Distinct Alleles Research Articles

Introgression of ASIP and TYRP1 Alleles Explains Coat Color Variation in Valais Goats.

The Valais Blackneck goat is a Swiss goat breed with a characteristic coat color phenotype. Before the revision of the breed standard in 1938, 4 different color varieties of Valais goats were known. Besides Blackneck animals resembling the modern breed standard, the brown and white Copperneck goat, the white Capra Sempione, and the greyish Grüenochte comprised the historic Valais goats. The brown pigmentation of Copperneck goats had previously been traced back to an introgression of a mutant TYRP1 allele from Toggenburg goats. In the present study, we identified additional introgression events of distinct ASIP alleles causing the remaining 2 rare coat color patterns within the Valais Blackneck goat breed. We identified the introgression of the AWt allele from Appenzell or Saanen goats in white Capra Sempione goats. Similarly, introgression of the Apc allele from Peacock goats resulted in the greyish Grüenochte phenotype. These results demonstrate past hybridization events between breeds that are separated today. A perfect genotype-phenotype association in 393 Valais goats supported the causality of the genotyped variants for the different coat color phenotypes. Our study gives insights into the introgression of functionally relevant copy number variant (CNV) alleles controlling pigmentation between goat breeds with strikingly different coat color patterns.

SCN5A compound heterozygosity mutation in Brugada syndrome: Functional consequences and the implication for pharmacological treatment

AimsSCN5A gene encodes the α-subunit of Nav1.5, mainly found in the human heart. SCN5A variants are the most common genetic alterations associated with Brugada syndrome (BrS). In rare cases, compound heterozygosity is observed; however, its functional consequences are poorly understood. We aimed to analyze the functional impact of de novo Nav1.5 mutations in compound heterozygosity in distinct alleles (G400R and T1461S positions) previously found in a patient with BrS. Moreover, we evaluated the potential benefits of quinidine to improve the phenotype of mutant Na+ channels in vitro. Materials and methodsThe functional properties of human wild-type and Nav1.5 variants were evaluated using whole-cell patch-clamp and immunofluorescence techniques in transiently expressed human embryonic kidney (HEK293) cells. Key findingsBoth variants occur in the highly conservative positions of SCN5A. Although all variants were expressed in the cell membrane, a significant reduction in the Na+ current density (except for G400R alone, which was undetected) was observed along with abnormal biophysical properties, once the variants were expressed in homozygosis and heterozygosis. Interestingly, the incubation of transfected cells with quinidine partially rescued the biophysical properties of the mutant Na+ channel. SignificanceDe novo compound heterozygosis mutations in SNC5A disrupt the Na+ macroscopic current. Quinidine could partially reverse the in vitro loss-of-function phenotype of Na+ current. Thus, our data provide, for the first time, a detailed biophysical characterization of dysfunctional Na+ channels linked to compound heterozygosity in BrS as well as the benefits of the pharmacological treatment using quinidine on the biophysical properties of Nav1.5.

Genetic diversity, forensic characteristic and phylogenetic analysis of the Qiongzhong aborigines residing in the tropical rainforests of Hainan Island via 19 autosomal STRs

Background The genetic landscape of the Qiongzhong aborigines, who reside in ‘the Heart of Hainan’, is still unclear. The Goldeneye™ DNA ID System 20A is available for forensic and population genetics applications. Aim To obtain genetic polymorphisms of 19 autosomal STR loci in the Qiongzhong aborigines, and to explore the genetic relationships with a total of 69,132 people from forty-five populations. Subjects and methods 724 Qiongzhong aborigines genotyped 19 autosomal STRs and phylogenetic relationships conducted by multidimensional scaling analysis (MDS), principal component analysis (PCA) and neighbor-joining (N-J) phylogenetic tree construction. Results No evidence of deviation from Hardy-Weinberg equilibrium was identified. A total of 233 distinct alleles were observed with allele frequencies ranging from 0.0007 to 0.5375. The combined power of discrimination (CPD) and combined power of exclusion (CPE) for the 19 autosomal STR loci were 1- 8.28 × 10−34 and 0.999999987, respectively. Conclusion Our phylogenetic results demonstrated that a) the populations of Southeast Asian countries have thorough integrations with southern China in terms of ethnicity and genetics due to long-term cultural and trade exchanges and b) based on genetic and linguistic analysis, the Qiongzhong aborigines have a close relationship with Fujian Han Chinese. Highlights The STR landscape of Qiongzhong aborigines inhabited in Hainan tropical rainforests was depicted by 19 autosomal STRs. A total of 69,132 people from forty-five populations were selected for a more extensive examination of genetic similarities and differences by multivariate statistical methods (MDS, PCA and N-J tree construction). The genetic analyses indicated that the populations of Southeast Asian countries are very genetically close to southern Chinese populations. From the genetic and linguistic perspective, the Qiongzhong aborigines has a close relationship with Han Chinese from Fujian Province.

