High-risk Haplotype Research Articles

Abstract P181: Class 1 human leukocyte antigens exhibit marked variability in capacity to present isolevuglandin adducts

Introduction: Isolevuglandins (isoLGs) are lipid peroxidation products that covalently modify lysine residues on self-proteins. These modified self-proteins are processed to peptides that are presented in the context of class I major histocompatibility complexes (MHC-I). In hypertension, this modification enhances CD8 + T cell activation, contributing to inflammation and elevated blood pressure. In mice we found that IsoLG-adduct presentation is highly dependent on class I MHC structure. We sought to determine if human class I MHC (HLAs) exhibit similar variability in their ability to present these adducts by screening alleles representing 90% of the population. Hypothesis: We hypothesize that HLA subtypes exhibit variable capacity for isoLG presentation. Methods: HLA alleles representing 90% demographic frequency were identified from the US National Merit Donor Program Allele Frequency Database. HLA-null human K562 cells were transduced with these alleles, expanded, and enriched for HLA expression by flow cytometry to produce single HLA allele lines. To induce isoLG adduct presentation, cells were treated with 1mM tert-Butyl hydroperoxide (tBHP). After 24 hours, we employed Forster resonance energy transfer (FRET) between HLAs and isoLG-adducts using antibodies specific for both, which we have shown is indicative of IsoLG-adduct presentation (Fig 1A-C). Results: Substantial variability for isoLG-adduct presentation was observed between HLA subtypes A, B and C (Figure 1A-1C). Scikit-learn’s KMeans clustering revealed four patterns of isoLG-adduct presentation among HLAs: Low baseline/low activation, low baseline/high activation, intermediate baseline/intermediate activation, and high baseline/high activation alleles (Fig 1D). Conclusions: These findings identify specific HLA alleles as high isoLG presenters, potentially associated with increased immune activation risk. Combining these findings, HLA alleles, and diagnosis code databases could aid in identifying high-risk haplotypes for immune activation in human hypertension.

Association of ADAMTS-5 gene polymorphisms with the susceptibility to knee osteoarthritis in a Chinese Han population

BackgroundOsteoarthritis (OA) is the most prevalent type of arthritis and the main reason for progressive disability in middle-aged and older people. Studies of candidate genes may provide a novel insight and treatment strategy for knee osteoarthritis (KOA). The aim of this study was to investigate the relationship between KOA susceptibility and single-nucleotide polymorphism (SNP) of the ADAMTS-5 gene.Materials and methodsThe case group included 188 patients from Luoyang Orthopedic Hospital with clinically and radiographically diagnosed primary KOA, and the control group included 100 age-matched individuals without KOA. Fifteen ADAMTS-5 SNPs were assayed using MALDI-TOF MS. Allelic and haplotypic frequencies were compared between the groups. The relationship between genotype distribution and risk of KOA was analyzed by multivariate logistic regression.ResultsThe frequency of A allele in rs2249350 site in the KOA group was significantly lower (odds ratio [OR]: 0.761; 95% confidence interval [95% CI]: 0.612–0.947; P = 0.016), while that of C allele was higher than that in the control group (OR: 1.176; 95% CI: 1.025–1.351; P = 0.016). AA genotype and gene model, especially recessive gene model at rs2249350 locus, negatively correlated with KOA risk after adjustment for sex, body mass index, age, and occupation (AA vs. CC: OR: 0.288; 95% CI: 0.124–0.669; P = 0.004; AA vs. CA + CC: OR: 0.348; 95% CI: 0.162–0.749; P = 0.007). Meanwhile, one protective haplotype, GA (rs229054, rs2249350) (OR: 0.763; 95% CI: 0.614–0.949; P = 0.017), and one high-risk haplotype, GC (rs229054, rs2249350) (OR: 1.259; 95% CI: 1.032–1.537; P = 0.019), were found in this study.ConclusionDespite a limited sample size, our study suggests that the rs2249350 polymorphism in the ADAMTS-5 gene is one of the genetic factors influencing the risk of KOA. The A allele and AA genotype of rs2249350 may protect from KOA, whereas C allele and CC genotype increase the risk of KOA. In addition, the GA haplotype (rs229054, rs2249350) might be associated with a decreased risk of KOA, whereas the GC haplotype (rs229054, rs2249350) may be a risk factor for KOA. Additional larger-sized studies in more ethnically diverse populations are needed to confirm these findings.

Association between HLA alleles and haplotypes with age at diagnosis of type 1 diabetes in an admixed Brazilian population: A nationwide study.

