Genome-wide association analysis for ß-hydroxybutyrate concentration in Milk in Holstein dairy cattle.

BACKGROUND: Ketosis in dairy cattle has been shown to cause a high morbidity in the farm and substantial financial losses to dairy farmers. Ketosis symptoms, however, are difficult to identify, therefore, the amount of ketone bodies (mainly ß-hydroxybutyric acid, BHB) is used as an indicator of subclinical ketosis in cows. It has also been shown that milk BHB concentrations have a strong correlation with ketosis in dairy cattle. Mid-infrared spectroscopy (MIR) has recently became a fast, cheap and high-throughput method for analyzing milk components. The aim of this study was to perform a genome-wide association study (GWAS) on the MIR-predicted milk BHB to identify genomic regions, genes and pathways potentially affecting subclinical ketosis in North American Holstein dairy cattle. RESULTS: Several significant regions were identified associated with MIR-predicted milk BHB concentrations (indicator of subclinical ketosis) in the first lactation (SCK1) and second and later lactations (SCK2) in Holstein dairy cows. The strongest association was located on BTA6 for SCK1 and BTA14 on SCK2. Several SNPs on BTA6 were identified in regions and variants reported previously to be associated with susceptibility to ketosis and clinical mastitis in Jersey and Holstein dairy cattle, respectively. One highly significant SNP on BTA14 was found within the DGAT1 gene with known functions on fat metabolism and inflammatory response in dairy cattle. A region on BTA6 and three SNPs on BTA20 were found to overlap between SCK1 and SCK2. However, a novel region on BTA20 (55-63 Mb) for SCK2 was also identified, which was not reported in previous association studies. Enrichment analysis of the list of candidate genes within the identified regions for MIR-predicted milk BHB concentrations yielded molecular functions and biological processes that may be involved in the inflammatory response and lipid metabolism in dairy cattle. CONCLUSIONS: The results of this study confirmed several SNPs and genes identified in previous studies as associated with ketosis susceptibility and immune response, and also found a novel region that can be used for further analysis to identify causal variations and key regulatory genes that affect clinical/ subclinical ketosis.

Estimating the effect of the deleterious recessive haplotypes AH1 and AH2 on reproduction performance of Ayrshire cattle.

The effects of 2 deleterious recessive haplotypes on reproduction performance of Ayrshire cattle, Ayrshire Haplotype 1 (AH1) and Ayrshire Haplotype 2 (AH2), were investigated in Canadian Ayrshire cattle. We calculated their phenotypic effects on stillbirth (SB) rate and 56-d nonreturn rate (NRR) by estimating the interaction of service sire carrier status with maternal grandsire carrier status using the official Canadian evaluation models for those 2 traits. The interaction term included 9 subclasses for the 3 possible statuses of each bull: haplotype carrier, noncarrier, or not genotyped. For AH1, 394 carriers and 1,433 noncarriers were available, whereas 313 carriers and 1,543 noncarriers were available for the AH2 haplotype. The number of matings considered for SB was 34,312 for heifers (first parity) and 115,935 for cows (later parities). For NRR, 49,479 matings for heifers and 160,528 for cows were used to estimate the haplotype effects. We observed a negative effect of AH1 on SB rates, which was 2.0% higher for matings of AH1-carrier sires to dams that had an AH1-carrier sire; this effect was found for both heifers and cows. However, AH1 had small, generally nonsignificant effects on NRR. The AH2 haplotype had a substantial negative effect on NRR, with 5.1% more heifers and 4.0% more cows returning to service, but the effects on SB rates were inconsistent and mostly small effects. Our results validate the harmful effects of AH1 and AH2 on reproduction traits in the Canadian Ayrshire population. This information will be of great interest for the dairy industry, allowing producers to make mating decisions that would reduce reproductive losses.

Candidate gene association analyses for ketosis resistance in Holsteins.

