Paternal Haplotypes Research Articles

Joint Analysis of Strain and Parent-of-Origin Effects for Recombinant Inbred Intercrosses Generated from Multiparent Populations with the Collaborative Cross as an Example.

Multiparent populations (MPP) have become popular resources for complex trait mapping because of their wider allelic diversity and larger population size compared with traditional two-way recombinant inbred (RI) strains. In mice, the collaborative cross (CC) is one of the most popular MPP and is derived from eight genetically diverse inbred founder strains. The strategy of generating RI intercrosses (RIX) from MPP in general and from the CC in particular can produce a large number of completely reproducible heterozygote genomes that better represent the (outbred) human population. Since both maternal and paternal haplotypes of each RIX are readily available, RIX is a powerful resource for studying both standing genetic and epigenetic variations of complex traits, in particular, the parent-of-origin (PoO) effects, which are important contributors to many complex traits. Furthermore, most complex traits are affected by >1 genes, where multiple quantitative trait locus mapping could be more advantageous. In this paper, for MPP-RIX data but taking CC-RIX as a working example, we propose a general Bayesian variable selection procedure to simultaneously search for multiple genes with founder allelic effects and PoO effects. The proposed model respects the complex relationship among RIX samples, and the performance of the proposed method is examined by extensive simulations.

Sex Chromosome Evolution, Heterochiasmy, and Physiological QTL in the Salmonid Brook Charr Salvelinus fontinalis.

Whole-genome duplication (WGD) can have large impacts on genome evolution, and much remains unknown about these impacts. This includes the mechanisms of coping with a duplicated sex determination system and whether this has an impact on increasing the diversity of sex determination mechanisms. Other impacts include sexual conflict, where alleles having different optimums in each sex can result in sequestration of genes into nonrecombining sex chromosomes. Sex chromosome development itself may involve sex-specific recombination rate (i.e., heterochiasmy), which is also poorly understood. The family Salmonidae is a model system for these phenomena, having undergone autotetraploidization and subsequent rediploidization in most of the genome at the base of the lineage. The salmonid master sex determining gene is known, and many species have nonhomologous sex chromosomes, putatively due to transposition of this gene. In this study, we identify the sex chromosome of Brook Charr Salvelinus fontinalis and compare sex chromosome identities across the lineage (eight species and four genera). Although nonhomology is frequent, homologous sex chromosomes and other consistencies are present in distantly related species, indicating probable convergence on specific sex and neo-sex chromosomes. We also characterize strong heterochiasmy with 2.7-fold more crossovers in maternal than paternal haplotypes with paternal crossovers biased to chromosome ends. When considering only rediploidized chromosomes, the overall heterochiasmy trend remains, although with only 1.9-fold more recombination in the female than the male. Y chromosome crossovers are restricted to a single end of the chromosome, and this chromosome contains a large interspecific inversion, although its status between males and females remains unknown. Finally, we identify quantitative trait loci (QTL) for 21 unique growth, reproductive, and stress-related phenotypes to improve knowledge of the genetic architecture of these traits important to aquaculture and evolution.

Non-invasive prenatal diagnosis of spinal muscular atrophy by relative haplotype dosage

Although technically possible, few clinical laboratories across the world have implemented non-invasive prenatal diagnosis (NIPD) for selected single-gene disorders, mostly owing to the elevated costs incurred. Having previously proven that NIPD for X-linked disorders can be feasibly implemented in clinical practice, we have now developed a test for the NIPD of an autosomal-recessive disorder, spinal muscular atrophy (SMA). Cell-free DNA was extracted from maternal blood and prepared for massively parallel sequencing on an Illumina MiSeq by targeted capture enrichment of single-nucleotide polymorphisms across a 6 Mb genomic window on chromosome 5 containing the SMN1 gene. Maternal, paternal and proband DNA samples were also tested for haplotyping purposes. Sequencing data was analysed by relative haplotype dosage (RHDO). Six pregnant SMA carriers and 10 healthy pregnant donors were recruited through the NIPSIGEN study. Inheritance of the maternally and paternally derived alleles of the affected SMN1 gene was determined in the foetus by RHDO analysis for autosomal-recessive disorders. DNA from the proband (for SMA carriers) or an invasively obtained foetal sample (for healthy pregnant donors) was used to identify the maternal and paternal reference haplotypes associated with the affected SMN1 gene. Results for all patients correlated with known outcomes and showed a testing specificity and sensitivity of 100%. On top of showing high accuracy and reliability throughout the stages of validation, our novel test for NIPD of SMA is also affordable and viable for implementation into clinical service.

