Involvement of molecules related to angiogenesis, proteolysis and apoptosis in implantation in rhesus monkey and mouse.

We have established the well-defined cycling, pseudo-pregnant and pregnant rhesus monkey models, and used these to analyze expression of the common molecules specifically related to angiogenesis, apoptosis or proteolysis, such as vascular endothelial growth factor (VEGF) and its receptors KDR, flt-1, flt-4 and flk-1, basic fibroblast growth factor (bFGF) and its receptors Flg, transforming growth factor-alpha and beta1 (TGF-a/beta1), and TGF-beta1 receptor type I (TbetaR-I) and type II (TbetaR-II), as well as steroidogenic acute regulatory protein (StAR), tissue type plasminogen activator/urokinase plasminogen activator/plasminogen activator inhibitor type 1 (tPA/uPA/PAI-1) and matrix matalloproteinase type 1, -3/tissue inhibitor matalloproteinase type 1, -2, -3 (MMP-1, -3/TIMP-1, -2, -3), Fas/FasL, BcL-2/Bax, in the corpus luteum (CL), in the functional layer of the endometrium and in the materno-fetal boundary of the implantation site. We have demonstrated that: expression of these molecules in the monkey CL, endometrium and materno-fetal boundary of the implantation site is correlated well with CL functional and vascular development and with the processes involved in the establishment of the implantation window as well as with the early stages of placentation. A coordinated increase in tPA and its inhibitor PAI-1 expression in the monkey and rat CL may be instrumental in initiating luteal regression in both species, and correlated well with the timing of the closure of the implantation window, whereas high uPA activity in the CL is important for the early formation of the CL and for maintaining its function which is closely correlated to the period of establishment of the implantation window. Apoptosis, proteolysis and angiogenesis occur in the CL and in the endometrium during the time of establishment of the implantation window, as well as in the materno-fetal boundary of the implantation site at the early stages of placentation. It seems that these processes occur in these tissues in a coordinated and time- and cell-dependent manner, and are reliant on each other. Based on these observations, we have designed experiments to test the actions of some related available compounds on mouse implantation, used alone or in combination. The preliminary data showed that the compounds which could effectively affect apoptosis, angiogenesis or proteolysis in the implantation site were capable of effectively inhibiting implantation by acting on the endometrium and/or on the CL. Furthermore, the combined use of these compounds produced an obvious additive effect on inhibiting implantation. This finding suggested this may be a good approach for developing an anti-implantation agent.

Association between VDR ApaI polymorphism and hip bone mineral density can be modified by body mass index: a study on postmenopausal Chinese women.

Osteoporosis is a major public health problem for old people. Genetic factors are considered to be major contributors to the pathogenesis of postmenopausal osteoporosis. The vitamin D receptor (VDR) gene is a prominent candidate gene for the regulation of postmenopausal bone mass; however, despite extensive studies, controversy remains regarding its association with postmenopausal body mineral density (BMD) variation. In this study, a total of 260 healthy postmenopausal Chinese women were genotyped at the VDR ApaI locus using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Raw hip BMD was significantly associated with VDR ApaI polymorphism with and without adjusting for age (P=0.015 and 0.040, respectively). This genetic effect can explain 3.32% of hip BMD variation. However, the significant association vanished after correcting for both age and body mass index (BMI) (P=0.169). In addition, we observed a significant association between VDR ApaI polymorphism with unadjusted BMI (P=0.042) or BMI adjusted for age (P=0.049). The raw hip BMD was also found to be significantly correlated with BMI (r=0.517, P=0.0001), with BMI explaining 26.35% of the variation of hip BMD. All of these facts prompt us to conclude that the significant association between the VDR ApaI genotype and hip BMD may be modified by BMI in postmenopausal Chinese women. Our findings may partially explain the earlier inconsistent association results concerning the VDR gene and BMD, and highlight the importance of incorporating covariates such as BMI into osteoporosis association studies.

SNPP: automating large-scale SNP genotype data management.

