Novel MC1R variants in Ligurian melanoma patients and controls

Several variant forms of the melanocortin-1 receptor gene (MC1R) have been associated with red hair, fair skin and an increased risk for melanoma. Their involvement in melanoma susceptibility is apparently linked both to skin sensitivity and to non-pigmentary pathways. We investigated the frequency of the MC1R variants in the Italian region of Liguria, where the occurrence and penetrance of melanoma are low and primary susceptibility is characterized by prevalence of the CDKN2A c.301G>T [p.G101W] founder mutation. Additionally, we attempted to establish the frequency of the red hair/fair skin phenotype in our region. As predicted by anecdotal evidence, the frequency of red hair/phototype I was very low (0.7%). Screening of 17 red-haired individuals and their red-haired relatives, 207 controls and 214 melanoma patients unselected for hair color but all of Ligurian descent, led to the detection of 8 novel substitutions (c.133T>C [p.F45L], c.248C>T [p.S83L], c.332C>T [p. A111V], c.479G>A [p.R160Q], c.637C>T [p.R213W], c.793G>A [p. V265I], c.923C>T [p. T308M], c.943T>C [p.C315R]), 1 novel deletion (c.520_523delGTC [p.V174del]) and 3 novel synonymous variants (c.366G>C [p. V122V], c.684G>A [p. Q228Q], c.726C>T [p.T241T]). Preliminary genotype/phenotype correlation seems to indicate that other genes involved in the regulation of human pigmentation may mask the recessive action of high-penetrance MC1R alleles, thus determining the low frequency of at-risk phototypes and of incidence and/or penetrance of melanoma in Liguria.     

A novel functionally deficient MYH variant in individuals with colorectal adenomatous polyposis

Inherited biallelic mutations in the base excision repair gene MYH confer susceptibility to colorectal adenomas and carcinoma. Approximately 85% of Caucasians with MYH mutations carry the (c.494A>G) p.Y165C and (c.1145G>A) p.G382D variants. Only a few other clearly pathogenic mutations have been identified, and mutation analyses tend to focus on the two founder mutations rather than the whole coding region of the gene. We sequenced the entire coding region of MYH in a population-based series of 24 Finnish APC-mutation negative polyposis patients in order to identify novel pathogenic MYH variants. A population-based series of 1,042 Finnish colorectal cancer patients and 85 cancer-free controls were available for further evaluation. A functional cleavage assay was designed to evaluate consequences of possible novel variants to protein function. Three novel MYH variants, (c.270C>T) p.Y90Y, (c.1376C>A) p.A459D, and (c.1389G>C) p.T469T, were observed. p.A459D variant in exon 14 was identified in two patients from the polyposis series, once in homozygosity and once in compound heterozygosity with p.Y165C. In the population-based series of 1,042 colorectal cancer patients, the p.A459D mutation was identified once, in homozygosity (allele frequency 0.1%). No p.A459D mutations were identified in the control individuals. In vitro cleavage assay showed significantly reduced repair activity in p.A459D cells. Interestingly, another variant in the same codon has previously been described in a British study, supporting a key role for the codon 459 in MYH function. We therefore suggest that screening of mutations in MYH exon 14 should be added to the molecular analysis at-risk individuals.     

Molecular scanning for mutations in the melanocortin-4 receptor gene in obese/diabetic Japanese

Decreased function of the melanocortin-4 receptor (MC4R) was reported to cause late-onset obesity and insulin resistance in rodents. Thus mutations in the MC4R gene drew strong attention as a possible cause of obesity and diabetes. We screened for mutations in the MC4R gene in extremely obese [body mass index (BMI) ≥ 35 kg/m²] Japanese with diabetes by direct sequencing. A heterozygous mutation (V103I) was detected in one case (2.0 %), however the frequency was not significantly different from that in non-obese (BMI ≤ 24 kg/m²) and non-diabetic subjects (2.7 %). No other mutations were detected. These results suggest that mutations including V103I in the MC4R gene are not a major cause of obesity or diabetes in Japanese.     