Mother Donors Improve Outcomes after HLA Haploidentical Hematopoietic Transplantation: A Retrospective Study By the Cell Therapy and Immunobiology Working Party of the EBMT

▪IntroductionTrans-placental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal micro-chimerism in both mother and child (Maloney et al., J Clin Invest, 104:41, 1999). As a consequence, the immune system of the mother may become sensitized to paternal histocompatibility antigens. In fact, antibodies directed against paternal HLA-antigens (van Rood JJ et al., Nature 181:1735, 1958) and T lymphocytes directed against paternal major and minor histocompatibility antigens (van Kampen CA et al., Hum Immunol 62:201, 2001; Verdijk RM et al., Blood 103:1961, 2004) were detected in multiparous women. More recently, it was hypothesized that mother's "exposure" to paternal HLA haplotype antigens during pregnancy may affect transplantation outcomes when the mother acts as donor for the child. Indeed, survival after T cell-depleted HLA haploidentical haematopoietic transplantation was improved using the mother as donor (vs all other family members) (Stern et al., Blood 112:2990, 2008; Kruchen et al., BMT 50:1367 2015). However, maternal donors were associated with increased incidence of GvHD and decreased survival after un-manipulated HLA haploidentical blood and marrow grafts (Wang Y et al., Blood 124:843, 2014).Patients and MethodsA retrospective EBMT registry-based study was performed in a combined series of adult (n=333) and pediatric (n=105) patients with acute leukemia (AML=268, ALL=160, Mixed phenotype=10) who received transplant from one MHC mismatched family donor. Forty-four percent (233) of patients were in I or II complete remission (CR) at the time of transplant, 193 were in chemo-resistant relapse or in CR > 2. Median age was 32.7 (range: 0.67-70). Seventy-one percent of patients received ex-vivo T cell depleted transplants. Twenty-two patients were given bone marrow and peripheral blood stem cells, the others were given peripheral blood cells only. The mother was used as donor in 98 patients.ResultsEighty patients developed acute GvHD ≥ grade II. At a median follow up of 58.7 months, 130 patients are alive without disease, 146 relapsed and 152 died of non-relapse mortality (NRM). Univariate analyses of factors influencing relapse-free survival (RFS) showed age, disease status at transplant, Karnofsky score and ex-vivo T cell depletion impacted significantly. As transplantation outcomes from family members other than mothers did not differ from one another, such transplants were combined for analyses. When compared with transplantation from all other family members (n=330), transplantation from mother donors (n=98) was associated with lower relapse incidence (RI) (28% vs 39%, P=0.06) and non-relapse mortality (NRM) (30% vs 40%, P=0.09) and, consequently, better relapse-free survival (RFS) (43% vs 21%, P < 0.001). Multivariate analyses showed transplantation from mother donors was an independent factor predicting better RFS (other donors vs mothers: HR: 1.42; CI: 1.01-2.00; P=0.04) and lower RI (other donors vs mothers: HR: 1.93; CI: 1.16-3.22; P = 0.01). In addition, transplantation in relapse (vs remission) predicted worse RFS (HR: 2.33, CI: 1.80-3.00, P < 0.001) such as ≥18 (vs < 18) adversely impacted on RFS (HR: 1.40, CI: 1.00-1.96, P=0.04).DiscussionOur retrospective analyses in 438 HLA haploidentical hematopoietic transplants for acute leukemia patients (pediatric and adult) show that transplantation from mother donors, when compared with transplantation from any other family member, is an independent factor predicting better outcomes, i.e., better RFS and lower RI. Mothers should therefore be preferred when selecting an HLA haploidentical family donor. Further clinical and preclinical studies are needed to unveil the mysteries underlying mother-to-child immune interaction during pregnancy and its bearing on the reproductive success of the human species. DisclosuresCiceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

A Genetic Algorithm for Diploid Genome Reconstruction Using Paired-End Sequencing.

