High-density SNP Array Research Articles

Mutant TET2 Allele Dosage Affects Response to 5-Azacitidine in Acute Myeloid Leukemia

Personalised medicine is predicted to significantly improve outcomes for cancer patients, but implementation requires comprehensive genetic characterisation of malignant cells to identify therapeutically exploitable vulnerabilities. Using an isogenic cell model system with CRISPR-inactivated TET2 in HEL acute myeloid leukemia (AML) cells and an orthotopic mouse xenograft model we demonstrate that mutant TET2 allele dosage significantly affects sensitivity to 5-azacitidine hypomethylating therapy in AML, with biallelic mutation conferring hypersensitivity relative to monoallelic mutation. In the presence of 5-azacitidine, cell clones with biallelic TET2 mutation had significantly lower cloning efficiency (P = 3 x 10-3) and proliferation in liquid culture (P < 1 x 10-4) compared to isogenic clones with monoallelic TET2 mutation. Mixed populations of monoallelic and biallelicTET2 mutated HEL AML cells were transplanted via intrafemoral injection into Rag2−/−Il2rg−/−129×Balb/c mice, and treatment with 5-azacitidine resulted in significant negative in vivo selection against TET2 null cells relative to cells with monoallelic TET2 mutation (P = 4 x 10-4). Methylation analysis revealed the acquisition of an overall hypermethylation phenotype in TET2 null cells and RNA sequencing identified significant down-regulation of ABCB1 transcript, resulting in concomitant pronounced down-regulation of the MDR1 drug efflux transporter at the protein level. RNA sequencing pathway analysis also identified a global effect on ribosome pathway (KEGG pathway ko03010) transcript levels (Padjusted = 0.002), evidenced by down-regulation of numerous RNA polymerase II components in cells with bi-allelic TET2 mutation compared to cells with monoallelic TET2 mutation. Consistent with our isogenic model data, we characterise biallelic somatic TET2 mutation in a patient with AML that was chemoresistant to anthracycline/cytarabine-based chemotherapy but acutely sensitive to 5-azacitidine, resulting in durable cytomorphological remission. Integration of next generation sequencing, interphase FISH and SNP array analysis of bone marrow at AML presentation, relapse and during remission was used to infer tumour phylogeny which indicated that disease pathogenesis was initiated by a TET2 nonsense mutation (c.2815C>T, Q939*) with subsequent deletion of the second TET2 allele and a NPM1 mutation (c.863_864ins, TCTG) that arose after the acquisition of bi-allelic TET2 mutation. Furthermore, our data demonstrate that 5-azacitidine treatment almost completely eliminated the TET2/NPM1-mutated clone. 5-azacitidine also induced a modest reduction in ancestral pre-leukemic cells carrying bi-allelic TET2 mutation but negative for the NPM1 mutation, although the majority retained viability and re-acquired the ability to differentiate and recapitulate normal haematopoiesis rendering a cytomorphological remission. These observations suggest that bi-allelic TET2 mutation confers sensitivity to the cytotoxic effects of 5-azacitidine, but that the major effect of 5-azacitidine is the induction of phenotypic re-programming. The frequency of TET2 mutation in primary AML is estimated at 10-20%, with the majority of these being monoallelic. We determined the frequency of TET2 alterations in AML patients presenting with a chromosome 4 abnormality discernible cytogenetically. TET2 copy number and mutational status were determined using high density SNP arrays and gene sequencing, respectively. In a panel of 30 AML cases with a chromosome 4 abnormality, four patients were heterozygous for TET2 mutation (all deletions resulting in reduced copy number) and three patients were homozygous for TET2 mutation (deletion plus base substitution in two cases and homozygous base substitution resulting from uniparental disomy in one case). Furthermore, all seven cases with TET2 mutation were characterised by cytogenetics that included loss or gain of material on chromosome 4. In contrast, only 1 case with a TET2 mutation had a translocation affecting chromosome 4. In summary, our data argue in favour of using 5-azacitidine in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of personalised medicine for cancer patients. Disclosures Stoelzel: JAZZ Pharmaceuticals: Consultancy; Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding. Jackson:Celgene, Amgen, Roche, Janssen, Sanofi: Honoraria. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Unraveling genomic associations with feed efficiency and body weight traits in chickens through an integrative approach