UNLABELLED: To manage high-throughput single nucleotide polymorphism (SNP) genotyping data efficiently, we developed a dynamic general database management system-SNPP (SNP Processor). It provides several functions, including data importing with comparison, Mendelian inheritance check within pedigrees, data compiling and exporting. Furthermore, SNPP may generate files for repeat genotyping and transform them into files that can be executed by a liquid handling system. AVAILABILITY: http://orclinux.creighton.edu/snpp/ CONTACT: lanjuanzhao@creighton.edu

Genetic dissection of human stature in a large sample of multiplex pedigrees.

Recently, we reported a whole genome scan on a sample of 630 Caucasian subjects from 53 human pedigrees. Several genomic regions were suggested to be linked to height. In an attempt to confirm the identified genomic regions, as well as to identify new genomic regions linked to height, we conducted a whole genome linkage study on an extended sample of 1,816 subjects from 79 pedigrees, which includes the 53 pedigrees containing the original 630 subjects from our previous whole genome study and an additional 128 new subjects, and 26 further pedigrees containing 1,058 subjects. Several regions achieved suggestive linkage signals, such as 9q22.32 [MLS (multipoint LOD score) = 2.74], 9q34.3 [MLS = 2.66], Xq24 [two-point LOD score = 2.64 at the marker DXS8067], and 7p14.2 [MLS = 2.05]. The importance of the above regions is supported either by other whole genome studies or by candidate genes within these regions relevant to linear growth or pathogenesis of short stature. In addition, this study has tentatively confirmed the Xq24 region's linkage to height, as this region was also detected in the previous whole genome study. To date, our study has achieved the largest sample size in the field of genetic linkage studies of human height. Together with the findings of other studies, the current study has further delineated the genetic basis of human stature.

A follow-up linkage study for bone size variation in an extended sample.

Bone size, which has strong genetic determination, is an important determinant of bone strength and a risk factor of osteoporotic fractures. We previously reported an approximately 10-cm genome-wide linkage scan in 630 subjects from 53 US Caucasian pedigrees. The strongest evidence of linkage was obtained on chromosome 17q22 near the marker D17S787, with a two-point LOD score of 3.98 and a multipoint maximum LOD score (MLS) of 3.01. Additionally, suggestive linkages (1.54 < MLS < 2.83) were found at the other four chromosomal regions. In the present study, with an attempt to further examine our previous findings, we perform a follow-up linkage analysis in an expanded sample of 79 pedigrees with 1816 subjects. The total sample contains >80,000 informative relative pairs for linkage analyses, including 3846 sib pairs. Fifteen markers covering the above five promising regions are genotyped, narrowing the average genomic distance from approximately 10 to 5 cm. In the total 79 pedigrees, support of linkage was achieved for the wrist bone size at 17q22 with a two-point LOD score of 2.27 (P = 0.0006) and MLS of 1.78 (P = 0.002). The genomic region 17q22 includes COL1A1, a strong candidate gene that is significantly associated with osteoporotic fracture risk. Our data suggest that this region is promising for further exploratory studies.

Quantifying the relationship between gene expressions and trait values in general pedigrees.

Treating mRNA transcript abundances as quantitative traits and examining their relationships with clinical traits have been pursued by using an analytical approach of quantitative genetics. Recently, Kraft et al. presented a family expression association test (FEXAT) for correlation between gene expressions and trait values with a family-based (sibships) design. This statistic did not account for biological relationships of related subjects, which may inflate type I error rate and/or decrease power of statistical tests. In this article, we propose two new test statistics based on a variance-components approach for analyses of microarray data obtained from general pedigrees. Our methods accommodate covariance between relatives for unmeasured genetic effects and directly model covariates of clinical importance. The efficacy and validity of our methods are investigated by using simulated data under different sample sizes, family sizes, and family structures. The proposed LR method has correct type I error rate with moderate to large sample sizes regardless of family structure and family sizes. It has higher power with complex pedigrees and similar power to the FEXAT with sibships. The other proposed FEXAT(R) method is favorable with large family sizes, regardless of sample sizes and family structure. Our methods, robust to population stratification, are complementary to the FEXAT in expression-trait association studies.

Test of linkage and/or association between the estrogen receptor alpha gene with bone mineral density in Caucasian nuclear families.