The genome of many species in the biosphere is a diploid consisting of paternal and maternal haplotypes. The differences between these two haplotypes range from single nucleotide polymorphisms (SNPs) to large-scale structural variations (SVs). Existing genome assemblers for next-generation sequencing platforms attempt to reconstruct one consensus sequence, which is a mosaic of two parental haplotypes. Reconstructing paternal and maternal haplotypes is an important task in linkage analysis and association studies. This study designs and implemented HapSVAssembler on the basis of Genetic Algorithm (GA) and paired-end sequencing. The proposed method builds a consensus sequence, identifies various types of heterozygous variants, and reconstructs the paternal and maternal haplotypes by solving an optimization problem with a GA algorithm. Experimental results indicate that the HapSVAssembler has high accuracy and contiguity under various sequencing coverage, error rates, and insert sizes. The program is tested on pilot sequencing of a highly heterozygous genome, and 12,781 heterozygous SNPs and 602 hemizygous SVs are identified. We observe that, although the number of SVs is much less than that of SNPs, the genomic regions occupied by SVs are much larger, implying the heterozygosity computed using SNPs or k-mer spectrum may be under-estimated.

Molecular genetics and bioinformatics analysis of EDG1 and AKIRIN2 genes in Iranian fat-tailed and nonfat-tailed sheep breeds

This study was conducted to detect different allelic forms and its association with fat-tail traits, and also investigation of domains variants in 5/- and 3/-UTR regions of EDG1 and AKIRIN2 genes in Iranian fat-tailed (Lori-Bakhtiari) and nonfat-tailed (Zel) sheep breeds. Blood samples were randomly collected from 300 sheep (150 samples per each breed) and genomic DNA was extracted by modified salting out method. Two primer pairs were designed by oligo7 software for amplification of fragments with the length of 516 and 454bp from EDG1 and AKIRIN2 genes, respectively. The results of genotyping by SSCP assay and silver nitrate staining showed that, in both breeds, there was no incidence of mutation in AKIRIN2 gene. But, the EDG1 was highly polymorph in studied sheep breeds, so that, six banding patterns with different frequencies of A (36.73 and 31.19%), B (8.84 and 21.6%), C (7.48 and 41.12%), D (29.93 and 34.50%), E (13.61 and 34.10%) and F (3.40 and 38.1%) were observed for Zel and Lori-Bakhtiari breeds, respectively. Comparison of banding pattern frequencies by Chi-square test showed significant difference between two breeds (P<0.01). One sample from each banding patterns were sequenced. The alignment and motif analysis of sequences by BioEdit and DNASIS MAX software showed incidence of 11 single nucleotide polymorphism (SNP) and 18 DNA motifs in EDG1 marker site. According to different SNPs, the paternal and maternal haplotypes were determined by phase software. The association study showed significant association between EDG1 gene haplotypes and banding patterns with fat-tail traits in Lori-Bakhtiari sheep except for height of fat-tail. This study was the first effort for evaluate of these genes in sheep. Therefore, more studies on other breeds of sheep and on coding sequence of these genes are recommended.

A genetic assessment of the English bulldog

BackgroundThis study examines genetic diversity among 102 registered English Bulldogs used for breeding based on maternal and paternal haplotypes, allele frequencies in 33 highly polymorphic short tandem repeat (STR) loci on 25 chromosomes, STR-linked dog leukocyte antigen (DLA) class I and II haplotypes, and the number and size of genome-wide runs of homozygosity (ROH) determined from high density SNP arrays. The objective was to assess whether the breed retains enough genetic diversity to correct the genotypic and phenotypic abnormalities associated with poor health, to allow for the elimination of deleterious recessive mutations, or to make further phenotypic changes in body structure or coat. An additional 37 English bulldogs presented to the UC Davis Veterinary Clinical Services for health problems were also genetically compared with the 102 registered dogs based on the perception that sickly English bulldogs are products of commercial breeders or puppy-mills and genetically different and inferior.ResultsFour paternal haplotypes, with one occurring in 93 % of dogs, were identified using six Y-short tandem repeat (STR) markers. Three major and two minor matrilines were identified by mitochondrial D-loop sequencing. Heterozygosity was determined from allele frequencies at genomic loci; the average number of alleles per locus was 6.45, with only 2.7 accounting for a majority of the diversity. However, observed and expected heterozygosity values were nearly identical, indicating that the population as a whole was in Hardy-Weinberg equilibrium (HWE). However, internal relatedness (IR) and adjusted IR (IRVD) values demonstrated that a number of individuals were the offspring of parents that were either more inbred or outbred than the population as a whole. The diversity of DLA class I and II haplotypes was low, with only 11 identified DLA class I and nine class II haplotypes. Forty one percent of the breed shared a single DLA class I and 62 % a single class II haplotype. Nineteen percent of the dogs were homozygous for the dominant DLA class I haplotype and 42 % for the dominant DLA class II haplotype. The extensive loss of genetic diversity is most likely the result of a small founder population and artificial genetic bottlenecks occurring in the past. The prominent phenotypic changes characteristic of the breed have also resulted in numerous large runs of homozygosity (ROH) throughout the genome compared to Standard Poodles, which were phenotypically more similar to indigenous-type dogs.ConclusionsEnglish bulldogs have very low genetic diversity resulting from a small founder population and artificial genetic bottlenecks. Although some phenotypic and genotypic diversity still exists within the breed, whether it is sufficient to use reverse selection to improve health, select against simple recessive deleterious traits, and/or to accommodate further genotypic/phenotypic manipulations without further decreasing existing genetic diversity is questionable.

