Loretta D. Spotila

First-stage autosomal genome screen in extended pedigrees suggests genes predisposing to low bone mineral density on chromosomes 1p, 2p and 4q

Osteoporosis is characterized by low bone density, and osteopenia is responsible for 1.5 million fractures in the United States annually.1 In order to identify regions of the genome which are likely to contain genes predisposing to osteopenia, we genotyped 149 members of seven large pedigrees having recurrence of low bone mineral density (BMD) with 330 DNA markers spread throughout the autosomal genome. Linkage analysis for this quantitative trait was carried out using spine and hip BMD values by the classical lod-score method using a genetic model with parameters estimated from the seven families. In addition, non-parametric analysis was performed using the traditional Haseman-Elston approach in 74 independent sib pairs from the same pedigrees. The maximum lod score obtained by parametric analysis in all families combined was +2.08 (θ = 0.05) for the marker CD3D on chromosome 11q. All other combined lod scores from the parametric analysis were less than +1.90, the threshold for suggestive linkage. Non-parametric analysis suggested linkage of low BMD to chromosomes 1p36 (Zmax = +3.51 for D1S450) and 2p23-24 (Zmax = +2.07 for D2S149). Maximum multi-point lod scores for these regions were +2.29 and +2.25, respectively. A third region with associated lod scores above the threshold of suggestive linkage in both single-point and multi-point non-parametric analysis was on chromosome 4qter (Zmax = +2.95 for D4S1539 and Zmax = +2.48 for D4S1554). Our data suggest the existence of multiple genes involved in controlling spine and hip BMD, and indicate several candidate regions for further screening in this and other independent samples.

Meta‐Analysis of Genome‐Wide Scans Provides Evidence for Sex‐ and Site‐Specific Regulation of Bone Mass

Several genome‐wide scans have been performed to detect loci that regulate BMD, but these have yielded inconsistent results, with limited replication of linkage peaks in different studies. In an effort to improve statistical power for detection of these loci, we performed a meta‐analysis of genome‐wide scans in which spine or hip BMD were studied. Evidence was gained to suggest that several chromosomal loci regulate BMD in a site‐specific and sex‐specific manner.

Sequence and expression analysis of WT1 and Sox9 in the red-eared slider turtle, Trachemys scripta.

Temperature-dependent sex-determination (TSD) is a phenomenon that has been characterized at the ecological, morphological, and endocrinological levels in some reptilian species. We have begun to investigate TSD at the level of molecular development by cloning, sequencing, and analyzing the expression of two genes, WT1 and Sox9, in the red-eared slider turtle Trachemys scripta. We obtained almost full-length cDNA clones for WT1 and Sox9 that were greater than 73% identical to the human homologues at the nucleotide level. WT1 was expressed in urogenital tissue at all developmental stages examined (Yntema stages 12-20) at incubation temperatures that produce males (26 degrees C) or females (32 degrees C). Sox9 was also expressed throughout these same stages, but some differences were observed. At both 26 degrees C and 32 degrees C Sox9 was expressed in the mesonephroi and the undifferentiated gonads until Yntema stage 20, when only the gonad from the 26 degrees C embryos expressed a high level. In addition, there were two transcripts of Sox9 at all stages, but the relative proportion of the two transcripts differed at the two temperatures. Although the similarities in gene expression between a TSD species and other species with genotypically determined sex probably reflect the common features of organogenesis, differences may illustrate unique mechanisms for TSD.

Univariate and bivariate variance component linkage analysis of a whole-genome scan for loci contributing to bone mineral density

Osteoporosis is a common condition characterized by reduced skeletal strength and increased susceptibility to fracture. The single major risk factor for osteoporosis is low bone mineral density (BMD) and strong evidence exists that genetic factors are in part responsible for an individuals BMD. A cohort of 40 multiplex Caucasian families selected through a proband with osteoporosis was genotyped for microsatellite markers spaced at an average of 10 cM, and linkage to femoral neck (FN), lumbar spine (LS) and trochanter (TR) BMD was analyzed using univariate and bivariate variance component linkage analysis. Maximum univariate multipoint lod-scores were 2.87 on chromosome 1p36 for FN BMD, 1.89 on 6q27 for TR BMD, and 2.15 on 7p15 for LS BMD. Results of bivariate linkage analysis were highly correlated with those of the univariate analysis, although generally less significant, suggesting the possibility that some of these susceptibility loci may exert pleiotropic effects on multiple skeletal sites.

