Allele Frequencies Of Variants Research Articles

ABCG2 and SLCO1B1 gene polymorphisms in the Croatian population

Background Organic anion-transporting polypeptide 1B1 (OATP1B1) and the ATP-binding cassette subfamily G member 2, ABCG2, are important transporters involved in the transport of endogenous substrates and xenobiotics, including drugs. Genetic polymorphisms of these transporters have effect on transporter activity. There is significant interethnic variability in the frequency of allele variants. Aim To determined allele and genotype frequencies of ABCG2 and SLCO1B1 genes in Croatian populations of European descent. Subjects and methods A total of 905 subjects (482 women) were included. Genotyping for ABCG2 c.421C > A (rs2231142) and for SLCO1B1 c.521T > C (rs4149056), was performed by real-time polymerase chain reaction (PCR) using TaqMan® DME Genotyping Assays. Results For ABCG2 c.421C > A, the frequency of CC, CA and AA genotypes was 81.4%, 17.8% and 0.8% respectively. The frequency of variant ABCG2 421 A allele was 9.7%. For SLCO1B1 c.521T > C, the frequency of TT, TC and CC genotypes was 61.7%, 34.8% and 3.5% respectively. The frequency of variant SLCO1B1 521 C allele was 20.9%. Conclusion The frequency of the ABCG2 and SLCO1B1 allelic variants and genotypes in the Croatian population is in accordance with other European populations. Pharmacogenetic analysis can serve to individualise drug therapy and minimise the risk of developing adverse drug reactions.

Autonomous BCR Signaling and Genetic Aberrations in CLL-Phenotype Monoclonal B Lymphocytosis in Siblings of CLL Patients

Clonal expansion of CD5-expressing B cells, termed monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL) with an increased incidence in CLL siblings. In app. 1% of MBL carriers, MBLprogresses eventually to clinically overt CLL. The mechanisms driving benign MBL expansion and subsequent malignant progression are unknown. Autonomous signaling of the clonotypic BCR of CLL cells, i.e. signaling without stimulation by cognate antigen, is an indispensable oncogenic signal in CLL. In order to investigate the early stages in CLL ontogeny, we sought to clarify whether MBL cells express BCR with autonomous signaling capacity and to compare the prevalence of inherited risk loci and acquired genetic aberrations in MBL-CLL sibling pairs. Peripheral blood of 191 siblings of CLL patients was screened with informed consent by six-color flow cytometry. A discrete CD19+CD5+CD20lowCD79low CLL-phenotype population of 0.2 to 1863 cells / µL blood was detected in 34 siblings (17.8%) of 26 CLL patients; 32 siblings (94%) had <500 clonal CLL-phenotype cells / µL. Clonal BCR were identified from highly purified MBL cells of 17 CLL siblings by ARTISAN PCR. Five MBL clones could be assigned to CLL stereotyped subsets, including two MBL BCR belonging to CLL subset #2 with expression of the IGLV3-21 gene with a G110R mutation and one IgG-expressing MBL belonging to CLL subset #4. Eleven MBL BCR, including all 5 assigned to a CLL stereotyped subset, were transduced into murine TKO cells arrested at the pro-B-cell stage by genetic deficiency of rag2, lambda5, and slp65. Slp65 function is reconstituted by a slp65-ERT2 fusion gene that enables BCR signaling in the presence of 4-hydroxytamoxifen. Calcium mobilization was measured in Indo-1 AM-loaded, live-gated TKO cells by flow cytometry as the ratio of signal intensities (SIR) at 485 and 405 nm prior and after tamoxifen supplementation and after BCR crosslinking with isotype-specific antibody. Robust calcium mobilization was observed in all MBL BCR-transduced TKO cells in the absence of external antigen and without experimental cross-linking, regardless of assignment to a CLL subtype. Calibrated quantification of this autonomous BCR signal, however, revealed lesser intensity in MBL BCR compared to the BCR of their CLL sibling (p=0.015, Wilcoxon's matched-pair signed rank test). BCR signaling strength was correlated to the degree of MBL clonal expansion (p=0.034). According to genotyping by single nucleotide polymorphism array, whole exome sequencing (WES), and targeted panel sequencing, a polygenic CLL risk score (PRS) of 24 risk loci was higher in both CLL and MBL siblings than in reference population data, but we found no evidence for a higher PRS in CLL cases in a matched-pair comparison to their MBL siblings. With respect to acquired genetic changes, quantitative analysis of SNP array data revealed recurrent CLL-associated copy number variations (CNV) at clonal levels in all of 11 informative CLL cases. In contrast, individual CNV were present in only a minor fraction of the highly purified MBL cells in six of ten samples (p=0.003; Fisher's exact test). Likewise, variant allele frequencies (VAF) of non-shared variants as detected by WES was significantly lower in MBL samples than in CLL cases (p<0.0001). In particular, subclonal variants defined by a VAF of 0.1-0.33 were more prevalent in MBL compared to CLL siblings (p=0.014), whereas clonal variants (VAF 0.43-0.55) were equally abundant (p=0.65) in both conditions. In this comprehensive comparison of MBL and CLL in siblings, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, suggesting that CLL risk loci predispose to clonal expansion of CLL phenotype cells in both low-count MBL and CLL. Vice versa, we did not find evidence for a particular association of CLL risk alleles with malignant progression from MBL to clinical CLL. Autonomous BCR signaling operates in both MBL and CLL as predicted by BCR stereotype, albeit at lesser strength in MBL. Acquired CLL-genetic changes, both copy number and sequence variants, are frequently subclonal in MBL but not in CLL. These findings support a stepwise CLL pathogenetic model wherein autonomous BCR signaling causes a non-malignant expansion of clonal CD5+ B cells in genetically predisposed individuals, followed by malignant progression to CLL after gradual acquisition of pathogenic genetic variants.

