Paternal Haplotypes Research Articles

Paternal HLA-Derived Epitopes and Live Birth in SecondaryRecurrent Pregnancy Loss: New Insights FromaClinical Trial.

Recurrent pregnancy loss (RPL), defined as two or more pregnancy losses before the 24th week of gestation, affects 1%-3% of women worldwide. Approximately, 40% of RPL cases are secondary RPL (sRPL), where women have given birth before facing pregnancy losses. The underlying causes of RPL remain unclear, but immune-related factors may play a role. Previously, a randomised controlled trial using immunoglobulin (IVIG) in sRPL women with a history of four pregnancy losses performed in our RPL unit did not show significant effects of IVIG treatment overall. Yet, some evidence suggests potential benefits for a subset of sRPL patients. In the cohort used for the randomised controlled trial, we examined the role of maternal HLA class II-presented fetal HLA-derived epitopes in sRPL using the predicted indirectly recognisable HLA epitopes (PIRCHE-II) algorithm. In the placebo group, sRPL mothers with an anti-HLA antibody response had higher PIRCHE-II scores when having a live birth compared with sRPL women who experienced another pregnancy loss. This difference was not observed in the IVIG-treated group. Furthermore, as a proxy for T-cell memory, the number of overlapping peptides between the two paternal haplotypes in couples having live births without treatment displayed a larger number of overlapping peptides. This effect was primarily driven by class II-derived peptides. These results suggest that specific combinations of sRPL mothers and fathers, particularly those with an anti-HLA antibody response, may generate higher PIRCHE-II scores, which could contribute to successful live births. Understanding these immune interactions may provide insights for personalised diagnostic and therapeutic strategies in sRPL.

Genetic complexity of killer-cell immunoglobulin-like receptor genes in human pangenome assemblies.

The killer-cell immunoglobulin-like receptor (KIR) gene complex, a highly polymorphic region of the human genome that encodes proteins involved in immune responses, poses strong challenges in genotyping owing to its remarkable genetic diversity and structural intricacy. Accurate analysis of KIR alleles, including their structural variations, is crucial for understanding their roles in various immune responses. Leveraging the high-quality genome assemblies from the Human Pangenome Reference Consortium (HPRC), we present a novel bioinformatic tool, the structural KIR annoTator (SKIRT), to investigate gene diversity and facilitate precise KIR allele analysis. In 47 HPRC-phased assemblies, SKIRT identifies a recurrent novel KIR2DS4/3DL1 fusion gene in the paternal haplotype of HG02630 and maternal haplotype of NA19240. Additionally, SKIRT accurately identifies eight structural variants and 15 novel nonsynonymous alleles, all of which are independently validated using short-read data or quantitative polymerase chain reaction. Our study has discovered a total of 570 novel alleles, among which eight haplotypes harbor at least one KIR gene duplication, six haplotypes have lost at least one framework gene, and 75 out of 94 haplotypes (79.8%) carry at least five novel alleles, thus confirming KIR genetic diversity. These findings are pivotal in providing insights into KIR gene diversity and serve as a solid foundation for understanding the functional consequences of KIR structural variations. High-resolution genome assemblies offer unprecedented opportunities to explore polymorphic regions that are challenging to investigate using short-read sequencing methods. The SKIRT pipeline emerges as a highly efficient tool, enabling the comprehensive detection of the complete spectrum of KIR alleles within human genome assemblies.

Haplotype-resolved T2T genome assemblies and pangenome graph of pear reveal diverse patterns of allele-specific expression and the genomic basis of fruit quality traits

Hybrid crops often exhibit increased yield and greater resilience, yet the genomic mechanism(s) underlying hybrid vigor or heterosis remain unclear, hindering our ability to predict the expression of phenotypic traits in hybrid breeding. Here, we generated haplotype-resolved T2T genome assemblies of two pear hybrid varieties, ‘Yuluxiang’ (YLX) and ‘Hongxiangsu’ (HXS), which share the same maternal parent but differ in their paternal parents. We then used these assemblies to explore the genome-scale landscape of allele-specific expression (ASE) and create a pangenome graph for pear. ASE was observed for close to 6000 genes in both hybrid cultivars. A subset of ASE genes related to aspects of fruit quality such as sugars, organic acids, and cuticular wax were identified, suggesting their important contributions to heterosis. Specifically, Ma1, a gene regulating fruit acidity, is absent in the paternal haplotypes of HXS and YLX. A pangenome graph was built based on our assemblies and seven published pear genomes. Resequencing data for 139 cultivated pear genotypes (including 97 genotypes sequenced here) were subsequently aligned to the pangenome graph, revealing numerous structural variant hotspots and selective sweeps during pear diversification. As predicted, the Ma1 allele was found to be absent in varieties with low organic acid content, and this association was functionally validated by Ma1 overexpression in pear fruit and calli. Overall, these results reveal the contributions of ASE to fruit-quality heterosis and provide a robust pangenome reference for high-resolution allele discovery and association mapping.