Computational Treatment Simulations to Assess the Need for Personalized Tamoxifen Dosing in Breast Cancer Patients of Different Biogeographical Groups.

Simple SummaryTamoxifen is a drug often used to treat the most common type of breast cancer. Its metabolite endoxifen is formed by the liver enzyme CYP2D6, whose activity is variable and depends on a patient’s genetic profile. The frequency of CYP2D6 variants with different functional enzymatic activity varies largely between populations. To ensure sufficient efficacy of tamoxifen, a certain target concentration of endoxifen is needed, and 20% of White breast cancer patients have been shown not to reach this target concentration. However, little is known about the risk of not attaining the endoxifen target amongst other ethnic populations. This study investigated the risk for suboptimal endoxifen concentration in nine different biogeographical populations based on their distinct CYP2D6 genetic profile. The variability between the populations was high (up to three-fold), and East Asian breast cancer patients were identified as the population with the highest need for personalized tamoxifen dosing.Tamoxifen is used worldwide to treat estrogen receptor-positive breast cancer. It is extensively metabolized, and minimum steady-state concentrations of its metabolite endoxifen (CSS,min ENDX) >5.97 ng/mL have been associated with favorable outcome. Endoxifen formation is mediated by the enzyme CYP2D6, and impaired CYP2D6 function has been associated with lower CSS,min ENDX. In the Women’s Healthy Eating and Living (WHEL) study proposing the target concentration, 20% of patients showed subtarget CSS,min ENDX at tamoxifen standard dosing. CYP2D6 allele frequencies vary largely between populations, and as 87% of the patients in the WHEL study were White, little is known about the risk for subtarget CSS,min ENDX in other populations. Applying pharmacokinetic simulations, this study investigated the risk for subtarget CSS,min ENDX at tamoxifen standard dosing and the need for dose individualization in nine different biogeographical groups with distinct CYP2D6 allele frequencies. The high variability in CYP2D6 allele frequencies amongst the biogeographical groups resulted in an up to three-fold difference in the percentages of patients with subtarget CSS,min ENDX. Based on their CYP2D6 allele frequencies, East Asian breast cancer patients were identified as the population for which personalized, model-informed precision dosing would be most beneficial (28% of patients with subtarget CSS,min ENDX).

KIR and HLA class I diversity in Pakistani and Yadav populations: a high-resolution study of immunogenetic diversity in South Asians

Abstract Killer cell immunoglobulin-like receptors (KIR) modulate the activity of human NK cells by interacting with HLA class I ligands, influencing their response to pathogens, cancer and during pregnancy. Combinatorial diversity of HLA and KIR allotypes impacts NK cell functions in health and disease. South Asians are under-studied for this immunogenetic diversity, despite representing almost one quarter of the world’s population. We analyzed KIR and HLA distribution of a cohort of healthy subjects comprising 81 from Karachi in Pakistan and 71 Yadav from Tamil Nadu in Southern India. To overcome genomic region complexity, we used our high throughput NGS and bioinformatics pipeline to derive high resolution gene content and allele level KIR and HLA data. We also developed a Cas9-targeted long read method to fully sequence specific structurally divergent KIR haplotypes. We identified 110 distinct HLA class I alleles in the Karachi population and 80 in the Yadav. We observed 96% individuals to have unique HLA class I genotypes and three were shown to be homozygous across all three HLA class I genes. While overall HLA diversity is lower in the Yadav compared to the Karachi, both encode approximately two KIR ligands per HLA haplotype. We identified 125 distinct KIR alleles, including 11 characterized for the first time in this study. We identified and sequenced multiple KIR haplotypes having large-scale deletions or duplications of complete genes. Most haplotypes were observed in more than one individual, but every individual studied has a unique KIR genotype. This is the first high-resolution study of KIR and HLA class I in a South Asian population, and this will lay the foundation for further immunogenic studies of subjects from this geographic region.