Pushing the limits of preimplantation genetic diagnosis (PGD): a Karyomapping and Next Generation Sequencing (NGS) stress test

We sought to examine the limits of embryo genetic analysis by performing PGD and aneuploidy screening (PGS) in 1) a consanguineous mating for, 2) two disease-causing point mutations and 3) HLA-genotyping to match 3 affected children while 4) detecting genetic recombination and potential maternal cell contamination, and 5) 24-chromosome aneuploidy screening with NGS. Karyomapping utilizes ∼300,000 Single Nucleotide Polymorphisms (SNPs) across the human genome and allows high-resolution analysis for nearly every gene. We used karyomapping to rapidly design a PGD test targeting the HBB gene and the highly recombinant HLA locus to identify embryos with one or both healthy HBB alleles and an HLA genotype matching the children in need of transplantation. Cheek-swab kits were used to collect DNA samples from the parents and affected children. Mutation status of each individual was confirmed by direct sequencing. DNA samples were analyzed using the Infinium HumanKaryomap-12 DNA Analysis Kit from Illumina. BluFuse Multi software was used to compare the SNP profiles of each family member to identify informative SNPs, regions of consanguinity, and determine HLA phase for each affected child. NGS was used for 24-chromosome aneuploidy screening. We identified sufficient informative SNPs surrounding the HBB gene and HLA region to track the inheritance of familial haplotypes. As expected, the same HBB haplotypes were observed in each affected child. Two children were found to have the same set of HLA haplotypes, while the third inherited the opposite paternal HLA haplotype. This is consistent with the HLA-typing reported by an external laboratory. Recombination and consanguineous regions were observed but did not impact our ability to determine genetic phase. We successfully used karyomapping to build a complex PGD test, which would have been highly challenging using previous methods. Moreover, we determined the HLA phases of all three children simultaneously and confirmed that recombination and consanguinity would not interfere with diagnosis or HLA matching. Our success addresses concerns about using karyomapping for HLA matching and diagnosing embryos of consanguineous families. Karyomapping combined with PGS by Next-Generation Sequencing represents a great advance to the field, 25 years after its inception.

Digging deep: Identification of a hidden crossover

Aim When patients requiring stem cell transplantation have multiple siblings as potential donors, HLA typing of all candidates is performed and haplotypes are ascertained. Recently, we began to routinely include HLA-DP typing in our analysis. Due to a hot spot of recombination between the HLA-DQ and HLA-DP loci, DP mismatches among otherwise HLA-A, B, C, DRB1 and DQB1 matched siblings are not an unexpected occurrence. In the family study shown below, a sibling who was HLA-DP mismatched to the patient was also mismatched at the HLA-DRB * 3 locus. Interestingly, this DRB3 * mismatch would not have been identified had high resolution typing for the HLA-DRB3 * /4 * /5 * loci not been performed. Methods High resolution typing for the HLA-A * , B * , C * , DRB1 * , DRB3 * , DRB4 * , DRB5 * , DQA * , DQB * , DPA * and DPB * loci was performed by SBT and/or SSOP. Results HLA typing results and haplotype assignments are shown below. Sibling 1 and the patient are HLA identical, sharing the “a” and “c” haplotypes. Sibling 2 inherited the alternative maternal and paternal haplotypes (“b” and “d”). Sibling 3 has a unique haplotype (“a/b” and “c”) resulting from a paternal recombination (represented by gray shading in the table below). Conclusions Although likely a rare event, this case illustrates that recombination between the HLA-B and HLA-DRB3 locus resulted in a HLA-DRB3 * mismatch between the patient and a potential donor. Due to sharing of the DRB1 * , DQA and DQB alleles on the “a” and “b” haplotypes, this sibling would have been considered to be only an HLA-DP mismatch with the patient. Collectively, this case illustrates the utility of performing high resolution HLA typing for all the polymorphic HLA loci. Download full-size image