Sperm Mosaic Variants and Their Influence on the Offspring

Genomic mosaicism arising from mosaic variants is a phenomenon that describes the presence of a cell or cell populations with different genome compositions from the germline cells of an individual. It comprises all types of genetic variants. A large proportion of childhood genetic disorders are defined as being de novo, meaning that the disease-causing mutations are only detected in the proband, not in any of the parents. Population studies show that 80% of the de novo mutations arise from the paternal haplotype, that is, from paternal sperm mosaicism. This review provides a summary of the types and detection strategies of sperm mosaicism. In addition, it provides discussions on how recent studies demonstrated that genomic mosaic mutations in parents, especially those in the paternal sperms, could be inherited by the offspring and cause childhood disorders. According to the previous findings of the author's research team, sperm mosaicism derived from early embryogenesis and primordial germ cell stages can explain 5% to 20% of the de novo mutations related to clinical phenotypes and can serve as an important predictor of both rare and complex disorders. Sperm mosaicism shows great potential for clinical genetic diagnosis and consultations. Based on the published literature, the author suggests that, large-scale screening for de novo sperm mosaic mutations and population-based genetic screening should be conducted in future studies, which will greatly enhance the risk assessment in the offspring and effectively improve the genetic health at the population level. Implementation of direct sperm detection for de novo mutations will significantly increase the efficiency of the stratification of patient cohorts and improve recurrence risk assessment for future births. Future research in the field should be focused on the impact of environmental and lifestyle factors on the health of the offspring through sperms and their modeling of mutation signatures. In addition, targeted in vitro modeling of sperm mutations will also be a promising direction.

Constructing telomere-to-telomere diploid genome by polishing haploid nanopore-based assembly.

Draft genomes generated from Oxford Nanopore Technologies (ONT) long reads are known to have a higher error rate. Although existing genome polishers can enhance their quality, the error rate (including mismatches, indels and switching errors between paternal and maternal haplotypes) can be significant. Here, we develop two polishers, hypo-short and hypo-hybrid to address this issue. Hypo-short utilizes Illumina short reads to polish an ONT-based draft assembly, resulting in a high-quality assembly with low error rates and switching errors. Expanding on this, hypo-hybrid incorporates ONT long reads to further refine the assembly into a diploid representation. Leveraging on hypo-hybrid, we have created a diploid genome assembly pipeline called hypo-assembler. Hypo-assembler automates the generation of highly accurate, contiguous and nearly complete diploid assemblies using ONT long reads, Illumina short reads and optionally Hi-C reads. Notably, our solution even allows for the production of telomere-to-telomere diploid genomes with additional manual steps. As a proof of concept, we successfully assembled a fully phased telomere-to-telomere diploid genome of HG00733, achieving a quality value exceeding 50.

Matrilineal and Patrilineal Genetic Continuity of Two Iron Age Individuals from a Pazyryk Culture Burial

In this study, geographic and linguistic distributions of contemporary and ancient matches with the
 paternal and maternal lineages of two individuals exhumed from the exemplary Pazyryk culture burial site of Ak-Alakha-1 mound 1 were investigated. Using the shared paternal and maternal haplotypes observed in both ancient individuals, extensive database and literature searches were conducted revealing numerous full matches among contemporary Eurasians, majority of whom speak Altaic Languages. Despite the current focus on the two Pazyryk individuals, a rare glimpse into the ancient migrations was gained through the discovery of paternal and maternal haplotype matches across an immense geography that spans from Yakutia to Turkey. In addition to a vast array of archaeological findings in such Scythian “frozen graves” across Central Asia, accumulating archaeogenetic data are expected to shed light on the anthropology of these otherwise mysterious people.