Thrombopoietin Receptor Signaling Promotes the Competitive Advantage of TET2-Mutated Hematopoietic Stem and Progenitor Cells in Clonal Hematopoiesis

Clonal Hematopoiesis of Indeterminate Potential (CHIP) is an age-related condition that predisposes individuals to blood cancers. The time interval from acquisition of CHIP to overt neoplasia can span many years, providing an opportunity to delay or prevent malignant transformation. A key to achieving this goal is identifying differential dependencies between mutant and normal hematopoietic stem and progenitor cells (HSPCs). Targeting such dependencies may prevent against CHIP-related health risks. One of the most commonly mutated genes in CHIP is TET2, which encodes a methylcytosine dioxygenase. In mouse models, Tet2 knockout (KO) HSPCs have a competitive advantage over wild-type (WT) HSPCs. In this study, we aimed to identify differential dependencies between Tet2KO and Tet2WT HSPCs. We first generated isogenic murine cell lines by overexpressing HOXB4, a transcription factor involved in self-renewal, in HSPCs derived from a Tet2KO mouse and Tet2WT littermate. Using the isogenic cell lines termed HPCHOXB4, we conducted an epigenetic drug screen and found SGC0946, a selective inhibitor of H3K79 methyltransferase DOT1L, reduced the advantage of Tet2KO HPCHOXB4 cells in competition assays by preferentially reducing their proliferation and viability. SGC0946 also reduced the competitive advantage of unmodified Tet2KO HSPCs in competition assays, indicating that the dependency was not a consequence of HOXB4 overexpression. To decipher the mechanism behind this differential dependency, we performed RNA-seq and ChIP-seq for H3K79me2 on Tet2KO and Tet2WT HPCHOXB4 cells at baseline and after treatment with SGC0946. Expression of Mpl, which encodes the thrombopoietin receptor TPOR, was 20-fold higher in Tet2KO HPCHOXB4 than in Tet2WT cells at baseline and decreased after drug treatment. Mpl expression at the mRNA and protein level was also elevated in unmodified Tet2KO HSPCs compared with Tet2WT cells. Importantly, ChIP-seq analysis revealed that the level of H3K79me2 which associates with active transcription at the Mpl gene locus was higher in Tet2KO than in Tet2WT HPCHOXB4 cells at baseline, a difference that was erased with SGC0946 treatment. Based on the above findings, we hypothesized that Tet2KO HSPCs are more dependent on TPOR signaling than Tet2WT cells. Consistent with our hypothesis, a higher concentration of TPO in the culture medium increased the competitive advantage of Tet2KO HPCHOXB4 cells. Moreover, overexpression of Mpl in Tet2KO HPCHOXB4 cells rescued the effects of DOT1L inhibition, and shRNA-mediated suppression of Mpl expression was sufficient to decrease the competitive advantage of Tet2KO HPCHOXB4 cells and unmodified Tet2KO HSPCs over their corresponding Tet2WT counterparts. To explore the translational relevance of our findings, we tested if inhibition of JAK2, a critical mediator of TPOR intracellular signaling, would also reduce the competitive advantage of Tet2KO HSPCs. In line with our hypothesis, treatment with JAK2 inhibitors (fedratinib and AZ960), but not JAK1 inhibitors, suppressed the competitive advantage of Tet2KO HPCHOXB4 cells in vitro. Importantly, competitive repopulation studies showed that treatment with ruxolitinib, a clinically approved JAK1/2 inhibitor, also reduced the competitive advantage of untransformed Tet2KO HSPCs in vivo. To determine if our findings apply to humans, we generated two TET2KO cell pools from cord blood HSPCs using CRISPR/Cas9. The TET2KO cells expressed higher levels of TPOR and were more sensitive to treatment with SGC0946 and ruxolitinib compared with control cells. In addition, we expanded HSPCs from TET2-mutated mobilized peripheral blood samples in short-term cultures and monitored the size of the mutant clones by measuring the allele frequency of the sample-specific TET2 variants. In untreated cultures, the relative size of the mutant clones increased over time, an effect that was diminished by treatment with either SGC0946 or ruxolitinib. In summary, our findings demonstrate that TET2-mutated HSPCs are more dependent on TPOR signaling than TET2WT HSPCs. This differential dependency is mediated through increased H3K79 methylation at the MPL gene locus and enhanced expression of MPL. Furthermore, inhibition of the TPOR/JAK2 signaling axis is effective in suppressing the competitive advantage of TET2-mutated HSPCs, thus identifying a tractable therapeutic target for TET2-mutated CHIP.