31. Maternal chromosome 18 meiosis II nondisjunction mosaic double trisomy rescue inferred from prenatal chromosomal microarray SNP data

SNP microarrays have the ability to detect imbalances in alleles due to cytogenetically invisible mechanisms, such as copy neutral regions of homozygosity. These regions can unmask recessive disease alleles and may also provide evidence of uniparental disomy (UPD) and the potential risk for imprinting disorders. UPD can arise from rescue of an aneuploid cell line, which may indicate the presence of occult aneuploid mosaicism. Here we report the results of SNP microarray performed on uncultured amniocytes of a 19 weeks gestational age fetus carried by a mother of advanced age with history of recurrent miscarriages. The pregnancy was uneventful and there were no abnormalities observed on ultrasound. The microarray showed a distinct allele pattern on chromosome 18 (CHR.18) with segments having seven B allele frequency tracks interrupted by segments having the expected normal three B allele frequency tracks, suggestive of greater than expected allele combinations. FISH to rule out trisomy 18 mosaicism in 200 nuclei was normal. By modeling the expected allele frequency values under different meiotic nondisjunction hypotheses, we determined that the data observed in the microarray is most consistent with a meiosis II nondisjunction event resolved by double trisomy rescue to two biparental cell lines. Further, the rescue events occurred at different times resulting in mixed proportions of the two cell lines. Parental SNP data revealed that the extra CHR.18 alleles were of maternal origin. This data is consistent with the known origins of CHR.18 aneuploidy, which is most often due to maternal meiosis II nondisjunction. The baby was born healthy and FISH on trophoblastic tissue from two different sites revealed two copies of CHR.18. This case highlights the power of SNP microarray to detect and infer rare events such as double trisomy rescue to two biparental cell lines, which allowed the family to be counseled appropriately.

Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as “headgear,” which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

'Off-the-shelf’ allogeneic antigen-specific adoptive T-cell therapy for the treatment of multiple EBV-associated malignancies

BackgroundEpstein-Barr virus (EBV), an oncogenic human gammaherpesvirus, is associated with a wide range of human malignancies of epithelial and B-cell origin. Recent studies have demonstrated promising safety and clinical efficacy...

The Clinical Utility of Determining the Allelic Background of Mutations Causing Alpha-1 Antitrypsin Deficiency: The Case with the Null Variant Q0(Mattawa)/Q0(Ourém).

Alpha-1 antitrypsin deficiency (AATD) is caused by genetic variants in the SERPINA1 gene conferring risk of developing emphysema. The clinical expression of AATD-related emphysema mostly occurs in carriers of 2 deficient alleles. By DNA sequencing of SERPINA1, numerous rare variants have been identified. Clarifying whether 2 mutations observed in 1 patient are on the same or distinct alleles has obvious clinical implications. We studied 7 carriers of a rare variant, Leu353Phe_fsTer24, known to lead to undetectable serum levels of AAT. Two of them were also carriers of the S or Z allele. We developed an allele-specific DNA sequencing method to characterize the allelic background of the Leu353Phe_fsTer24 variant. The Leu353Phe_fsTer24 variant was transmitted on the same allele as the M3 variant (E376D) in all patients. This mutation is thus named Q0Ourém on the conventional PI system. We demonstrated that individuals harboring the E264V (S) and E342K (Z) mutations had them on distinct alleles from Q0Ourém and are, thus, compound heterozygotes. The 7 Q0Ourém carriers had AAT levels ranging from 0.18g/l to 0.82g/l. The lowest AAT serum levels were observed in compound heterozygotes (S/Q0Ourém and Z/Q0Ourém) suggesting higher risk of developing emphysema. For the 7 patients, Leu353Phe_fsTer24 is transmitted on the M3 background and they are, thus, carriers of the Q0Ourém allele. Allele-specific DNA sequencing was useful to distinguish 1 or 2 deficient alleles in carriers of 2 mutations. In rare cases, this method is important to understand the clinical significance of genetic variants found in SERPINA1.