BackgroundFeed efficiency and growth rate have been targets for selection to improve chicken production. The incorporation of genomic tools may help to accelerate selection. We genotyped 529 individuals using a high-density SNP chip (600 K, Affymetrix®) to estimate genomic heritability of performance traits and to identify genomic regions and their positional candidate genes associated with performance traits in a Brazilian F2 Chicken Resource population. Regions exhibiting selection signatures and a SNP dataset from resequencing were integrated with the genomic regions identified using the chip to refine the list of positional candidate genes and identify potential causative mutations.ResultsFeed intake (FI), feed conversion ratio (FC), feed efficiency (FE) and weight gain (WG) exhibited low genomic heritability values (i.e. from 0.0002 to 0.13), while body weight at hatch (BW1), 35 days-of-age (BW35), and 41 days-of-age (BW41) exhibited high genomic heritability values (i.e. from 0.60 to 0.73) in this F2 population. Twenty unique 1-Mb genomic windows were associated with BW1, BW35 or BW41, located on GGA1–4, 6–7, 10, 14, 24, 27 and 28. Thirty-eight positional candidate genes were identified within these windows, and three of them overlapped with selection signature regions. Thirteen predicted deleterious and three high impact sequence SNPs in these QTL regions were annotated in 11 positional candidate genes related to osteogenesis, skeletal muscle development, growth, energy metabolism and lipid metabolism, which may be associated with body weight in chickens.ConclusionsThe use of a high-density SNP array to identify QTL which were integrated with whole genome sequence signatures of selection allowed the identification of candidate genes and candidate causal variants. One novel QTL was detected providing additional information to understand the genetic architecture of body weight traits. We identified QTL for body weight traits, which were also associated with fatness in the same population. Our findings form a basis for further functional studies to elucidate the role of specific genes in regulating body weight and fat deposition in chickens, generating useful information for poultry breeding programs.

A genomewide association study in divergently selected lines in rabbits reveals novel genomic regions associated with litter size traits.

Uterine capacity (UC), defined as the total number of kits from unilaterally ovariectomized does at birth, has a high genetic correlation with litter size. The aim of our research was to identify genomic regions associated with litter size traits through a genomewide association study using rabbits from a divergent selection experiment for UC. A high-density SNP array (200K) was used to genotype 181 does from a control population, high and low UC lines. Traits included total number born (TNB), number born alive (NBA), number born dead, ovulation rate (OR), implanted embryos (IE) and embryo, foetal and prenatal survivals at second parity. We implemented the Bayes B method and the associations were tested by Bayes factors and the percentage of genomic variance (GV) explained by windows. Different genomic regions associated with TNB, NBA, IE and OR were found. These regions explained 7.36%, 1.27%, 15.87% and 3.95% of GV, respectively. Two consecutive windows on chromosome 17 were associated with TNB, NBA and IE. This genomic region accounted for 6.32% of GV of TNB. In this region, we found the BMP4, PTDGR, PTGER2, STYX and CDKN3 candidate genes which presented functional annotations linked to some reproductive processes. Our findings suggest that a genomic region on chromosome 17 has an important effect on litter size traits. However, further analyses are needed to validate this region in other maternal rabbit lines.

SNP Marker Selection for Dog Breed Identification from Genotypes of High-density SNP Array and Machine Learning

SNP Marker Selection for Dog Breed Identification from Genotypes of High-density SNP Array and Machine Learning

Genome-wide association scan for QTL and their positional candidate genes associated with internal organ traits in chickens

BackgroundPoultry breeding programs have been focused on improvement of growth and carcass traits, however, this has resulted in correlated changes in internal organ weights and increased incidence of metabolic disorders. These disorders can affect feed efficiency or even cause death. We used a high density SNP array (600 K, Affymetrix) to estimate genomic heritability, perform genome-wide association analysis, and identify genomic regions and positional candidate genes (PCGs) associated with internal organ traits in an F2 chicken population. We integrated knowledge of haplotype blocks, selection signature regions and sequencing data to refine the list of PCGs.ResultsEstimated genomic heritability for internal organ traits in chickens ranged from low (LUNGWT, 0.06) to high (GIZZWT, 0.45). A total of 20 unique 1 Mb windows identified on GGA1, 2, 4, 7, 12, 15, 18, 19, 21, 27 and 28 were significantly associated with intestine length, and weights or percentages of liver, gizzard or lungs. Within these windows, 14 PCGs were identified based on their biological functions: TNFSF11, GTF2F2, SPERT, KCTD4, HTR2A, RB1, PCDH7, LCORL, LDB2, NR4A2, GPD2, PTPN11, ITGB4 and SLC6A4. From those genes, two were located within haplotype blocks and three overlapped with selection signature regions. A total of 13,748 annotated sequence SNPs were in the 14 PCGs, including 156 SNPs in coding regions (124 synonymous, 26 non-synonymous, and 6 splice variants). Seven deleterious SNPs were identified in TNFSF11, NR4A2 or ITGB4 genes.ConclusionsThe results from this study provide novel insights to understand the genetic architecture of internal organ traits in chickens. The QTL detection performed using a high density SNP array covered the whole genome allowing the discovery of novel QTL associated with organ traits. We identified PCGs within the QTL involved in biological processes that may regulate internal organ growth and development. Potential functional genetic variations were identified generating crucial information that, after validation, might be used in poultry breeding programs to reduce the occurrence of metabolic disorders.

