José Carlos Rodríguez-Rey

Pitx2 Participates in the Late Phase of the Pathway Controlling Left-Right Asymmetry

Pitx2, a member of the bicoid-related family of homeobox-containing genes, is asymmetrically expressed in the left lateral plate mesoderm and derived tissues during chick and mouse development. Modifications of Pitx2 pattern of expression in the iv mouse mutation correlate with the situs alterations characteristic of the mutation. Misexpression experiments demonstrate that Shh and nodal positively regulate Pitx2 expression. Our results are compatible with a Pitx2 function in the late phase of the gene cascade controlling laterality.

DNA methylation contributes to the regulation of sclerostin expression in human osteocytes

Sclerostin, encoded by the SOST gene, is a potent inhibitor of bone formation, produced by osteocytes, not by osteoblasts, but little is known about the molecular mechanisms controlling its expression. We aimed to test the hypothesis that epigenetic mechanisms, specifically DNA methylation, modulate SOST expression. We found two CpG‐rich regions in SOST: region 1, located in the proximal promoter, and region 2, around exon 1. qMSP and pyrosequencing analysis of DNA methylation showed that region 2 was largely methylated in all samples analyzed. In contrast, marked differences were observed in region 1. Whereas the CpG‐rich region 1 was hypermethylated in osteoblasts, this region was largely hypomethylated in microdissected human osteocytes. Bone lining cells showed a methylation profile between primary osteoblasts and osteocytes. Whereas SOST expression was detected at very low level or not at all by RT‐qPCR in several human osteoblastic and nonosteoblastic cell lines, and human primary osteoblasts under basal conditions, it was dramatically upregulated (up to 1300‐fold) by the demethylating agent AzadC. Experiments using reporter vectors demonstrated the functional importance of the region −581/+30 of the SOST gene, which contains the CpG‐rich region 1. In vitro methylation of this CpG‐island impaired nuclear protein binding and led to a 75 ± 12% inhibition of promoter activity. In addition, BMP‐2‐induced expression of SOST was markedly enhanced in cells demethylated by AzadC. Overall, these results strongly suggest that DNA methylation is involved in the regulation of SOST expression during osteoblast–osteocyte transition, presumably by preventing the binding of transcription factors to the proximal promoter. To our knowledge, our data provide first ever evidence of the involvement of DNA methylation in the regulation of SOST expression and may help to establish convenient experimental models for further studies of human sclerostin.

Identification of recurrent and novel mutations in the LDL receptor gene in Spanish patients with familial hypercholesterolemia

We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(‐18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706‐10G→A and 2390‐1G→A, and the other one has been reported recently, 313+1G→C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G→C; C95R and D679E; W(‐18)X and E256K, and C281Y and 1706‐10G→A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population. Hum Mutat 11:413, 1998.

Exacerbation of type II collagen–induced arthritis in apolipoprotein E–deficient mice in association with the expansion of Th1 and Th17 cells

OBJECTIVE To explore the bidirectional relationship between the development of rheumatoid arthritis (RA) and atherosclerosis using bovine type II collagen (CII)-immunized B10.RIII apoE(-/-) mice, a murine model of spontaneous atherosclerosis and collagen-induced arthritis (CIA). METHODS Male B10.RIII apoE(-/-) mice and wild-type controls were immunized with 150 μg of CII emulsified in Freunds complete adjuvant (CFA). The clinical, radiologic, and histopathologic severity of CIA, the levels of circulating IgG1 and IgG2a anti-CII antibodies, the expression of proinflammatory and antiinflammatory cytokines in the joints, and the percentages of Th1, Th17, and Treg lymphocytes in the draining lymph nodes were evaluated during CIA induction. In addition, the size of atherosclerotic lesions was assessed in these mice 8 weeks after CIA induction. RESULTS B10.RIII apoE(-/-) mice that were immunized with CII and CFA developed an exacerbated CIA that was accompanied by increased joint expression of multiple proinflammatory cytokines and by the expansion in the draining lymph nodes of Th1 and Th17 cells. In contrast, the size of vascular lesions in B10.RIII apoE(-/-) mice was not affected by the development of CIA. CONCLUSION Our findings indicate that a deficiency in apolipoprotein E and/or its consequences in cholesterol metabolism act as accelerating factors in autoimmunity by promoting Th1 and Th17 inflammatory responses.