Assessment of Somaclonal Variations in Embryo-derived Axillary Shoots of Chickpea using Molecular Markers

Background: Somaclonal variation is considered as a source of genetic variation for crop improvement. It has been investigated using cytological, biochemical and molecular techniques.Methods: Genetic stability in the embryo-derived axillary shoots of 4 chickpea genotypes was assessed using eight inter-simple sequence repeat (ISSR) and 19 random amplified polymorphic DNA (RAPD) primers. Result: RAPD primers produced 94 and ISSR primers produced 38 distinct and scorable alleles, with an average of 4.9 alleles for RAPD and 4.75 for ISSR primers. The polymorphic information content (PIC) ranged from 0.36 to 0.90 for RAPD and from 0.50 to 0.87 for ISSR. ISSR recognized a 90%, but RAPD recognized 82% similarity value. No absolute similarity value was between the mother plant and the regenerated shoots for the overall genotypes. At a 90% similarity value, 15 out of the 20 regenerated shoots from ‘Giza 88’ group with their mother plant using ISSR markers; however, 11 regenerated shoots grouped with their mother plant in one central cluster for ‘Giza 4’ using RAPD markers. The observed variations in the total number of polymorphic RAPD and ISSR bands and the number of bands specific to the mother and regenerated shoots, detected intra-clonal variation and genetic instability seem to be genotype-dependent.

Inferring the Allelic Series at QTL in Multiparental Populations.

Multiparental populations (MPPs) are experimental populations in which the genome of every individual is a mosaic of known founder haplotypes. These populations are useful for detecting quantitative trait loci (QTL) because tests of association can leverage inferred founder haplotype descent. It is difficult, however, to determine how haplotypes at a locus group into distinct functional alleles, termed the allelic series. The allelic series is important because it provides information about the number of causal variants at a QTL and their combined effects. In this study, we introduce a fully Bayesian model selection framework for inferring the allelic series. This framework accounts for sources of uncertainty found in typical MPPs, including the number and composition of functional alleles. Our prior distribution for the allelic series is based on the Chinese restaurant process, a relative of the Dirichlet process, and we leverage its connection to the coalescent to introduce additional prior information about haplotype relatedness via a phylogenetic tree. We evaluate our approach via simulation and apply it to QTL from two MPPs: the Collaborative Cross (CC) and the Drosophila Synthetic Population Resource (DSPR). We find that, although posterior inference of the exact allelic series is often uncertain, we are able to distinguish biallelic QTL from more complex multiallelic cases. Additionally, our allele-based approach improves haplotype effect estimation when the true number of functional alleles is small. Our method, Tree-Based Inference of Multiallelism via Bayesian Regression (TIMBR), provides new insight into the genetic architecture of QTL in MPPs.

Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.

Clonal Hematopoiesis Before, During, and After Human Spaceflight

SUMMARYClonal hematopoiesis (CH) occurs when blood cells harboring an advantageous mutation propagate faster than others. These mutations confer a risk for hematological cancers and cardiovascular disease. Here, we analyze CH in blood samples from a pair of twin astronauts over 4 years in bulk and fractionated cell populations using a targeted CH panel, linked-read whole-genome sequencing, and deep RNA sequencing. We show CH with distinct mutational profiles and increasing allelic fraction that includes a high-risk, TET2 clone in one subject and two DNMT3A mutations on distinct alleles in the other twin. These astronauts exhibit CH almost two decades prior to the mean age at which it is typically detected and show larger shifts in clone size than age-matched controls or radiotherapy patients, based on a longitudinal cohort of 157 cancer patients. As such, longitudinal monitoring of CH may serve as an important metric for overall cancer and cardiovascular risk in astronauts.