Discovery and characterization of functional modules associated with body weight in broilers

Aim of the present study was to investigate whether body weight (BW) in broilers is associated with functional modular genes. To this end, first a GWAS for BW was conducted using 6,598 broilers and the high density SNP array. The next step was to search for positional candidate genes and QTLs within strong LD genomic regions around the significant SNPs. Using all positional candidate genes, a network was then constructed and community structure analysis was performed. Finally, functional enrichment analysis was applied to infer the functional relevance of modular genes. A total number of 645 positional candidate genes were identified in strong LD genomic regions around 11 genome-wide significant markers. 428 of the positional candidate genes were located within growth related QTLs. Community structure analysis detected 5 modules while functional enrichment analysis showed that 52 modular genes participated in developmental processes such as skeletal system development. An additional number of 14 modular genes (GABRG1, NGF, APOBEC2, STAT5B, STAT3, SMAD4, MED1, CACNB1, SLAIN2, LEMD2, ZC3H18, TMEM132D, FRYL and SGCB) were also identified as related to body weight. Taken together, current results suggested a total number of 66 genes as most plausible functional candidates for the trait examined.

Genome-Wide Scan Identifies Selection Signatures in Chinese Wagyu Cattle Using a High-Density SNP Array.

Simple SummaryAnalysis of the genomic regions under consideration can provide important insights into the genetic basis of complex traits. In this study, we utilized a high-density SNP (Single nucleotide polymorphism) array for analysis of genome selection signatures in Chinese Wagyu cattle. In total, 239 selected regions responding to 162 candidate genes were identified, which related to economic traits, including fatty acid, meat quality, growth and developmental traits. Moreover, we found that these selected genes were enriched for the four Gene Ontology (GO) terms of biological regulation and metabolic process. These results also contribute to the understanding of genetic basis of these traits during the formation of this population.Selective breeding can lead to genetic diversity and diverse phenotypes in farm animals. Analysis of the genomic regions under selection can provide important insights into the genetic basis of complex traits. In this study, a high-density SNP array was used for analysis of genome selection signatures in Chinese Wagyu cattle. In total, we obtained 478,903 SNPs and 24,820 no-overlap regions for |iHS| (integrated haplotype score) estimations. Under the threshold of the top 1%, 239 regions were finally identified as candidate selected regions and 162 candidate genes were found based on the UMD3.1 genome assembly. These genes were reported to be associated with fatty acids, such as Bos taurus nitric oxide synthase 1 adaptor protein (NOS1AP), Bos taurus hydroxysteroid 17-beta dehydrogenase 7 (HSD17B7), Bos taurus WD repeat domain 7 (WDR7), Bos taurus ELOVL fatty acid elongase 2 (ELOVL2), Bos taurus calpain 1 (CAPN1), Bos taurus parkin RBR E3 ubiquitin protein ligase (PRKN, also known as PARK2), Bos taurus mitogen-activated protein kinase kinase 6 (MAP2K6), meat quality, including Bos taurus ADAM metallopeptidase domain 12 (ADAM12), Bos taurus 5′-aminolevulinate synthase 1 (ALAS1), Bos taurus small integral membrane protein 13 (SMIM13) and Bos taurus potassium two pore domain channel subfamily K member 2 (KCNK2), growth, and developmental traits, such as Bos taurus insulin like growth factor 2 receptor (IGF2R), Bos taurus RAR related orphan receptor A (RORA), Bos taurus fibroblast growth factor 14 (FGF14), Bos taurus paired box 6 (PAX6) and Bos taurus LIM homeobox 6 (LHX6). In addition, we identified several genes that are associated with body size and weight, including Bos taurus sorting nexin 29 (SNX29), Bos taurus zinc finger imprinted 2 (ZIM2), Bos taurus family with sequence similarity 110 member A (FAM110A), immune system, including Bos taurus toll like receptor 9 (TLR9), Bos taurus TAFA chemokine like family member 1 (TAFA1), Bos taurus glutathione peroxidase 8 (putative) (GPX8), Bos taurus interleukin 5 (IL5), Bos taurus PR domain containing 9 (PRDM9), Bos taurus glutamate ionotropic receptor kainate type subunit 2 (GRIK2) and feed intake efficiency, Bos taurus sodium voltage-gated channel alpha subunit 9 (SCN9A), Bos taurus relaxin family peptide/INSL5 receptor 4 (RXFP4), Bos taurus RNA polymerase II associated protein 3 (RPAP3). Moreover, four GO terms of biological regulation (GO:0009987, GO:0008152) and metabolic process (GO:0003824, GO:0005488) were found based on these genes. In addition, we found that 232 candidate regions (~18 Mb) overlapped with the Quantitative trait loci (QTL)regions extracted from cattle QTLdb. Our findings imply that many genes were selected for important traits in Chinese Wagyu cattle. Moreover, these results can contribute to the understanding of the genetic basis of the studied traits during the formation of this population.

