Col1a1 Locus Research Articles

Chromosomal localization of the chicken and mammalian orthologues of the orphan phosphatase PHOSPHO1 gene.

PHOSPHO1 is a recently identified phosphatase expressed at high levels in the chicken growth plate and which may be involved in generating inorganic phosphate for skeletal matrix mineralization. Using a degenerate RT-PCR approach a fragment of human PHOSPHO1 was cloned. This enabled the identification of the human orthologue on HSA17q21, and the mouse orthologue on a region of MMU11 that exhibits conservation of synteny with HSA17q21. Chicken PHOSPHO1 was mapped by SSCP analysis to position 44 cM on GGA27, adjacent to the HOXB@ (44 cM) and COL1A1 (36 cM) loci. Comparison of genes on GGA27 with their orthologues on the preliminary draft of the human genome identifies regions of conserved synteny equivalent to 25 Mb on HSA17q21.2-23.3 and approximately 20 Mb on GGA27 in which the gene order appears to be conserved. Mapping of the PHOSPHO1 genes to regions of HSA17q21.3, MMU11 and GGA27 that exhibit conservation of synteny provides strong evidence that they are orthologous.

Genetic control of bone density and turnover: role of the collagen 1alpha1, estrogen receptor, and vitamin D receptor genes.

Genetic factors are known to influence both the peak bone mass and probably the rate of change in bone density. A range of regulatory and structural genes has been proposed to be involved including collagen 1alpha (COL1A1), the estrogen receptor (ER), and the vitamin D receptor (VDR), but the actual genes involved are uncertain. We therefore studied the role of the COL1A1 and VDR loci in control of bone density by linkage in 45 dizygotic twin pairs and 29 nuclear families comprising 120 individuals. The influences on bone density of polymorphisms of COL1A1, VDR, and ER were studied by association both cross-sectionally and longitudinally in 193 elderly postmenopausal women (average age, 69 years) over a mean follow-up time of 6.3 years. Weak linkage of the COL1A1 locus with bone density was observed in both twins and families (p = 0.02 in both data sets), confirming previous observations of linkage of this locus with bone density. Association between the MscI polymorphism of COL1A1 and rate of lumbar spine bone loss was observed with significant gene-environment interaction related to dietary calcium intake (p = 0.0006). In the lowest tertile of dietary calcium intake, carriers of "s" alleles lost more bone than "SS" homozygotes (p = 0.01), whereas the opposite was observed in the highest dietary calcium intake (p = 0.003). Association also was observed between rate of bone loss at both the femoral neck and the lumbar spine and the TaqI VDR polymorphism (p = 0.03). This association was strongest in those in the lowest tertile of calcium intake, also suggesting the presence of gene-environment interaction involving dietary calcium and VDR, influencing bone turnover. No significant association was observed between the PvuII ER polymorphism alone or in combination with VDR or COL1A1 genotypes, with either bone density or its rate of change. These data support the involvement of COL1A1 in determination of bone density and the interaction of both COL1A1 and VDR with calcium intake in regulation of change of bone density over time.

Susceptibility to osteoporotic fracture is determined by allelic variation at the Sp1 site, rather than other polymorphic sites at the COL1A1 locus.

Previous studies have identified an association between osteoporotic fracture and a polymorphism affecting a Sp1 binding site in the first intron of the collagen type I alpha 1 gene (COL1A1). It is currently unclear, however, whether this association is direct or the result of linkage disequilibrium with other polymorphisms situated nearby. In this study we analyzed the relationship between four well-characterized single-nucleotide polymorphisms at the COL1A1 locus and osteoporotic fracture in 93 patients with vertebral fracture and 88 age-matched controls randomly selected from the community. We studied a MspI polymorphism 26 kb upstream of the COLIA1 gene, the Sp1 binding site polymorphism in intron 1, a RsaI polymorphism in intron 5 and a MnlI polymorphism in exon 52. The Sp1 and RsaI polymorphisms were in strong linkage disequilibrium (chi 2 = 77.87, p < 0.001) and weaker linkage disequilibrium was detected between the Sp1 and MnlI polymorphisms (chi 2 = 5.54, p < 0.025). There was a significant association between COL1A1 haplotypes that included the Sp1 and RsaI polymorphisms and fracture (p < 0.05-0.001), but no association with haplotypes that included only the MspI and MnlI polymorphisms. This association with fracture was strongest when haplotypes were grouped by Sp1 alleles (chi 2 = 11.15, d.f. = 1; p = 0.001). Furthermore, logistic regression analysis showed that of all the polymorphisms tested, only the Sp1 binding site polymorphism acted as an independent predictor of fracture: odds ratio [95% CI] = 2.26 [1.09-4.69]. These data suggest that it is the Sp1 polymorphism rather than other polymorphisms at the COL1A1 locus which act as a marker for osteoporotic fractures.