High-yielding dairy cattle are susceptible to ketosis, a metabolic disease that negatively affects the health, fertility, and milk production of the cow. Interest in breeding for more robust dairy cattle with improved resistance to disease is global; however, genetic evaluations for ketosis would benefit from the additional information provided by genetic markers. Candidate genes that are proposed to have a biological role in the pathogenesis of ketosis were investigated in silico and a custom panel of 998 putative single nucleotide polymorphism (SNP) markers was developed. The objective of this study was to test the associations of these new markers with deregressed estimated breeding values (EBV) for ketosis. A sample of 653 Canadian Holstein cows that had been previously genotyped with a medium-density SNP chip were regenotyped with the custom panel. The EBV for ketosis in first and later lactations were obtained for each animal and deregressed for use as pseudo-phenotypes for association analyses. Results of the mixed inheritance model for single SNP association analyses suggested 15 markers in 6 unique candidate genes were associated with the studied trait. Genes encoding proteins involved in metabolic processes, including the synthesis and degradation of fatty acids and ketone bodies, gluconeogenesis, lipid mobilization, and the citric acid cycle, were identified to contain SNP associated with ketosis resistance. This work confirmed the presence of previously described quantitative trait loci for dairy cattle, suggested novel markers for ketosis-resistance, and provided insight into the underlying biology of this disease.

Molecular cloning and localization to chromosome 6 of mouse INT1L1 gene.

The human INT1L1 gene, which exhibits homology to the protooncogene INT1 is very closely linked to the MET gene and cystic fibrosis locus on human chromosome 7. In the present study we have isolated overlapping genomic clones that correspond to the mouse homolog of the INT1L1 gene and have used the cloned DNA as probes to examine the distribution of the mouse INT1L1 gene within a series of 35 mouse-hamster somatic cell hybrids. These analyses have localized the INT1L1 gene to mouse chromosome 6. In addition, we demonstrate that the mouse INT1L1 and MET genes are coamplified in lines of spontaneously transformed mouse NIH3T3 cells, indicating that these genes may remain closely linked within the mouse genome.

Characterization of the mouse met proto-oncogene.

The DNA sequence of cDNA clones prepared from transcripts of the mouse met proto-oncogene reveals that the mouse met gene encodes a 1380 amino acid protein with the characteristics of a growth factor receptor. This protein can be divided into several putative domains, including an intracellular protein tyrosine kinase domain, a transmembrane domain and a 929 amino acid extracellular domain, possessing a potential proteolytic cleavage site with the sequence Lys-Arg-Arg-Lys-Arg-Ser. To gain additional insights into the function of the met protein we have examined the level of met transcripts in tissues of the late-gestation mouse conceptus. Transcription of met was observed in most of the tissues analysed, but the highest levels of met mRNA were detected in the yolk sac, amnion and kidney; no transcripts were detectable in the calvaria. Chromosomal localization using a series of mouse-hamster hybrid cell lines has demonstrated that met is located on mouse chromosome 6.

Chromosomal organization of the cytochrome P450-2C gene family in the mouse: a locus associated with constitutive aryl hydrocarbon hydroxylase.

Cytochromes P-450 represent a superfamily of enzymes with a central role in the metabolism of drugs, chemical toxins, and carcinogens. We have used genetic analysis to establish the complexity and catalytic function of a recently identified constitutively expressed murine hepatic cytochrome P-450 encoded by P450-2C. Southern blotting analysis shows that there are at least seven or eight genes within this family in the mouse and rat and that DNA restriction fragment length variants between different mouse inbred strains are observed. Analysis of recombinant inbred strains derived from these parent strains shows (i) these genes are clustered within 1 centimorgan, (ii) this gene family does not correspond to any of the known cytochrome P-450 loci or map near any well-characterized genomic markers, and (iii) this gene family segregates to within 1-2 centimorgans of a locus controlling constitutive aryl hydrocarbon hydroxylase activity in mice. With use of Chinese hamster/mouse somatic cell hybrids, the P450-2C locus was assigned to a region of mouse chromosome 19 that appears to be syntenic with the previously mapped human P450C2C locus on human chromosome 10. By in situ hybridization to mitotic mouse chromosomes, we have localized this region to the tip of chromosome 19. These results are discussed in relation to the physiological roles of this P-450 family in foreign compound metabolism and steroid oxidations.