Whole Genome Linkage Disequilibrium and Effective Population Size in a Coho Salmon (Oncorhynchus kisutch) Breeding Population Using a High-Density SNP Array.

The estimation of linkage disequilibrium between molecular markers within a population is critical when establishing the minimum number of markers required for association studies, genomic selection, and inferring historical events influencing different populations. This work aimed to evaluate the extent and decay of linkage disequilibrium in a coho salmon breeding population using a high-density SNP array. Linkage disequilibrium was estimated between a total of 93,502 SNPs found in 64 individuals (33 dams and 31 sires) from the breeding population. The markers encompass all 30 coho salmon chromosomes and comprise 1,684.62 Mb of the genome. The average density of markers per chromosome ranged from 48.31 to 66 per 1 Mb. The minor allele frequency averaged 0.26 (with a range from 0.22 to 0.27). The overall average linkage disequilibrium among SNPs pairs measured as r2 was 0.10. The Average r2 value decreased with increasing physical distance, with values ranging from 0.21 to 0.07 at a distance lower than 1 kb and up to 10 Mb, respectively. An r2 threshold of 0.2 was reached at distance of approximately 40 Kb. Chromosomes Okis05, Okis15 and Okis28 showed high levels of linkage disequilibrium (>0.20 at distances lower than 1 Mb). Average r2 values were lower than 0.15 for all chromosomes at distances greater than 4 Mb. An effective population size of 43 was estimated for the population 10 generations ago, and 325, for 139 generations ago. Based on the effective number of chromosome segments, we suggest that at least 74,000 SNPs would be necessary for an association mapping study and genomic predictions. Therefore, the SNP panel used allowed us to capture high-resolution information in the farmed coho salmon population. Furthermore, based on the contemporary Ne, a new mate allocation strategy is suggested to increase the effective population size.

The SYNBREED chicken diversity panel: a global resource to assess chicken diversity at high genomic resolution

BackgroundSince domestication, chickens did not only disperse into the different parts of the world but they have also undergone significant genomic changes in this process. Many breeds, strains or lines have been formed and those represent the diversity of the species. However, other than the natural evolutionary forces, management practices (including those that threaten the persistence of genetic diversity) following domestication have shaped the genetic make-up of and diversity between today’s chicken breeds. As part of the SYNBREED project, samples from a wide variety of chicken populations have been collected across the globe and were genotyped with a high density SNP array. The panel consists of the wild type, commercial layers and broilers, indigenous village/local type and fancy chicken breeds. The SYNBREED chicken diversity panel (SCDP) is made available to serve as a public basis to study the genetic structure of chicken diversity. In the current study we analyzed the genetic diversity between and within the populations in the SCDP, which is important for making informed decisions for effective management of farm animal genetic resources.ResultsMany of the fancy breeds cover a wide spectrum and clustered with other breeds of similar supposed origin as shown by the phylogenetic tree and principal component analysis. However, the fancy breeds as well as the highly selected commercial layer lines have reduced genetic diversity within the population, with the average observed heterozygosity estimates lower than 0.205 across their breeds’ categories and the average proportion of polymorphic loci lower than 0.680. We show that there is still a lot of genetic diversity preserved within the wild and less selected African, South American and some local Asian and European breeds with the average observed heterozygosity greater than 0.225 and the average proportion of polymorphic loci larger than 0.720 within their breeds’ categories.ConclusionsIt is important that such highly diverse breeds are maintained for the sustainability and flexibility of future chicken breeding. This diversity panel provides opportunities for exploitation for further chicken molecular genetic studies. With the possibility to further expand, it constitutes a very useful community resource for chicken genetic diversity research.