Mitochondrial DNA copy number changes, heteroplasmy, and mutations in plasma-derived exosomes and brain tissue of glioblastoma patients

Glioblastoma is the most common malignant tumor of the central nervous system (CNS) in adults. Glioblastoma cells show increased glucose consumption associated with poor prognosis. Since mitochondria play a crucial role in energy metabolism, mutations and copy number changes of mitochondrial DNA may serve as biomarkers. As the brain is difficult to access, analysis of mitochondria directly from the brain tissue represents a challenge. Exosome analysis is an alternative (still poorly explored) approach to investigate molecular changes in CNS tumors.We analyzed brain tissue DNA and plasma-derived exosomal DNA (exoDNA) of 44 glioblastoma patients and 40 control individuals. Quantitative real-time PCR was performed to determine mtDNA copy numbers and the Kruskal-Wallis and Mann-Whitney U test were used for statistical analysis of data. Subsequently, sequencing libraries were prepared and sequenced on the MiSeq platform to identify mtDNA point mutations.Tissue mtDNA copy number was different among controls and patients in multiple comparisons. A similar tendency was detected in exosomes. Based on NGS analysis, several mtDNA point mutations showed slightly different frequencies between cases and controls, but the clinical relevance of these observations is difficult to assess and likely less than that of overall mtDNA copy number changes. Allele frequencies of variants were used to determine the level of heteroplasmy (found to be higher in exo-mtDNA of control individuals).Despite the suggested potential, the use of such biomarkers for the screening and/or diagnosis of glioblastomas is still limited, thus further studies are needed.

Identification of genetic characteristics in pediatric epilepsy with focal cortical dysplasia type 2 using deep whole-exome sequencing.

Focal cortical dysplasia type 2 (FCD2) is a malformation of cortical development that constitutes a common cause of pediatric focal epilepsy. Germline or somatic variants in the mammalian target of rapamycin (mTOR) signaling pathway genes are the pathogenesis of FCD2. In this study, whole-exome deep sequencing was performed on dysplastic cortex from focal epilepsy in children to explore genetic characteristics in FCD2. Resected core lesions of FCD2 were confirmed by pathology, and peripheral blood was collected from 11 patients. Deep whole-exome sequencing (>500X) was performed on derived genomic DNA, germline, or somatic variants in brain-specific genes were analyzed and identified. In 11 patients, a heterozygous likely pathogenic germline variant of DEPDC5 was identified in one case, while somatic variants were found in four brain samples. The frequencies of the somatic variant allele were 2.52%-5.12%. Somatic variants in AKT3, TSC2, and MTOR (mTOR signaling pathway genes) were found in three samples. Besides, one somatic variant was detected in MED12 which has not been reported to associate with FCD2. Our study expanded the variant spectrum in the mTOR-GATOR pathway, and also detected a somatic variant in MED12 which was potentially associated with FCD 2.

Molecular Study of STin2 (Intron 2) Variant of the SLC6A4 Gene in Children and Adolescents with Attention-deficit Hyperactivity Disorder

Background: Attention-deficit hyperactivity disorder (ADHD) is one of the most familiar childhood psychiatric disorders. Various molecular genetic reviews indicate that genes are crucial in susceptibility to ADHD. The serotonin transporter gene (SLC6A4) has polymorphisms that correlate with ADHD. The association between ADHD and SLC6A4 gene variants in the Iranian population has not been investigated yet. Objectives: This study analyzed the STin2 (intron 2) variant of the SLC6A4 gene in Iranian children and adolescents with ADHD. Methods: In this retrospective case-control study, 86 ADHD patients and 99 healthy volunteers aged five to 14 were enrolled as the case and control groups, respectively. The STin2 (intron2) fragment of the SLC6A4 gene was amplified using specific primers by conventional PCR, and three STin2 alleles of the SLC6A4 gene (STin2.9, STin2.10, and STin2.12) were examined using the acrylamide gel method. Results: We found no significant difference between the ADHD and control groups in STin2.9 (34.9% vs. 39.4%, P-value = 0.824), STin2.10 (29.1% vs. 23.2%, P-value = 1.354), and STin2.12 (36% vs. 36.4%, P-value = 0.986) variants. Conclusions: There was no association between the frequency of STin2 variant alleles of the SLC6A4 gene and ADHD, but in the study of risk estimation, allele 10 of this variant was a risk allele in ADHD patients.

