Mutations in the HFE, TFR2, and SLC40A1 genes in patients with hemochromatosis

Hereditary hemochromatosis causes iron overload and is associated with a variety of genetic and phenotypic conditions. Early diagnosis is important so that effective treatment can be administered and the risk of tissue damage avoided. Most patients are homozygous for the c.845G>A (p.C282Y) mutation in the HFE gene; however, rare forms of genetic iron overload must be diagnosed using a specific genetic analysis. We studied the genotype of 5 patients who had hyperferritinemia and an iron overload phenotype, but not classic mutations in the HFE gene. Two patients were undergoing phlebotomy and had no iron overload, 1 with metabolic syndrome and no phlebotomy had mild iron overload, and 2 patients had severe iron overload despite phlebotomy. The patients' first-degree relatives also underwent the analysis. We found 5 not previously published mutations: c.-408_-406delCAA in HFE, c.1118G>A (p.G373D), c.1473G>A (p.E491E) and c.2085G>C (p.S695S) in TFR2; and c.-428_-427GG>TT in SLC40A1. Moreover, we found 3 previously published mutations: c.221C>T (p.R71X) in HFE; c.1127C>A (p.A376D) in TFR2; and c.539T>C (p.I180T) in SLC40A1. Four patients were double heterozygous or compound heterozygous for the mutations mentioned above, and the patient with metabolic syndrome was heterozygous for a mutation in the TFR2 gene. Our findings show that hereditary hemochromatosis is clinically and genetically heterogeneous and that acquired factors may modify or determine the phenotype.     

Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer

Genome-wide association studies (GWAS) have identified 14 tagging single nucleotide polymorphisms (tagSNPs) that are associated with the risk of colorectal cancer (CRC), and several of these tagSNPs are near bone morphogenetic protein (BMP) pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3), BMP4 (14q22.2), and BMP2 (20p12.3) using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10)) and BMP2 (rs4813802, P = 4.65×10(-11)). Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8)) and rs11632715 (P = 2.30×10(-10)). As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.     

Common genetic variants and modification of penetrance of BRCA2-associated breast cancer

The considerable uncertainty regarding cancer risks associated with inherited mutations of BRCA2 is due to unknown factors. To investigate whether common genetic variants modify penetrance for BRCA2 mutation carriers, we undertook a two-staged genome-wide association study in BRCA2 mutation carriers. In stage 1 using the Affymetrix 6.0 platform, 592,163 filtered SNPs genotyped were available on 899 young (<40 years) affected and 804 unaffected carriers of European ancestry. Associations were evaluated using a survival-based score test adjusted for familial correlations and stratified by country of the study and BRCA2*6174delT mutation status. The genomic inflation factor (λ) was 1.011. The stage 1 association analysis revealed multiple variants associated with breast cancer risk: 3 SNPs had p-values<10(-5) and 39 SNPs had p-values<10(-4). These variants included several previously associated with sporadic breast cancer risk and two novel loci on chromosome 20 (rs311499) and chromosome 10 (rs16917302). The chromosome 10 locus was in ZNF365, which contains another variant that has recently been associated with breast cancer in an independent study of unselected cases. In stage 2, the top 85 loci from stage 1 were genotyped in 1,264 cases and 1,222 controls. Hazard ratios (HR) and 95% confidence intervals (CI) for stage 1 and 2 were combined and estimated using a retrospective likelihood approach, stratified by country of residence and the most common mutation, BRCA2*6174delT. The combined per allele HR of the minor allele for the novel loci rs16917302 was 0.75 (95% CI 0.66-0.86, ) and for rs311499 was 0.72 (95% CI 0.61-0.85, ). FGFR2 rs2981575 had the strongest association with breast cancer risk (per allele HR = 1.28, 95% CI 1.18-1.39, ). These results indicate that SNPs that modify BRCA2 penetrance identified by an agnostic approach thus far are limited to variants that also modify risk of sporadic BRCA2 wild-type breast cancer.     