A mouse homeobox containing gene on chromosome 11: sequence and tissue-specific expression.

We have molecularly cloned a mouse homeobox containing gene by isolating cDNA and genomic clones. The gene is located in a previously described cluster on chromosome 11 (Hart et al. (1985) Cell 43, 9-18) and was identified as the Hox2.3 gene. We present the complete mRNA sequence of this gene and describe similarities to other homeobox containing genes, among which its human homologue, the cl gene. High expression of the Hox2.3 gene was found in kidney, testis, and spinal cord of adult mice, in the spinal cord of 12.5-17.5 day embryos and in differentiating EC cells depending on their treatment. Three different treatments of the pluripotent EC cell line P19, each leading to the induction of a specific differentiation pathway, resulted in all cases in induction of Hox2.3; however, major quantitative differences in this response were observed.

Comparison of circulating MAM-6 and CEA levels and correlation with the estrogen receptor in patients with breast cancer.

MAM-6 and CEA serum levels of 136 staged breast cancer patients were determined concomitantly. The sensitivities of the MAM-6 assay using monoclonal antibody (MAb) 115D8 and a polyclonal CEA assay were equally low and only a limited number of patients with early stages of breast cancer showed elevated antigen levels. However, the sensitivity rose to 75% for MAM-6 and to 60% for CEA in stage-IV patients. The levels of both antigens correlated well in the sera of these patients, although MAM-6 serum levels were elevated more frequently, while only in a few cases were MAM-6-negative sera CEA-positive. A group of stage-II breast cancer patients who eventually developed distant metastases was followed in a longitudinal study. Tumor progression or regression was clinically determined and compared with the MAM-6 and CEA serum levels in order to establish the value of each assay for the monitoring of breast cancer. The course of the disease correlated significantly better with changes in MAM-6 antigen levels than with changes in CEA levels (p less than 0.05), being 79% and 42% respectively. The lower correlation of CEA levels with the course of the disease was mainly due to a lower sensitivity of the CEA assay for advanced breast cancer. The specificity of changing MAM-6 and CEA levels was not significantly different. The main advantage of the MAM-6 assay over the CEA assay is the higher sensitivity of the former. In a preliminary study among stage-IV patients a correlation was found between elevated MAM-6 levels and the presence of the estrogen receptor in the primary tumor.

MAM-6 antigen, a new serum marker for breast cancer monitoring.

Almost all carcinomas contain a cell surface antigen, MAM-6, which has been defined by several monoclonal antibodies, including 115D8 (Hilkens et al., Int. J. Cancer, 34: 197-206, 1984). A quantitative sandwich radioimmunoassay, using 115D8 as catcher and as tracer antibody, has been developed to detect MAM-6 in serum. To quantitate the MAM-6 level, pooled human milk was used as a standard, and arbitrary units were chosen. Less than 5% of the sera of apparently healthy individuals contained more than 5 units/ml. In sera of patients with benign breast lesions, the same low levels were detected. However, concentrations over 5 units/ml were found in 24, 21, 43, and 79% of the sera of patients with pathological Stages I, II, III, and IV breast cancer, respectively. MAM-6 levels were also increased in almost all sera tested from patients with advanced stages of ovarian carcinoma, but in a low percentage of sera from patients with other advanced cancers. A longitudinal study was carried out to test the MAM-6 assay as clinical marker to monitor the therapeutic response of breast cancer. Increasing or decreasing MAM-6 serum levels correlated in 93% of the cases with breast cancer progression or regression, indicating that the assay can be used to monitor the course of the disease during therapy. In some breast cancer patients, elevated MAM-6 levels were observed prior to any clinical indication of tumor recurrence.

Genetic mapping of Pim-1 putative oncogene to mouse chromosome 17.