Investigation of allele frequencies of polymorphic variants in genes that are related to polycystic ovary syndrome.

Polycystic ovary syndrome is a hormonal disorder that normally affects women of reproductive age in the range of 18-44 years. This study aimed to investigate the allelic frequencies of two polymorphisms, IRS rs18012781 and INSR rs1799817, which are suspected to be involved in polycystic ovary syndrome. The samples were obtained from the patients admitted to the Near East University Hospital, Department of Gynecology and Obstetrics. The samples were divided into two groups: control and polycystic ovary syndrome groups. Blood samples were collected from 55 women in the control group and 65 samples from the patient group. DNA from whole blood was obtained. The allelic frequencies of single-nucleotide polymorphisms were determined using real-time PCR. Results were presented as the heterozygous and homozygous state of the single-nucleotide polymorphisms. There were no significant differences in the allelic frequencies of the single-nucleotide polymorphisms between the patient and control groups. Further statistical analysis investigating the INSR Tm using the Mann-Whitney U test value revealed that there was no difference in the homozygous and heterozygous state of INSR rs1799817. The result of this study showed that there was no statistically significant difference between the allelic frequencies of IRS1 rs1801278 and INSR rs1799817 between the patient and control groups. These single-nucleotide polymorphisms do not seem to modify the risk of polycystic ovary syndrome, and they cannot be used as a marker in clinical circumstances to evaluate the possible occurrence of polycystic ovary syndrome.

Unique ESR1 and ESR2 estrogen receptor gene variants associated with altered risk of triple-negative breast cancer: A case-control study

BackgroundWe previously reported on the association between ESR1 and ESR2 gene variants and heightened risk of breast cancer (BC). Here we investigated the association of common ESR1 and ESR2 gene variants with triple negative BC (TNBC). MethodsThis retrospective case-control study involved 488 BC patients (130 TNBC, 358 non-TNBC patients). ESR1 (rs2234693, rs9340799, rs3020314, rs3798577) and ESR2 (rs928554, rs944459, rs4986938, rs1256049, rs1256030, rs1271572) genotyping was done by real-time PCR. ResultsWhile minor allele frequencies (MAF) of ESR1 variants were comparable between TNBC and non-TNBC subjects, significantly higher ESR2 rs1256049 MAF was seen in TNBC patients. Significantly higher frequency of ESR1 rs3798577 T/C and C/C genotypes were noted in TNBC cases, and significant differences were seen in ESR2 rs928554, rs1256049, and rs1271572 genotype distribution. Increased TNBC risk was associated with ESR1 rs3798577 T/C and C/C genotypes according to codominant and dominant models, while positive association of ESR2 rs928554 with TNBC was seen according to codominant and recessive models, and positive association of ESR2 rs1256049 with TNBC was seen according to codominant and dominant models. Positive interactions were noted between ESR2 rs1271572-ESR1 rs3020314, ESR2 rs1271572-ESR1 rs9340799, and ESR2 rs1271572-ESR1 rs2234693, ESR2 rs4986938-ESR1 rs2234693, and ESR2 rs928554-ESR1 rs9340799. Haplotype analysis confirmed the positive association of ESR1 CATT with TNBC, while ACGGCTC and ACGGTT ESR2 haplotypes were positively associated with TNBC. ConclusionResults of this study confirmed the association of unique ESR1 and ESR2 genetic variants with altered risk of TNBC. This suggests possible diagnostic and prognostic role of these variants with TNBC independent of their association with BC.

P.98 Calculating the genetic prevalence of congenital myasthenic syndromes based on data from genomic databases

Congenital myasthenic syndromes (CMS) are a heterogeneous group of rare inherited neuromuscular disorders characterized by fatigable weakness owing to compromised function of the neuromuscular junction. Next-generation sequencing has dramatically increased the number of genetic defects reported as causative of CMS, with over 30 genes implicated. Although all CMS subtypes share common features, their clinical and genetic heterogeneity, as well as referral and ascertainment biases in diagnostic practice, have made phenotype-based prevalence estimations difficult, with publications in the last decade providing estimates ranging from 1.8 cases per million total population to 22.2 cases per million children. This lack of accurate data hampers diagnosis, healthcare provision and therapy development. Most CMS subtypes are recessive, meaning that carriers of single variants that are pathogenic when present in homozygosity or compound heterozygosity can be found in the healthy general population. The new large-scale population databases of genomic data that have come online in recent years thus provide the opportunity to calculate the genetic prevalence or lifetime risk of autosomal recessive CMS based on the allele frequency of individual variants in an unaffected population. Based on reported pathogenic/likely pathogenic variants in databases including ClinVar, LOVD, HGMD and RD-Connect and predicted pathogenic variants from in silico predictions, we established a comprehensive curated list of known and likely pathogenic variants across 31 disease genes. We then queried the Genome Aggregation Database (gnomAD), the UK Biobank WES Cohort and the RD-Connect Genome-Phenome Analysis Platform to establish the allele frequency of disease-causing variants in genes associated with autosomal recessive congenital myasthenic syndromes. Based on this, we provide a population-genetic estimation of the genetic prevalence of CMS and compare it to existing published estimates. This data may be used to assign diagnostic probabilities, provide insights into numbers of patients amenable to clinical research and novel therapies, and aid resource allocation in therapy development and healthcare provision.