Quercetin prevents ethanol-induced iron overload by regulating hepcidin through the BMP6/SMAD4 signaling pathway

Emerging evidence has demonstrated that chronic ethanol exposure induces iron overload, enhancing ethanol-mediated liver damage. The purpose of this study was to explore the effects of the naturally occurring compound quercetin on ethanol-induced iron overload and liver damage, focusing on the signaling pathway of the iron regulatory hormone hepcidin. Adult male C57BL/6J mice were pair-fed with isocaloric-Lieber De Carli diets containing ethanol (accounting for 30% of total calories) and/or carbonyl iron (0.2%) and treated with quecertin (100 mg/kg body weight) for 15 weeks. Mouse primary hepatocytes were incubated with ethanol (100 mM) and quercetin (100 μM) for 24 h. Mice exposed to either ethanol or iron presented significant fatty infiltration and iron deposition in the liver; these symptoms were exacerbated in mice cotreated with ethanol and iron. Quercetin attenuated the abnormity induced by ethanol and/or iron. Ethanol suppressed BMP6 and intranuclear SMAD4 as well as decreased hepcidin expression. These effects were partially alleviated by quercetin supplementation in mice and hepatocytes. Importantly, ethanol caused suppression of SMAD4 binding to the HAMP promoter and of hepcidin messenger RNA expression. These effects were exacerbated by anti-BMP6 antibody and partially alleviated by quercetin or human recombinant BMP6 in cultured hepatocytes. In contrast, co-treatment with iron and ethanol, especially exposure of iron alone, activated BMP6/SMAD4 pathway and up-regulated hepcidin expression, which was also normalized by quercetin in vivo. Quercetin prevented ethanol-induced hepatic iron overload different from what carbonyl iron diet elicited in the mechanism, by regulating hepcidin expression via the BMP6/SMAD4 signaling pathway.     

BMP2 variants in the risk of ankylosing spondylitis

The purpose of the study was to explore the genetic effects of bone morphogenetic protein (BMP2) polymorphisms on the susceptibility to ankylosing spondylitis (AS) in Chinese Han population. The case-control study included 120 AS cases and 110 healthy controls. Hardy-Weinberg equilibrium test was performed in control group. BMP2 rs235768 and rs3178250 polymorphisms were analyzed by polymerase chain reaction and direct sequencing. Additionally, the χ² test was used to estimate association strength between BMP2 genetic polymorphisms and AS susceptibility, and the results were assessed via odds ratio (OR) with the corresponding 95% confidence interval (95%CI). Results adjustment was performed using logistic regression analysis. AA, AT, TT genotype and A, T allele frequencies of BMP2 rs235768 polymorphism presented no significant differences between case and control groups (P > .05 for all). TC genotype of rs3178250 polymorphism showed significantly higher in case group than that in control group (P = .048). After adjusting, TC genotype was a risk factor for AS (OR = 2.095; 95%CI = 1.086-4.038; P = .027). BMP2 rs3178250 polymorphism may increase individual susceptibility to AS in Chinese Han population.     

Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis

Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.     

Serum regulation of Id1 expression by a BMP pathway and BMP responsive element

Immediate early genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP responsive element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad 4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs.     

Genetic variants in the vitamin D pathway genes VDBP and RXRA modulate cutaneous melanoma disease‐specific survival

Single nucleotide polymorphisms (SNPs) in the vitamin D pathway genes have been implicated in cutaneous melanoma (CM) risk, but their role in CM disease‐specific survival (DSS) remains obscure. We comprehensively analyzed the prognostic roles of 2669 common SNPs in the vitamin D pathway genes using data from a published genome‐wide association study (GWAS) at The University of Texas M.D. Anderson Cancer Center (MDACC) and then validated the SNPs of interest in another GWAS from the Nurses’ Health Study and Health Professionals Follow‐up Study. Among the 2669 SNPs, 203 were significantly associated with DSS in MDACC dataset (P < 0.05 and false‐positive report probability < 0.2), of which 18 were the tag SNPs. In the replication, two of these 18 SNPs showed nominal significance: the VDBP rs12512631 T > C was associated with a better DSS [combined hazards ratio (HR) = 0.66]; and the same for RXRA rs7850212 C > A (combined HR = 0.38), which were further confirmed by the Fine and Gray competing‐risks regression model. Further bioinformatics analyses indicated that these loci may modulate corresponding gene methylation status.     