To investigate the potential relationship between HLA alleles and haplotypes and the age at diagnosis of type 1 diabetes (T1DAgeD) in an admixed Brazilian population. This nationwide study was conducted in public clinics across 12 Brazilian cities. We collected demographic and genetic data from 1,600 patients with T1D. DNA samples were utilised to determine genomic ancestry (GA) and perform HLA typings for DRB1, DQA1 and DQB1. We explored allele and haplotype frequencies and GA in patients grouped by T1DAgeD categories (<6 years, ≥6-<11 years, ≥11-<19 years and ≥19 years) through univariate and multivariate analyses and primary component analyses. Additionally, we considered self-reported colour-raceand identified a familiar history of T1D in first-degree relatives. The homozygosity index for DRB1~DQA1~DQB1 haplotypes exhibited the highest variation among T1DAgeD groups, and the percentages of Sub-Saharan African and European ancestries showed opposite trends in principal component analysis (PCA) analyses. Regarding the association of alleles and haplotypes with T1DAgeD, risk alleles such as HLA-DQB1*03:02g, -DQA1*03:01g, -02:01g, DRB1*04:05g and -04:02g were more frequently observed in heterozygosity or homozygosity in T1D patients with an early disease onset. Conversely, alleles such as DRB1*07:01g, -13:03g, DQB1*06:02g and DQA1*02:01 were more prevalent in older T1D patients. The combination DR3/DR4.5 was significantly associated with early disease onset. However, gender, GA, familiar history of T1D and self-reported colour-race identity did not exhibit significant associations with the onset of T1D. It is worth noting that the very common risk haplotype DRB1*03:01g~DQA1*05:01g~DQB1*02:01g did not differentiate between T1DAgeD groups. In the admixed Brazilian population, the high-risk haplotype DRB1*04:05~DQA1*03:01~DQB1*03:02 was more prevalent in individuals diagnosed before 6 years of age. In contrast, the protective alleles DQA1*01:02g, DQB1*06:02g, DRB1*07:01g and DRB1*13:03g and haplotypes DRB1*13:03g~DQA1*05:01g~DQB1*03:01g and DRB1*16:02g~DQA1*01:02g~DQB1*05:02g were more frequently observed in patients diagnosed in adulthood. Notably, these associations were independent of factors such as sex, economic status, GA, familiar history of T1D and region of birth in Brazil. These alleles and haplotypes contribute to our understanding of the disease onset heterogeneity and may have implications for early interventions when detected in association with well-known genomic risk or protection factors for T1D.

Whole Exome Sequencing in Children With Type 1 Diabetes Before Age 6 Years Reveals Insights Into Disease Heterogeneity.

Aims: This study is aimed at comparing whole exome sequencing (WES) data with the clinical presentation in children with type 1 diabetes onset ≤ 5 years of age (EOT1D). Methods: WES was performed in 99 unrelated children with EOT1D with subsequent analysis to identify potentially deleterious rare variants in MODY genes. High-resolution HLA class II haplotyping, SNP genotyping, and T1D-genetic risk score (T1D-GRS) were also evaluated. Results: Eight of the ninety-nine EOT1D participants carried a potentially deleterious rare variant in a MODY gene. Rare variants affected five genes: GCK (n = 1), HNF1B (n = 2), HNF4A (n = 1), PDX1 (n = 2), and RFX6 (n = 2). At diagnosis, these children had a mean age of 3.0 years, a mean HbA1c of 10.5%, a detectable C-peptide in 5/8, and a positive islet autoantibody in 6/7. Children with MODY variants tend to exhibit a lower number of pancreatic autoantibodies and a lower fasting C-peptide compared to EOT1D without MODY rare variants. They also carried at least one high-risk DR3-DQ2 or DR4-DQ8 haplotype and exhibited a T1D-GRS similar to the other individuals in the EOT1D cohort, but higher than healthy controls. Conclusions: WES found potentially deleterious rare variants in MODY genes in 8.1% of EOT1D, occurring in the context of a T1D genetic background. Such genetic variants may contribute to disease precipitation by a β-cell dysfunction mechanism. This supports the concept of different endotypes of T1D, and WES at T1D onset may be a prerequisite for the implementation of precision therapies in children with autoimmune diabetes.

Robust preimplantation genetic testing of the common F8 Inv22 pathogenic variant of severe hemophilia A using a highly polymorphic multi-marker panel encompassing the paracentric inversion

BackgroundHemophilia A (HEMA) is an X-linked bleeding disorder caused by reduced/absent coagulation factor VIII expression, as a result of pathogenic variants in the F8 gene. Preimplantation prevention of HEMA should ideally include direct pathogenic F8 variant detection, complemented by linkage analysis of flanking markers to identify the high-risk F8 allele. Linkage analysis is particularly indispensable when the pathogenic variant cannot be detected directly or identified. This study evaluated the suitability of a panel of F8 intragenic and extragenic short tandem repeat markers for standalone linkage-based preimplantation genetic testing for monogenic disorder (PGT-M) of the Inv22 pathogenic variant, an almost 600 kb paracentric inversion responsible for almost half of all severe HEMA globally, for which direct detection is challenging.MethodsThirteen markers spanning 1 Mb and encompassing both F8 and the Inv22 inversion interval were genotyped in 153 unrelated females of Viet Kinh ethnicity.ResultsAll individuals were heterozygous for ≥ 1 marker, ~ 90% were heterozygous for ≥ 1 of the five F8 intragenic markers, and almost 98% were heterozygous for ≥ 1 upstream (telomeric) and ≥ 1 downstream (centromeric) markers. A prospective PGT-M couple at risk of transmitting F8 Inv22 were fully informative at four marker loci (2 intra-inversion, 1 centromeric, 1 telomeric) and partially informative at another five (2 intra-inversion, 3 centromeric), allowing robust phasing of low- and high-risk haplotypes. In vitro fertilization produced three embryos, all of which clearly inherited the low-risk maternal allele, enabling reliable unaffected diagnoses. A single embryo transfer produced a clinical pregnancy, which was confirmed as unaffected by amniocentesis and long-range PCR, and a healthy baby girl was delivered at term.ConclusionRobust and reliable PGT-M of HEMA, including the common F8 Inv22 pathogenic variant, can be achieved with sufficient informative intragenic and flanking markers.