Pancreatitis-Associated PRSS1-PRSS2 Haplotype Alters T-Cell Receptor Beta (TRB) Repertoire More Strongly Than PRSS1 Expression

Pancreatitis-Associated PRSS1-PRSS2 Haplotype Alters T-Cell Receptor Beta (TRB) Repertoire More Strongly Than PRSS1 Expression

Insights into the architecture of human-induced polygenic selection in Duroc pigs

BackgroundAs one of the most utilized commercial composite boar lines, Duroc pigs have been introduced to China and undergone strongly human-induced selection over the past decades. However, the efficiencies and limitations of previous breeding of Chinese Duroc pigs are largely understudied. The objective of this study was to uncover directional polygenic selection in the Duroc pig genome, and investigate points overlooked in the past breeding process.ResultsHere, we utilized the Generation Proxy Selection Mapping (GPSM) on a dataset of 1067 Duroc pigs with 8,766,074 imputed SNPs. GPSM detected a total of 5649 putative SNPs actively under selection in the Chinese Duroc pig population, and the potential functions of the selection regions were mainly related to production, meat and carcass traits. Meanwhile, we observed that the allele frequency of variants related to teat number (NT) relevant traits was also changed, which might be influenced by genes that had pleiotropic effects. First, we identified the direction of selection on NT traits by hat{G}, and further pinpointed large-effect genomic regions associated with NT relevant traits by selection signature and GWAS. Combining results of NT relevant traits-specific selection signatures and GWAS, we found three common genome regions, which were overlapped with QTLs related to production, meat and carcass traits besides “teat number” QTLs. This implied that there were some pleiotropic variants underlying NT and economic traits. We further found that rs346331089 has pleiotropic effects on NT and economic traits, e.g., litter size at weaning (LSW), litter weight at weaning (LWW), days to 100 kg (D100), backfat thickness at 100 kg (B100), and loin muscle area at 100 kg (L100) traits.ConclusionsThe selected loci that we identified across methods displayed the past breeding process of Chinese Duroc pigs, and our findings could be used to inform future breeding decision.

Breed Distribution and Allele Frequencies of Base Coat Color, Dilution, and White Patterning Variants across 28 Horse Breeds.

Since domestication, horses have been selectively bred for various coat colors and white spotting patterns. To investigate breed distribution, allele frequencies, and potential lethal variants for recommendations on genetic testing, 29 variants within 14 genes were investigated in 11,281 horses from 28 breeds. The recessive chestnut ea allele in melanocortin 1 receptor (MC1R) (p.D84N) was identified in four breeds: Knabstrupper, Paint Horse, Percheron, and Quarter Horse. After filtering for relatedness, ea allele frequency in Knabstruppers was estimated at 0.035, thus illustrating the importance of testing for mate selection for base coat color. The Rocky Mountain Horse breed had the highest allele frequency for two of the dilution variants under investigation (Za.f. = 0.32 and Cha.f. = 0.026); marker-assisted selection in this breed could aid in the production of horses with desirable dilute coats with less severe ocular anomalies caused by the silver (Z) allele. With regard to white patterning, nine horses homozygous for the paired box 3 (PAX3) splashed white 2 (SW2) allele (p.C70Y) and six horses homozygous for the KIT proto-oncogene, receptor tyrosine kinase (KIT) sabino 1 (SB1) allele (ECA3g.79544206A>T) were identified, thus determining they are rare and confirming that homozygosity for SW2 is not embryonic lethal. The KIT dominant white 20 (W20) allele (p.R682H) was identified in all but three breeds: Arabian (n = 151), Icelandic Horse (n = 66), and Norwegian Fjord Horse (n = 90). The role of W20 in pigmentation across breeds is not well understood; given the different selection regimes of the breeds investigated, these data provide justification for further evaluating the functional role of this allele in pigmentation. Here, we present the largest dataset reported for coat color variants in horses to date, and these data highlight the importance of breed-specific studies to inform on the proper use of marker-assisted selection and to develop hypotheses related to pigmentation for further testing in horses.

Association of gallstone and polymorphisms of UGT1A1*27 and UGT1A1*28 in patients with hepatitis B virus-related liver failure.

