SUMMARY

Chronic kidney disease (CKD) represents a major health burden1. Its central feature of renal fibrosis is not well understood. By whole exome resequencing in a model disorder for renal fibrosis, nephronophthisis (NPHP), we identified mutations of Fanconi anemia-associated nuclease 1 (FAN1) as causing karyomegalic interstitial nephritis (KIN). Renal histology of KIN is indistinguishable from NPHP except for the presence of karyomegaly2. FAN1 has nuclease activity, acting in DNA interstrand crosslinking (ICL) repair within the Fanconi anemia pathway of DNA damage response (DDR)3–6. We demonstrate that cells from individuals with FAN1 mutations exhibit sensitivity to the ICL agent mitomycin C. However, they do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from patients with Fanconi anemia. We complement ICL sensitivity with wild type FAN1 but not mutant cDNA from individuals with KIN. Depletion of fan1 in zebrafish revealed increased DDR, apoptosis, and kidney cysts akin to NPHP. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms of renal fibrosis and CKD.

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is an autosomal dominantly inherited central nervous system white matter disease with variable clinical presentations including personality and behavioral changes, dementia, depression, parkinsonism, seizures, and others1,2. We combined genome-wide linkage analysis with exome sequencing and identified 14 different mutations affecting the tyrosine kinase domain of the colony stimulating factor receptor 1 (encoded by CSF1R) in 14 families affected by HDLS. In one kindred, the de novo occurrence of the mutation was confirmed. Follow-up sequencing analyses identified an additional CSF1R mutation in a patient clinically diagnosed with corticobasal syndrome (CBS). In vitro, CSF-1 stimulation resulted in the rapid autophosphorylation of selected tyrosine-residues in the kinase domain of wild-type but not mutant CSF1R, suggesting that HDLS may result from a partial loss of CSF1R function. Since CSF1R is a critical mediator of microglial proliferation and differentiation in the brain, our findings suggest an important role for microglial dysfunction in HDLS pathogenesis.

BACKGROUND & AIMS

Staging inadequately predicts metastatic risk in patients with colon cancer. We used a gene expression profile derived from invasive, murine colon cancer cells that were highly metastatic in an immunocompetent mouse model to identify patients with colon cancer at risk of recurrence.

METHODS

This phase 1, exploratory biomarker study used 55 patients with colorectal cancer from Vanderbilt Medical Center (VMC) as the training dataset and 177 patients from the Moffitt Cancer Center as the independent dataset. The metastasis-associated gene expression profile developed from the mouse model was refined with comparative functional genomics in the VMC gene expression profiles to identify a 34-gene classifier associated with high risk of metastasis and death from colon cancer. A metastasis score derived from the biologically based classifier was tested in the Moffitt dataset.

RESULTS

A high score was significantly associated with increased risk of metastasis and death from colon cancer across all pathologic stages and specifically in stage II and stage III patients. The metastasis score was shown to independently predict risk of cancer recurrence and death in univariate and multivariate models. For example, among stage III patients, a high score translated to increased relative risk of cancer recurrence (hazard ratio, 4.7; 95% confidence interval, 1.566–14.05). Furthermore, the metastasis score identified patients with stage III disease whose 5-year recurrence-free survival was >88% and for whom adjuvant chemotherapy did not increase survival time.

CONCLUSION

A gene expression profile identified from an experimental model of colon cancer metastasis predicted cancer recurrence and death, independently of conventional measures, in patients with colon cancer.

Context

Genetic testing for monogenic diabetes is important for patient care. Given the extensive genetic and clinical heterogeneity of diabetes, exome sequencing might provide additional diagnostic potential when standard Sanger sequencing-based diagnostics is inconclusive.

Objective

The aim of the study was to examine the performance of exome sequencing for a molecular diagnosis of MODY in patients who have undergone conventional diagnostic sequencing of candidate genes with negative results.