HLA-A, -B, and -DRB1 haplotype frequencies in 5-6/6 HLA matched parents

Aim In our previous study, we suggested that 19% of parents could be donors with HLA zero or one antigen mismatch of HLA-A, -B, and -DRB1 in the graft-versus-host direction, and 6.2% of parents could be 5–6/6 antigen matched donors in Japanese families. A previous study on a German population indicated that 3.5% of parents were 5–6/6 antigen matched donors. Our hypothesis was that linkage disequilibrium caused this difference, and we analyzed the relation between the haplotypes and the numbers of HLA matched loci. Methods The subjects were 1153 Japanese quartet families. Their HLA-A, -B, and -DRB1 alleles were typed by SSO method. Each family has a father, a mother, and a sibling of the patient requiring hematopoietic stem cell transplantation. The parents were divided into two groups: (i) 5–6/6 antigen matched parents (n = 143), and (ii) 0–4/6 antigen matched parents (n = 2163). In each group, NIAH (non-inherited paternal or maternal antigen haplotype) were counted and these frequencies were calculated. Results Top eight NIAH frequencies of all parents, group (i), and group (ii) are shown below. Download : Download full-size image Conclusion There were not many tendencies for NIAH of 5-6/6 antigen matched parents to be biased against frequent haplotypes in the Japanese population.

The sooner the better: Our laboratory experience with chorionic villus sampling (CVS) HLA typing prior to HSCT

Aim Allogeneic Hematopoietic stem cell transplantation(HSCT) will prolong and improve the quality of life of babies born affected by inherited metabolic diseases. Genetic testing and preparation for a potential HSCT starts at 10 weeks of mother’s pregnancy. This study aims to describe our strategy to obtain an HLA typing from the minuscule tissue fragments submitted for HLA typing in preparation for an HSCT just after birth. Methods Mothers carrying babies that may be affected with Hurler or Krabbe A syndromes are the most frequent challenges at Duke Hospital. As needed, CVS is submitted to our laboratory and DNA is extracted using a micro method developed specifically for these samples to allow High Resolution HLA typing. Since maternal blood contamination of the CVS is a common finding, High Resolution HLA typing on the parents is also done to assist with the identification of all HLA alleles present on the CVS. Our laboratory sequencing strategy for HLA-A, B, C, DRB1 & DQB1 identifies most alleles in a hemizygous form and duplication of the same allele (potential homozygosis) is always resolved by both parents HLA typing. All positive SBT amplification reactions obtained for the CVS DNA are sequenced, and based on parents typing results we are able to identify, first the paternal haplotype present followed by the maternal one. The software SeqPilot®, used for high resolution data analysis, allows the analysis of all positive reactions chosen to be sequenced. Results The CVS samples we have tested (total of 20 cases) have provided the correct pool of alleles expected to be present and it facilitated cord blood searches for these unborn babies, providing an increased chance for successful treatment. Conclusions The HLA typing strategy we apply to CVS is only possible due to our level of expertise extracting DNA from small tissue fragments and the flexibility and data quality offered by the High Resolution HLA Typing methodology and software data analysis we use. The affected and genetic carrier unborn babies get a much higher chance at cure and survival when their diagnosis and treatment can be done earlier in their lives.

Extreme-Depth Re-sequencing of Mitochondrial DNA Finds No Evidence of Paternal Transmission in Humans

Recent reports have questioned the accepted dogma that mammalian mitochondrial DNA (mtDNA) is strictly maternally inherited. In humans, the argument hinges on detecting a signature of inter-molecular recombination in mtDNA sequences sampled at the population level, inferring a paternal source for the mixed haplotypes. However, interpreting these data is fraught with difficulty, and direct experimental evidence is lacking. Using extreme-high depth mtDNA re-sequencing up to ~1.2 million-fold coverage, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans, thus excluding a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals. Our findings indicate that an active mechanism eliminates paternal mtDNA which likely acts at the molecular level.