Extensive studies have been performed on the association between the estrogen receptor alpha (ER-alpha) gene and bone mineral density (BMD). Despite considerable efforts, the studies using limited markers and relatively small sample size have yielded largely inconsistent results. In this study, 1873 Caucasian subjects from 405 nuclear families containing 1512 sib pairs were recruited. BMD at the lumbar spine (LS) and femoral neck (FN) was measured by dual-energy X-ray absorptiometry (DXA). Seven single-nucleotide polymorphisms (SNPs) spanning from exon 1 to 8 in the ER-alpha gene were genotyped. The program QTDT (quantitative transmission disequilibrium test) was applied to test linkage and/or association of the ER-alpha gene and BMD variation using individual SNP markers and reconstructed haplotypes. Linkage disequilibrium (LD) was generally detected for SNPs in the ER-a gene (P < 0.05). Associations were observed between SNP rs932477 and FN BMD (P = 0.028), and between the most predominant three-marker haplotype (GCG) containing SNP rs932477 and FN BMD (P = 0.010). Within-family association (present only with both linkage and association) between SNP rs2228480 (G2014A) and FN BMD (P = 0.015) was observed. The most predominant seven-SNP haplotype (TCGCGGG) was associated with higher LS BMD (P = 0.015). However, after correction for multiple testing, these associations did not reach statistical significance. Denser markers may be necessary to better define the relationship between the ER-alpha gene and BMD variation in our sample.

Lack of association between the HindIII RFLP of the osteocalcin (BGP) gene and bone mineral density (BMD) in healthy pre- and postmenopausal Chinese women.

In Caucasian populations, the polymorphic restriction endonuclease HindIII marker of the osteocalcin (also known as BGP, for bone Gla protein) gene has recently been reported to be associated with bone mass, a major risk determinant of osteoporosis. In this study, we investigated the relationship between the BGP HindIII polymorphism and bone mineral density (BMD) in 388 premenopausal (31.18 +/- 5.92 years) and 169 postmenopausal (58.90 +/- 6.27 years) Chinese women. The BMD of spine and hip was measured by dual-energy X-ray absorptiometry (DEXA). All the study subjects were genotyped at the HindIII site of the BGP gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) detecting methods. The BGP alleles were designated according to the absence ( H) or presence ( h) of the HindIII restriction site. We did not find any significant difference in spine and hip BMD across BGP genotypes in either pre- or postmenopausal women or the combined group. Our result is not consistent with recent reports that the HindIII marker of the BGP gene is associated with osteoporosis. The different findings may reflect inter-population differences in the association (i.e., linkage disequilibrium) of molecular markers with BMD, and indicate the limit of using the HindIII marker of the BGP gene as a genetic marker to discern women susceptible to low BMD and thus osteoporosis in Chinese.

Genome scan for QTLs underlying bone size variation at 10 refined skeletal sites: genetic heterogeneity and the significance of phenotype refinement.

To identify quantitative trait loci (QTLs) underlying variation in bone size, we conducted a whole-genome linkage scan in 53 pedigrees with 630 subjects using 380 microsatellite markers. Lumbar area 1, 2, 3, and 4 at the spine, femoral neck, trochanter, intertrochanter areas at the hip, ultradistal, mid-distal, and one-third distal areas at the wrist were measured by dual-energy X-ray absorptiometry (DXA), and adjusted for age, height, weight, and sex. Two-point and multipoint linkage analyses were performed for skeletal bone size at each site and their composite measurements using the SOLAR package. Two chromosomal regions (1q22 and 10q21) were identified with significant evidence of linkage (LOD > 4.32) to one-third distal area, and three were identified with suggestive evidence of linkage (LOD > 2.93) to bone size in one skeletal site. Our results indicated that the low power of QTLs mapping for composite phenotypic measurements may result from genetic heterogeneity of complex traits.

A follow-up linkage study for quantitative trait loci contributing to obesity-related phenotypes.