Research Design and Methods

We performed exome enrichment followed by high-throughput sequencing in nine patients with suspected MODY. They were Sanger sequencing-negative for mutations in the HNF1A, HNF4A, GCK, HNF1B and INS genes. We excluded common, non-coding and synonymous gene variants, and performed in-depth analysis on filtered sequence variants in a pre-defined set of 111 genes implicated in glucose metabolism.

Results

On average, we obtained 45 X median coverage of the entire targeted exome and found 199 rare coding variants per individual. We identified 0–4 rare non-synonymous and nonsense variants per individual in our a priori list of 111 candidate genes. Three of the variants were considered pathogenic (in ABCC8, HNF4A and PPARG, respectively), thus exome sequencing led to a genetic diagnosis in at least three of the nine patients. Approximately 91% of known heterozygous SNPs in the target exomes were detected, but we also found low coverage in some key diabetes genes using our current exome sequencing approach. Novel variants in the genes ARAP1, GLIS3, MADD, NOTCH2 and WFS1 need further investigation to reveal their possible role in diabetes.

Conclusion

Our results demonstrate that exome sequencing can improve molecular diagnostics of MODY when used as a complement to Sanger sequencing. However, improvements will be needed, especially concerning coverage, before the full potential of exome sequencing can be realized.

Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological diseases. We have reconstructed two-dimensional images of gene expression for 20,000 genes in a coronal slice of the mouse brain at the level of the striatum by using microarrays in combination with voxelation at a resolution of 1 mm3. Good reliability of the microarray results were confirmed using multiple replicates, subsequent quantitative RT-PCR voxelation, mass spectrometry voxelation, and publicly available in situ hybridization data. Known and novel genes were identified with expression patterns localized to defined substructures within the brain. In addition, genes with unexpected patterns were identified, and cluster analysis identified a set of genes with a gradient of dorsal/ventral expression not restricted to known anatomical boundaries. The genome-scale maps of gene expression obtained using voxelation will be a valuable tool for the neuroscience community.

Despite high heritability, a large fraction of cases with schizophrenia do not have a family history of the disease (sporadic cases). Here, we examine the possibility that rare de novo protein-altering mutations contribute to the genetic component of schizophrenia by sequencing the exome of 53 sporadic cases, 22 unaffected controls and their parents. We identified 40 de novo mutations in 27 patients affecting 40 genes including a potentially disruptive mutation in DGCR2, a gene removed by the recurrent schizophrenia-predisposing 22q11.2 microdeletion. Comparison to rare inherited variants revealed that the identified de novo mutations show a large excess of nonsynonymous changes in cases, as well as a greater potential to affect protein structure and function. Our analysis reveals a major role of de novo mutations in schizophrenia and also a large mutational target, which together provide a plausible explanation for the high global incidence and persistence of the disease.

Susceptibility to peripheral neuropathy during antiretroviral therapy with nucleoside reverse transcriptase inhibitors (NRTIs) was previously associated with a European mitochondrial DNA (mtDNA) haplogroup among non-Hispanic white persons. To determine if NRTI-associated peripheral neuropathy was related to mtDNA variation in non-Hispanic black persons, we sequenced mtDNA of participants from AIDS Clinical Trials Group study 384. Of 156 non-Hispanic blacks with genomic data, 51 (33%) developed peripheral neuropathy. In a multivariate model, African mtDNA subhaplogroup L1c was an independent predictor of peripheral neuropathy (OR=3.7, 95% CI 1.1-12.0). An African mtDNA subhaplogroup is for the first time implicated in susceptibility to NRTI-associated toxicity.