Possible roles of the hereditary hemochromatosis protein, HFE, in regulating cellular iron homeostasis

Hereditary hemochromatosis (HH) is the most common inherited disorder in people of Northern European descent. Over 83% of the cases of HH result from a single mutation of a Cys to Tyr in the HH protein. HFE. This mutation causes a recessive disease resulting in an accumulation of iron in selected tissues. Iron overload damages these organs leading to cirrhosis of the liver, diabetes, cardiomyopathy, and arthritis. The mechanism by which HFE influences iron homeostasis in cells and in the body remains elusive. Lack of functional HFE in humans produces the opposite effects in different cell types in the body. In the early stages of the disease. Kupffer cells in the liver and enterocytes in the intestine cells are iron depleted and have low intracellular ferritin levels, whereas hepatocytes in the liver are iron overloaded and have high intracellular iron levels. This review gives the background and a model as to possible mechanisms of how HFE could exert different effects on iron homeostasis in different cell types.     

Bone morphogenetic protein: chromosomal localization of human genes for BMP1, BMP2A, and BMP3

Bone morphogenetic protein (BMP) induces endochondral bone formation in vivo. The human genes have been cloned for a group of proteins containing BMP activity (BMP1, BMP2A, and BMP3). Two of the proteins are members of the transforming growth factor-beta supergene family (BMP2A and BMP3), while BMP1 is a novel regulatory protein. Using somatic cell hybrid lines, cDNA probes were used to map BMP1 to chromosome 8, BMP2A to chromosome 20, and BMP3 to the p14-q21 region of chromosome 4. This analysis reveals that the BMP2A and BMP3 genes map to conserved regions between mouse and human, while the BMP1 gene does not. The locations of the BMP genes were found to overlap with the loci for several disorders of cartilage and bone formation.     

The hereditary hemochromatosis protein, HFE, inhibits iron uptake via down-regulation of Zip14 in HepG2 cells

Lack of functional hereditary hemochromatosis protein, HFE, causes iron overload predominantly in hepatocytes, the major site of HFE expression in the liver. In this study, we investigated the role of HFE in the regulation of both transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI) uptake in HepG2 cells, a human hepatoma cell line. Expression of HFE decreased both TBI and NTBI uptake. It also resulted in a decrease in the protein levels of Zip14 with no evident change in the mRNA level of Zip14. Zip14 (Slc39a14) is a metal transporter that mediates NTBI into cells (Liuzzi, J. P., Aydemir, F., Nam, H., Knutson, M. D., and Cousins, R. J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 13612-13617). Knockdown of Zip14 with siRNA abolished the effect of HFE on NTBI uptake. To determine if HFE had a similar effect on Zip14 in another cell line, HeLa cells expressing HFE under the tetracycline-repressible promoter were transfected with Zip14. As in HepG2 cells, HFE expression inhibited NTBI uptake by approximately 50% and decreased Zip14 protein levels. Further analysis of protein turnover indicated that the half-life of Zip14 is lower in cells that express HFE. These results suggest that HFE decreases the stability of Zip14 and therefore reduces the iron loading in HepG2 cells.     

Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells

HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and beta(2)-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the (59)Fe uptake and release, and internalization and recycling of (125)I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ((59)Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4. 3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5x10(5)/cell to 1. 2x10(5)/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with (125)I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.     

Transcriptional regulation of BMP4 synexpression in transgenic Xenopus

Synexpression groups are genetic modules composed of genes that share both a complex expression pattern and the biological process in which they function. Here we investigate the regulation of BMP4 synexpression by studying the enhancers of bambi, smad7 and vent2 in Xenopus. We find that a BMP4 synexpression promoter module is compact and (i) requires direct BMP responsiveness through Smad and Smad-cofactor binding motifs, (ii) may contain an evolutionary conserved BMP-responsive element, bre7 (TGGCGCC), that is crucial for expression of bambi and smad7 and is highly prognostic for novel BMP-responsive enhancers (BREs); and (iii) requires a narrow window of BMP inducibility, because minor enhancement or reduction of BMP responsiveness abolishes synexpression. Furthermore, we used a bioinformatic model to predict in silico 13 novel BREs, and tested five of them that were found in the id1-4 genes. The results highlight that in vivo analysis is required to reveal the physiological, spatio-temporal regulation of BMP-responsive genes.     