Genetic variation in UDP-glucuronosyltransferase 1A1 gene (UGT1A1) is a lithogenic risk factor for gallstone formation. This study aimed to assess genotype and allele frequencies of common UGT1A1 variants in patients with gallstone and hepatitis B virus (HBV)-related hepatic failure. This study enrolled 113 healthy individuals (CTRL), 54 patients with HBV infection (HBV), 134 patients with gallstone-free hepatic failure and HBV infection, and 34 patients with gallstone-related hepatic failure and HBV infection (GRHF). Peripheral venous blood samples were collected for genomic DNA isolation. Polymerase chain reaction amplification was carried out for UGT1A1, followed by direct sequencing. Analysis for genotype and allele frequencies of UGT1A1 variants (UGT1A1*6, UGT1A1*27, UGT1A1*28, and UGT1A1*60) was performed. The allele distributions of the four groups did not deviate from Hardy–Weinberg equilibrium. Allele (A) and genotype (CA) frequency distributions of UGT1A1*27 were significantly different between GRHF and CTRL, or between GRHF and HBV. GRHF and CTRL exhibited significant differences in allele (A) and genotype (CA) frequency distributions of UGT1A1*28. Linkage disequilibrium analysis suggested that haplotype G-G-[TA]7-T may be associated with gallstone in HBV-related hepatic failure. Our data reveal that UGT1A1*27 and UGT1A1*28 variants are significantly observed in patients with GRHF compared to healthy individuals.

FMR1 alleles in women with idiopathic infertility

Introduction: The frequency of the premutated alleles of the FMR1 gene varies from 1: 100 to 1: 260 Israeli, Canadian, Finnish and American women, but it is unknown in Brazil. Premutation carriers may have reduced reproductive age and are at risk of transmitting the expanded allele to their offspring, and consequently fragile X syndrome. Objective: To observe the distribution range of the FMR1 gene alleles in a population of women with idiopathic infertility, without symptoms of premature ovarian insufficiency. Methods: The presence of premutation in FMR1 was assessed by conventional PCR, agarose and acrylamide gel and analysis of fragments in capillary electrophoresis. From the lymphocyte DNA obtained from 283 women undergoing infertility treatment. Results: It was observed that 169 patients had the normal heterozygous allele (59.7%), 114 had the normal homozygous allele (40.6%) and no patient had the premutation. Premature ovarian insufficiency is seen in 20 to 30% of women with the premutated allele. Thus, the condition can be asymptomatic in a large part of the premutation carriers. Brazil has a diverse population and, therefore, the allele frequencies of many gene variants are unknown. Previous Brazilian studies have shown a low frequency of the premutated allele in different patient cohorts. Corroborating these articles, the results demonstrated that the frequency of the premutated allele is low in the infertile women population studied. Conclusion: Tracking the size of the FMR1 gene alleles allows the expansion of knowledge about the frequency of risk alleles associated to genetic diseases in the Brazilian population.

Frequency of β-Casein Gene Polymorphisms in Jersey Cows in Western Japan.

Simple SummaryReports presenting survey results for β-casein gene polymorphisms have increased, but none have been about Jersey cows in Asia. This study examined the CSN2 gene variants for 590 Jersey cows in Okayama Prefecture, located in the western region of Japan. Blood samples were collected at eight farms, and nucleotide substitutions were determined by sequencing exon 7 regions of chromosome 6 of the CSN2 gene. Blood biochemical analyses were also performed to clarify if A1A1, A1A2, and A2A2 cows differ in their metabolic profiles. The frequency of the A2 allele found in this study was numerically higher than those reported for Holsteins, crossbreeds, and Mexican and Danish Jerseys. The β-casein genotypes did not affect the metabolism of the major nutrients.This study aimed to investigate β-casein gene polymorphisms in Jersey cows in Japan. Blood samples were collected from 590 cows from eight Jersey farms in Okayama Prefecture, western Japan. Sequence analysis of exon 7 regions in chromosome 6 of the CSN2 gene revealed the genotype and allele frequencies of the β-casein variants. Considering that variant B belongs to the A1 group and variant I to the A2 group, plasma metabolite concentrations were compared among the A1A1, A1A2, and A2A2 group-based genotypes. The most frequent genotype was A2A2 (0.558), followed by A2B (0.190) and A2I (0.103). No variants of A3, F, G, H1, or H2 were found. The frequencies of group-based genotypes were A1A1 (0.032), A1A2 (0.303), and A2A2 (0.665). Although farm-to-farm differences were observed in the plasma concentrations of urea nitrogen, calcium, and phosphorus, no differences were found between the A1A1, A1A2, and A2A2 group-based genotypes; hence, the β-casein genotypes did not affect the metabolism of major nutrients. Owing to the high frequency of the A2 variant, Jersey cows can be considered an attractive breed for marker-assisted selection to create A2A2 herds.

Identification of Pathogenic Variant Burden and Selection of Optimal Diagnostic Method Is a Way to Improve Carrier Screening for Autosomal Recessive Diseases.