Role of BMPs in the regulation of sclerostin as revealed by an epigenetic modifier of human bone cells

Sclerostin, encoded by the SOST gene, is specifically expressed by osteocytes. However osteoblasts bear a heavily methylated SOST promoter and therefore do not express SOST. Thus, studying the regulation of human SOST is challenged by the absence of human osteocytic cell lines. Herein, we explore the feasibility of using the induction of SOST expression in osteoblasts by a demethylating agent to study the mechanisms underlying SOST transcription, and specifically, the influence of bone morphogenetic proteins (BMPs). Microarray analysis and quantitative PCR showed that AzadC up-regulated the expression of several BMPs, including BMP-2, BMP-4 and BMP-6, as well as several BMP downstream targets. Recombinant BMP-2 increased the transcriptional activity of the SOST promoter cloned into a reporter vector. Likewise, exposing cells transfected with the vector to AzadC also resulted in increased transcription. On the other hand, inhibition of the canonical BMP signaling blunted the effect of AzadC on SOST. These results show that the AzadC-induced demethylation of the SOST promoter in human osteoblastic cells may be a valuable tool to study the regulation of SOST expression. As a proof of concept, it allowed us to demonstrate that BMPs stimulate SOST expression by a mechanism involving BMPR1A receptors and downstream Smad-dependent pathways.

Association of ACACB polymorphisms with obesity and diabetes

Acetyl-CoA carboxylase beta, encoded by the ACAB gene, plays an important role in the oxidation of fatty acids. The aim of this study was to check the hypothesis that allelic variants of ACACB influence the risk of obesity and type 2 diabetes mellitus. Twenty five tagging single nucleotide polymorphisms (SNPs) capturing common variants of the ACACB gene were selected and analyzed in two cohorts including 1695 postmenopausal women of the general population and in 161 women with severe obesity (BMI>35). In vitro binding of transcription factors was explored by electrophoretic mobility shift assays (EMSA). T alleles at the rs2268388 locus were overrepresented in women with severe obesity (18% vs. 10% in controls; OR 1.74 [95% confidence interval 1.30-2.47]), which was statistically significant after multiple-test adjustment (p=0.0004). Likewise, T alleles at the rs2268388 locus and C alleles at the rs2239607 locus were associated with diabetes, in the discovery as well as in the replication cohorts, even after women with severe obesity were excluded (OR 3.6 and 2.8, for TT and CC homozygotes, respectively). Allelic differences in the binding affinity for nuclear proteins were revealed in vitro by EMSA and competition experiments were consistent with the binding of glucorticoid receptor and serum response factor. In conclusion, common polymorphisms of ACACB gene are associated with obesity and, independently, with type 2 diabetes in postmenopausal women, suggesting that the activity of acetyl-CoA carboxylase beta plays an important role in these disorders related to energy metabolism.

Functional analysis of LDLR promoter and 5' UTR mutations in subjects with clinical diagnosis of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a dominant disorder due to mutations in the LDLR gene. Several mutations in the LDLR promoter are associated with FH. Screening of 3,705 Spanish FH patients identified 10 variants in the promoter and 5′ UTR. Here, we analyse the functionality of six newly identified LDLR variants. Mutations located in the LDLR promoter regulatory elements R2 and R3 (c.−155_‐150delACCCCinsTTCTGCAAACTCCTCCC, c.−136C>G, c.−140C>G, and c.−140C>T) resulted in 6 to 15% residual activity in reporter expression experiments and changes in nuclear protein binding affinity compared to wild type. No reduction was observed when cells were transfected with c.−208T, c.−88A, and c.−36G mutant fragments. Our results indicate that mutations localized in R2 and R3 are associated with hypercholesterolemia, whereas mutations outside the LDLR response elements are not a cause of FH. This data emphasizes the importance of functional analysis of variants in the LDLR promoter to determine their association with the FH phenotype.Hum Mutat 32:1–5, 2011.