Clinical, biochemical and molecular characterization of cystinuria in a cohort of 12 patients

Cystinuria is a rare autosomal inherited disorder characterized by impaired transport of cystine and dibasic aminoacids in the proximal renal tubule. Classically, cystinuria is classified as type I (silent heterozygotes) and non-type I (heterozygotes with urinary hyperexcretion of cystine). Molecularly, cystinuria is classified as type A (mutations on SLC3A1 gene) and type B (mutations on SLC7A9 gene). The goal of this study is to provide a comprehensive clinical, biochemical and molecular characterization of a cohort of 12 Portuguese patients affected with cystinuria in order to provide insight into genotype-phenotype correlations. We describe seven type I and five non-type I patients. Regarding the molecular classification, seven patients were type A and five were type B. In SLC3A1 gene, two large genomic rearrangements and 13 sequence variants, including four new variants c.611-2A>C; c.1136+44G>A; c.1597T (p.Y533N); c.*70A>G, were found. One large genomic rearrangement was found in SLC7A9 gene as well as 24 sequence variants including 3 novel variants: c.216C>T (p.C72C), c.1119G>A (p.S373S) and c.*82C>T. In our cohort the most frequent pathogenic mutations were: large rearrangements (33.3% of mutant alleles) and a missense mutation c.1400T>C (p.M467T) (11.1%). This report expands the spectrum of SLC3A1 and SLC7A9 mutations and provides guidance in the clinical implementation of molecular assays in routine genetic counseling of Portuguese patients affected with cystinuria.     

Mutation analysis of NR0B2 among 1545 Danish men identifies a novel c.278G>A (p.G93D) variant with reduced functional activity

Variations of the small heterodimer partner (SHP, NR0B2) gene, an atypical nuclear receptor that inhibits transactivation by hepatocyte nuclear factor (HNF)-4alpha, are associated with obesity among Japanese. The purpose of the study was to evaluate the prevalence of SHP variants among obese Danish men. Using combined SSCP and heteroduplex analysis, we analyzed the entire coding region of SHP for variants in a cohort of 750 Danish men with early-onset obesity and genotyped a cohort of 795 nonobese control subjects using PCR-RFLP. Functional analyses of the identified coding region variants were performed in both MIN6-m9 and HepG2 cell lines. A total of five novel variants, including three missense variants (c.100C>G [p.R34G], c.278G>A [p.G93D], and c.415C>A [p.P139H]) and two silent variants (c.65C>T [p.Y22Y] and c.339G>A [p.P113P]) were identified. Moreover, the previously reported c.512G>C [p.G171A] polymorphism was identified. The 171A allele was not associated with obesity (p = 0.07). The 34G, 93D, and 139H-alleles were rare variants, which were found only among obese subjects. Among the four coding region variants, the 93D-allele showed a reduced in vitro inhibition of the HNF-4alpha transactivation of the HNF-1alpha promoter expression when expressed in MIN6-m9 and HepG2 cell lines (p<0.01). In contrast to reported findings among obese Japanese, functional variants are rare among Danish men. A functional 93D variant of SHP was identified in 1 out of 750 obese and in none of 795 nonobese control subjects. Further large-scale population studies are necessary to assess the clinical impact of this rare variant on obesity risk among European subjects.     

Mutational analysis of the N-ras, p53, p16INK4a, CDK4, and MC1R genes in human congenital melanocytic naevi

Eighteen human congenital melanocytic naevi (CMN) from 17 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 and for sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis. In addition, all lesions were screened for deletions and point mutations in the tumour suppressor genes p53 and p16INK4a (CDKN2A) by combined multiplex PCR/SSCP analysis. Positive screening data were specified by sequencing of the corresponding PCR product. Activating point mutations in the N-ras gene (nine CAA (Gln) to AAA (Lys) transversions and one CAA (Gln) to CGA (Arg) transition at codon 61) were detected at high frequency (56%). Furthermore, three missense mutations (V92M) and two silent mutations (CGA (Arg) to CGG (Arg), codon 213, exon 6) were found in the MC1R and p53 genes, respectively. No mutations were found in p16 or CDK4. The activated N-ras oncogene, which is also found in human cutaneous melanomas, may constitute a potential risk factor for melanoma formation within CMN.     

The ATM missense mutation p.Ser49Cys (c.146C>G) and the risk of breast cancer

Homozygous mutation in the ATM gene causes ataxia telangiectasia and heterozygous mutation carriers may be at increased risk of breast cancer. We studied a total of 22 ATM variants; 18 variants were analyzed in one of two large population-based studies from the U.S. and Poland, and four variants were analyzed in all 2,856 breast cancer cases and 3,344 controls from the two studies. The missense mutation Ser49Cys (c.146C>G, p.S49C), carried by approximately 2% of subjects, was more common in cases than controls in both study populations, combined odds ratio (OR) 1.69 (95% CI, 1.19-2.40; P=0.004). Another missense mutation at approximately 2% frequency, Phe858Leu (c.2572T>C, p.F858L), was associated with a significant increased risk in the U.S. study but not in Poland, and had a combined OR of 1.44 (95% CI, 0.98-2.11; P=0.06). These analyses provide the most convincing evidence thus far that missense mutations in ATM, particularly p.S49C, may be breast cancer susceptibility alleles. Because of their low frequency, even larger sample sizes are required to more firmly establish these associations.     