Plasma homocysteine (Hcy) level is associated with cardiovascular disease and may play an etiologic role in vascular damage, a precursor for atherosclerosis. We performed a genome-wide association study for Hcy in 1786 unrelated Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS). The most strongly associated single-nucleotide polymorphism (SNP) (rs7422339, P = 4.7 × 10−13) encodes Thr1405Asn in the gene CPS1 and explained 3.0% of variation in the Hcy level. The widely studied MTHFR C677T SNP (rs1801133) was also highly significant (P = 8.7 × 10−10) and explained 1.6% of the trait variation. We also genotyped these two SNPs in 1679 CLHNS young adult offspring. The MTHFR C677T SNP was strongly associated with Hcy (P = 1.9 × 10−26) and explained ∼5.1% of the variation in the offspring. In contrast, the CPS1 variant was significant only in females (P = 0.11 in all; P = 0.0087 in females). Combined analysis of all samples confirmed that the MTHFR variant was more strongly associated with Hcy in the offspring (interaction P = 1.2 × 10−5). Furthermore, although there was evidence for a positive synergistic effect between the CPS1 and MTHFR SNPs in the offspring (interaction P = 0.0046), there was no significant evidence for an interaction in the mothers (P = 0.55). These data confirm a recent finding that CPS1 is a locus influencing Hcy levels in women and suggest that genetic effects on Hcy may differ across developmental stages.

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders featuring dysplasia or degeneration preferentially in kidney, retina, and cerebellum. Here we combine homozygosity mapping with candidate gene analysis by performing “ciliopathy candidate exome capture” followed by massively-parallel sequencing. We detect 12 different truncating mutations of SDCCAG8 in 10 NPHP-RC families. We demonstrate that SDCCAG8 is localized at both centrioles and directly interacts with NPHP-RC-associated OFD1. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in 3D renal cell cultures. This work identifies SDCCAG8 loss of function as a novel cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.

A sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition, is a common feature of apoptosis and is mediated by the mitochondrial permeability transition pore (mtPTP). It is thought that the mtPTP is a protein complex formed by the voltage-dependent anion channel, members of the pro- and anti-apoptotic BAX-BCL2 protein family, cyclophilin D, and the adenine nucleotide (ADP/ATP) translocators (ANTs)1,2. The latter exchange mitochondrial ATP for cytosolic ADP and have been implicated in cell death. To investigate the role of the ANTs in the mtPTP, we genetically inactivated the two isoforms of ANT3–5 in mouse liver and analysed mtPTP activation in isolated mitochondria and the induction of cell death in hepatocytes. Mitochondria lacking ANT could still be induced to undergo permeability transition, resulting in release of cytochrome c. However, more Ca2+ than usual was required to activate the mtPTP, and the pore could no longer be regulated by ANT ligands. Moreover, hepatocytes without ANT remained competent to respond to various initiators of cell death. Therefore, ANTs are non-essential structural components of the mtPTP, although they do contribute to its regulation.

The mitochondrial DNA (mtDNA) chloramphenicol (CAP)-resistance (CAPR) mutation has been introduced into the tissues of adult mice via female embryonic stem (ES) cells. The endogenous CAP-sensitive (CAPS) mtDNAs were eliminated by treatment of the ES cells with the lipophilic dye Rhodamine-6-G (R-6-G). The ES cells were then fused to enucleated cell cytoplasts prepared from the CAPR mouse cell line 501-1. This procedure converted the ES cell mtDNA from 100% wild-type to 100% mutant. The CAPR ES cells were then injected into blastocysts and viable chimeric mice were isolated. Molecular testing for the CAPR mutant mtDNAs revealed that the percentage of mutant mtDNAs varied from zero to approximately 50% in the tissues analyzed. The highest percentage of mutant mtDNA was found in the kidney in three of the chimeric animals tested. These data suggest that, with improved efficiency, it may be possible to transmit exogenous mtDNA mutants through the mouse germ-line.

Introduction

The objective of this study was to identify genetic variants that are associated with adult leisure-time exercise behavior using genome-wide association in two independent samples.