A long-read sequencing and SNP haplotype-based novel preimplantation genetic testing method for female ADPKD patient with de novo PKD1 mutation

The autosomal dominant form of polycystic kidney disease (ADPKD) is the most common hereditary disease that causes late-onset renal cyst development and end-stage renal disease. Preimplantation genetic testing for monogenic disease (PGT-M) has emerged as an effective strategy to prevent pathogenic mutation transmission rely on SNP linkage analysis between pedigree members. Yet, it remains challenging to establish reliable PGT-M methods for ADPKD cases or other monogenic diseases with de novo mutations or without a family history. Here we reported the application of long-read sequencing for direct haplotyping in a female patient with de novo PKD1 c.11,526 G > C mutation and successfully established the high-risk haplotype. Together with targeted short-read sequencing of SNPs for the couple and embryos, the carrier status for embryos was identified. A healthy baby was born without the PKD1 pathogenic mutation. Our PGT-M strategy based on long-read sequencing for direct haplotyping combined with targeted SNP haplotype can be widely applied to other monogenic disease carriers with de novo mutation.

Two-Sample Mendelian Randomization detects bidirectional causality between gut microbiota and celiac disease in individuals with high genetic risk.

Celiac Disease (CeD) is an autoimmune disorder triggered by gluten intake in genetically susceptible individuals. Highest risk individuals are homozygous for the Human Leucocyte Antigen (HLA) DQ2.5 haplotype or DQ2.5/DQ2.2 heterozygous. Both the HLA-DQ2-positive high genetic risk individuals and those that have developed the disease have altered intestinal microbiota, but it remains unclear whether these alterations are a cause or a consequence of CeD. To investigate a potential bidirectional causality between gut microbiota (GM) and CeD in HLA-DQ2 high genetic risk individuals. We performed a bidirectional Two-Sample Mendelian Randomization (2SMR) test using summary statistics from the largest publicly available Genome-Wide Association Study (GWAS) of GM and the summary statistics of the Immunochip CeD study of those individuals with the HLA-DQ2 high-risk haplotype. To test whether changes in GM composition were causally linked to CeD, GM data were used as exposure and CeD data as outcome; to test for reverse causation, the exposure and outcome datasets were inverted. We identified several bacteria from Ruminococcaceae and Lachnospiraceae families of the Firmicutes phylum as potentially causal in both directions. In addition, our results suggest that changes in the abundance of Veillonellaceae family might be causal in the development of CeD, while alterations in Pasteurellaceae family might be a consequence of the disease itself. Our results suggest that the relationship between GM and HLA-DQ2 high risk individuals is highly complex and bidirectional.

#5124 GENETIC GLOMERULAR DISEASES IN THE ADULT PATIENT: A WORK IN PROGRESS – SINGLE CENTRE COHORT

Abstract Background and Aims Recent advances in genetic molecular techniques have allowed the expansion of knowledge in glomerular diseases of genetic cause, including the identification of genes involved in focal and segmental glomerular sclerosis (FSGS). The prevalence of adult-onset genetic FSGS varies widely depending on the population studied. In central Europe, NPHS2 and COL4A3, COL4A4 e COL4A5 genes are postulated to be the most frequent genes implicated in genetic causes of adult FSGS. This study aims to characterize patients submitted to genetic study of glomerular diseases in the Nephrogenetics Clinic of our centre. Method We conducted a single-centre retrospective analysis of the patients submitted to genetic study of glomerular diseases in the Nephrogenetics Clinic of our centre from 2016 to 2022. Adult patients with a glomerular disease phenotype suggestive of a genetic cause were studied, using a predetermined phenotype-targeted panel of genes, through massive parallel sequencing. The number of genes included in this panel has increased (from 21 genes in 2016 to 49 genes since 2018); given this change, patients with a negative result on the first approach repeated screening with the broader panel. Additionally, patients presenting with advanced chronic kidney disease (CKD) without an identifiable phenotype, studied with a larger panel for CKD causing genes, are reported. In patients with a negative result but highly suggestive presentation, whole exome sequencing (WES) was considered, after multidisciplinary discussion with a Geneticist's evaluation. Patients with variants of unknown significance (VUS) were referred to a Genetics’ consultation for genetic counselling and segregation studies. Results Of the 80 patients studied (comprising 74 families), 52.5% were female (n=42) and 79% (n=63) of Caucasian ascent. Mean age was 45.4 ± 16.0 years. Nine patients were studied with the larger gene panel for CKD. 85% (n=68) had CKD (all stages) at presentation, with proteinuria (ranging from sub-nephrotic to nephrotic range) and 15% had asymptomatic urinary changes. 50% (n=40) of patients had positive family history, and 31% (n=25) had a syndromic presentation and/or extra renal manifestations. 27.5% (n=22) had a renal biopsy, and the most prevalent result was FSGS (11%; n=9). Genetic variants were identified in 70% (n=56) of the patients; but pathogenicity is not fully established in all: genotype-phenotype correlations, including inheritance pattern and histological correlation, and segregation studies (when indicated and possible) are ongoing. In 5 patients results are pending. Pathogenic / likely pathogenic variants were identified in at least 47.5% (n=38) patients. The most frequently implicated genes were: COL4A3, COL4A4 e COL4A5 (n=21), high risk APOL1 (n=8); NPSH2 (n=5), IFN2 (n=6, 3 pathogenic, 1 variant of unknown significance (VUS), 2 under segregation studies) and MYH9 (n=3). COL4A3, COL4A4 e COL4A5 mutations occur with Alport syndrome, but also with histologically documented FSGS, sub-nephrotic and nephrotic proteinuria, nephrotic syndrome and asymptomatic urinary changes. In our cohort, NPHS2 p.R229Q was identified in 5 patients (6%), but only with pathogenic significance in 3 patients, and APOL1 high risk haplotype was co-identified with COL4 variants in 3 patients. Conclusion The genetic study of a cohort of patients suspected of genetic FSGS allowed the identification of genetic variants in the genes of interest in a high percentage of cases. The most frequently implicated genes were COL4A3, COL4A4 e COL4A5, NPHS2, INF2 and APOL1 high risk haplotype. The high percentage of gene variants identified in our cohort is probably due to the selection of a population with a high likelihood of genetic FSGS. Further studies will clarify the causal and/or modifier effect of some genes, namely COL4A3, COL4A4 e COL4A5. The future use of WES in cases of high likelihood of genetic cause will probably allow the identification on novel candidate genes and increase the clinical benefits of a genetic diagnosis of FSGS.