Genome-wide selection for reproductive traits in swine

The introduction of high-density SNP arrays in livestock species has enabled genomic evaluations on a scale not possible just a few years ago. Faster genetic gains are realized from application of genomics in pigs by increasing the accuracy of selection. This is especially important for lowly heritable reproductive traits, where female selection candidates have not yet expressed a phenotype at the point of selection and male candidates have no phenotype at all. Litter size was one of the first traits for which the breeding company PIC implemented genome assisted selection, using a 196 SNP panel in 2010. Significant improvements in accuracy for all selection index traits are now achieved using single step genomic evaluation incorporating genomic information into multivariate mixed model evaluations. Genotyping costs have been a barrier to fullscale implementation, but this challenge has been overcome by imputing high density genotypes from low density panels on selection candidates. Next generation sequencing technology is revolutionizing genomics research. The recently published draft pig genome sequence, along with the availability of phenotypes and tissue samples on thousands of animals, often with complete pedigree, facilitate discovery of genomic regions, genes and causative mutations for traits such as disease resistance. In the future, genome editing has the potential to introgress beneficial alleles from rare or indigenous breeds not present in improved commercial lines, as well as increasing the expression of beneficial genes through directed gene duplication. Additionally, nonlinear and nonparametric methods may be applied to further improve statistical predictions of genetic merit and performance.

Identification of gene expression determinants of radiosensitivity in bladder cancer (BCa) cell lines.

470 Background: Trimodality therapy with TURBT followed by concurrent chemoradiation is an alternative to cystectomy in selected patients with muscle invasive BCa. Identifying genetic determinants of radiation response may help select patient for organ-sparing treatments with curative intent. Methods: Molecular characterization of 20 cell lines was previously performed, including mutation analysis, CNA (high density SNP arrays) and gene expression (RNAseq). Cell line identity was authenticated by genomic fingerprinting. We performed colony forming assays (CFA) and cumulative survival was quantified using the area under the cell survival curve (AUC, range 0-8Gy) to create a radiosensitivity index. Pre and post-radiation proliferative capacity was determined by cell titer glo (CTG) assay. Gene (R v3.5.1) and network (Ingenuity Pathyway Analysis) level expression differences were determined as a function of AUC to identify biologically relevant associations with radiation response. Results: Of 20 cell lines (7 basal, 13 luminal subtype), both CFA and CTG data was successfully obtained for 13 cell lines (6 basal, 7 luminal) treated with high dose rate (HDR) radiation. AUCs for the colony forming assay (CFA) survival curves ranged 1.79-3.27. RNAseq identified 18,634 unique transcripts mapping to distinct loci and 196 genes were identified with AUC correlation p values <0.01 (FDR <0.5, mean FPKM>0.5). These genes were strongly enriched for membership in the peroxisome proliferator (PPAR) pathway (IPA, p = 9.40E-03) and STAT3 pathway (IPA, p = 1.56E-3). Validation studies confirmed PPARγ, ICAM2, JAK3, IL1B, OAS1 and OAS2 genes to be associated with differential response to radiation, with expression upregulated in radio-resistant cells. A basal subtype was strongly associated with a radiosensitive phenotype (Chi-Squared χ2 p=0.0083). Conclusions: BCa radio-sensitivity was associated with baseline gene expression differences in vitro. The basal subtype and repression of either the PPAR or STAT3 pathways predicted increased radiosensitivity. This study nominates candidate biomarkers for mechanistic studies and clinical validation in BCa.

Five genomic regions have a major impact on fat composition in Iberian pigs

The adipogenic nature of the Iberian pig defines many quality attributes of its fresh meat and dry-cured products. The distinct varieties of Iberian pig exhibit great variability in the genetic parameters for fat deposition and composition in muscle. The aim of this work is to identify common and distinct genomic regions related to fatty acid composition in Retinto, Torbiscal, and Entrepelado Iberian varieties and their reciprocal crosses through a diallelic experiment. In this study, we performed GWAS using a high density SNP array on 382 pigs with the multimarker regression Bayes B method implemented in GenSel. A number of genomic regions showed strong associations with the percentage of saturated and unsaturated fatty acid in intramuscular fat. In particular, five regions with Bayes Factor >100 (SSC2 and SSC7) or >50 (SSC2 and SSC12) explained an important fraction of the genetic variance for miristic, palmitoleic, monounsaturated (>14%), oleic (>10%) and polyunsaturated (>5%) fatty acids. Six genes (RXRB, PSMB8, CHGA, ACACA, PLIN4, PLIN5) located in these regions have been investigated in relation to intramuscular composition variability in Iberian pigs, with two SNPs at the RXRB gene giving the most consistent results on oleic and monounsaturated fatty acid content.