Revealing Distinctions in Genetic Diversity and Adaptive Evolution Between Two Varieties of Camellia sinensis by Whole-Genome Resequencing

Camellia sinensis var. sinensis (CSS) and C. sinensis var. assamica (CSA) are the two most economically important tea varieties. They have different characteristics and geographical distribution. Their genetic diversity and differentiation are unclear. Here, we identified 18,903,625 single nucleotide polymorphisms (SNPs) and 7,314,133 insertion–deletion mutations (indels) by whole-genome resequencing of 30 cultivated and three wild related species. Population structure and phylogenetic tree analyses divided the cultivated accessions into CSS and CSA containing 6,440,419 and 6,176,510 unique variations, respectively. The CSS subgroup possessed higher genetic diversity and was enriched for rare alleles. The CSA subgroup had more non-synonymous mutations and might have experienced a greater degree of balancing selection. The evolution rate (dN/dS) and KEGG enrichment indicated that genes involved in the synthesis and metabolism of flavor substances were positively selected in both CSS and CSA subpopulations. However, there are extensive genome differentiation regions (2959 bins and approximately 148 M in size) between the two subgroups. Compared with CSA (141 selected regions containing 124 genes), the CSS subgroup (830 selected regions containing 687 genes) displayed more selection regions potentially related to environmental adaptability. Fifty-three pairs of polymorphic indel markers were developed. Some markers were located in hormone-related genes with distinct alleles in the two cultivated subgroups. These identified variations and selected regions provide clues for the differentiation and adaptive evolution of tea varieties. The newly developed indel markers will be valuable in further genetic research on tea plants.

Mutant RNA polymerase can reduce susceptibility to antibiotics via ppGpp-independent induction of a stringent-like response.

BackgroundMutations in RNA polymerase (RNAP) can reduce susceptibility to ciprofloxacin in Escherichia coli, but the mechanism of transcriptional reprogramming responsible is unknown. Strains carrying ciprofloxacin-resistant (CipR) rpoB mutations have reduced growth fitness and their impact on clinical resistance development is unclear.ObjectivesTo assess the potential for CipRrpoB mutations to contribute to resistance development by estimating the number of distinct alleles. To identify fitness-compensatory mutations that ameliorate the fitness costs of CipRrpoB mutations. To understand how CipRrpoB mutations reprogramme RNAP.Methods E. coli strains carrying five different CipRrpoB alleles were evolved with selection for improved fitness and characterized for acquired mutations, relative fitness and MICCip. The effects of dksA mutations and a ppGpp0 background on growth and susceptibility phenotypes associated with CipRrpoB alleles were determined.ResultsThe number of distinct CipRrpoB mutations was estimated to be >100. Mutations in RNAP genes and in dksA can compensate for the fitness cost of CipRrpoB mutations. Deletion of dksA reduced the MICCip for strains carrying CipRrpoB alleles. A ppGpp0 phenotype had no effect on drug susceptibility.ConclusionsCipRrpoB mutations induce an ppGpp-independent stringent-like response. Approximately half of the reduction in ciprofloxacin susceptibility is caused by an increased affinity of RNAP to DksA while the other half is independent of DksA. Stringent-like response activating mutations might be the most diverse class of mutations reducing susceptibility to antibiotics.

Functional Characterization of Compound DDX41 Germline and Somatic R525H Mutations in the Development of Myeloid Malignancies

DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~7 % of sporadic cases of high-risk MDS, conferring the largest germline risk for myeloid malignancies. In typical cases, a germline loss-of-function allele (most commonly p.A500fs or p.D140fs, depending on the ethnicity) is compounded by a somatic missense mutation affecting the helicase domain in the remaining allele (p.R525H). However, the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms have not been elucidated. To clarify the role of these distinct DDX41 alleles, we generated mice models carrying either or both of conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles. Vav1-Cre mediated homozygous deletion of Ddx41 resulted in embryonic lethality, suggesting that Ddx41 is indispensable for normal hematopoiesis. Next, by crossing these mice and further breeding with Rosa26-CreERT2 transgenic mice, we engineered mice that were wild-type for Ddx41 (Ddx41+/+), heterozygous Ddx41 KO (Ddx41+/-), heterozygous for the Ddx41 R525H mutation (Ddx41R525H/+), or hemizygous for the Ddx41 R525H mutation (Ddx41R525H/-), in which expression of the mutant allele was induced by tamoxifen administration. First, we assessed cell intrinsic effects of these Ddx41 alleles, using noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were reconstituted with BM from Ddx41R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF) with no development of myeloid neoplasms. However, about 20% of mice transplanted with BM derived from Ddx41R525H/- mice survived longer without showing BMF. These mice exhibited macrocytic anemia and increased platelet counts four months after tamoxifen-induction. In contrast, mice transplanted with BM from Ddx41+/- and Ddx41R525H/+ animals showed increased white blood cell counts compared to those with BM from Ddx41+/+ mice. In flow cytometry, Ddx41R525H/--derived BM-transplanted mice showed a significant increase in the number of long-term and short-term hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs) and granulocyte/macrophage lineage-restricted progenitors (GMPs), compared to those transplanted with BM from Ddx41+/+, Ddx41+/- or Ddx41R525H/+ mice. Single cell RNA-seq of lineage negative cell fractions from these mice also revealed expanded stem cell fractions in mice transplanted with BM from Ddx41R525H/- mice, even though there was impaired formation of mature peripheral blood cells, which was suggestive of impaired HSPC differentiation. We also assessed the reconstitution capacity of whole BM cells from different Ddx41 mutant mice in competitive transplantation experiments. The donor chimerism of Ddx41R525H/- mice-derived cells in PB was reduced compared to that of cells derived from Ddx41+/+, Ddx41+/- or Ddx41R525H/+ mice. Transcriptome analysis of stem cells (Kit+Sca-1-Linlow cells) from different Ddx41 mutant mice revealed significant changes in gene expression and splicing patterns in many genes in stem cells from all the mutant mice, with larger changes for Ddx41R525H/- than Ddx41+/- or Ddx41 R525H/+ cells. Notably, Ddx41R525H/- cells exhibited a significant upregulation of genes involved in innate immunity, whereas there was a downregulation of genes related to RNA metabolism and ribosome biogenesis. Proteomics analysis confirmed the significant downregulation of ribosomal proteins in hematopoietic cells derived from Ddx41R525H/- mice. In summary, our results revealed an essential role of Ddx41 in normal hematopoiesis. While both heterozygous Ddx41 KO and heterozygous R525H knock-in did not develop myeloid neoplasm, compound biallelic loss-of function and R525 alleles led to a compromised function of hematopoietic stem cells, which was evident from reduced competitive repopulation capacity and impaired hematopoietic differentiation, where activated innate immunity and impaired ribosome functions may play important roles. Their roles in myeloid neoplasms need further evaluation. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Kataoka:Takeda Pharmaceutical Company: Research Funding; Asahi Genomics: Current equity holder in private company; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Otsuka Pharmaceutical: Research Funding. Ogawa:KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company.