We have recently reported a whole-genome scan in a sample of 630 subjects from 53 extended pedigrees, in which several genomic regions that may contain quantitative trait loci (QTLs) for obesity were suggested. In the present study, with an attempt to confirm our previous findings, we performed a follow-up linkage study in an expanded sample of 79 pedigrees with 1816 subjects (including expanded previous 53 pedigrees and 26 newly recruited pedigrees containing 1058 subjects). A new set of microsatellite markers spanning previously identified regions were selected, with the average genomic distance narrowed from approximately 10 cM to approximately 5 cM in this study. Using a variance component method, we performed two- and multipoint linkage analyses in the following three sample sets: expanded previous 53 pedigrees (758 subjects), 26 new pedigrees, and 79 total pedigrees. For body mass index, analyses of the expanded 53 pedigrees attained a LOD score of 2.32 near marker D1S468 in two-point analysis and a maximum LOD score (MLS) of 2.21 in multipoint analysis; 2q14 near marker D2S347 attained a LOD score of 3.42 in two-point analysis and a MLS of 3.93 in multipoint analysis. The linkage peaks at 1p36 and 2q14 were further supported in the analyses of all 79 pedigrees, with multipoint MLS being 1.38 and 0.90, respectively. For fat mass, genomic region 6q27 achieved a LOD score of 1.24 in two-point analysis and an MLS of 0.92 in multipoint analysis in all 79 pedigrees. Our data support that 1p36, 2q14, and 6q27 are promising regions that may harbor QTLs for obesity phenotypes.

Several genomic regions potentially containing QTLs for bone size variation were identified in a whole-genome linkage scan.

Bone size is an important determinant of osteoporotic fractures. For a sample of 53 pedigrees that contains more than 10,000 relative pairs informative for linkage analyses, we performed a whole-genome linkage scan using 380 microsatellite markers to identify genomic regions that may contain QTLs of bone size (two dimensional measurement by dual energy X-ray absorptiometry). We conducted two- and multi-point linkage analyses. Several potentially important genomic regions were identified. For example, the genomic region 17q23 may contain a QTL for wrist (ultra distal) bone size variation; a LOD score of 3.98 is achieved at D17S787 in two-point analyses and a maximum LOD score (MLS) of 3.01 is achieved in multi-point analyses in 17q23. 19p13 may contain a QTL for hip bone size variation; a LOD score of 1.99 is achieved at D19S226 in two-point analyses and a MLS of 2.83 is achieved in 19p13 in multi-point analyses. The genomic region identified on chromosome 17 for wrist bone size seems to be consistent with that identified for femur head width variation in an earlier whole-genome scan study. The genomic regions identified in this study and an earlier investigation on one-dimensional bone size measurement by radiography are compared. The two studies may form a basis for further exploration with larger samples and/or denser markers for confirmation and fine mapping studies to eventually identify major functional genes and the associated etiology for osteoporosis.

A whole-genome linkage scan suggests several genomic regions potentially containing quantitative trait Loci for osteoporosis.

Osteoporosis is an important health problem, particularly in the elderly women. Bone mineral density (BMD) is a major determinant of osteoporosis. For a sample of 53 pedigrees that contain 1249 sibling pairs, 1098 grandparent-grandchildren pairs, and 2589 first cousin pairs, we performed a whole- genome linkage scan using 380 microsatellite markers to identify genomic regions that may contain quantitative trait loci (QTL) of BMD. Each pedigree was ascertained through a proband with BMD values belonging to the bottom 10% of the population. We conducted two-point and multipoint linkage analyses. Several potentially important genomic regions were suggested. For example, the genomic region near the marker D10S1651 may contain a QTL for hip BMD variation (with two-point analysis LOD score of 1.97 and multipoint analysis LOD score of 2.29). The genomic regions near the markers D4S413 and D12S1723 may contain QTLs for spine BMD variation (with two-point analysis LOD score of 2.12 and 2.17 and multipoint analysis LOD score of 3.08 and 2.96, respectively). The genomic regions identified in this and some earlier reports are compared for exploration in extension studies with larger samples and/or denser markers for confirmation and fine mapping to eventually identify major functional genes involved in osteoporosis.

Differences in bone mineral density, bone mineral content, and bone areal size in fracturing and non-fracturing women, and their interrelationships at the spine and hip.