Methods

Exercise behavior was measured in 1,772 unrelated Dutch and 978 unrelated American adults with detailed questions about type, frequency and duration of exercise. Individuals were classified into regular exercisers or non-exercisers using a threshold of 4 METhours (metabolic equivalents*hours per week). Regular exercisers were further divided into 5 categories of METhours, ranging from moderate (>=4 METhours) to highly vigorous (>=40 METhours) exercisers. Genome-wide association analyses with a total of 470,719 SNPs were conducted in both samples independently using regression-based techniques in SNPtest, including sex, age and BMI as covariates.

Results

SNPs located in SGIP1, DNASE2B, PRSS16, ERCC2 and PAPSS2 were associated with exercise participation (combined p-value between 0.0004 and 4.5*10-6 with the same direction of allelic effects in both samples). Associations of candidate genes based on existing literature were replicated for the LEPR gene in the American sample (rs12405556, p=0.0005) and for the CYP19A1 gene in the Dutch sample (rs2470158, 0.0098).

Conclusion

Two genes (SGIP1 and LEPR) are expressed in the hypothalamus and involved in the regulation of energy homeostasis. Their effects were independent of BMI, suggesting a direct role of hypothalamic factors in the drive to exercise.

Background

Approximately 5-10% of persons infected with M. tuberculosis develop tuberculosis, but the factors associated with disease progression are incompletely understood. Both linkage and association studies have identified human genetic variants associated with susceptibility to pulmonary tuberculosis, but few genetic studies have evaluated extrapulmonary disease. Because extrapulmonary and pulmonary tuberculosis likely have different underlying pathophysiology, identification of genetic mutations associated with extrapulmonary disease is important.

Findings

We performed a pilot genome-wide association study among 24 persons with previous extrapulmonary tuberculosis and well-characterized immune defects; 24 pulmonary tuberculosis patients and 57 patients with M. tuberculosis infection served as controls. The Affymetrix GeneChip Human Mapping Xba Array was used for genotyping; after careful quality control, genotypes at 44,175 single nucleotide polymorphisms (SNPs) were available for analysis. Eigenstrat quantified population stratification within our sample; logistic regression, using results of the Eigenstrat analysis as a covariate, identified significant associations between groups. Permutation testing controlled the family-wise error rate for each comparison between groups. Four SNPs were significantly associated with extrapulmonary tuberculosis compared to controls with M. tuberculosis infection; one (rs4893980) in the gene PDE11A, one (rs10488286) in KCND2, and one (rs2026414) in PCDH15; one was in chromosome 7 but not associated with a known gene. Two additional variants were significantly associated with extrapulmonary tuberculosis compared with pulmonary tuberculosis; one (rs340708) in the gene FAM135B and one in chromosome 13 but not associated with a known gene. The function of all four genes affects cell signaling and activity, including in the brain.

Conclusions

In this pilot study, we identified 6 novel variants not previously known to be associated with extrapulmonary tuberculosis, including two SNPs more common in persons with extrapulmonary than pulmonary tuberculosis. This provides some support for the hypothesis that the pathogenesis and genetic predisposition to extrapulmonary tuberculosis differs from pulmonary tuberculosis. Further study of these novel SNPs, and more well-powered genome-wide studies of extrapulmonary tuberculosis, is warranted.

Human stature, as an important physical index in clinical practice and a usual covariate in gene mapping of complex disorders, is a highly heritable complex trait. To identify specific genes underlying stature, a genome-wide association study was performed in 1000 unrelated homogeneous Caucasian subjects using Affymetrix 500K arrays. A group of seven contiguous markers in the region of SBF2 gene (Set-binding factor 2) are associated with stature, significantly so at the genome-wide level after false discovery rate (FDR) correction (FDR q = 0.034–0.042). Three SNPs in another SNP group in the Filamin B (FLNB) gene were also associated with stature, significantly so with FDR q = 0.042–0.048. In follow-up independent replication studies, rs10734652 in the SBF2 gene was significantly (P = 0.036) and suggestively (P = 0.07) associated with stature in Caucasian families and 1306 unrelated Caucasian subjects, respectively, and rs9834312 in the FLNB gene was also associated with stature in such two independent Caucasian populations (P = 0.008 in unrelated sample and P = 0.049 in family sample). Particularly, additional significant replication association signals were detected in Chinese, an ethnic population different from Caucasian, between rs9834312 and stature in 619 unrelated northern Chinese subjects (P = 0.017), as well as between rs10734652 and stature in 2953 unrelated southern Chinese subjects (P = 0.048). This study also provides additional replication evidence for some of the already published stature loci. These results, together with the known functional relevance of the SBF2 and FLNB genes to skeletal linear growth and bone formation, support that two regions containing FLNB and SBF2 genes are two novel loci underlying stature variation.