Characteristics of de novo structural changes in the human genome.

Small insertions and deletions (indels) and large structural variations (SVs) are major contributors to human genetic diversity and disease. However, mutation rates and characteristics of de novo indels and SVs in the general population have remained largely unexplored. We report 332 validated de novo structural changes identified in whole genomes of 250 families, including complex indels, retrotransposon insertions, and interchromosomal events. These data indicate a mutation rate of 2.94 indels (1–20 bp) and 0.16 SVs (>20 bp) per generation. De novo structural changes affect on average 4.1 kbp of genomic sequence and 29 coding bases per generation, which is 91 and 52 times more nucleotides than de novo substitutions, respectively. This contrasts with the equal genomic footprint of inherited SVs and substitutions. An excess of structural changes originated on paternal haplotypes. Additionally, we observed a nonuniform distribution of de novo SVs across offspring. These results reveal the importance of different mutational mechanisms to changes in human genome structure across generations.

Mother Donors Confer Protection Against Infectious Mortality after Haploidentical T Cell-Depleted Hematopoietic Transplantation

Transplacental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal microchimerism in both mother and child because of exposure of the two immune systems to the non-self alloantigens (Maloney et al., J Clin Invest. 1999;104:41-47). Studies show the immune system in the mother is capable of being sensitized by paternal histocompatibility antigens. For example, antibodies directed against paternal HLA-antigens (van Rood et al., Nature. 1958;181:1735-1736) and T lymphocytes directed against paternal major (van Kampen et al., Hum Immunol. 2001;62:201-207) and minor histocompatibility antigens (Verdijk et al., Blood. 2004;103:1961-1964) are frequently detected in multiparous women. We previously demonstrated mother/child immune interactions positively influenced the outcome of mother to child HLA haploidentical T cell-depleted hematopoietic transplantation. In a series of adult and pediatric patients we demonstrated mother donors conferred protection against leukemia relapse and improved transplant related mortality (TRM), which was largely due to infection, and improved survival (Stern et al., Blood 2008, Oct 1;112:2990-5). However, in unmanipulated haploidentical transplantation, it has been recently shown that transplantation from mother donors increases the incidence of GvHD and decreases survival (Huang et al., EBMT 2014).Here, we analyzed the outcomes of 238 adult acute leukemia patients after T cell-depleted haploidentical transplantation. When compared with transplantation from all other family members, transplantation from mother donors was associated with significantly lower TRM (largely infectious) (27% vs 50% from all other donors, P = 0.01). Multivariate analyses demonstrated transplantation from mother donors was an independent factor predicting improved survival (hazard ratio 0.41, 95% confidence interval 0.12 to 0.95, P = 0.03). In an attempt to elucidate the mechanism, we analyzed donor T cell repertoires that were specific for CMV antigens presented by recipient APCs (by ELISPOT and by limiting dilution cloning). Unlike all other donor/recipient pairs, mothers possessed CMV-specific CD8 cell clones that killed child’s and father’s CMV-pulsed dendritic cells (DCs). Such clones were non-alloreactive as they did not kill the child’s or father’s non-CMV-pulsed DCs. Mothers also possessed CD4 T cell clones that produced IFN-gamma in response to child’s and father’s CMV-loaded antigen-presenting cells (APCs). Such clones were non-alloreactive as they did not respond to child’s or father’s non-CMV-pulsed APCs. Thus, mothers possessed a T cell repertoire that recognized CMV antigens also when presented by the unshared, father’s, HLA haplotype. In fact, they showed twice as many T cells that recognized CMV antigens presented by the child’s APCs than all other donor/recipient pairs (p<0.05).Apparently, therefore, pregnancy resulted in the generation of an additional T cell repertoire that specifically recognized pathogen antigens presented by the unshared paternal HLA haplotype antigens on the child’s APCs. Apparently, upon mother to child T cell-depleted hematopoietic transplantation, such repertoire expands over time and helps reduce infectious mortality. Further studies are needed to elucidate the mechanisms underlying mother T cell selection/education by paternal HLA haplotype antigens on the child’s APCs. DisclosuresNo relevant conflicts of interest to declare.