OR14-6 Probiotic Supplementation with Lactiplantibacillus Plantarum 299v Modulates β-Cell Endoplasmic Reticulum Stress and Prevents Type 1 Diabetes in Biobreeding Rats

Abstract Type 1 diabetes (T1D) is a complex disease characterized by the loss of pancreatic beta-cells and lifelong dependence on insulin replacement therapy. As the worldwide incidence of T1D has increased and the prevalence of high-risk HLA haplotypes has declined among new diagnoses, a greater focus has been placed on environmental factors contributing to T1D pathogenesis. Lactiplantibacillus plantarum 299v (Lp299v) supplement is reported to increase plasma and stool levels of anti-inflammatory short-chain fatty acids (SCFAs) and promote IL-10 signaling in colonic derived macrophages and T-cells. Therefore, we investigated the effect of Lp299v supplement on T1D pathogenesis in diabetes-prone BioBreeding DRlyp/lyp rats. Rats were weaned at 21 days of age (D21) onto a normal cereal diet (ND) or a gluten-free hydrolyzed casein diet (HCD), with or without daily Lp299v supplementation. All rats provided ND developed diabetes by D83 and supplementing ND with Lp299v did not significantly delay progression. Rats provided HCD showed a significant delay in diabetes onset over ND, while progression was even more delayed in the HCD+Lp299v group, with 25% of animals remaining diabetes-free to D130 (study end). Relative to the other groups, HCD+Lp299v rats exhibited significant increases in the plasma levels of the SCFAs propionate and butyrate (beneficial metabolites produced by intestinal bacteria). In addition, stool microbiota showed increased abundances of SCFA-producing taxa at D40. Gene expression analysis of pancreatic islets was conducted at D40, prior to insulitis. Lp299v supplementation favorably modulated islet expression levels of pathways related to endoplasmic reticulum (ER) stress, and the major arms of the unfolded protein response (UPR), especially in combination with the HCD. Notably, ER stress has been implicated in the formation of islet neoantigens that may underlie the initial loss of immune tolerance in T1D. Lp299v supplement differentially modulated the UPR to favor the expression of the transcription factor Atf6, increase expression of transcripts related to cell survival/proliferation, and the ER-associated protein degradation (ERAD) pathway of the UPR. This contrasted with HCD alone, which promoted a transcriptional program related to autophagy. To colocalize and confirm activities detected in the transcriptional analyses to β-cells, pancreata of D40 rats were subjected to dual immunofluorescence staining for insulin and UPR-related proteins. Consistent with elevated ERAD activity, significantly greater EIF4G1 and OS9 expression were observed in HCD+Lp299v islets. Phospho-PERK staining was significantly higher in islets of the HCD-alone group, consistent with the elevated expression of autophagy transcripts. Analysis of plasma proinsulin: C-peptide ratios revealed that Lp299v alone could improve islet function, as significant decreases were measured in all groups relative to ND. While ongoing studies aim to define the bacterial metabolites underlying the favorable outcomes, these studies suggest that Lp299v supplementation improves proteostasis capacity in β-cells and slows T1D progression in DRlyp/lyp rats. Presentation: Sunday, June 12, 2022 12:15 p.m. - 12:30 p.m.