Background

Human genetic variants may affect tuberculosis susceptibility, but the immunologic correlates of the genetic variants identified are often unclear.

Methods

We conducted a pilot case-control study to identify genetic variants associated with extrapulmonary tuberculosis in patients with previously characterized immune defects: low CD4+ lymphocytes and low unstimulated cytokine production. Two genetic association approaches were used: 1) variants previously associated with tuberculosis risk; 2) single nucleotide polymorphisms (SNPs) in candidate genes involved in tuberculosis pathogenesis. Single locus association tests and multifactor dimensionality reduction (MDR) assessed main effects and multi-locus interactions.

Results

There were 24 extrapulmonary tuberculosis cases (18 black), 24 pulmonary tuberculosis controls (19 black) and 57 PPD+ controls (49 black). In approach 1, 22 SNPs and 3 microsatellites were assessed. In single locus association tests, interleukin (IL)-1β +3953 C/T was associated with extrapulmonary tuberculosis compared to PPD+ controls (P = 0.049). Among the sub-set of patients who were black, genotype frequencies of the vitamin D receptor (VDR) Fok1 A/G SNP were significantly different in extrapulmonary vs. pulmonary TB patients (P = 0.018). In MDR analysis, the toll-like receptor (TLR) 2 microsatellite had 76% prediction accuracy for extrapulmonary tuberculosis in blacks (P = 0.002). In approach 2, 613 SNPs in 26 genes were assessed. None were associated with extrapulmonary tuberculosis.

Conclusions

In this pilot study among extrapulmonary tuberculosis patients with well-characterized immune defects, genetic variants in IL-1β, VDR Fok1, and TLR2 were associated with an increased risk of extrapulmonary disease. Additional studies of the underlying mechanism of these genetic variants are warranted.

Cigarette smoking is the leading preventable cause of death in the US. Although smoking behavior has a significant genetic determination, the specific genes and associated mechanisms underlying smoking behavior are largely unknown. Here, we performed a genome-wide association study on smoking behavior in 840 Caucasians, including 417 males and 423 females, in which we examined ∼380,000 SNPs. We found that a cluster of nine SNPs upstream from the IL15 gene were associated with smoking status in males, with the most significant SNP, rs4956302, achieving a p value (8.80×10−8) of genome-wide significance. Another SNP, rs17354547, that is highly conserved across multiple species, achieved a p value of 5.65×10−5. These two SNPs, together with two additional SNPs (rs1402812 and rs4956396) were selected from the above nine SNPs for replication in an African-American sample containing 1,251 subjects, including 412 males and 839 females. The SNP rs17354547 was successfully replicated in the male subgroup of the replication sample; it was associated with smoking quantity (SQ), the Heaviness of Smoking Index (HSI) and the Fagerstrom Test for Nicotine Dependence (FTND), with p values of 0.031, 0.0046 and 0.019, respectively. In addition, a haplotype formed by rs17354547, rs1402812 and rs4956396 was also associated with SQ, HSI and FTND, achieving p values of 0.039, 0.0093 and 0.0093, respectively. To further confirm our findings, we performed an in silico replication study of the nine SNPs in a Framingham Heart Study sample containing 7,623 Caucasians from 1,731 families, among which, 3,491 subjects are males and 4,132 are females. Again, male-specific association with smoking status was observed, for which seven of the nine SNPs achieved significant p values (p<0.05) and two achieved marginally significant p values (p<0.10) in males. Several of the nine SNPs, including the highly conserved one across species, rs17354547, are located at potential transcription factor binding sites, suggesting transcription regulation as a possible function for these SNPs. Through this function, the SNPs may modulate gene expression of IL15, a key cytokine regulating immune function. As the immune system has long been recognized to influence drug addiction behavior, our association findings suggest a novel mechanism for smoking addiction involving immune modulation via the IL15 pathway.

