were analyzed statistically. ResultsAmong the studied SNPs, the COL4A3 rs55703767 GT genotype (dominant model (DM): odds ratio (OR) = 0.243, (95 %CI) = 0.16–0.36, p=>0.0001), and allele-G (OR = 0.35, 95 %CI = 0.26–0.48, p < 0.000)), showed protective association against KC development. While COL4A3 rs10178458 CT genotype (DM: OR = 2.11(95 %CI = 1.16–3.85), COL4A4 rs2229814 TT genotype (RM: OR = 147.778(95 %CI = 20.401–1070.439), (p > 0.05) and allele-T (OR = 2.351(95 %CI = 1.826–3.028), (p > 0.05); COL4A4 rs2228555 AG genotype (DM: OR = 2.370(95 %CI = 1.594–3.524) (<0.0001) and GG genotype (RM: OR = 2.347(95 %CI = 1.587–3.472), (p < 0.0001); and allele-G (OR = 2.024(95 %CI = 1.577–2.597), (p > 0.0001) were observed to be disease associated. ConclusionCOL4A3 rs10178458 and COL4A4 SNPs rs2229814 and rs2228555 were found to be pathogenic for KC, whereas COL4A3 rs55703767 was found to play a protective role against KC development in South-Asian (Pakistani) Cohort.

Systematic Identification of Autosomal and Y-Encoded Minor Histocompatibility Antigens Reveals Predictors of Chronic Gvhd and Candidate GVL Targets

At the core of the therapeutic effect of HLA-matched allogeneic hematopoietic cell transplantation (allo-HCT) is T cell alloreactivity against minor histocompatibility antigens (mHAgs), polymorphic peptides resulting from donor-recipient disparity at sites of single nucleotide gene polymorphisms (SNPs). Despite their crucial role in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD), only few mHAgs have been characterized to date. To systematically identify autosomal and Y-chromosome encoded HLA-I restricted mHAgs, we devised a computational pipeline based on: (i) comparison of whole exomes (WES) from paired donor-recipient DNA to define recipient-restricted exonic non-synonymous polymorphisms; and (ii) generation of expression filters by incorporating published single cell expression profiles from normal and malignant hematopoietic cells and GvHD target organs. For each GvHD tissue (skin, liver, GI, lung, oral mucosa and lacrimal gland), a large single cell database combining ≥1 independent datasets was created to cluster and annotate tissue-resident cell types. Cell-type specific genes were defined based on their total number of reads (CPM>5 in any cluster), thus creating an expression atlas capturing less frequent albeit biologically relevant cell types underestimated in bulk expression profiles. For GvL predictions, we built an AML-specific filter by defining AML-expressed genes from single cell and bulk data, and by subtracting genes with expression in non-hemopoietic tissues, per transcriptome and proteome GTEx databases. Candidate GvL/GvHD mHAgs prediction was performed by applying the HLAthena binding prediction tool on recipient-restricted SNPs expressed in GvL or GvHD tissues. To validate the pipeline predictive power, we used a panel of 95 HLA class I monoallelic lines generated from B721.221 cells, which we previously profiled by WES, RNASeq and immunopeptidomics. By assigning the B721.221 genome as surrogate 'HCT recipient’ and the reference hg19 genome as surrogate 'donor', we identified recipient-restricted SNPs, whose class I-restricted presentation we confirmed by mass spectrometry for a median of 5 (range 1-16) predicted epitopes/ allele. For 2 HLA-A0201 epitopes encompassing SNPs with an allelic frequency <0.001, we validated immunogenicity through stimulation of PBMCs from HLA-A0201 healthy donors (HDs) with corresponding synthetic peptides. For Y-encoded mHAgs, we predicted a median of 62 (range 24-107) epitopes/HLA-I allele from 9 genes expressed (>1 TPM) in ≥1 non germline tissue. Analysis of 12 male immunopeptidomes confirmed presentation of 3 known Y mHAgs and 26 novel predicted epitopes across 18 alleles. To estimate the fraction of immunogenic predicted binders, we tested all Y epitopes predicted across 3 common alleles (A0201, B1801, C0501). Of 215 Y peptides tested, 53 (~25%) elicited an antigen-specific T cell response in PBMCs from female HDs, as per dextramer-based detection of ≥0.2% CD8 cells. To test the prognostic potential of the mHAg load on cGvHD risk, we performed WES on 220 donor-recipient pairs treated with matched-related-donor allo-HCT for myeloid disease at DFCI between 2013-2020. Our logistic regression analysis used organ-specific NIH moderate/severe cGvHD as outcome; as covariates, we included tissue-specific mHAg load and standard HCT variables (disease type and prognostic risk, conditioning intensity, graft type, GvHD prophylaxis, female into male [F→M] transplant, relapse). The burden of lung-expressed mHAgs was correlated with lung GvHD occurrence (P=0.023). Liver GvHD was associated with F→M transplants (P=0.023). Subgroup analysis further showed liver GvHD to be correlated with the number of epitopes predicted from the RPS14Y gene (P=0.037). For identification of AML-specific mHAgs, a median of 31 (range 13-50) mHAgs/patient were predicted in our cohort from 269 genes preferentially expressed in AML, with a trend for a protective effect of their load against relapse (P=0.058). Of note, 39 SNPs generated putative mHAgs shared by >10 patients and covering 8 common HLA-I alleles in Caucasians, thereby representing potential targets for post-HCT immunotherapy. Overall, we report the generation of a robust analytic pipeline which enables the broadening of the repertoire of mHAgs available for donor selection and for predicting, monitoring or manipulating GvL and GvHD after allo-HCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Silico Evaluation of Nonsynonymous SNPs in Human ADAM33: The Most Common Form of Genetic Association to Asthma Susceptibility.