An NPC1L1 gene promoter variant is associated with autosomal dominant hypercholesterolemia

BACKGROUND AND AIMS A substantial number of subjects with autosomal dominant hypercholesterolemia (ADH) do not have LDL receptor (LDLR) or apolipoprotein B (APOB) mutations. Some ADH subjects appear to hyperabsorb sterols from the intestine, thus we hypothesized that they could have variants of the Niemann-Pick C1-Like 1 gene (NPC1L1). NPC1L1 encodes a crucial protein involved in intestinal sterol absorption. METHODS AND RESULTS Four NPC1L1 variants (-133A>G, -18C>A, 1679C>G, 28650A>G) were analyzed in 271 (155 women and 116 men) ADH bearers without mutations in LDLR or APOB aged 30-70years and 274 (180 women and 94 men) control subjects aged 25-65years. The AC haplotype determined by the -133A>G and -18C>A variants was underrepresented in ADH subjects compared to controls (p=0.01). In the ADH group, cholesterol absorption/synthesis markers were significantly lower in AC homozygotes that in all others haplotypes. Electrophoretic mobility shift assay (EMSA) results revealed that the -133A-specific oligonucleotide produced a retarded band stronger than the -133G allele. Luciferase activity with NPC1L1 -133G variant was 2.5-fold higher than with the -133A variant. CONCLUSION The -133A>G polymorphism exerts a significant effect on NPC1L1 promoter activity. NPC1L1 promoter variants might explain in part the hypercholesterolemic phenotype of some subjects with nonLDLR/nonAPOB ADH.

Functional analysis of regulatory single-nucleotide polymorphisms.

Purpose of review The identification of regulatory polymorphisms has become a key problem in human genetics. In the past few years there has been a conceptual change in the way in which regulatory single-nucleotide polymorphisms are studied. We revise the new approaches and discuss how gene expression studies can contribute to a better knowledge of the genetics of common diseases. Recent findings New techniques for the association of single-nucleotide polymorphisms with changes in gene expression have been recently developed. This, together with a more comprehensive use of the old in-vitro methods, has produced a great amount of genetic information. When added to current databases, it will help to design better tools for the detection of regulatory single-nucleotide polymorphisms. Summary The identification of functional regulatory single-nucleotide polymorphisms cannot be done by the simple inspection of DNA sequence. In-vivo techniques, based on primer-extension, and the more recently developed ‘haploChIP’ allow the association of gene variants to changes in gene expression. Gene expression analysis by conventional in-vitro techniques is the only way to identify the functional consequences of regulatory single-nucleotide polymorphisms. The amount of information produced in the last few years will help to refine the tools for the future analysis of regulatory gene variants.

Association study of sirtuin 1 polymorphisms with bone mineral density and body mass index

BACKGROUND AND AIMS Sirtuin 1, encoded by the SIRT1 gene, is an emerging modulator of carbohydrate and lipid metabolism and may also influence the differentiation of bone cells. Our objective was to test the hypothesis that polymorphisms of SIRT1 are associated with body mass index (BMI) and bone mineral density (BMD). METHODS We carried out a cross-sectional genetic association study with genotyping of ten single nucleotide polymorphisms of the SIRT1 region. The discovery cohort included 1394 individuals (342 males, 1052 females). Significant results were replicated in an independent cohort of 408 males. RESULTS We did not find a significant association of genotypes with BMD. There were also no significant BMI differences across genotypes in females. However, in males, two polymorphisms tended to be associated with BMI in the discovery cohort (p 0.03 and 0.05). A similar trend was also observed in the replication cohort. Thus, in the combined analysis of both cohorts, males with C alleles at the rs12049646 locus had a lower BMI than TT homozygotes, with a mean difference of 0.82 kg/m(2) (95% confidence interval 0.15-1.48; p = 0.016). Differences in the DNA binding of nuclear proteins between C and T alleles were also observed in vitro. CONCLUSIONS These results suggest that common variants of the SIRT1 gene influence BMI but not BMD.