Association of a Polymorphism in the TNFR2 Gene with Low Bone Mineral Density

Previous genetic linkage data suggested that a gene on chromosome 1p36.2–36.3 might be linked to low bone mineral density (BMD). Here, we examine the gene for tumor necrosis factor receptor 2 (TNFR2), a candidate gene within that interval, for association with low BMD in a group of 159 unrelated individuals. We assess two polymorphic sites within the gene, a microsatellite repeat within intron 4, and a three‐nucleotide variation in the 3′ untranslated region (UTR) of the gene. The latter has five alleles of which the rarest allele is associated with low spinal BMD Z score (p = 0.008). Lowest mean spinal BMD Z scores were observed for individuals having genotypes that were heterozygous for the rarest allele. No homozygotes for the rarest allele were observed. Preliminary analysis suggests that there is a difference in the genotype frequency distribution between the group with low BMD and a control group.

Vitamin D receptor genotype is not associated with bone mineral density in three ethnic/regional groups.

Abstract. We report a cross-sectional study of 48 men, 56 premenopausal women, and 80 postmenopausal women who were of three ethnic/regional backgrounds: southern European (Greek, Italian), eastern European (Jewish, Polish, Hungarian), and western European (French, British). We determined bone mineral density (BMD) at four skeletal sites and assessed the vitamin D receptor (VDR) genotype by the Bsml restriction site polymorphism. Age and body mass index had significant effects on BMD by multiple regression analysis. In addition, ethnic/regional group had a significant effect on spinal BMD in premenopausal females (P= 0.014) and in males (P= 0.039). However, VDR genotype had no significant effect on BMD in any of the three study groups.

Association Analysis of Bone Mineral Density and Single Nucleotide Polymorphisms in Two Candidate Genes on Chromosome 1p36

Two candidate genes for bone mineral density (BMD), tumor necrosis factor alpha receptor 2 (TNFRSF1B) and lysyl hydroxylase (PLOD1), have been scanned for single nucleotide polymorphisms (SNPs) within their coding and promoter regions. These two genes, separated by about 200 kb, are located within the chromosomal interval 1p36.2–1p36.3 that has been linked to femoral neck BMD. In a patient population (n = 104) of European descent, there were four SNPs within TNFRSF1B and six SNPs within PLOD1 that occurred with greater than 5% frequency. There was significant linkage disequilibrium within both genes. Single marker analysis revealed significant association for one SNP located in intron 6 of PLOD1 and lumbar spine BMD (P = 0.01). Allelic haplotypes that encompassed the four SNPs in TNFRSF1B or the six SNPs in PLOD1 were assigned using a Bayesian algorithm as implemented in the program Haplotyper. Association of TNFRSF1B haplotypes with femoral neck BMD was statistically significant (P = 0.01). Similarly, PLOD1 haplotypes demonstrated a statistically significant association with spinal BMD (P = 0.04). These findings strengthen the potential importance of chromosome 1p36.2–1p36.3 in contributing to BMD variation, and are consistent with genetic variation in either PLOD1, TNFRSF1B or nearby genes playing a role in the phenotype.

Expression of a new RNA-splice isoform of WT1 in developing kidney–gonadal complexes of the turtle, Trachemys scripta

WT1 is a tumor suppressor gene encoding a zinc finger DNA-binding protein required for normal vertebrate kidney and gonad development. Although the sequence and function of this gene has been studied mostly in mammals, comparative analysis in other vertebrates may suggest regions of conservation of function as well as evolution of function. We have initiated a study of this gene in the freshwater turtle, Trachemys scripta, a species that demonstrates temperature dependent sex determination. The turtle WT1 amino acid sequence (GenBank Accession No. AF019779) is over 85% identical to that of other species overall, but there are some major differences. The greatest differences are in the N-terminal portion of the peptide which is thought to mediate transcriptional repression by interaction with other proteins. Turtle WT1, like those of the alligator, chicken, and Xenopus lacks the proline- and glycine-rich stretches that are present in mammalian WT1. Exon 5, which is alternatively spliced in mammals, is altogether absent in the non-mammalian vertebrates. In addition, turtle WT1 is alternatively spliced so that exon 4 is either present or absent. These differences suggest that the interaction of reptilian WT1 with other factor required for mediation of activity may be different than the interaction of mammalian WT1. It also suggests that alternative splicing is a conserved regulatory mechanism of vertebrate WT1. Expression of WT1 in turtle embryonic kidney-gonadal complexes begins after the mesonephroi have formed and continues at least until the bipotential gonad begins to differentiate. Although the proportions of the different splice isoforms are relatively constant during these stages of kidney development, the level of steady state expression is increased in embryos incubated at 26 degrees C, the testis-producing temperature.