The effect of mitochondrial recombination on fertilization success in blue mussels

The presence of doubly uniparental inheritance (DUI) in bivalves represents a unique mode of mitochondrial transmission, whereby paternal (male-transmitted M-type) and maternal (female-transmitted F-type) haplotypes are transmitted to offspring separately. Male embryos retain both haplotypes, but the M-type is selectively removed from females. Due to the presence of heteroplasmy in males, mtDNA can recombine resulting in a ‘masculinized’ haplotype referred to as Mf-type. While mtDNA recombination is usually rare, it has been recorded in multiple mussel species across the Northern Hemisphere. Given that mitochondria are the powerhouse of the cell, different mtDNA haplotypes may have different selective advantages under diverse environmental conditions. This may be particularly important for sperm fitness and fertilization success. In this study we aimed to i) determine the presence, prevalence of the Mf-type in Australian blue mussels (Mytilus sp.) and ii) investigate the effect of Mf-mtDNA on sperm performance (a fitness correlate). We found a high prevalence of recombined mtDNA (≈35 %) located within the control region of the mitochondrial genome, which occurred only in specimens that contained Southern Hemisphere mtDNA. The presence of two female mitotypes were identified in the studied mussels, one likely originating from the Northern Hemisphere, and the other either representing the endemic M. planulatus species or introduced genotypes from the Southern Hemisphere. Despite having recombination events present in a third of the studied population, analysis of sperm performance indicated no difference in fertilization success related to mitotype.

Investigation of genetic diversity and polyandry of Leptinotarsa decemlineata using X-linked microsatellite markers

A panel of X-linked microsatellite markers was newly designed using the data from a previous sequencing project available in NCBI and used for a study of the Colorado potato beetle (CPB, Leptinotarsa decemlineata) X-haplotype variability. The analysis of scaffolds 49 and 61 (newly identified as fragments of CPB chromosome X) found ten high-quality markers, which were arranged in two PCR multiplexes and evaluated in both 420 CPB adults, collected from 14 localities of Czechia and Slovakia, and 866 larvae from five single-female families from two more Czech localities. Length polymorphisms found in 6 loci have predicted 192 potential X-haplotypes, however, only 36 combinations were detected in the adult males (N = 189), and seven additional ones in the larvae. The X-haplotypes were also generally unevenly distributed; five of the most frequent haplotypes were detected in 55% of males, 19 repeating up to ten-times in 38.7% of males and the remained 12 occurred uniquely in 6.3% of males. Bulk analysis of X-haplotypes dissimilarity indicated seven haplotype groups diversified by mutations and recombinations. Two haplotypes showed a distinctive regional distribution, which indicates an east–west disruption of CPB migration probably caused by different environments of localities in the South Bohemia region and Vysocina region. On the contrary, the results indicate a south–north migration corridor alongside the Vltava River. In the single-female families, from 6 to 13 distinct paternal haplotypes were detected, which proved and quantified a frequented polyandry in CPB.

Haplotype-resolved assemblies and variant benchmark of a Chinese Quartet

BackgroundRecent state-of-the-art sequencing technologies enable the investigation of challenging regions in the human genome and expand the scope of variant benchmarking datasets. Herein, we sequence a Chinese Quartet, comprising two monozygotic twin daughters and their biological parents, using four short and long sequencing platforms (Illumina, BGI, PacBio, and Oxford Nanopore Technology).ResultsThe long reads from the monozygotic twin daughters are phased into paternal and maternal haplotypes using the parent–child genetic map and for each haplotype. We also use long reads to generate haplotype-resolved whole-genome assemblies with completeness and continuity exceeding that of GRCh38. Using this Quartet, we comprehensively catalogue the human variant landscape, generating a dataset of 3,962,453 SNVs, 886,648 indels (< 50 bp), 9726 large deletions (≥ 50 bp), 15,600 large insertions (≥ 50 bp), 40 inversions, 31 complex structural variants, and 68 de novo mutations which are shared between the monozygotic twin daughters. Variants underrepresented in previous benchmarks owing to their complexity—including those located at long repeat regions, complex structural variants, and de novo mutations—are systematically examined in this study.ConclusionsIn summary, this study provides high-quality haplotype-resolved assemblies and a comprehensive set of benchmarking resources for two Chinese monozygotic twin samples which, relative to existing benchmarks, offers expanded genomic coverage and insight into complex variant categories.