Common variants of xeroderma pigmentosum genes and prostate cancer risk

The genetic basis of prostate cancer (PC) is complex and appears to involve multiple susceptibility genes. A number of studies have evaluated a possible correlation between several NER gene polymorphisms and PC risk, but most of them evaluated only single SNPs among XP genes and the results remain inconsistent. Out of 94 SNPs located in seven XP genes (XPA–XPG) a total of 15 SNPs were assayed in 720 unselected patients with PC and compared to 1121 healthy adults. An increased risk of disease was associated with the XPD SNP, rs1799793 (Asp312Asn) AG genotype (OR = 2.60; p < 0.001) and with the AA genotype (OR = 531; p < 0.0001) compared to the control population. Haplotype analysis of XPD revealed one protective haplotype and four associated with an increased disease risk, which showed that the A allele (XPD rs1799793) appeared to drive the main effect on promoting prostate cancer risk. Polymorphism in XPD gene appears to be associated with the risk of prostate cancer.     

Detection of an unusual combination of mutations in the HFE gene for hemochromatosis

In the present paper, we describe an individual, found as part of a screening study, being homozygous for the C282Y mutation and at the same time heterozygous for the H63D mutation in the HFE gene. Identical results were obtained by three different methods, i.e., by PCR-RFLP, by sequencing, and by melting curve analysis. Thus, the common conception that the C282Y and the H63D mutations are mutually exclusive is not valid. Clinical symptoms and laboratory data on the individual were similar to hemochromatosis patients homozygous for the C282Y mutation. The implications of our finding for diagnostic analytical laboratory procedures are briefly discussed.     

Polymorphism of the HFE gene associated with hereditary hemochromatosis in populations of Russia

Expression of hereditary hemochromatosis as well as predisposition to iron overload syndrome and sporadic porphyria cutanea tarda are currently believed to be associated with the inheritance of certain allelic variants of the HFE gene. Allele frequencies of the C282Y (845A) and H63D (187G) mutations in the HFE gene in human populations of different races are remarkably different, and the prevalence of the S65C (193T) mutation is still poorly studied. In the present study we estimated allele frequencies of HFE mutations in Russians and in a number of Siberian ethnic indigenous populations. In Russians, allele frequencies of the C282Y, H63D and S65C mutations were 3.7, 13.3 and 1.7%, respectively. These values were similar to those observed in populations of Europe. The C282Y mutation was not detected in the population samples of Siberian ethnic groups, including Mansis, Khantys (Finno-Ugric group), Altaians, and Nivkhs (Mongoloids), suggesting that the frequency of this allele in the populations examined was lower than 1%. The frequency of the C282Y allele in the Tuvinian and Chukchi samples (Mongoloids) constituted 0.45 and 0.8%, respectively. Furthermore, pedigree analysis of both Chukchi carriers discovered showed that some of their ancestors were from other ethnic groups. Low frequencies of this allelic variant is typical of many Eastern Asian populations, which are also characterized by rather low frequencies of the H63D variant. In contrast, in some ethnic groups of Western Siberia allelic frequency of the H63D mutation is rather high, constituting 8.7% in Altaians, 15.5% in Mansis, and 11.3% in Khantys. The frequency of this allele in Tuvinians, Nivkhs, and Chukchis constituted 5, 4.7, and 0.8%, respectively. These findings make it possible to estimate the proportion of individuals predisposed to iron overload syndrome in different Russian ethnic groups. The HFE allele frequency distribution patterns observed in the populations examined pointed to pre-Celtic appearance of the CY82 allele. It also provides elucidation of the evolutionary genetic relationships between Siberian ethnic groups and the contemporary populations of Eastern and Western Europe.