Elucidating the genetic background of the early-flowering transgenic genetic stock T1190 with a high-density SNP array

Apple trees have a long juvenile period, which makes apple genetic improvement via breeding costly and time-consuming. Transgenic genetic stocks carrying the early-flowering gene BpMADS4 have been used to reduce the juvenility of apple from five or more years to less than 10 months. One such genetic stock, T1190, has been used widely in breeding and research. It was reported to be a seedling of ‘Pinova’ but the other parent was unknown. Not knowing the alleles that this unknown parent contributed to T1190 brings uncertainties to breeding programs and research studies. In this study, the full pedigree of the genetic stock T1190 was reconstructed using an apple 20K SNP array and a panel of 530 reference cultivars and breeding selections. T1190 was determined to be an offspring of ‘Pinova’ and ‘Idared’. The full pedigree of T1190 was used to deduce the mosaic ancestor composition of the transgene-hosting chromosome. Such knowledge is useful to ensure breeding programs and research studies achieve their expected objectives.

Genomic characterization of mithun (Bos frontalis) populations using high density SNP array

Mithun (Bos frontalis) is a bovine species found in the four North-Eastern Hilly States of India viz., Nagaland, Arunachal, Manipur and Mizoram. The 770k BovineHDBeadChip (Illumina, CA), which is basically a highdensity SNP array was utilized for genomic characterization of four mithun populations and to study genetic diversity present in the populations. SNP genotyping of a total 24 samples representing four mithun populations as per their geographic locations were carried out. The results indicated that the majority of polymorphic SNPs were found to be in HWE. Observed heterozygosity ranged between 0.263±0.040 (Nagaland) to 0.299±0.044 (Arunachal). Inbreeding coefficient ranged from 0.087±0.139 for Nagaland to 0.186±0.053 for Manipur mithun. The result of the Principal Component Analysis (PCA) indicated all the mithuns were distributed together in close proximity and no clear separation of the four populations was observed.The first and second principal components (PC1 and PC2) explained 9.59% and 88.0% of the total variation, respectively. The results of the STRUCTURE analysis also showed no population substructure as inferred from an increasing plot of cross validation error, similar to PCA. In our knowledge, this is the first study on genomic characterization of any Indian livestock species, through analysis of population structure and genetic diversity using high density SNP array. The baseline information generated in our study will help in developing proper strategies for implementing genomic selection and conservation of mithun.

Two novel genomic regions associated with fearfulness in dogs overlap human neuropsychiatric loci

Anxiety disorders are among the leading health issues in human medicine. The complex phenotypic and allelic nature of these traits as well as the challenge of establishing reliable measures of the heritable component of behaviour from the associated environmental factors hampers progress in their molecular aetiology. Dogs exhibit large natural variation in fearful and anxious behaviour and could facilitate progress in the molecular aetiology due to their unique genetic architecture. We have performed a genome-wide association study with a canine high-density SNP array in a cohort of 330 German Shepherds for two phenotypes, fear of loud noises (noise sensitivity) and fear of strangers or in novel situations. Genome-widely significant loci were discovered for the traits on chromosomes 20 and 7, respectively. The regions overlap human neuropsychiatric loci, including 18p11.2, with physiologically relevant candidate genes that contribute to glutamatergic and dopaminergic neurotransmission in the brain. In addition, the noise-sensitivity locus includes hearing-related candidate genes. These results indicate a genetic contribution for canine fear and suggest a shared molecular aetiology of anxiety across species. Further characterisation of the identified loci will pave the way to molecular understanding of the conditions as a prerequisite for improved therapy.

Genomic Studies in a Large Cohort of Hearing Impaired Italian Patients Revealed Several New Alleles, a Rare Case of Uniparental Disomy (UPD) and the Importance to Search for Copy Number Variations.

Hereditary hearing loss (HHL) is a common disorder characterized by a huge genetic heterogeneity. The definition of a correct molecular diagnosis is essential for proper genetic counseling, recurrence risk estimation, and therapeutic options. From 20 to 40% of patients carry mutations in GJB2 gene, thus, in more than half of cases it is necessary to look for causative variants in the other genes so far identified (~100). In this light, the use of next-generation sequencing technologies has proved to be the best solution for mutational screening, even though it is not always conclusive. Here we describe a combined approach, based on targeted re-sequencing (TRS) of 96 HHL genes followed by high-density SNP arrays, aimed at the identification of the molecular causes of non-syndromic HHL (NSHL). This strategy has been applied to study 103 Italian unrelated cases, negative for mutations in GJB2, and led to the characterization of 31% of them (i.e., 37% of familial and 26.3% of sporadic cases). In particular, TRS revealed TECTA and ACTG1 genes as major players in the Italian population. Furthermore, two de novo missense variants in ACTG1 have been identified and investigated through protein modeling and molecular dynamics simulations, confirming their likely pathogenic effect. Among the selected patients analyzed by SNP arrays (negative to TRS, or with a single variant in a recessive gene) a molecular diagnosis was reached in ~36% of cases, highlighting the importance to look for large insertions/deletions. Moreover, copy number variants analysis led to the identification of the first case of uniparental disomy involving LOXHD1 gene. Overall, taking into account the contribution of GJB2, plus the results from TRS and SNP arrays, it was possible to reach a molecular diagnosis in ~51% of NSHL cases. These data proved the usefulness of a combined approach for the analysis of NSHL and for the definition of the epidemiological picture of HHL in the Italian population.