Contemporary hybridization between female mule deer and male white-tailed deer in west Texas differs from the hypothesized sex mating patterns recovered from ancient hybridization events

Introgressive hybridization between members of Odocoileus was examined using the mitochondrial cytochrome-b (maternal marker) and paternal sex-determining region Y ( Sry) genes. Eight out of 130 free-ranging individuals from the Panhandle and Trans-Pecos regions of Texas were determined to possess the mitochondrial haplotype of mule deer ( Odocoileus hemionus (Rafinesque, 1817)) and the paternal haplotype of white-tailed deer ( Odocoileus virginianus Rafinesque, 1832). Results indicated that hybridization between deer species in Texas (6.15%) was more broadly distributed than previously reported. Previous studies demonstrated that ancient hybridization events (1.32 mya) involved the capture of the white-tailed deer mitochondrial genome by mule deer, indicating a male mule deer × a female white-tailed deer directionality relevant to hybridization. Alternatively, contemporary hybridization events indicated a reversal in directionality and suggested a cross between a female mule deer × a male white-tailed deer. The Sry gene and species assignment based on morphological characters consistently were in agreement. Further, phylogenetic relationships between Odocoileus virginianus couesi and Odocoileus hemionus eremicus warrant additional investigation as recent hybridization (&gt;200 years) may be a mechanism that allowed these two subspecies to evolve a unique evolutionary trajectory.

Last-percent improvement in eligibility rates of crop seeds based on quality evaluation using near-infrared imaging spectrometry.

As the world population continues to grow, the need for high-quality crop seeds that promise stable food production is increasing. Conversely, excessive demand for high quality is causing "seed loss and waste" due to slight shortfalls in eligibility rates. In this study, we applied near-infrared imaging spectrometry combined with machine learning techniques to evaluate germinability and paternal haplotype in crop seeds from 6 species and 8 cultivars. Candidate discriminants for quality evaluation were derived by linear sparse modeling using the seed reflectance spectra as explanatory variables. To systematically proceed with model selection, we defined the sorting condition where the recovery rate of seeds matches the initial eligibility rate (iP) as "standard condition". How much the eligibility rate after sorting (P) increases from iP under this condition offers a reasonable criterion for ranking candidate models. Moreover, the model performance under conditions with adjusted discrimination strength was verified using a metric "relative precision" (rP) defined as (P-iP)/(1-iP). Because rP, compared to precision (= P), is less dependent on iP in relation to recall (R), i.e., recovery rate of eligible seeds, the rP-R curve and area under the curve also offer useful criteria for spotting better discriminant models. We confirmed that the batches of seeds given higher discriminant scores by the models selected with reference to these criteria were more enriched with eligible seeds. The method presented can be readily implemented in developing a sorting device that enables "last-percent improvement" in eligibility rates of crop seeds.

P-795 Identification of a specific paternal haplotype in the germline and perpetuating progenies by male gamete replication