At the southeast fringe of the Bantu expansion: genetic diversity and phylogenetic relationships to other sub-Saharan tribes

Here, we present 12 loci paternal haplotypes (Y-STR profiles) against the backdrop of the Y-SNP marker system of Bantu males from the Maputo Province of Southeast Africa, a region believed to represent the southeastern fringe of the Bantu expansion. Our Maputo Bantu group was analyzed within the context of 27 geographically relevant reference populations in order to ascertain its genetic relationship to other Bantu and non Bantu (Pygmy, Khoisan and Nilotic) sub-equatorial tribes from West and East Africa. This study entails statistical pair wise comparisons and multidimensional scaling based on YSTR Rst distances, network analyses of Bantu (B2a-M150) and Pygmy (B2b-M112) lineages as well as an assessment of Y-SNP distribution patterns. Several notable findings include the following: 1) the Maputo Province Bantu exhibits a relatively close paternal affinity with both east and west Bantu tribes due to high proportion of Bantu Y chromosomal markers, 2) only traces of Khoisan (1.3%) and Pygmy (1.3%) markers persist in the Maputo Province Bantu gene pool, 3) the occurrence of R1a1a-M17/M198, a member of the Eurasian R1a-M420 branch in the population of the Maputo Province, may represent back migration events and/or recent admixture events, 4) the shared presence of E1b1b1-M35 in all Tanzanian tribes examined, including Bantu and non-Bantu groups, in conjunction with its nearly complete absence in the West African populations indicate that, in addition to a shared linguistic, cultural and genetic heritage, geography (e.g., east vs. west) may have impacted the paternal landscape of sub-Saharan Africa, 5) the admixture and assimilation processes of Bantu elements were both highly complex and region-specific.

A saturated genetic linkage map of autotetraploid alfalfa (Medicago sativa L.) developed using genotyping-by-sequencing is highly syntenous with the Medicago truncatula genome.

A genetic linkage map is a valuable tool for quantitative trait locus mapping, map-based gene cloning, comparative mapping, and whole-genome assembly. Alfalfa, one of the most important forage crops in the world, is autotetraploid, allogamous, and highly heterozygous, characteristics that have impeded the construction of a high-density linkage map using traditional genetic marker systems. Using genotyping-by-sequencing (GBS), we constructed low-cost, reasonably high-density linkage maps for both maternal and paternal parental genomes of an autotetraploid alfalfa F1 population. The resulting maps contain 3591 single-nucleotide polymorphism markers on 64 linkage groups across both parents, with an average density of one marker per 1.5 and 1.0 cM for the maternal and paternal haplotype maps, respectively. Chromosome assignments were made based on homology of markers to the M. truncatula genome. Four linkage groups representing the four haplotypes of each alfalfa chromosome were assigned to each of the eight Medicago chromosomes in both the maternal and paternal parents. The alfalfa linkage groups were highly syntenous with M. truncatula, and clearly identified the known translocation between Chromosomes 4 and 8. In addition, a small inversion on Chromosome 1 was identified between M. truncatula and M. sativa. GBS enabled us to develop a saturated linkage map for alfalfa that greatly improved genome coverage relative to previous maps and that will facilitate investigation of genome structure. GBS could be used in breeding populations to accelerate molecular breeding in alfalfa.

Noninvasive prenatal testing for autosomal recessive conditions by maternal plasma sequencing in a case of congenital deafness.

The goals of our study were to develop a noninvasive prenatal test for autosomal recessive monogenic conditions and to prove its overall feasibility and potential for clinical integration. We recruited a pregnant woman and her spouse, who had a proband child suffering from congenital deafness, and obtained the target-region sequencing data from a semicustom array that used genomic and maternal plasma DNA from three generations of this family. A haplotype-assisted strategy was developed to detect whether the fetus inherited the pathogenic mutations in the causative gene, GJB2. The parental haplotype was constructed using a trio strategy through two different processes, namely, the grandparent-assisted haplotype phasing process and the proband-assisted haplotype phasing process. The fetal haplotype was deduced afterward based on both the maternal plasma sequencing data and the parental haplotype. The accuracy levels of paternal and maternal haplotypes obtained by grandparent-assisted haplotype phasing were 99.01 and 97.36%, respectively, and the proband-assisted haplotype phasing process yielded slightly lower accuracies of 98.73 and 96.79%, respectively. Fetal inheritance of the pathogenic gene was deduced correctly in both processes. Our study indicates that the strategy of haplotype-based noninvasive prenatal testing for monogenic conditions has potential applications in clinical practice.