Determinants of bone mineral density in postmenopausal white Iowans.

Osteoporosis is a major health problem for older individuals. For women, development of osteoporosis is a function of the accretion of "peak" bone mass in the third decade, age at menopause, and rate of bone loss with aging. Low bone mineral density (BMD) is a major risk factor for osteoporosis and fracture. The purpose of this study was to identify life style, nutritional, medical, and genetic predictors of low BMD in postmenopausal Iowa women. One hundred thirty-four postmenopausal White women ranging in age from 57 to 81 years were included in this case-control study. Bone mineral density was measured at the femoral neck, using dual photon X-ray absorptiometry (Hologic 2000 QDR). Sixty-six women with BMD measurements below 0.68 g/cm2 (the bottom quartile of the BMD distribution in the population from which participants were recruited), and 68 women with values at or above 0.83 g/cm2 (the top quartile of the BMD distribution in the same population) were included. Information about environmental, nutritional, medical, and life style modifiers of BMD was obtained by written questionnaire and telephone interview. To assess familial factors that might influence BMD, we obtained a detailed family history for each participant. In addition, we tested the hypothesis that allelic variation at the Vitamin D receptor (VDR), and the type I collagen gene (COL1A1 and COL1A2) loci influence BMD. Weight, loss of height, age, and age at menopause were strong predictors of BMD in our population. After adjustment for these differences, we found no effect of genotype at the COL1A1, COL1A2, and VDR loci on BMD. Bone mineral density is a complex trait that is influenced by several different modifiers; in the present study, weight was the best predictor of postmenopausal BMD. While several studies suggest that VDR genotype is an important determinant of BMD, we did not find this association in our population, nor did we identify an association between allelic variation at the type I collagen gene loci and BMD. Identification of genes that determine body mass index may provide additional insight into risk factors for low BMD, and osteoporotic fractures.

Characterization of Multilocus Lesions in Human Cells Exposed to X Radiation and Radon

Human TK6 lymphoblasts were exposed to X radiation or radon, and thymidine kinase negative (TK-/-) mutants were selected, isolated and harvested for analysis of structural changes in the TK gene. A large majority (82%) of the radon-induced mutants, 74% of the X-radiation-induced mutants and 45% of the spontaneous mutants lost the entire active TK allele. To analyze these mutants further we measured the loss of heterozygosity at several loci neighboring the TK locus on chromosome 17q. A greater proportion (61%) of the radon-induced mutants than X-radiation-induced or spontaneous mutants harbored the smaller lesions involving the TK allele alone or extending from the TK locus to one or both of the closest neighboring sequences tested. Further, 21% of the X-radiation-induced mutants but only 5% of the radon-induced mutants lost heterozygosity at the col1A1 locus, 31 Mb from the TK gene. These results are in agreement with a recent analysis of radon- and X-radiation-induced lesions inactivating the HPRT gene of TK6 cells, in which we reported that a lower percentage of radon- than X-radiation-induced mutants showed lesions extending to markers 800 kb or more from the HPRT gene on the X chromosome (Bao et al., Mutat. Res. 326, 1-13, 1995). In the present study, we observed that the percentage of slowly growing and very slowly growing TK-/- mutants was greater after treatment with radon than after treatment with X radiation, regardless of the type of lesion present. It is possible, therefore, that the radon-induced lesions are complex and/or less easily repaired, leading to slow growth in a large proportion of the surviving mutant cells.

Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells.

A subtle mutation that rendered type I collagen resistant to mammalian collagenase has been introduced into the murine Col1a-1 (recently redesignated Cola-1) gene by homologous recombination in embryonic stem (ES) cells. Initially, a "hit and run" procedure was used. Since two steps were required for introducing each mutation and more than one mutation was to be introduced in the same genomic region independently, we have developed a streamlined procedure that involves two sequential replacement-type homologous recombination events. In the first step, an internal deletion was introduced into the Col1a-1 locus along with the positive and negative selectable markers, neo and tk, to mark the region of interest. G418-resistant homologous recombinants were isolated and used in the second step in which the deleted Col1a-1 allele was replaced with a construct containing the desired mutation. Homologous recombinants containing the mutation were identified among the Tk- ES clones after selection with FIAU [1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (called fialuridine)]. Approximately 10% of such clones contained the desired mutation. The double replacement procedure greatly reduces the time and amount of work required to introduce mutations independently into the same or closely linked regions. Once the homologous recombinants derived from the first step are established, the introduction of other mutations into the deleted region becomes a one-step procedure. For X number of introduced mutations, 2X selections are required with the "hit and run" approach, but only X + 1 are required with the double-replacement method. This innovative procedure could be very useful in studies of gene structure and function as well as gene expression and regulation.

Osteogenesis imperfecta type III: mutations in the type I collagen structural genes, COL1A1 and COL1A2, are not necessarily responsible.