Abstract Study question Can we identify and replicate a male germ cell genome to generate progenies carrying a specific genotype? Summary answer We were able to replicate male genome and identify the androgenote with a specific genotype to generate conceptuses with identical paternal haplotype. What is known already For patients with heritable genetic disorders, current treatment relies on preimplantation genetic testing for monogenic disorders (PGT-M) to select unaffected embryos for replacement; however, this can lead to embryo wastage, causing emotional distress. Although genomic editing of gametes is being attempted, CRISPR-based technologies are inaccurate and can lead to loss of heterozygosity in cases where the embryo uses the maternal genome as a template to edit the paternal genome. Therefore, the use of haploid androgenotes may offer a useful tool to select male gametes with genes of interest or a successfully edited genome before the creation of an embryo. Study design, size, duration To generate haploid androgenotes, spermatozoa from hemizygous transgenic mice with GFP expression (B6-EGFP x B6D2F1) were used to inseminate enucleated wildtype oocytes. Constructs were cultured to the 8-cell stage. Two pseudo-blastomeres were isolated for genotyping by DNAseq. A blastomere with GFP expression was used as a male gamete. Experiments were repeated using mice with the Fbn1tm1Hcd mutation as a model for Marfan syndrome. Biparental embryos were created to preserve the mutation as a disease model. Participants/materials, setting, methods To generate haploid androgenotes, metaphase II oocytes from wildtype B6 mice were enucleated and inseminated using spermatozoa from hemizygous GFP transgenic mice. Haploid androgenetic embryos were cultured in a time-lapse incubator up to the 8-cell stage. Biparental embryos were created by fusing a selected androgenote with an activated oocyte from another cohort. Resulting embryos were genotyped to confirm the paternal haplotype. Experiments were reproduced using spermatozoa from heterozygous Fbn1tm1Hcd-mutant mice. Main results and the role of chance In the first set of experiments using spermatozoa from transgenic mice hemizygous of GFP, 200 wildtype oocytes were enucleated and generated 195 (97.5%) ooplasts. Insemination of those ooplasts yielded 168 (86.2%) monopronucleated androgenetic embryos, and subsequently generated 116 (69.0%) 8-cell haploid androgenetic embryos, of which 59 (50.8%) expressed GFP. Up to 2 sibling blastomeres were genotyped individually by DNAseq and confirmed to have an identical paternal haplotype. The remaining blastomeres were used to create biparental embryos and yielded a 97.2% fertilization rate after grafting into 145 oocytes, comparable with the fertilization rate of ICSI controls at 95.6%. Although control zygotes yielded an 88.0% blastulation rate, the experimental embryos yielded a lower blastulation rate of 58.2% (P&amp;lt;0.0001). Albeit with a lower developmental rate, all experimental blastocysts expressed GFP and displayed normal embryo morphokinesis. For the experiment using spermatozoa from Fbn1tm1Hcd mice, 50 8-cell stage haploid androgenetic embryos were generated. Two individual pseudo-blastomeres from 35 haploid embryos were used for genotyping. The remaining 210 haploid blastomeres were used to construct biparental embryos and yielded 119 (56.7%) blastocysts. PCR on embryo biopsies confirmed that the resulting blastocysts inherited the mutant (n = 63) or wildtype (n = 56) paternal haplotype from the original gamete. Limitations, reasons for caution This technique allowed the selection of male gametes with the desired genotype to generate conceptuses with a known haplotype in a reproducible manner. A more prospective experiment is ongoing to simulate clinical application by cryopreserving haploid androgenetic embryos and using only androgenotes with a confirmed genotype to create biparental embryos. Wider implications of the findings This approach to utilize gamete substitutes before fertilization ensures the maintenance of rare and unique genetic materials to generate conceptuses with the desired genotype in transgenic mice in disease models. Indeed, once this technique is implemented in humans, genotyped androgenotes can be used to characterize germline heterozygosity. Trial registration number N/A

P-723 Pre-fertilization genetic testing to select healthy gametes in a mammalian Marfan syndrome model

Abstract Study question Is it possible to identify disease-free gametes before insemination to prevent vertical transmission of an autosomal dominant disease? Summary answer In a mammalian Marfan syndrome model, we successfully generated and identified healthy androgenotes to be utilized as male gametes to produce unaffected conceptuses. What is known already In patients with autosomal dominant genetic disorders such as Marfan syndrome, germline heterozygosity poses a risk to offspring health. For these patients, preimplantation genetic testing for monogenic disorders (PGT-M) allows us to select unaffected conceptuses for embryo replacement. However, PGT-M is inherently inefficient and can lead to embryo wastage. This has sparked interest in replicating the genome of individual gametes for (1) pre-fertilization genetic testing and (2) subsequent use as genotyped gamete substitutes during IVF. Study design, size, duration For this study, we selected 4 male B6 mice that were heterozygous for the Fbn1tm1Hcd mutation and displayed classic manifestations of Marfan syndrome. Spermatozoa from these mice were used to inseminate enucleated wildtype mouse oocytes and generate haploid androgenetic embryos for (1) Fbn1tm1Hcd genotyping and (2) use as unaffected male gamete substitutes. Participants/materials, setting, methods Using tail clippings and embryo biopsy specimens, a Fbn1tm1Hcd allele-specific PCR primer was developed and validated. Haploid androgenetic embryos were allowed to progress to the 8-cell stage. Subsequently, a single-haploid pseudo-blastomere was isolated for genotyping, while the remainder were individually grafted into activated recipient wildtype oocytes and fused using inactivated Sendai virus to generate biparental conceptuses. Resulting diploid conceptuses were cultured in a time-lapse incubator and then genotyped to confirm the healthy paternal haplotype. Main results and the role of chance A total of 175 wildtype B6 oocytes were enucleated yielding 169 ooplasts (96.6% survival rate). The ooplasts were inseminated with spermatozoa from mutant Fbn1tm1Hcd mice, yielding 144 (85.2%) monopronucleated haploid androgenetic embryos. Of those, 100 (69.4%) progressed to the 8-cell stage. In 47 (47.0%) of them, genetic analysis by PGT-M on an individual pseudo-blastomere characterized the embryo as unaffected because it harbored the wildtype Fbn1 allele. The remaining 7 haploid pseudo-blastomeres from 5 Fbn1 wildtype androgenetic embryos were individually isolated and grafted into 35 recipient oocytes to generate 31 (88.6%) biparental conceptuses with the desired paternal haplotype. These achieved a 61.3% (19/31) blastocyst development rate after 96 hours, displaying normal embryo morphokinesis. A blastocyst biopsy was carried out (n = 19), confirming the unaffected paternal haplotype void of the Fbn1tm1Hcd mutation. Limitations, reasons for caution Although this technique is feasible for pre-fertilization genetic testing, it needs to be reproduced with the same efficiency in humans. A major concern is related to the dynamics and roles of the sperm centrosome in the first mitotic division of human zygotes, which may be missing in the pseudo-gametes. Wider implications of the findings This approach to pre-fertilization genetic testing will benefit patients with heritable genetic conditions. In addition, the inherited decondensation of the male genome by the ooplasm may enable genome editing experiments to be performed. Trial registration number Not applicable