African-specific alleles modify risk for asthma at the 17q12-q21 locus in African Americans

BackgroundAsthma is the most common chronic disease in children, occurring at higher frequencies and with more severe disease in children with African ancestry.MethodsWe tested for association with haplotypes at the most replicated and significant childhood-onset asthma locus at 17q12-q21 and asthma in European American and African American children. Following this, we used whole-genome sequencing data from 1060 African American and 100 European American individuals to identify novel variants on a high-risk African American–specific haplotype. We characterized these variants in silico using gene expression and ATAC-seq data from airway epithelial cells, functional annotations from ENCODE, and promoter capture (pc)Hi-C maps in airway epithelial cells. Candidate causal variants were then assessed for correlation with asthma-associated phenotypes in African American children and adults.ResultsOur studies revealed nine novel African-specific common variants, enriched on a high-risk asthma haplotype, which regulated the expression of GSDMA in airway epithelial cells and were associated with features of severe asthma. Using ENCODE annotations, ATAC-seq, and pcHi-C, we narrowed the associations to two candidate causal variants that are associated with features of T2 low severe asthma.ConclusionsPreviously unknown genetic variation at the 17q12-21 childhood-onset asthma locus contributes to asthma severity in individuals with African ancestries. We suggest that many other population-specific variants that have not been discovered in GWAS contribute to the genetic risk for asthma and other common diseases.

1254-P: Genetic Prediction of C-Peptide Trajectory before Type 1 Diabetes Diagnosis in TrialNet

Modeling the course of C-peptide decline in autoantibody (Ab) positive individuals is important for type 1 diabetes (T1D) prediction and implementation of T1D prevention trials. Single nucleotide polymorphisms in TCF7L2, JAZF1, SLC30A8, INS, PTPRK, G6CP2, CLEC16, PTPN22 and HLA genes are associated with persistent C-peptide at or after T1D diagnosis. We sought to determine whether 12 SNPs in these genes and the T1D genetic risk score-2 (GRS2) can predict C-peptide trajectory before diagnosis of T1D. We studied Ab positive at-risk participants in the TrialNet Pathway to Prevention Study who had ImmunoChip data (N=1217, age at initial screen (mean±SD) 16.1±12.7 years, 51.5% female, 81.0% non-Hispanic white) . Over a mean follow up of 3.4±2. years, 255 (21.0%) developed multiple Ab and 336 (27.6%) developed clinical T1D. We analyzed the influence of these 12 SNPs and the T1D GRS2 on C-peptide AUC during progression from single to multiple Abs, from single Ab to clinical T1D, and from multiple Abs to clinical T1D. Analyses were adjusted for baseline C-peptide AUC, age, glucose AUC, BMI Z-score and HbA1c; the presence of high-risk HLA haplotypes (DR3 and DR4-DQ8) ; and the first 3 principal components. The type 2 diabetes (T2D) -associated TCF7L2 rs7901695 and rs4506565 SNPs were significantly associated with higher C-peptide AUC during progression from multiple Abs to T1D (p&amp;lt;0.04) and neared significance for the single-to-multiple Ab transition (p=0.05) . Additionally, lower T1D GRS2 was significantly associated with higher C-peptide AUC during progression from multiple Abs to T1D (p=0.01) . We observed trend associations of lower C-peptide AUC with JAZF1 SNP rs864745 in both single Ab-to-T1D and multiple Ab-to-T1D progression (both p=0.06) , and with SNPs in SLC30A8 and INS in multiple Abs to T1D (p=0.and 0.08, respectively) . In conclusion, T2D-associated SNPs in the TCF7L2 gene and lower T1D GRS2 predict higher C-peptide particularly in progression from multiple Abs to clinical T1D. Disclosure T.M.Triolo: None. M.J.Redondo: Advisory Panel; Provention Bio, Inc. H.M.Parikh: None. M.Tosur: Advisory Panel; Provention Bio, Inc. P.Gottlieb: Advisory Panel; Janssen Research &amp; Development, LLC, ViaCyte, Inc., Other Relationship; IM Therapeutics, Research Support; Caladrius Biosciences, Inc., Immune Tolerance Network, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk, Precigen, Inc., Tolerion, Inc. R.A.Oram: Consultant; Janssen Research &amp; Development, LLC, Research Support; Randox R &amp; D. S.Onengut-gumuscu: None. J.Krischer: None. S.S.Rich: None. A.Steck: None. Funding NIH K12 DK094712NIH RDK121843NIH RDK124395

Genomic landscape of Epstein-Barr virus in familial nasopharyngeal carcinoma.

To better understand the genomic characteristics of Epstein-Barr virus (EBV) in familial nasopharyngeal carcinoma (NPC), we sequenced the EBV genomes by whole-genome capture in 38 unrelated patients with NPC family history in first-degree relatives and 47 healthy controls, including 13 with family history and 34 without. Compared with type 1 reference genome, mutation hotspots were observed in the latent gene regions of EBV in familial NPC cases. Population structure analysis showed that one cluster has a higher frequency in familial cases than in controls (OR=5.33, 95 % CI 2.50-11.33, P=1.42×10-5), and similar population structure composition was observed among familial and sporadic NPC cases in high-endemic areas. By genome-wide association analysis, four variants were found to be significantly associated with familial NPC. Consistent results were observed in the meta-analysis integrating two published case-control EBV sequencing studies in NPC high-endemic areas. High-risk haplotypes of EBV composed of 34 variants were associated with familial NPC risk (OR=13.85, 95 % CI 4.13-46.44, P=2.06×10-5), and higher frequency was observed in healthy blood-relative controls with NPC family history (9/13, 69.23 %) than those without family history (16/34, 47.06%). This study suggested the potential contribution of EBV high-risk subtypes to familial aggregation of NPC.