Signatures of Selection in Admixed Dairy Cattle in Tanzania.

Multiple studies have investigated selection signatures in domestic cattle and other species. However, there is a dearth of information about the response to selection in genomes of highly admixed crossbred cattle in relation to production and adaptation to tropical environments. In this study, we evaluated 839 admixed crossbred cows sampled from two major dairy regions in Tanzania namely Rungwe and Lushoto districts, in order to understand their genetic architecture and detect genomic regions showing preferential selection. Animals were genotyped at 150,000 SNP loci using the Geneseek Genomic Profiler (GGP) High Density (HD) SNP array. Population structure analysis showed a large within-population genetic diversity in the study animals with a high degree of variation in admixture ranging between 7 and 100% taurine genes (dairyness) of mostly Holstein and Friesian ancestry. We explored evidence of selection signatures using three statistical methods (iHS, XP-EHH, and pcadapt). Selection signature analysis identified 108 candidate selection regions in the study population. Annotation of these regions yielded interesting genes potentially under strong positive selection including ABCG2, ABCC2, XKR4, LYN, TGS1, TOX, HERC6, KIT, PLAG1, CHCHD7, NCAPG, and LCORL that are involved in multiple biological pathways underlying production and adaptation processes. Several candidate selection regions showed an excess of African taurine ancestral allele dosage. Our results provide further useful insight into potential selective sweeps in the genome of admixed cattle with possible adaptive and productive importance. Further investigations will be necessary to better characterize these candidate regions with respect to their functional significance to tropical adaptations for dairy cattle.

155 Whole genome association analysis suggests an influence of inbreeding on bull sperm morphometry

Inbreeding depression, the genetic condition caused by mating related individuals, is particularly important in several cattle breeds with limited effective populations. This condition is often associated with decreases in performance, including fertility. Furthermore, sperm head morphometry was associated with fertility in several species. To our knowledge, the influence of inbreeding on sperm morphometry has not been widely reported in cattle. In this study, a Sperm Class Analyzer (SCA™, Microptic S.L., Barcelona, Spain) was used to characterise 7 sperm morphometry parameters in 59 Retinta bulls, including sperm head length, width, perimeter, ellipticity, elongation, regularity and rugosity. Two replicates of at least 100 sperm heads, from 2 frozen semen samples, were assessed per individual (n=200 sperm per bull). Additionally, all individuals were genotyped with the Axiom Bos1 high density SNP Array (Affymetrix, Santa Clara, CA, USA). The molecular-based inbreeding factor (FROH; mean 12.5%, range 1.75 to 33.0) had very low correlations with all sperm morphometry parameters. On average, sperm heads from bulls with higher FROH had a smaller area, but variability was high. Correlations between inbreeding and sperm shape were low and negative, length (r=−0.1449; P<0.01), width (r=−0.2494; P<0.01), and rugosity (r=−0.1086; P>0.01) being the most informative. Whole-genome association study (GWAS) analysis, performed using molecular inbreeding as co-factor, revealed 8 SNP, located on chromosomes BTA2, BTA5, BTA7, and BTA11, significantly associated with sperm head regularity and rugosity. Genomic analysis revealed that genes SLF1 and TMEFF2 are located close to enriched SNP. Gene SLF1 (SMC5-SMC6 complex localization factor 1) is associated with the regulation of protein complex and cytoskeleton assembly, whereas TMEFF2 (transmembrane protein with EGF like and 2 follistatin like domains 2) is associated with integral components of cell membrane and cell spreading and development. Therefore, we inferred that SLF1 and TMEFF2 may be involved in variations of sperm head shape. In this preliminary study, there was evidence of a potential influence of inbreeding on sperm morphometry in a beef cattle breed. However, additional studies, ideally including more individuals and additional breeds, are necessary to determine the validity of this potential association.