Identification and characterisation of de novo germline structural variants in two commercial pig lines using trio-based whole genome sequencing

BackgroundDe novo mutations arising in the germline are a source of genetic variation and their discovery broadens our understanding of genetic disorders and evolutionary patterns. Although the number of de novo single nucleotide variants (dnSNVs) has been studied in a number of species, relatively little is known about the occurrence of de novo structural variants (dnSVs). In this study, we investigated 37 deeply sequenced pig trios from two commercial lines to identify dnSVs present in the offspring. The identified dnSVs were characterised by identifying their parent of origin, their functional annotations and characterizing sequence homology at the breakpoints.ResultsWe identified four swine germline dnSVs, all located in intronic regions of protein-coding genes. Our conservative, first estimate of the swine germline dnSV rate is 0.108 (95% CI 0.038–0.255) per generation (one dnSV per nine offspring), detected using short-read sequencing. Two detected dnSVs are clusters of mutations. Mutation cluster 1 contains a de novo duplication, a dnSNV and a de novo deletion. Mutation cluster 2 contains a de novo deletion and three de novo duplications, of which one is inverted. Mutation cluster 2 is 25 kb in size, whereas mutation cluster 1 (197 bp) and the other two individual dnSVs (64 and 573 bp) are smaller. Only mutation cluster 2 could be phased and is located on the paternal haplotype. Mutation cluster 2 originates from both micro-homology as well as non-homology mutation mechanisms, where mutation cluster 1 and the other two dnSVs are caused by mutation mechanisms lacking sequence homology. The 64 bp deletion and mutation cluster 1 were validated through PCR. Lastly, the 64 bp deletion and the 573 bp duplication were validated in sequenced offspring of probands with three generations of sequence data.ConclusionsOur estimate of 0.108 dnSVs per generation in the swine germline is conservative, due to our small sample size and restricted possibilities of dnSV detection from short-read sequencing. The current study highlights the complexity of dnSVs and shows the potential of breeding programs for pigs and livestock species in general, to provide a suitable population structure for identification and characterisation of dnSVs.

Snowflake epitope matching correlates with child-specific antibodies during pregnancy and donor-specific antibodies after kidney transplantation.