Towards Identifying the Target of Autoimmunity in Aplastic Anemia

Acquired aplastic anemia (AA) is an autoimmune bone marrow aplasia caused by T cell-mediated destruction of hematopoietic stem and progenitor cells. The antigenic target in AA remains unknown. Recently, we and others identified frequent somatic loss of function of several human leukocyte antigen (HLA) class I alleles in hematopoietic cells that survive the AA immune attack, suggesting these alleles ("risk alleles") present autoantigen in the affected patients. We hypothesize that risk alleles share structural features and peptide-binding characteristics that may inform our understanding of the immune mechanism of AA. To identify additional AA HLA class I risk alleles, we have partnered with two multi-institutional consortia, the North American Pediatric Aplastic Anemia Consortium (NAPAAC) and the Center for International Blood and Marrow Transplant Research (CIBMTR), to evaluate 507 AA patients for somatic HLA loss. Using a combination of targeted massively parallel sequencing of HLA class I genes and single nucleotide polymorphism array genotyping, we identified HLA loss in 19% of the NAPAAC cohort (30 of 156 unselected AA patients) and 13% of the CIBMTR cohort (46 of 351 patients selected to maximize discovery of less common risk alleles). Copy number-neutral loss of heterozygosity of chromosome arm 6p (6p CN-LOH) was the most common cause of HLA loss, occurring in 50 patients (10% of total cohort, 12% NAAPAC and 9% CIBMTR). Somatic mutations were present in 45 patients (9% of total cohort, 12% of NAPAAC and 7% CIBMTR). Patients with HLA loss had a median of 1 mutant clone per patient (range 1-7). Of the somatic mutations, 80% were predicted to disrupt expression of the affected allele (through loss of start, nonsense, or frameshift mutations). The other 20% were missense mutations affecting residues in the peptide binding groove (6 mutations) and the α3 domain (9 mutations) of the HLA class I protein. A total of 19 distinct AA HLA risk alleles were identified, of which 13 were newly identified in this study. Several risk alleles (HLA-B*14:02, HLA-B*40:02, HLA-A*02:01) were enriched in AA patients compared to ethnicity matched controls. Among the 507 patients in the study, at least one risk allele was present in 436/493 (88%) of AA patients and in 34/39 (87%) of the 6p CN-LOH events. Mutations clustered in several groups of alleles (supertypes) known to bind overlapping peptide repertoires, with alleles in B27 and B44 supertypes being most commonly affected. There were no mutations in A01, A01A03, A01A24, A24, B58, B62, or in any of the HLA-C alleles analyzed. Using several metrics to estimate the strength of the autoimmune selection on a given allele, including the frequency of somatic loss and similarity to other HLA risk alleles, we developed an AA HLA risk allele pathogenicity index ranging from very high (B*14:02 and B*40:02) and high pathogenicity (A*33:03, B*08:01, B*13:02, B*14:01, B*27:03, B*27:05, B*38:02, B*41:02 and B*49:01) to those unlikely to be pathogenic. Interestingly, even within high risk supertypes such as B44, there was a wide variation in predicted pathogenicity, ranging from high (for B*40:02 and B*41:02) to low pathogenicity (for B*44:02 and B*44:03), suggesting differences in autopeptide binding. Our study provides a comprehensive analysis of AA HLA risk alleles in a large diverse cohort of AA patients. Our results suggest that HLA risk alleles have shared autoantigen binding specificities that define their pathogenicity in AA, which can be used to identify candidate AA autoantigens. HLA risk allele pathogenicity may have future clinical utility as an adjunctive diagnostic test, as well as for prognostic assessment and haploidentical donor selection. Disclosures Lee: AstraZeneca: Research Funding; Kadmon: Research Funding; Takeda: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Syndax: Research Funding; Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Research Funding. Monos:Omixon: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Disentangling the Effects of HLA DRB1*15:01 and DQB1*06:02 to Establish the True HLA Risk Allele for Inhibitor Development in the Treatment of Hemophilia A

The HLA haplotype DRB1*15:01/DQB1*06:02 has been shown to be associated with the development of inhibitory antibodies ("inhibitors") against therapeutic FVIII (tFVIII) proteins in Hemophilia A (HA) patients. Moreover, this same haplotype has also been implicated with an increased risk for the development of multiple sclerosis, narcolepsy, and drug-induced-hypersensitivity and -liver-injury. Recent reports in the literature have indicated that the alleles of this haplotype may have differential effects on immunogenicity despite the fact that they are in strong linkage disequilibrium (LD). Thus, the question arises as to which of the two constituent alleles of this haplotype is the true HLA risk allele. To address this question we analyzed peptidomic profiling data on HLA-class-II (HLAcII)-presented peptides from dendritic cell (DC)-protein processing and presentation assays (PPPAs) performed in two independent experiments performed by Peyron et al. (2018) and Diego et al. (2020) which reported results on both the HLA-DQ and -DR isomers. The tFVIII in both experiments is full-length recombinant FVIII (FL-rFVIII). We performed log-linear mixed model analyses under two models, where the dependent variable in both models is the logarithm of the expected peptide count. Under Model 1, we analyzed in the fixed effect component of the model a single HLA allele predictor variable consisting of 10 levels represented by distinct DRB1 and DQB1 alleles (4 and 6 levels, respectively). Under this model, we analyzed in the random effects component of the model variables for individuals (8 and 9 levels from Peyron et al. and Diego et al., respectively), experiments (2 levels for Peyron et al. and Diego et al.), and HLA isomers (2 levels for DQ and DR). The random effects component of the model serves to reduce the error (or "noise") in our estimation of the predictive effect of HLA alleles for the peptide counts by accounting for the clustering due to individuals, experiments, and isomers. This approach therefore can be understood as maximizing the signal-to-noise ratio. Model 2 was more focused in that the single HLA allele predictor variable in the fixed effect component consisted of only DQB1*06:02 and DRB1*15:01. Model 2 was also simpler in that we only accounted for two random effect variables, namely for individuals (in this case 2 and 6 levels for Peyron et al. and Diego et al., respectively) and experiments. Thus, Model 2 is a direct head-to-head comparison of the two main HLA alleles of interest. The Model 1 results are reported in Figure 1A and B, where it can be seen that relative to the baseline reference allele provided by DQB1*02:01, both DQB1*06:02 and DRB1*15:01 are at significantly increased risk of contributing to the overall peptide count. Results are reported as risk ratios (RRs) and their associated 95% confidence interval lower and upper bounds (95% CI LB and 95% CI UB). For DQB1*06:02 and DRB1*15:01 respectively, we found a RR (95% CI LB, 95% CI UB) of 1.76 (1.24, 2.50) and 14.16 (10.38, 19.33). Because they are compared to the same baseline, the two RRs may also be directly compared, thus showing that DRB1*15:01 contributes significantly more to the overall peptide count than DQB1*06:02. Under Model 2 (the head-to-head comparison), the RR for the DRB1*15:01 allele against the baseline DQB1*06:02 allele is 7.00 (5.80, 8.44) (Figure 2A and B). These results contribute to the field of precision medicine because they demonstrate that even in the case of alleles in tight LD, their effects can be effectively disentangled by a DC-PPPA and a log-linear mixed model analysis. Our results support the conclusion that in regard to the well-known risk haplotype of DQB1*06:02/DRB1*15:01, the true HLA risk allele seems to be DRB1*15:01. Disclosures Luu: Haplogenics Corporation: Current Employment. Hofmann:CSL Behring: Current Employment. Powell:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees. Dinh:Haplogenics Corporation: Current Employment. Escobar:National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maraskovsky:CSL Behring: Current Employment. Howard:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees.