Osteoporotic fractures are a major public health problem, particularly in women. Bone mineral density (BMD), bone mineral content (BMC), and bone size have been regarded as important determinants of osteoporotic fractures. In 1449 women over age 30 years, we studied the detailed relationship, at the spine and hip, between BMD, BMC, and bone areal size (all measured by dual-energy X-ray absorptiometry) and compared their relative magnitudes in fracturing and non-fracturing individuals. We find that, (1) BMD and BMC are significantly higher at the spine and hip in non-fracturing women. Bone areal size is significantly larger at the spine in non-fracturing women; however, the significance disappears when adjustment is made for the significant difference of height (stature) between fracturing and non-fracturing women. In contrast to the spine, bone areal size is always significantly largerin fracturing women at the hip. (2) The relationship among BMD, BMC, and bone areal size is different at the spine and hip. Specifically, at the spine, BMD increases with bone areal size linearly. At the hip, BMD has a quadratic relationship with bone areal size, so that BMD increases at lower bone areal sizes, then (after an intermediate zone of values) decreases with increasing bone areal size. However, BMD adjusted for BMC always decreases with increasing bone areal size, as expected by the definition of BMD. With no adjustment for BMC, the increase in BMD with bone areal size is due to a more rapid increase of BMC than increasing bone areal size, thus explaining the observations of association of both larger BMD and larger bone areal size with stronger bone. (3) At the spine, 86.2% of BMD variation is attributable to BMC and 12.6% to bone areal size. At the hip, 98.0% of BMD variation is due to BMC and 1.1% due to bone areal size. The current study may be important in understanding the relationship among BMD, BMC, and bone size as risk determinants of osteoporotic fractures.

A whole-genome linkage scan suggests several genomic regions potentially containing QTLs underlying the variation of stature.

Human height is a complex trait under the control of both genetic and environment factors. In order to identify genomic regions underlying the variation of stature, we performed a whole-genome linkage analysis on a sample of 53 human pedigrees containing 1,249 sib pairs, 1,098 grandparent-grandchildren pairs, 1,993 avuncular pairs, and 1,172 first-cousin pairs. Several genomic regions were suggested by our study to be linked with human height variation. These regions include 5q31 at 144 cM from pter on chromosome 5 (with a maximum LOD score of 2.14 in multipoint linkage analyses), Xp22 at the marker DXS1060, and Xq25 at DXS1001 on the X chromosome (with LOD scores of 1.95 and 1.91, respectively, in two-point linkage analyses). Noticeably, Xp22 happens to be the very region where a newly identified gene underlying idiopathic short stature, SHOX, maps. Based on our findings, further confirmation and fine-mapping studies are to be pursued on expanded samples and/or with denser markers for eventual identification of major functional genes involved in human height variation.

Determination of bone size of hip, spine, and wrist in human pedigrees by genetic and lifestyle factors.

Osteoporosis is a major public health problem defined as a loss of bone strength, of which bone size is an important determinant. Compared with extensive studies on bone mass, studies on the importance of factors determining variation in bone size are relatively few. In particular, the significance of genetic factors is largely unknown. In 49 pedigrees with 703 subjects bone sizes of the hip, spine, and wrist were measured by dual X-ray absorptiometry. We evaluated the contribution of genetic factors in determining variation in bone size of the hip, spine, and wrist while studying age, sex, weight, height, exercise, smoking, alcohol consumption, and the interaction among these factors as covariates for their effects on bone size. We found that, on average, males have larger bone sizes. Male bone sizes at the spine and hip increased with age; however, the effect of age in our female subjects was nonsignificant. Height invariably affected bone size at all the sites studied. Alcohol consumption and exercise generally had significant effects in increasing bone size at the spine and/or hip in both males and females. After adjusting for sex, age, weight, height, lifestyle factors, and the significant interactions among these factors, heritabilities (+/-SE) were, respectively, 0.48 (0.09), 0.64 (0.08), and 0.60 (0.09) for bone size at the hip, spine, and wrist.

A genomewide linkage scan for quantitative-trait loci for obesity phenotypes.