Cystic fibrosis, phenylketonuria, alpha-1 antitrypsin deficiency, and sensorineural hearing loss are among the most common autosomal recessive diseases, which require carrier screening. The evaluation of population allele frequencies (AF) of pathogenic variants in genes associated with these conditions and the choice of the best genotyping method are the necessary steps toward development and practical implementation of carrier-screening programs. We performed custom panel genotyping of 3821 unrelated participants from two Russian population representative samples and three patient groups using real-time polymerase chain reaction (PCR) and next generation sequencing (NGS). The custom panel included 115 known pathogenic variants in the CFTR, PAH, SERPINA1, and GJB2 genes. Overall, 38 variants were detected. The comparison of genotyping platforms revealed the following advantages of real-time PCR: relatively low cost, simple genotyping data analysis, and easier detection of large indels, while NGS showed better accuracy of variants identification and capability for detection of additional pathogenic variants in adjacent regions. A total of 23 variants had significant differences in estimated AF comparing with non-Finnish Europeans from gnomAD. This study provides new AF data for variants associated with the studied disorders and the comparison of genotyping methods for carrier screening.

P793: SCREENING OF NATURALLY OCCURRING CXCR4 VARIANTS FOR IDENTIFICATION OF NOVEL PATHOGENIC MUTATIONS FOR WHIM SYNDROME

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare, autosomal-dominant primary immunodeficiency marked by neutropenia and lymphopenia. Classically, WHIM syndrome pathogenesis is causally linked to a variety of heterozygous gain-of-function mutations in the C-terminus of chemokine receptor CXCR4, a master regulator of immune cell trafficking and homeostasis. As of February 2022, 18 variants in CXCR4 have been implicated in WHIM syndrome, while a substantial number remain unexplored for their association with the disease. Using in vitro functional assays, we previously found that impaired receptor internalization and enhanced chemotaxis are conserved in all CXCR4WHIM variants and that defective internalization correlates with neutropenia, the most penetrant phenotype of patients with WHIM syndrome. We used this approach to confirm functional defects in primary cells isolated from patients harboring a novel variant, suggesting that the in vitro profiling approach can potentially drive identification of pathogenic variants. Aims: We aimed to functionally characterize multiple previously uncharacterized variants of CXCR4 using in vitro assays. We also examined allele frequencies of these novel variants to estimate the potential number of individuals harboring these variants with a long-term goal of determining the actual prevalence of WHIM syndrome. Methods: The CXCR4-negative K562 cell line was used as a model system to express 53 novel CXCR4 variants identified in patient and population databases (ClinVar, Ensembl, CentoMD, GnomAD) and genetic screening initiatives (Invitae PATH4WARD) found in the C-terminus (hotspot of CXCR4WHIM mutations) as well as throughout the protein. The effects of these mutations on CXCR4 internalization and chemotaxis were studied in cells stimulated with the C-X-C chemokine ligand 12 (CXCL12). The in vitro functional parameters were compared with known pathogenic CXCR4 variants and were used to assign a cumulative score (impaired internalization + enhanced chemotaxis) of functional defect severity. Results: Out of 53 selected variants, 42 were missense (ms), 6 were frameshift (fs), 3 were nonsense (ns), and 2 were transcript variants (tv). Eighteen of the screened variants led to a decreased internalization of CXCR4 in comparison to the wild-type (WT) receptor upon stimulation with CXCL12 while 20 variants demonstrated enhanced chemotaxis of cells toward CXCL12. Overall, 34 variants showed defects in at least one CXCR4-dependent marker. Correlation analysis and scoring of the functional parameters in cells expressing mutated or CXCR4WT revealed 17 variants (9 ms, 5 fs, 2 ns, 1 tv) that co-segregated with the known pathogenic WHIM variants. Finally, white blood cell counts in 3 patients harboring novel variants showed that CXCR4 internalization defects in vitro correlated with the severity of neutropenia. Summary/Conclusion: This is the first study characterizing functional impairments in naturally occurring CXCR4 variants via a pipeline of assays intended to predict pathogenicity. Seventeen CXCR4 variants were identified (several outside the C-terminus of CXCR4) that caused in vitro functional defects resembling those exhibited by known WHIM variants. Many (10/17) of these newly identified variants are present at high frequencies (4.4 × 10-4–8.0 × 10-6) in genomic databases. Potential association of these variants with WHIM syndrome is under investigation and is likely to expand current estimates of WHIM syndrome prevalence.