Most forms of osteogenesis imperfecta are caused by dominant mutations in either of the two genes, COL1A1 and COL1A2, that encode the pro alpha 1(I) and pro alpha 2(I) chains of type I collagen, respectively. However, a severe, autosomal recessive form of OI type III with a comparatively high frequency has been recognised in the black populations of southern Africa. We preformed linkage analyses in eight OI type III families using RFLPs associated with the COL1A1 and COL1A2 loci to determine whether mutations in the genes for type I collagen were responsible for this form of OI. Recombination between the OI phenotype and polymorphic markers at both loci was shown in three of the eight families investigated. The combined lod scores for the eight families were -10.6 for COL1A1 and -11.2 for COL1A2. Further, we examined the type I procollagen produced by skin fibroblast cultures derived from 15 affected and 12 unaffected subjects from the above eight families plus one further family. We found no evidence for defects in the synthesis, structure, secretion, or post-translational modification of the chains of type I procollagen produced by any of the family members. These results suggest that mutations within or near the type I collagen structural genes are not responsible for this form of OI.

Haplotype analysis of collagen type I genes in the general population and in osteogenesis imperfecta families

The allele frequencies of 2 new polymorphic markers of collagen type I proalpha 1 (COL1A1) and proalpha 2 (COL1A2) genes were determined in a random sample of chromosomes by polymerase chain reaction. The minor allele frequencies were 0.27 for COL1A1/+88Mn1I, and 0.39 for COL1A2/1446 PvuII RFLPs, respectively. These 2 polymorphisms increased the combined (PIC) values we previously determined in the Italian population with Southern blotting procedures, from 0.71 at the COL1A1 locus to 0.81, and from 0.73 at the COL1A2 locus to 0.88, respectively. With a combination of these markers, we have carried out the segregation analysis of 4 new families in which osteogenesis imperfecta (OI) segregated as a dominant trait. The disease segregated with COL1A1 in 2 OI type I families, and with COL1A2 in one OI type IV family. In one OI type I family the concordant locus was uncertain. This analysis was extended to the 7 dominant OI families we previously reported: in 3 out of 11 pedigrees either locus still could not be excluded, indicating the need for more genetic markers. COL1A1 and COL1A2 haplotype frequencies were compared in normal and OI chromosomes: no preferential association of the disease with a given haplotype was detected. The correlation between affected locus and clinical aspects is discussed.

Molecular complementation of a collagen mutation in mammalian cells using yeast artificial chromosomes.

The cloning of large contiguous segments of mammalian DNA in Saccharomyces cerevisiae has become possible with the advent of Yeast Artificial Chromosomes (YACs). We are interested in extending the technique of genetic complementation analysis to the molecular level through the introduction of YACs into mammalian cells and the mammalian germline. We report the successful introduction of homogeneous DNA derived from a 150 kbp YAC spanning the murine Col1a1 locus into murine fibroblasts carrying a mutation at this locus. The YAC DNA was fractionated by pulse field electrophoresis, condensed with polyamines, and introduced into mutant fibroblasts via DNA-lipid micelles. The DNA was integrated as a stable intact unit in 10% of the transfected clones conferring collagen RNA expression to the mutant cells.

Haplotype frequencies of the collagen type-I genes in the Italian population

We have determined the frequencies of six restriction fragment length polymorphisms (RFLPs) of type-I collagen genes in a random sample of 100 subjects. Alpha 1 gene (COL1A1) DNA polymorphisms, FG2/MspI, 2FC6/RsaI, and NST70/RsaI, had polymorphism information content (PIC) values of 0.35, 0.32, and 0.26, respectively. Alpha 2 gene (COL1A2) RFLPs, NJ3/EcoRI, Hf32/RsaI, and Hf32/MspI had PIC values of 0.36, 0.35, and 0.25, respectively. The combined haplotype PIC values were 0.71 at the COL1A1 locus and 0.73 for COL1A2. Two COL1A1 and two COL1A2 RFLPs were more polymorphic than in the English population, making them better markers for the analysis of Italian families affected by osteogenesis imperfecta and some other inherited collagen diseases.

Chromosome 17 markers and von Recklinghausen neurofibromatosis: A genetic linkage study in a British population

A genetic linkage study of the RFLPs identified by nine DNA probes localized to the pericentromeric region and long arm of chromosome 17 has been undertaken in 16 families with von Recklinghausen neurofibromatosis (NF1). Close linkage has been shown with the markers CRI-L946 (D17S36), CRI-L581 (D17S37), p17H8 (D17Z1), and pA10–41 (D17S71). The ERBA1 and COL1A1 loci may also be closely linked, but the data are limited. The results for HOX2 and NGFR suggest only loose linkage with the NF1 gone, while no linkage was found between NF1 and the growth hormone locus. No suggestion of nonallelic heterogeneity of NF1 was found in this study.