On the use of dense sets of SNP markers and their potential in relationship inference

With the advent of high density SNP arrays and the progress of next generation sequencing, demands for new methods to handle the subsequent data analysis have exploded. Forensic laboratories are generally hesitant to implement new methods in casework unless they are thoroughly tested and validated. This is particularly true when a third party contractor is involved in the analysis. In this paper we explore data from dense sets of SNP markers and study how different errors could potentially affect the results. Particularly, we study the effects of genotyping errors, linkage disequilibrium as well as the use of inappropriate population frequencies. We demonstrate that ignoring these concepts may lead to false conclusions for some different relationship cases and outline solutions to mitigate these problems.

Chromosome 21 Gain Is Dispensable for Transient Myeloproliferative Disorder (TMD) Development

Transient myeloproliferative disorder (TMD) is a hematopoietic disease, characterized by a clonal proliferation of immature megakaryoblasts in the neonatal period occurring in approximately 10% of newborns with Down syndrome (DS). Rarely, TMD occurs in non-DS newborns but then it is associated with somatic trisomy 21 (tri21). Tri21 together with in-utero gained mutations in the GATA1 gene encoding a myeloid transcription factor are thus considered essential in TMD.Recently, we have identified a TMD with a typical manifestation and course in a newborn without DS/somatic tri21, which admits that tri21 is dispensable for TMD development. To elucidate the alternative TMD pathogenesis, we performed comprehensive genomic/transcriptomic profiling of this TMD case. We utilized high-density SNP array and whole exome and transcriptome sequencing (WES/RNAseq) to detect copy number changes, mutations and fusion genes. We did not find any aberrations on chromosome 21 and any fusion genes. Two focal intronic losses, likely representing benign germline variants, were found on chromosome X. In addition to 6 missense mutations affecting genes without established roles in hematopoietic disorders, we found in-frame deletions in the GATA1 and JAK1 genes. Both mutations are novel. The GATA1 D65_C228del mutation is predicted to result in an internally truncated protein - GATA1aber. Unlike GATA1s (resulting from GATA1 mutations in DS-TMD) which lacks the transactivation domain (TAD) but retains both Zinc fingers (ZF), GATA1aber lacks part of TAD and the N-terminal ZF. Nevertheless, we hypothesize that GATA1aber substitutes the pathogenetic role of GATA1s. The JAK1 gene encodes a non-receptor tyrosine-kinase engaged in the JAK/STAT signaling pathway. The identified mutation results in the loss of phenylalanine 636 (F636del), which is located in the pseudokinase domain and belongs to a conserved amino acid triad (F636-F575-V658) that is believed to mediate a structural switch controlling the JAK1 catalytic activity (Toms, Nat Struct Mol Biol, 2013). JAK1 mutations are implicated in various hematological malignancies including acute megakaryocytic leukemia, and we hypothesize that JAK1 F636del co-operates with GATA1aber on TMD pathogenesis via deregulation of cytokine/growth factor signaling.We cloned the coding sequences of GATA1aber and JAK1 F636del and transfected them into a model cell line in which we confirmed the expression of both in-silico predicted proteins. Their subcellular trafficking was analogous to that of their wild type counterparts; GATA1aber was found in the nucleus and JAK1 F636del in both the nucleus and cytoplasm. Next, we assessed the kinase activity of JAK1 F636del. To distinguish auto- from trans-phosphorylation, we utilized the JAK1 F636del construct harboring an inactivating mutation of an ATP-binding site (K908G). The JAK1 F636del (but not JAK1 F636del + K908G) was autophosphorylated on Y1034/Y1035 and induced STATs phosphorylation both under steady-state conditions and following non-specific stimulation with PMA. However, at all studied time points all phosphorylation levels were lower compared to wild-type JAK1. Moreover, unlike constitutively active JAK1 V658I, JAK1 F636del did not confer IL3-independent growth to the murine B-cell progenitor cell line BAF3. Interestingly, the transforming potential of double-mutated JAK1 (JAK1 V658I + F636del) was enforced compared to JAK1 V658I. These data show that F636del does not lead to constitutive activation, but in the same time it is not functionally neutral. As the impact of F636del on JAK1 function may vary depending on upstream signaling, we are currently assessing JAK1 F636 kinase activity/transforming potential in BAF3 cells stably expressing the IL6 receptor, which (unlike the IL3 receptor) directly activates JAK1 upon ligand binding. In the future, we plan to study the impact of JAK1 F636del on GATA1s induced deregulation of erythroid/megakaryocytic lineage development and to demonstrate “GATA1s-like” function of GATA1aber.To conclude, we identified two novel mutations affecting GATA1 and JAK1 as likely drivers in an alternative tri21-independent TMD pathogenesis. As the pathogenetic role of tri21 has been poorly understood so far, we believe that by clarifying an alternative mechanism of TMD development, we could improve our understanding of this intriguing disease in general.Support: GAUK 86218 DisclosuresNo relevant conflicts of interest to declare.