Development of donor-specific human leukocyte antigen (HLA) antibodies (DSA) remains a major risk factor for graft loss following organ transplantation, where DSA are directed towards patches on the three-dimensional structure of the respective organ donor's HLA proteins. Matching donors and recipients based on HLA epitopes appears beneficial for the avoidance of DSA. Defining surface epitopes however remains challenging and the concepts underlying their characterization are not fully understood. Based on our recently implemented computational deep learning pipeline to define HLA Class I protein-specific surface residues, we hypothesized a correlation between the number of HLA protein-specific solvent-accessible interlocus amino acid mismatches (arbitrarily called Snowflake) and the incidence of DSA. To validate our hypothesis, we considered two cohorts simultaneously. The kidney transplant cohort (KTC) considers 305 kidney-transplanted patients without DSA prior to transplantation. During the follow-up, HLA antibody screening was performed regularly to identify DSA. The pregnancy cohort (PC) considers 231 women without major sensitization events prior to pregnancy who gave live birth. Post-delivery serum was screened for HLA antibodies directed against the child's inherited paternal haplotype (CSA). Based on the involved individuals' HLA typings, the numbers of interlocus-mismatched antibody-verified eplets (AbvEPS), the T cell epitope PIRCHE-II model and Snowflake were calculated locus-specific (HLA-A, -B and -C), normalized and pooled. In both cohorts, Snowflake numbers were significantly elevated in recipients/mothers that developed DSA/CSA. Univariable regression revealed significant positive correlation between DSA/CSA and AbvEPS, PIRCHE-II and Snowflake. Snowflake numbers showed stronger correlation with numbers of AbvEPS compared to Snowflake numbers with PIRCHE-II. Our data shows correlation between Snowflake scores and the incidence of DSA after allo-immunization. Given both AbvEPS and Snowflake are B cell epitope models, their stronger correlation compared to PIRCHE-II and Snowflake appears plausible. Our data confirms that exploring solvent accessibility is a valuable approach for refining B cell epitope definitions.

A feasibility study of noninvasive prenatal diagnosis in facioscapulohumeral muscular dystrophy type 1 in a Chinese family.

Objective: To demonstrate the feasibility of haplotype-based noninvasive prenatal diagnosis of Facioscapulohumeral Muscular Dystrophy type 1 (FSHD1). Methods: Bionano optical mapping was used to identify the D4Z4 structural variation of the genomic DNA sample from the proband affected with FSHD1. In addition, based on the technique of next generation sequencing, the pathogenic haplotype was determined by using trio strategy through genotyping his parents, and also fetal inheritance of paternal haplotypes was then deduced using the Hidden Markov Model. Results: Bionano optical mapping analysis revealed that the proband has only three D4Z4 repeats left in the 4q35 chromosomal region and a disease-permitting 4qA haplotype. The other normal allele of the proband contains 29 D4Z4 repeats and also a 4qA haplotype. The noninvasive cell-free fetal DNA (cffDNA)-based haplotype analysis suggested that the fetus inherited the pathogenic allele from his father and thus was predicted to be affected by FSHD1. In addition, Bionano optical mapping also demonstrated the presence of the pathogenic allele in the fetus by interrogating the genomic DNA from the amniotic fluid cells. Conclusion: Our study showed the cffDNA-based haplotyping was feasible for the noninvasive prenatal diagnosis of FSHD1, which is able to provide earlier testing results with a lower risk of miscarriage and infection than invasive techniques.

UTILIZATION OF HAPLOID ANDROGENETIC BLASTOMERES AS FUNCTIONAL REPLICATES OF THE MALE GENOME

To generate offspring with identical paternal haplotypes from haploid male genome replicates initiated through the ooplasmic machinery of enucleated oocytes.

Genome architecture and tetrasomic inheritance of autotetraploid potato

Potato (Solanum tuberosum) is the most consumed non-cereal food crop. Most commercial potato cultivars are autotetraploids with highly heterozygous genomes, severely hampering genetic analyses and improvement. By leveraging the state-of-the-art sequencing technologies and polyploid graph binning, we achieved a chromosome-scale, haplotype-resolved genome assembly of a cultivated potato, Cooperation-88 (C88). Intra-haplotype comparative analyses revealed extensive sequence and expression differences in this tetraploid genome. We identified haplotype-specific pericentromeres on chromosomes, suggesting a distinct evolutionary trajectory of potato homologous centromeres. Furthermore, we detected double reduction events that are unevenly distributed on haplotypes in 1021 of 1034 selfing progeny, a feature of autopolyploid inheritance. By distinguishing maternal and paternal haplotype sets in C88, we simulated the origin of heterosis in cultivated tetraploid with a survey of 3110 tetra-allelic loci with deleterious mutations, which were masked in the heterozygous condition by two parents. This study provides insights into the genomic architecture of autopolyploids and will guide their breeding.