Obesity is an increasingly serious health problem in the world. Body mass index (BMI), percentage fat mass, and body fat mass are important indices of obesity. For a sample of pedigrees that contains >10,000 relative pairs (including 1,249 sib pairs) that are useful for linkage analyses, we performed a whole-genome linkage scan, using 380 microsatellite markers to identify genomic regions that may contain quantitative-trait loci (QTLs) for obesity. Each pedigree was ascertained through a proband who has extremely low bone mass, which translates into a low BMI. A major QTL for BMI was identified on 2q14 near the marker D2S347 with a LOD score of 4.04 in two-point analysis and a maximum LOD score (MLS) of 4.44 in multipoint analysis. The genomic region near 2q14 also achieved an MLS >2.0 for percentage of fat mass and body fat mass. For the putative QTL on 2q14, as much as 28.2% of BMI variation (after adjustment for age and sex) may be attributable to this locus. In addition, several other genomic regions that may contain obesity-related QTLs are suggested. For example, 1p36 near the marker D1S468 may contain a QTL for BMI variation, with a LOD score of 2.75 in two-point analysis and an MLS of 2.09 in multipoint analysis. The genomic regions identified in this and earlier reports are compared for further exploration in extension studies that use larger samples and/or denser markers for confirmation and fine-mapping studies, to eventually identify major functional genes involved in obesity.

Tests of linkage and/or association of genes for vitamin D receptor, osteocalcin, and parathyroid hormone with bone mineral density.

Bone mineral density (BMD) is a major determinant of osteoporotic fractures (OFs). The heritability of BMD ranges from 50% to 90% in human populations. Extensive molecular genetic analyses have been performed through traditional linkage or association approaches to test and identify genes or genomic regions underlying BMD variation. The results, particularly those concerning the vitamin D receptor (VDR) gene, have been inconsistent and controversial. In this study, we simultaneously test linkage and/or association of the genes for VDR, osteocalcin (also known as bone Gla protein [BGP]), and parathyroid hormone (PTH) with BMD in 630 subjects from 53 human pedigrees. Each of these pedigrees was ascertained through a proband with an extreme BMD value at the hip or spine (Z score < or = -1.28). For the raw BMD values, adjusting for significant covariate effects of age, sex, and weight, we performed tests for linkage alone, association alone, and then both linkage and association. For the spine BMD, at the two markers (ApaI and FokI) inside the VDR gene we found evidence for linkage (p < 0.05) and for both linkage and association by the transmission disequilibrium test (TDT; p < 0.05); association was detected (p < 0.07) with regular statistical testing by analyses of variance (ANOVA). In addition, significant results were found for association alone (p < 0.05), linkage alone (p = 0.0005), and for linkage and association (p = 0.0019) for the intragenic marker HindIII of the BGP gene for the hip BMD. Through testing for association, linkage, and linkage and association simultaneously, our data support the VDR gene as a quantitative trait locus (QTL) underlying spine BMD variation and the BGP gene as a QTL underlying hip BMD variation. However, our data do not support the PTH gene as a QTL underlying hip or spine BMD variation. This is the first study in the broad field of bone genetics that tests candidate genes as QTLs for BMD by testing simultaneously for association alone, for linkage alone, and for association and linkage (via the TDT).

Mutation patterns at dinucleotide microsatellite loci in humans.

Microsatellites are a major type of molecular markers in genetics studies. Their mutational dynamics are not clear. We investigated the patterns and characteristics of 97 mutation events unambiguously identified, from 53 multigenerational pedigrees with 630 subjects, at 362 autosomal dinucleotide microsatellite loci. A size-dependent mutation bias (in which long alleles are biased toward contraction, whereas short alleles are biased toward expansion) is observed. There is a statistically significant negative relationship between the magnitude (repeat numbers changed during mutation) and direction (contraction or expansion) of mutations and standardized allele size. Contrasting with earlier findings in humans, most mutation events (63%) in our study are multistep events that involve changes of more than one repeat unit. There was no correlation between mutation rate and recombination rate. Our data indicate that mutational dynamics at microsatellite loci are more complicated than the generalized stepwise mutation models.