Although copy number variation (CNV) has recently received much attention as a form of structure variation within the human genome, knowledge is still inadequate on fundamental CNV characteristics such as occurrence rate, genomic distribution and ethnic differentiation. In the present study, we used the Affymetrix GeneChip® Mapping 500K Array to discover and characterize CNVs in the human genome and to study ethnic differences of CNVs between Caucasians and Asians. Three thousand and nineteen CNVs, including 2381 CNVs in autosomes and 638 CNVs in X chromosome, from 985 Caucasian and 692 Asian individuals were identified, with a mean length of 296 kb. Among these CNVs, 190 had frequencies greater than 1% in at least one ethnic group, and 109 showed significant ethnic differences in frequencies (p<0.01). After merging overlapping CNVs, 1135 copy number variation regions (CNVRs), covering approximately 439 Mb (14.3%) of the human genome, were obtained. Our findings of ethnic differentiation of CNVs, along with the newly constructed CNV genomic map, extend our knowledge on the structural variation in the human genome and may furnish a basis for understanding the genomic differentiation of complex traits across ethnic groups.

A genome-wide association study was conducted among Chinese women to identify risk variants for breast cancer. By analyzing 607,728 SNPs in 1505 cases and 1522 controls, we selected 29 promising SNPs for a fast-track replication in an independent set of 1554 cases and 1576 controls. Four replicated loci were further investigated in a third set of samples including 3472 cases and 900 controls. SNP rs2046210 at 6q25.1, located upstream of the estrogen receptor 1 gene (ESR1), exhibited strong and consistent association with breast cancer across all three stages. Adjusted odds ratio (95% CI) were 1.36 (1.24–1.49) and 1.59 (1.40–1.82), respectively, for genotypes A/G and A/A versus G/G (P for trend, 2.0×10−15) in the pooled analysis of samples from all three stages. A similar, although weaker, association was also found in an independent study including 1591 cases and 1466 controls of European ancestry (Ptrend, 0.01). These results provide strong evidence implicating 6q25.1 as a susceptibility locus for breast cancer.

Background

Current genome-wide association studies (GWAS) are normally implemented in a univariate framework and analyze different phenotypes in isolation. This univariate approach ignores the potential genetic correlation between important disease traits. Hence this approach is difficult to detect pleiotropic genes, which may exist for obesity and osteoporosis, two common diseases of major public health importance that are closely correlated genetically.

Principal Findings

To identify such pleiotropic genes and the key mechanistic links between the two diseases, we here performed the first bivariate GWAS of obesity and osteoporosis. We searched for genes underlying co-variation of the obesity phenotype, body mass index (BMI), with the osteoporosis risk phenotype, hip bone mineral density (BMD), scanning ∼380,000 SNPs in 1,000 unrelated homogeneous Caucasians, including 499 males and 501 females. We identified in the male subjects two SNPs in intron 1 of the SOX6 (SRY-box 6) gene, rs297325 and rs4756846, which were bivariately associated with both BMI and hip BMD, achieving p values of 6.82×10−7 and 1.47×10−6, respectively. The two SNPs ranked at the top in significance for bivariate association with BMI and hip BMD in the male subjects among all the ∼380,000 SNPs examined genome-wide. The two SNPs were replicated in a Framingham Heart Study (FHS) cohort containing 3,355 Caucasians (1,370 males and 1,985 females) from 975 families. In the FHS male subjects, the two SNPs achieved p values of 0.03 and 0.02, respectively, for bivariate association with BMI and femoral neck BMD. Interestingly, SOX6 was previously found to be essential to both cartilage formation/chondrogenesis and obesity-related insulin resistance, suggesting the gene's dual role in both bone and fat.