Pim-1 is a putative oncogene activated in T-cell lymphomas induced by Moloney and AKR mink cell focus forming (MCF) viruses. We have determined the chromosomal localization of the Pim-1 gene in mice by Southern blot analysis of DNAs obtained from a panel of mouse-Chinese hamster somatic cell hybrids. The Pim-1 gene was localized on chromosome 17, a chromosome frequently aberrant in T-cell lymphomas. Two chromosomal regions, containing sequences homologous to regions within the Pim-1 locus, were localized on chromosome 6 and 16.

Mouse interferon alpha and beta genes are linked at the centromere proximal region of chromosome 4.

In order to determine the chromosomal localization of the murine interferon-alpha (MuIFN-alpha) and murine interferon-beta (MuIFN-beta) genes the DNAs of a panel of somatic cell hybrids were analysed by Southern blot hybridization. The hybrid cells were derived from E36 Chinese hamster cells and GRSL or GR MaTu mouse cells and retained all hamster chromosomes but segregated mouse chromosomes. The MuIFN-alpha probe used was a 0.7 kb HindIII-EcoRI fragment derived from the MuIFN-alpha 1 gene which hybridized with both mouse and hamster DNA. However, four fragments present in EcoRI digests of mouse DNA were clearly absent from the hybridization profile of EcoRI-digested hamster DNA and could be used for detection of MuIFN-alpha sequences in the hybrid cells. The MuIFN-beta probe, a 0.5 kb BglII-BamHI fragment derived from the MuIFN-beta gene, hybridized with a 2.6 kb EcoRI fragment of mouse DNA and only weakly cross-hybridized with a 4.8 kb EcoRI fragment in hamster DNA. Southern blot analysis of DNA from mouse/hamster hybrids compared with the analysis of chromosome markers showed that both the MuIFN-alpha and the MuIFN-beta genes are located on chromosome 4. Analysis of DNA from hybrids that contained only part of chromosome 4 indicated that the MuIFN-alpha gene family and the MuIFN-beta gene are situated at the centromere-proximal region of the chromosome.

Genetic marker patterns of inbred strains of mice at the Cancer Research Institute, Bombay.

36 genetic markers were examined in 14 inbred strains of mice maintained at the Cancer Research Institute, Bombay. The genetic marker profiles of these strains when compared with the profiles of similar strains maintained elsewhere revealed discrepancies in 4 of them which are reported and discussed. The results emphasize the importance of genetic monitoring of inbred strains.

Localization of a gene controlling the expression of the human transferrin receptor to the region q12 leads to qter of chromosome 3.

A monoclonal antiserum, 66-IG10, raised against human thymocytes was found to be directed against the human transferrin receptor. A panel of human X Chinese hamster somatic cell hybrids, in conjunction with the 66-IG10 reagent, was used to assign the gene(s) coding for the transferrin receptor to the q12 leads to qter region of human chromosome 3.

Identification of a cellular receptor for mouse mammary tumor virus and mapping of its gene to chromosome 16.

Pseudotypes of vesicular stomatitis virus (VSV) containing envelope glycoproteins provided by C3H mammary tumor virus (MTV) instead of the normal VSV G-proteins were prepared and used to assay the presence of an MTV receptor on cells. The assay was specific as demonstrated by competition studies with excess MTV particles and neutralization of the pseudotypes with anti-MTV serum or monoclonal antibodies directed against MTV gp52. The MTV receptor was abundantly present on mouse cells but hardly detectable on nonmurine cells, including the Chinese hamster cell line E36. Somatic cell hybrids between E36 cells and GRS/A spontaneous leukemia cells (GRSL cells) and between E36 and GRS/A primary mammary tumor cells were made. The hybrids retained all Chinese hamster chromosomes but segregated mouse chromosomes. From the analysis of the isoenzymes and chromosomes of the hybrid cell lines we conclude that the gene for the receptor (MTVR-1) is located on mouse chromosome 16.