Human Leukocyte Antigens and Biomarkers in Type 1 Diabetes Mellitus Induced by Immune-Checkpoint Inhibitors.

BackgroundType 1 diabetes mellitus induced by immune-checkpoint inhibitors (ICI-T1DM) is a rare critical entity. However, the etiology of ICI-T1DM remains unclear.MethodsIn order to elucidate risk factors for ICI-T1DM, we evaluated the clinical course and immunological status of patients with ICI-T1DM who had been diagnosed during 2016 to 2021.ResultsSeven of 871 (0.8%, six men and one woman) patients developed ICI-T1DM. We revealed that the allele frequencies of human leukocyte antigen (HLA)-DPA1*02:02 and DPB1*05:01 were significantly higher in the patients with ICI-T1DM In comparison to the controls who received ICI (11/14 vs. 10/26, P=0.022; 11/14 vs. 7/26, P=0.0027, respectively). HLA-DRB1*04:05, which has been found to be a T1DM susceptibility allele in Asians, was also observed as a high-risk allele for ICI-T1DM. The significance of the HLA-DPB1*05:01 and DRB1*04:05 alleles was confirmed by an analysis of four additional patients. The absolute/relative neutrophil count, neutrophils-lymphocyte ratio, and neutrophil-eosinophil ratio increased, and the absolute lymphocyte count and absolute/relative eosinophil count decreased at the onset as compared with 6 weeks before. In two patients, alterations in cytokines and chemokines were found at the onset.ConclusionNovel high-risk HLA alleles and haplotypes were identified in ICI-T1DM, and peripheral blood factors may be utilized as biomarkers.

Genetic markers for inherited thrombophilia related pregnancy loss and implantation failure in Indian population – implications for diagnosis and clinical management

Aim The biology of recurrent pregnancy loss and recurrent implantation failure (RPL-RIF) is complex with multi-factorial etiology, with defective thrombosis being one of the most important and highly prevalent causes. The role of several thrombophilia related genes and variants associated with RPL-RIF is widely reported, and this study aimed to identify the risk associated with these genes in the Indian population. Methods Next generation sequencing (NGS) was employed for the current study. NGS enables sequencing of multiple genes, identification of new variants, and establishment of genetic correlations with reproductive failure in diverse population groups. The present NGS based study evaluates association of twenty-nine genotypes of ten coagulation pathway genes (F2, F5, F13, MTR, MTRR, MTHFR, ANXA5, PROZ, SERPINE1 and VEGFA) with RPL-RIF in 540 female subjects − 474 patients with early recurrent pregnancy loss, late pregnancy loss, pregnancy complications in late gestation and recurrent implantation failure, with 66 controls. Results The results emphasize inclusion of genotypes of seven thrombophilia genes (MTHFR, MTRR, MTR, ANXA5, PROZ, SERPINE1, VEGFA) for diagnosis of inherited thrombophilia risk for RPL-RIF in Indian population, as against the common practice of testing limited to F2, F5 and MTHFR genes. Conclusion Deriving risk magnitude from Combined Risk Analysis and interpretation of high-risk haplotypes are crucial components for evidence based personalized management such as selection of drugs and dosage, and prenatal or pre-implantation recommendations, for high-risk patients in fertility and obstetric clinics.

Molecular Genetic Testing for Kidney Disorders During the COVID-19 Pandemic.

Chronic kidney disease (CKD) has major morbidity and mortality for children and adults. While in adults CKD often is associated with diabetic complications, genetic variants can be the underlying cause in both populations. Beginning in 2016 with the emergence of more affordable next-generation sequencing (NGS) technologies, the Molecular Diagnostics Lab at Nemours Children’s Hospital-Delaware developed the first clinically actionable pediatric NGS kidney panel comprised of 46 genes including APOL1. Apolipoprotein L1 (APOL1) associated nephropathy is reported along a spectrum of non-diabetic kidney disease. It is significantly associated with two “risk alleles” defined as G1 and G2 and typically found in individuals of African descent. In early 2020, as COVID-19 spread across the globe, reports of patients with kidney failure began to emerge. A collapsing glomerulopathy in Black patients with COVID-19 was found to be associated with the APOL1 predisposition of the known G1 and/or G2 risk variants. We identified genetic variants in 11 genes (NPHS1; NPHS2; LAMB2; WT1; COL4A4; COL4A5; COQ8B; CUBN; MEFV; PMM2; SMARCAL1) known to be associated with pediatric onset nephrotic syndrome, or detection of the high-risk haplotype of APOL1, in the majority (78%) of patients tested. These clinically actionable results guided medical care and improved patient outcomes.