Conclusions

Our findings, together with the prior biological evidence, suggest the SOX6 gene's importance in co-regulation of obesity and osteoporosis.

Matrix metalloproteinase-7 (MMP-7) is a small secreted proteolytic enzyme with broad substrate specificity against extracellular matrix (ECM) and non-ECM components. Known to be vital for tumor invasion and metastasis, accumulating evidence also implicates MMP-7 in cancer development. Using data from the Shanghai Breast Cancer Study (SBCS), we conducted a two-stage study to evaluate the association of MMP-7 single nucleotide polymorphisms (SNPs) with breast cancer risk. Additionally, associated SNPs were characterized by laboratory assays. In Stage 1, 11 SNPs were genotyped among 1,079 incident cases and 1,082 community controls using an Affymetrix Genotyping System. Promising SNPs were selected for Stage 2 evaluation and genotyped by TaqMan allelic discrimination assays in an independent set of 1,911 cases and 1,811 controls. Three SNPs were selected for Stage 2 validation (rs880197, rs10895304, and rs12184413); one had highly consistent results between the two stages of the study. In combined analysis, homozygosity for the variant T allele for rs12184413 was associated with an odds ratio of 0.7 (95% CI: 0.6–0.9) compared to the common C allele. This effect was slightly more pronounced in postmenopausal women (OR: 0.6, 95% CI: 0.4–0.8) than in pre-menopausal women (OR: 0.8, 95% CI: 0.6–1.1). This SNP is located 3′ of the MMP-7 gene, in an area enriched with CTCF binding sites. In silico analysis suggested a regulatory role for this region, and our in vitro assays demonstrated an allelic difference in nuclear protein binding capacity. Results from our study suggest that common MMP-7 genetic polymorphisms may contribute to breast cancer susceptibility.

Colon cancer progression is characterized by activating mutations in Ras and by the emergence of the tumor-promoting effects of transforming growth factor-β (TGF-β) signaling. Ras-inducible rat intestinal epithelial cells (RIE:iRas) undergo a well-described epithelial to mesenchymal transition and invasive phenotype in response to H-RasV12 expression and TGF-β treatment, modeling tumor progression. We characterized global gene expression profiles accompanying Ras-induced and TGF-β–induced epithelial to mesenchymal transition in RIE:iRas cells by microarray analysis and found that the regulation of gene expression by the combined activation of Ras and TGF-β signaling was associated with enrichment of a class of mRNAs containing 3′ AU-rich element (ARE) motifs known to regulate mRNA stability. Regulation of ARE-containing mRNA transcripts was validated at the mRNA level, including genes important for tumor progression. Ras and TGF-β synergistically increased the expression and mRNA stability of vascular endothelial growth factor (VEGF), a key regulator of tumor angiogenesis, in both RIE:iRas cells and an independent cell culture model (young adult mouse colonocyte). Expression profiling of human colorectal cancers (CRC) further revealed that many of these genes, including VEGF and PAI-1, were differentially expressed in stage IV human colon adenocarcinomas compared with adenomas. Furthermore, genes differentially expressed in CRC are also significantly enriched with ARE-containing transcripts. These studies show that oncogenic Ras and TGF-β synergistically regulate genes containing AREs in cultured rodent intestinal epithelial cells and suggest that posttranscriptional regulation of gene expression is an important mechanism involved in cellular transformation and CRC tumor progression.