Identification of More Than Two Paternal Haplotypes of the Ovine Fatty Acid-Binding Protein 4 (FABP4) Gene in Half-Sib Families: Evidence of Intragenic Meiotic Recombination

The fatty acid binding protein 4 (FABP4) plays an important role in the regulation of lipid metabolism in mammals. In this study, two regions of ovine FABP4 spanning exon 2-intron 2 and exon 3-intron 3 were investigated in four hundred and twenty lambs derived from seven sires that were previously typed as having heterozygous genotypes in both these regions of the gene. These regions have been shown to be variable, with three SNPs plus one indel and four SNPs respectively constituting five and four allele variants in the two regions. Across these regions, fourteen haplotypes have been identified. The lambs were typed using a Polymerase Chain Reaction Single-Stranded Conformational Polymorphism (PCR-SSCP) method to identify the haplotypes inherited from the sires. Between three and four paternally-derived haplotypes were identified in the progeny of six of the seven sires, suggesting that meiotic recombination occurs within ovine FABP4. A number of sequence motifs associated with recombination “hotspots” were detected in the two regions of the gene that were analyzed and these may facilitate the recombination.

Detection of recombination events, haplotype reconstruction and imputation of sires using half-sib SNP genotypes

BackgroundIdentifying recombination events and the chromosomal segments that constitute a gamete is useful for a number of applications in genomic analyses. In livestock, genotypic data are commonly available for half-sib families. We propose a straightforward but computationally efficient method to use single nucleotide polymorphism marker genotypes on half-sibs to reconstruct the recombination and segregation events that occurred during meiosis in a sire to form the haplotypes observed in its offspring. These meiosis events determine a block structure in paternal haplotypes of the progeny and this can be used to phase the genotypes of individuals in single half-sib families, to impute haplotypes of the sire if they are not genotyped or to impute the paternal strand of the offspring’s sequence based on sequence data of the sire.MethodsThe hsphase algorithm exploits information from opposing homozygotes among half-sibs to identify recombination events, and the chromosomal regions from the paternal and maternal strands of the sire (blocks) that were inherited by its progeny. This information is then used to impute the sire’s genotype, which, in turn, is used to phase the half-sib family. Accuracy (defined as R2) and performance of this approach were evaluated by using simulated and real datasets. Phasing results for the half-sibs were benchmarked to other commonly used phasing programs – AlphaPhase, BEAGLE and PedPhase 3.ResultsUsing a simulated dataset with 20 markers per cM, and for a half-sib family size of 4 and 40, the accuracy of block detection, was 0.58 and 0.96, respectively. The accuracy of inferring sire genotypes was 0.75 and 1.00 and the accuracy of phasing was around 0.97, respectively. hsphase was more robust to genotyping errors than PedPhase 3, AlphaPhase and BEAGLE. Computationally, hsphase was much faster than AlphaPhase and BEAGLE.ConclusionsIn half-sib families of size 8 and above, hsphase can accurately detect block structure of paternal haplotypes, impute genotypes of ungenotyped sires and reconstruct haplotypes in progeny. The method is much faster and more accurate than other widely used population-based phasing programs. A program implementing the method is freely available as an R package (hsphase).

Efficient fast heuristic algorithms for minimum error correction haplotyping from SNP fragments

Availability of complete human genome is a crucial factor for genetic studies to explore possible association between the genome and complex diseases. Haplotype, as a set of single nucleotide polymorphisms (SNPs) on a single chromosome, is believed to contain promising data for disease association studies, detecting natural positive selection and recombination hotspots. Various computational methods for haplotype reconstruction from aligned fragment of SNPs have already been proposed. This study presents a novel approach to obtain paternal and maternal haplotypes form the SNP fragments on minimum error correction (MEC) model. Reconstructing haplotypes in MEC model is an NP-hard problem. Therefore, our proposed methods employ two fast and accurate clustering techniques as the core of their procedure to efficiently solve this ill-defined problem. The assessment of our approaches, compared to conventional methods, on two real benchmark datasets, i.e., ACE and DALY, proves the efficiency and accuracy.