ADAM33 is a zinc-dependent metalloprotease of the ADAM family, which plays a vital biological role as an activator of Th2 cytokines and growth factors. Moreover, this protein is crucial for the normal development of the lung in the fetus two months after gestation leading to determining lung functions all over life. In this regard, mutations in ADAM33 have been linked with asthma risk factors. Consequently, identifying ADAM33 pathogenic nonsynonymous single-nucleotide polymorphisms (nsSNPs) can be very important in asthma treatment. In the present study, 1055 nsSNPs of human ADAM33 were analyzed using biocomputational software, 31 of which were found to be detrimental mutations. Precise structural and stability analysis revealed D219V, C669G, and C606S as the most destabilizing SNPs. Furthermore, MD simulations disclosed higher overall fluctuation and alteration in intramolecular interactions compared with the wild-type structure. Overall, the results suggest D219V, C669G, and C606S detrimental mutations as a starting point for further case-control studies on the ADAM33 protein as well as an essential source for future targeted mechanisms.

Low Genetic Polymorphism in the Immunogenic Sequences of Rhipicephalus microplus Clade C.

Rhipicephalus microplus tick highly affects the veterinary sector throughout the world. Different tick control methods have been adopted, and the identification of tick-derived highly immunogenic sequences for the development of an anti-tick vaccine has emerged as a successful alternate. This study aimed to characterize immunogenic sequences from R. microplus ticks prevalent in Pakistan. Ticks collected in the field were morphologically identified and subjected to DNA and RNA extraction. Ticks were molecularly identified based on the partial mitochondrial cytochrome C oxidase subunit (cox) sequence and screened for piroplasms (Theileria/Babesia spp.), Rickettsia spp., and Anaplasma spp. PCR-based pathogens-free R. microplus-derived cDNA was used for the amplification of full-length cysteine protease inhibitor (cystatin 2b), cathepsin L-like cysteine proteinase (cathepsin-L), glutathione S-transferase (GST), ferritin 1, 60S acidic ribosomal protein (P0), aquaporin 2, ATAQ, and R. microplus 05 antigen (Rm05Uy) coding sequences. The cox sequence revealed 100% identity with the nucleotide sequences of Pakistan's formerly reported R. microplus, and full-length immunogenic sequences revealed maximum identities to the most similar sequences reported from India, China, Cuba, USA, Brazil, Egypt, Mexico, Israel, and Uruguay. Low nonsynonymous polymorphisms were observed in ATAQ (1.5%), cathepsin-L (0.6%), and aquaporin 2 (0.4%) sequences compared to the homologous sequences from Mexico, India, and the USA, respectively. Based on the cox sequence, R. microplus was phylogenetically assembled in clade C, which includes R. microplus from Pakistan, Myanmar, Malaysia, Thailand, Bangladesh, and India. In the phylogenetic trees, the cystatin 2b, cathepsin-L, ferritin 1, and aquaporin 2 sequences were clustered with the most similar available sequences of R. microplus, P0 with R. microplus, R. sanguineus and R. haemaphysaloides, and GST, ATAQ, and Rm05Uy with R. microplus and R. annulatus. This is the first report on the molecular characterization of clade C R. microplus-derived immunogenic sequences.