Abstract 1213: Uncovering HLA loss of heterozygosity and allelic imbalance in cancer for the improvement of personalized neoTCR immunotherapy with PACT-ESCAPE

Abstract Introduction: PACT Pharma is a clinical-stage adoptive cell therapy company that develops personalized neoTCR-T cells using a state-of-the-art approach to discover and validate predicted neoepitopes and their cognate T cell receptors (clinical trial NCT03970382). An important mechanism of resistance to adoptive cell therapy targeting tumor-specific neoantigens is the genetic loss of human leukocyte antigen (HLA) alleles or alterations in HLA expression in tumor cells. Therapies targeting neoantigens presented by HLA alleles that have either been deleted, mutated or are strongly downregulated are likely to lack efficacy due to the absence or reduction of epitope presentation. Surveying neoepitope presentation escape mechanisms is key for personalized immunotherapy. Methods: PACT Pharma has developed PACT-ESCAPE, a method that integrates DNA and RNA sequencing data to quantify allelic imbalance at HLA loci, including the most extreme case of loss of heterozygosity (LOH). Allelic imbalance is first determined at the DNA level by determining the patient’s maternal and paternal haplotypes across chromosome 6p to infer their copy number states. Twenty-six HLA Class I and II loci from chromosome 6 are then genotyped at high resolution, enabling accurate assignment of copy number states to the patient’s alleles, including any alleles that have been deleted in the tumor. Unlike other currently available methods, PACT-ESCAPE also measures the relative RNA expression of HLA alleles to provide an orthogonal estimate of allelic imbalance across chromosome 6 and support loss of heterozygosity calls made at the DNA level. Presentation machinery genes are also surveyed for loss of function mutations. To benchmark PACT-ESCAPE, we compared loss of heterozygosity calls at HLA-A, HLA-B, and HLA-C between our method and a previously published DNA-based HLA LOH classifier for 17 samples for which we had matched tumor/normal whole exome sequencing. Results: We found 100% concordance between PACT-ESCAPE and the benchmark, with four samples showing LOH at HLA-A, HLA-B, and HLA-C at the DNA level. Next, we evaluated RNA sequencing from tumor samples and confirmed that the relative expression of the lost allele compared to the kept allele was lower when LOH was called. High levels of allelic imbalance in gene expression, however, were not unique to samples with LOH, suggesting that differential expression of HLA alleles might also be an important contributor to immune escape in cancer. Conclusions: Evaluation of allelic imbalance in DNA copy number and RNA expression in PACT-ESCAPE provides new insights into immune escape in cancer. Adoptive cell therapy targeting neoantigens will benefit by enabling the accurate selection of targets most likely to be presented by the tumor. Citation Format: Chad C. Smith, Yan Ma, Katie Campbell, Zheng Pan, Eric Stawiski. Uncovering HLA loss of heterozygosity and allelic imbalance in cancer for the improvement of personalized neoTCR immunotherapy with PACT-ESCAPE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1213.

Noninvasive prenatal prediction of fetal haplotype with Spearman rank correlation analysis model.

BackgroundNoninvasive prenatal testing (NIPT) has been widely used clinically to detect fetal chromosomal aneuploidy with high accuracy rates, gradually replacing traditional serological screening. However, the application of NIPT for monogenic diseases is still in an immature stage of exploration. The detection of mutations in peripheral blood of pregnant women requires precise qualitative and quantitative techniques, which limits its application. The bioinformatic strategies based on the SNP (single nucleotide polymorphism) linkage analysis are more practical, which can be divided into two types depending on whether proband information is needed. Hidden Markov Mode (HMM) and Sequential probability ratio test (SPRT) are suitable for families with probands. In contrast, methods based on databases and population demographic information are suitable for families without probands.MethodsIn this study, we proposed a Spearman rank correlation analysis method to infer the fetal haplotypes based on core family information. Allele frequencies of SNPs that were used to construct parental haplotypes were calculated as sets of nonparametric variables, in contrast to their theoretical values represented by a fetal fraction (FF). The effects on the calculation of the fetal concentration of two DNA enrichment methods, multiple‐PCR amplification, and targeted hybrid capture, were compared, and the heterozygosity distribution of SNPs within pedigrees was analyzed to reveal the best conditions for the model application.ResultsPredictions of the paternal haplotype inheritance were in line with expectations for both DNA library construction methods, while for maternal haplotype inheritance prediction, the rates were 96.55% for method multiple‐PCR amplification and 95.8% for method targeted hybrid capture.ConclusionPositive prediction rates showed that the maternal haplotype prediction was not as accurate as paternal one, due to the large amount of maternal noise in the mother's peripheral blood. Although this model is relatively immature, it provides a new perspective for noninvasive prenatal clinical tests of monogenic diseases.