High allelic diversity of the centromere-specific histone H3 (CENH3) in the legume sainfoin (Onobrychis viciifolia).

The centromere is a structurally and functionally specialized region on each eukaryotic chromosome and is essential for accurate and complete segregation of chromosomes during cell division. Centromeric nucleosomes differ from canonical nucleosomes by replacement of the histone H3 with its centromere-specific variant CENH3. CENH3 is essential for active centromeres in most eukaryotes. Homologs of CENH3 are identified in many organisms. Sainfoin (Onobrychis viciifolia) is an agriculturally important perennial forage and is a legume of the Fabaceae family. There is very limited information on the structure of the sainfoin genome and no data are available on its centromere structure. Here, we aim to characterize the sainfoin CENH3 homolog (OvCENH3). Using a sequence homology-based strategy with gene-specific primers, we were able to clone transcripts from sainfoin total RNA. The amplified clones were sequenced and compared by bioinformatics tools. Four distinct alleles of OvCENH3 were detected. Our study provides the first structural features on sainfoin centromeres with a possible allotetraploid origin for sainfoin. We discuss and compare our findings with that for other important legume species.

A novel algorithm comprehensively characterizes human RH genes using whole-genome sequencing data

AbstractRHD and RHCE genes encode Rh blood group antigens and exhibit extensive single-nucleotide polymorphisms and chromosome structural changes in patients with sickle cell disease (SCD). RH variation can drive loss of antigen epitopes or expression of new epitopes, predisposing patients with SCD to Rh alloimmunization. Serologic antigen typing is limited to common Rh antigens, necessitating a genetic approach to detect variant antigen expression. We developed a novel algorithm termed RHtyper for RH genotyping from existing whole-genome sequencing (WGS) data. RHtyper determined RH genotypes in an average of 3.4 and 3.3 minutes per sample for RHD and RHCE, respectively. In a validation cohort consisting of 57 patients with SCD, RHtyper achieved 100% accuracy for RHD and 98.2% accuracy for RHCE, when compared with genotypes obtained by RH BeadChip and targeted molecular assays and after verification by Sanger sequencing and independent next-generation sequencing assays. RHtyper was next applied to WGS data from an additional 827 patients with SCD. In the total cohort of 884 patients, RHtyper identified 38 RHD and 28 RHCE distinct alleles, including a novel RHD DAU allele, RHD* 602G, 733C, 744T 1136T. RHtyper provides comprehensive and high-throughput RH genotyping from WGS data, facilitating deconvolution of the extensive RH genetic variation among patients with SCD. We have implemented RHtyper as a cloud-based public access application in DNAnexus (https://platform.dnanexus.com/app/RHtyper), enabling clinicians and researchers to perform RH genotyping with next-generation sequencing data.