Multiplex Epstein-Barr Virus Genotyping PCR Detects High-Risk Variants in Plasma for Population-Level Nasopharyngeal Carcinoma Screening

<h3>Purpose/Objective(s)</h3> Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is unusually geographically and ethnically restricted. Without screening, most patients present at an advanced stage. Population-level screening in high-risk populations can detect most cases at an early stage, but existing serologic or molecular diagnostics are limited by low positive predictive value secondary to benign EBV reactivation. In contrast to human genome-wide association studies, recent EBV genome-wide association studies have identified polymorphisms with much greater attributable risk for NPC. We hypothesized that a noninvasive molecular diagnostic could detect high-risk EBV haplotypes in plasma to triage patients who screen positive for plasma EBV DNA. <h3>Materials/Methods</h3> We designed a multiplex allele-specific real-time polymerase chain reaction (qPCR) genotyping assay to detect three non-synonymous polymorphisms within the EBV BALF2 gene (162215C > A, 162476T > C, 163364C > T). Two dsDNA gene fragments served as either the high-risk or low-risk variant controls. Supernatant from the EBV-infected B95-8 cell line served as an additional wild-type whole-virus control. Positive clinical specimens sent for routine plasma EBV EBNA-1 testing were collected from patients with a variety of benign and neoplastic EBV-associated disorders. Three sets of conserved primers flanked the polymorphisms, with one allele-specific hydrolysis probe for each of the three high-risk alleles. A fourth allele-specific probe (162215-mt) served as an internal control. The 95% lower limit of detection (LLOD) was assessed in replicates of 20; linearity was assessed from 0.0-6.0 log10 cp/µL. Mutant allele frequencies from 0-100% were assessed by mixing dsDNA controls. <h3>Results</h3> The assay's limit of quantitation and 95% LLOD were 50 and 5 copies per well, respectively. At 100 copies per well, the assay could detect mutant allele frequencies as low as 5%, below the intra-host heterozygosity threshold. Wild-type B95-8 DNA confirmed assay specificity. Forty EBV-positive plasma specimens were genotyped from patients with non-transplant-associated viremia, post-transplant lymphoproliferative disorders, EBV-associated lymphomas (diffuse large-cell, Hodgkin, NK/T-cell, peripheral T-cell), and EBV-positive NPC. High-risk haplotypes were more common among patients with NPC (64% vs. 10%, <i>P</i> = 0.002). Compared to screening with plasma BamHI-W PCR alone, triaging screen-detected EBV DNA with this genotyping assay modestly reduced screening sensitivity (97% to 91%) but could decrease false positives and unnecessary nasoendoscopies by 63% in a modeled high-risk population. <h3>Conclusion</h3> Triaging screen-detected plasma EBV DNA with a multiplex BALF2 genotyping assay has the potential to decrease false positives and resource utilization in high-risk populations screened for NPC. This may be a low-cost and accessible alternative to higher-complexity sequencing assays in low- and middle-income countries.

Genetic variation in CYP1A1 and AHRR genes increase the risk of systemic lupus erythematosus and exacerbate disease severity in smoker patients.

Genetic variations of aryl hydrocarbon receptor (AHR) pathway genes could influence the imbalanced immune response to xenobiotics. Therefore, we aimed to investigate the polymorphism of AHR pathway genes in systemic lupus erythematosus (SLE) patients in association with smoking. Genomic DNA from patients (N = 107) and controls (N = 105) of a population from northeast of Iran was used for genotyping of CYP1A1 T>C (rs4646903) and AHRR C>G (rs2292596) variants. The SLEDAI score and smoking status of the patients were registered. The AHR activity was estimated by CYP1A1 and CYP1B1 geneexpression in peripheral blood mononuclear cells (PBMC). The C allele in rs4646903 (odds ratio [OR] = 2.67) and G allele in rs2292596 (OR = 1.79) SNPs were significantly associated with the increased risk of SLE. The AHR pathway was more active in high-risk CYP1A1/AHRR: C/G haplotype. The most severe disease was observed in smoker patients with high-risk haplotype and both smoking (Exp (β) = 9.5) and high-risk CYP1A1/AHRR (C/G) haplotype (Exp (β) = 3.7) can significantly increase the likelihood of having severe (SLEDAI ≥ 20) SLE disease activity. Our findings indicated the association of xenobiotic-metabolizing genes (CYP1A1, AHRR) polymorphisms with the susceptibility to SLE and disease severity regarding the smoking background, suggesting the interaction of gene and environmental risk factors in SLE pathogenesis.

DR15-DQ6 remains dominantly protective against type 1 diabetes throughout the first five decades of life

Aims/hypothesisAmong white European children developing type 1 diabetes, the otherwise common HLA haplotype DR15-DQ6 is rare, and highly protective. Adult-onset type 1 diabetes is now known to represent more overall cases than childhood onset, but it is not known whether DR15-DQ6 is protective in older-adult-onset type 1 diabetes. We sought to quantify DR15-DQ6 protection against type 1 diabetes as age of onset increased.MethodsIn two independent cohorts we assessed the proportion of type 1 diabetes cases presenting through the first 50 years of life with DR15-DQ6, compared with population controls. In the After Diabetes Diagnosis Research Support System-2 (ADDRESS-2) cohort (n = 1458) clinician-diagnosed type 1 diabetes was confirmed by positivity for one or more islet-specific autoantibodies. In UK Biobank (n = 2502), we estimated type 1 diabetes incidence rates relative to baseline HLA risk for each HLA group using Poisson regression. Analyses were restricted to white Europeans and were performed in three groups according to age at type 1 diabetes onset: 0–18 years, 19–30 years and 31–50 years.ResultsDR15-DQ6 was protective against type 1 diabetes through to age 50 years (OR < 1 for each age group, all p < 0.001). The following ORs for type 1 diabetes, relative to a neutral HLA genotype, were observed in ADDRESS-2: age 5–18 years OR 0.16 (95% CI 0.08, 0.31); age 19–30 years OR 0.10 (0.04, 0.23); and age 31–50 years OR 0.37 (0.21, 0.68). DR15-DQ6 also remained highly protective at all ages in UK Biobank. Without DR15-DQ6, the presence of major type 1 diabetes high-risk haplotype (either DR3-DQ2 or DR4-DQ8) was associated with increased risk of type 1 diabetes.Conclusions/interpretationHLA DR15-DQ6 confers dominant protection from type 1 diabetes across the first five decades of life.Graphical abstract