Distinct characteristics of central serous chorioretinopathy according to gender

To investigate the differences in clinical and genetic characteristics between males and females with central serous chorioretinopathy (CSC). Consecutive 302 patients (mean age; 56.3 ± 11.7, male/female: 249/53) with CSC were evaluated on the initial presentation. All CSC patients underwent fluorescein angiography and indocyanine green angiography (FA/ICGA), swept-source or spectral-domain optical coherence tomography (OCT), and fundus autofluorescence (FAF) to confirm a diagnosis. All patients were genotyped for rs800292 and rs1329428 variants of CFH using TaqMan technology. On the initial presentation, female patients were significantly older (p = 2.1 × 10–4, female 61.6 ± 12.4 vs male 55.1 ± 11.3) and had thinner subfoveal choroidal thickness (p = 3.8 × 10–5) and higher central retinal thickness (p = 3.0 × 10–3) compared to males. A descending tract was more frequently seen in males than in females (p = 8.0 × 10–4, 18.1% vs 0%). Other clinical characteristics were comparable between the sexes. The risk allele frequency of both variants including CFH rs800292 and CFH rs1329428 was comparable between males and females (CFH rs800292 A allele male 51.2% vs female 47.2%, CFH rs1329428 T allele male 56.2% vs 52.8%). On the initial presentation, age, subfoveal choroidal thickness and central retinal thickness differ between males and females in eyes with CSC. A descending tract may be a strong male finding in CSC.

Genetic epidemiology of blood type, disease and trait variants, and genome-wide genetic diversity in over 11,000 domestic cats.

In the largest DNA-based study of domestic cats to date, 11,036 individuals (10,419 pedigreed cats and 617 non-pedigreed cats) were genotyped via commercial panel testing elucidating the distribution and frequency of known disease, blood type, and physical trait associated genetic variants across cat breeds. This study provides allele frequencies for many disease-associated variants for the first time and provides updates on previously reported information with evidence suggesting that DNA testing has been effectively used to reduce disease associated variants within certain pedigreed cat populations over time. We identified 13 disease-associated variants in 47 breeds or breed types in which the variant had not previously been documented, highlighting the relevance of comprehensive genetic screening across breeds. Three disease-associated variants were discovered in non-pedigreed cats only. To investigate the causality of nine disease-associated variants in cats of different breed backgrounds our veterinarians conducted owner interviews, reviewed clinical records, and invited cats to have follow-up clinical examinations. Additionally, genetic variants determining blood types A, B and AB, which are relevant clinically and in cat breeding, were genotyped. Appearance-associated genetic variation in all cats is also discussed. Lastly, genome-wide SNP heterozygosity levels were calculated to obtain a comparable measure of the genetic diversity in different cat breeds. This study represents the first comprehensive exploration of informative Mendelian variants in felines by screening over 10,000 pedigreed cats. The results qualitatively contribute to the understanding of feline variant heritage and genetic diversity and demonstrate the clinical utility and importance of such information in supporting breeding programs and the research community. The work also highlights the crucial commitment of pedigreed cat breeders and registries in supporting the establishment of large genomic databases, that when combined with phenotype information can advance scientific understanding and provide insights that can be applied to improve the health and welfare of cats.

The interplay between XPG-Asp1104His polymorphism and reproductive risk factors elevates risk of breast cancer in Tanzanian women: A multiple interaction analysis.

Reproductive history and genetics are well-known risk factors of breast cancer (BC). Little is known about how these factors interact to effect BC. This study investigated the association of ten polymorphisms in DNA repair genes with BC susceptibility in the Tanzanian samples and further analyzed the association between reproductive risk factors and disease risk METHODS: A hospital-based case-control study in 263 histopathological confirmed BC patients and 250 age-matched cancer-free controls was carried out. Allelic, genotypic, and haplotype association analyses were executed. Also, multifactor dimensionality reduction (MDR), and interaction dendrogram approaches were performed. The frequency of genotypic and allelic variants of XRCC1-Arg399Gln (rs25487), XRCC2-Arg188His (rs3218536), XRCC3-Thr241Met (rs861539), XPG-Asp1104His (rs17655), and MSH2-Gly322Asp (rs4987188) were significantly different between the groups (p < 0.05). Moreover, XRCC1-Arg399Gln (rs25487), XRCC3-Thr241Met (rs861539), and XPG-Asp1104His (rs17655) were associated with the increased risk of BC in co-dominant, dominant, recessive, and additive genetic-inheritance models (p < 0.05). XRCC1-Arg/Gln genotype indicated a 3.1-fold increased risk of BC in pre-menopausal patients (p=0.001) while XPG-His/His genotype showed a 1.2-fold increased risk in younger BC patients (<40 years) (p=0.028). Asp/His+His/His genotypes indicated a 1.3-fold increased risk of BC in PR+ patients and a 1.1-fold decreased risk of BC in luminal-A patients (p=0.014, p=0.020, respectively). MDR analysis revealed a positive interaction between BC and the XPG-Asp1104His (rs17655) together with family history of cancer in the first-degree relatives. Dendrogram analysis indicated that the XPG-Asp1104His (rs17655) and family history of cancer in first-degree relatives were significantly synergistic and might be associated with an elevated risk of BC in Tanzania. The XPG-Asp1104His (rs17655) might exert both independent and interactive effects on BC development in the Tanzanian women.