Obesity is a major public health problem with strong genetic determination; however, the genetic factors underlying obesity are largely unknown. In this study, we performed a genome-wide association scan for obesity by examining approximately 500 000 single-nucleotide polymorphisms (SNPs) in a sample of 1000 unrelated US Caucasians. We identified a novel gene, CTNNBL1, which has multiple SNPs associated with body mass index (BMI) and fat mass. The most significant SNP, rs6013029, achieved experiment-wise P-values of 2.69 × 10−7 for BMI and of 4.99 × 10−8 for fat mass, respectively. The SNP rs6013029 minor allele T confers an average increase in BMI and fat mass of 2.67 kg/m2 and 5.96 kg, respectively, compared with the alternative allele G. We further genotyped the five most significant CTNNBL1 SNPs in a French case–control sample comprising 896 class III obese adults (BMI ≥ 40 kg/m2) and 2916 lean adults (BMI < 25 kg/m2). All five SNPs showed consistent associations with obesity (8.83 × 10−3 < P < 6.96 × 10−4). Those subjects who were homozygous for the rs6013029 T allele had 1.42-fold increased odds of obesity compared with those without the T allele. The protein structure of CTNNBL1 is homologous to β-catenin, a family of proteins containing armadillo repeats, suggesting similar biological functions. β-Catenin is involved in the Wnt/β-catenin-signaling pathway which appears to contribute to maintaining the undifferentiated state of pre-adipocytes by inhibiting adipogenic gene expression. Our study hence suggests a novel mechanism for the development of obesity, where CTNNBL1 may play an important role. Our study also provided supportive evidence for previously identified associations between obesity and INSIG2 and PFKP, but not FTO.

For females, menarche is a most significant physiological event. Age at menarche (AAM) is a trait with high genetic determination and is associated with major complex diseases in women. However, specific genes for AAM variation are largely unknown. To identify genetic factors underlying AAM variation, a genome-wide association study (GWAS) examining about 380,000 SNPs was conducted in 477 Caucasian women. A follow-up replication study was performed to validate our major GWAS findings using two independent Caucasian cohorts with 854 siblings and 762 unrelated subjects, respectively, and one Chinese cohort of 1,387 unrelated subjects—all females. Our GWAS identified a novel gene, SPOCK (Sparc/Osteonectin, CWCV, and Kazal-like domains proteoglycan), which had seven SNPs associated with AAM with genome-wide false discovery rate (FDR) q<0.05. Six most significant SNPs of the gene were selected for validation in three independent replication cohorts. All of the six SNPs were replicated in at least one cohort. In particular, SNPs rs13357391 and rs1859345 were replicated both within and across different ethnic groups in all three cohorts, with p values of 5.09×10−3 and 4.37×10−3, respectively, in the Chinese cohort and combined p values (obtained by Fisher's method) of 5.19×10−5 and 1.02×10−4, respectively, in all three replication cohorts. Interestingly, SPOCK can inhibit activation of MMP-2 (matrix metalloproteinase-2), a key factor promoting endometrial menstrual breakdown and onset of menstrual bleeding. Our findings, together with the functional relevance, strongly supported that the SPOCK gene underlies variation of AAM.

Author Summary

Menarche is a physical milestone in a woman's life. Age at menarche (AAM) is related to many common female health problems. AAM is mainly determined by genetic factors. However, the specific genes and the associated mechanisms underlying AAM are largely unknown. Here, taking advantage of the most recent technological advances in the field of human genetics, we identified multiple genetic variants in a gene, SPOCK, which are associated with AAM variation in a group of Caucasian women. This association was subsequently confirmed not only in two independent groups of Caucasian women but also across ethnic boundaries in one group of Chinese women. In addition, SPOCK has a function in regulating a key factor involved in menstrual cycles, MMP-2, which provides further support to our findings. Our study provides a solid basis for further investigation of the gene, which may help to reveal the underlying mechanisms for the timing of menarche and for AAM's relationship with women's health in general.