Probiotic Supplementation with Lactobacillus plantarum 299v Lowers Systemic Inflammation, Reduces Islet ER Stress and Prevents Type 1 Diabetes in BioBreeding Rats

Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of the pancreatic β-cells. T1D pathogenesis has a strong genetic basis. However, in recent decades, the prevalence of high-risk HLA haplotypes among new diagnoses has declined, the age of onset has decreased, and T1D incidence has increased. These changes are consistent with increased environmental pressure and coincide with introduction of the Western diet, widespread antibiotic use and reduced breast feeding. These factors are thought to drive intestinal dysbiosis, increased gut permeability and systemic inflammation. Notably, our studies of T1D families and the BioBreeding (BB) rat have identified a peripheral inflammatory state associated with diabetes susceptibility that is consistent with microbial antigen exposure and pattern recognition receptor ligation. Lactobacillus plantarum 299v (Lp299v), a probiotic strain, is reported to increase plasma and stool levels of anti-inflammatory short chain fatty acids (SCFA) and promote IL-10 signaling in colonic derived macrophages and T-cells. Here we investigated the effect of Lp299v supplement on T1D progression and inflammatory phenotypes in diabetes prone BB DRlyp/lyp rats. Rats were weaned at 21 days onto a normal cereal diet (ND) or a gluten-free hydrolyzed casein diet (HCD), with and without daily Lp299v supplementation. All DRlyp/lyp ND rats developed T1D by day 83 (mean time to onset of 62.8+/-7.9 days). DRlyp/lyp ND+Lp299v rats exhibited an insignificant delay in T1D onset (62.6+/-6.5 days), however 8% remained diabetes-free to day 130. Providing DRlyp/lyp rats HCD prevented T1D in 17% of rats (to age 130 days) and significantly delayed onset (mean time to onset 72.8+/-7.3 days, p<0.001). Providing DRlyp/lyp rats HCD+Lp299v prevented T1D in 25% of rats and more robustly delayed onset (mean time to onset 84.9 +/-14.3 days, p<0.001). While multiplex ELISA failed to detect significantly altered plasma cytokine/chemokine levels at 40 days of life, plasma induced transcription revealed the greatest normalization of systemic inflammation in the HCD+Lp299v group. Plasma SCFA levels (propionate and butyrate, p<0.01) were elevated in the HCD+Lp299v group compared to the ND group. Global gene expression analysis of pancreatic islets was conducted at 40 days, prior to insulitis. Endoplasmic reticulum (ER) stress has been implicated in the formation of islet neoantigens that may underlie the initial loss of immune tolerance in T1D. Under one or both diets, Lp299v favorably modulated islet expression levels of pathways and transcripts related to inflammation and innate immunity (Cxcl9, Cxcl10), oxidative stress (Gsta1, Gsta4, Gstp1, Gstk1), as well as ER stress and unfolded protein response (Cirbp, Edem1, Hspa1a, Atf4). These ongoing studies add to a growing understanding that inherited susceptibility can be modulated by diet and microbiota.

Residual β cell function in long-term type 1 diabetes associates with reduced incidence of hypoglycemia.

BACKGROUNDWe investigated residual β cell function in Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study participants with an average 35-year duration of type 1 diabetes mellitus (T1DM).METHODSSerum C-peptide was measured during a 4-hour mixed-meal tolerance test. Associations with metabolic outcomes and complications were explored among nonresponders (all C-peptide values after meal <0.003 nmol/L) and 3 categories of responders, classified by peak C-peptide concentration (nmol/L) as high (>0.2), intermediate (>0.03 to ≤0.2), and low (≥ 0.003 to ≤0.03).RESULTSOf the 944 participants, 117 (12.4%) were classified as responders. Residual C-peptide concentrations were associated with higher DCCT baseline concentrations of stimulated C-peptide (P value for trend = 0.0001). Residual C-peptide secretion was not associated with current or mean HbA1c, HLA high-risk haplotypes for T1DM, or the current presence of T1DM autoantibodies. The proportion of subjects with a history of severe hypoglycemia was lower with high (27%) and intermediate (48%) residual C-peptide concentrations than with low (74%) and no (70%) residual C-peptide concentrations (P value for trend = 0.0001). Responders and nonresponders demonstrated similar rates of advanced microvascular complications.CONCLUSIONβ Cell function can persist in long-duration T1DM. With a peak C-peptide concentration of >0.03 nmol/L, we observed clinically meaningful reductions in the prevalence of severe hypoglycemia.TRIAL REGISTRATIONClinicalTrials.gov NCT00360815 and NCT00360893.FUNDINGDivision of Diabetes Endocrinology and Metabolic Diseases of the National Institute of Diabetes and Digestive and Kidney Diseases (DP3-DK104438, U01 DK094176, and U01 DK094157).