Germline mutations of the MSR1 gene in prostate cancer families from Germany

The MSR1 gene at 8p22 has been suggested as a candidate gene for hereditary prostate cancer because germline variants have been found to be associated with the disease. Aside from a single nonsense mutation (R293X) that was found repeatedly at low frequencies in several samples, little evidence has been gained by follow-up studies to confirm the gene's relevance for prostate cancer. Prompted by reasonable support for a linkage to 8p22, we sought to determine the mutation spectrum of MSR1 in our family sample. Screening of 139 probands (representing 139 prostate cancer families) revealed 15 novel and a total of 20 sequence variants within the 10 coding exons and their intronic proximities. Aside from the known mutation c.877C>T (R293X) present in two of our families, we identified a second nonsense allele (c.251C>G; S84X) and a splice-site mutation (c.818-1G>A) that results in mRNA instability (each in a single pedigree). The novel missense alleles were c.703C>T (H235Y), c.856C>T (P286S), c.905C>T (P302L), c.1193C>G (A398G), and c.1289A>G (K430R). Of the eight variants that affect the encoded protein (splice site, nonsense, and missense), only R293X as well as the polymorphism c.823C>G (P275A) were additionally present at remarkable frequencies in further samples of sporadic prostate cancer and controls. Of note, carriers of R293X were equally frequent in 367 sporadic prostate cancer cases (1.9%) and in 197 controls (2.0%). To our knowledge, our study is the first to demonstrate further loss of function variants of MSR1 apart from R293X. Nevertheless, the low frequencies of deleterious alleles, in addition to an apparently moderate penetrance, does not support MSR1 as a major susceptibility gene in this family sample.     

Evaluation of germline BMP4 mutation as a cause of colorectal cancer

Transforming growth factor-β (TGF-β) signalling plays a key role in colorectal cancer (CRC). Bone morphogenetic protein-4 (BMP4) is a member of the TGF-β family of signal transduction molecules. To examine if germline mutation in BMP4 causes CRC we analysed 504 genetically enriched CRC cases (by virtue of early-onset disease, family history of CRC) for mutations in the coding sequence of BMP4. We identified three pathogenic mutations, p.R286X (g.8330C>T), p.W325C (g.8449G>T) and p.C373S (g.8592G>C), amongst the CRC cases which were not observed in 524 healthy controls. p.R286X localizes to the N-terminal of the TGF-β1 prodomain truncating the protein prior to the active domain. p.W325C and p.C373S mutations are predicted from protein homology modelling with BMP2 to impact deleteriously on BMP4 function. Segregation of p.C373S with adenoma and hyperplastic polyp in first-degree relatives of the case suggests germline mutations may confer a juvenile polyposis-type phenotype. These findings suggest mutation of BMP4is a cause of CRC and the value of protein-based modelling in the elucidation of rare disease-causing variants.     

Seven novel mutations of the ADAR gene in Chinese families and sporadic patients with dyschromatosis symmetrica hereditaria (DSH)

Dyschromatosis symmetrica hereditaria (DSH) is an autosomal dominant pigmentary genodermatosis characterized by hyperpigmented and hypopigmented macules of on the extremities and caused by the mutations in the ADAR gene(also called DSRAD) encoding for RNA-specific adenosine deaminase. Here we reported clinical and molecular findings of 6 Chinese multi-generation families and 2 sporadic patients with DSH. We found that the same mutation could lead to different phenotypes even in the same family and we did not establish a clear correlation between genotypes and phenotypes. Seven novel heterozygous mutations of ADAR were identified, which were c.2433_2434delAG (p.T811fs), c.2197G>T (p.E733X), c.3286C>T (p.R1096X), c.2897G>T (p.C966F), c.2797C>T (p.Q933X), c.2375delT (p.L792fs) and c.3203-2A>G respectively. Our data add new variants to the repertoire of ADAR mutations in DSH.     

Sporadic mutations in melanocortin receptor 3 in morbid obese individuals

Several mutations in the melanocortin receptor 4 gene have been identified in humans and account for 3-6% of morbid obesity. In contrast, strong evidence of a causative role for melanocortin receptor 3 (MC3R) mutations are still lacking. In MC3R knockout mice, high feed efficiency rather than hyperphagia seems to contribute to increased fat mass. On the basis of this evidence, the objective of the present study was to investigate the presence of MC3R mutations in a group of 290 obese subjects (mean BMI 44.2+/-5.9 kg/m2). As a control, a group of 215 normal-weight subjects (mean BMI 22.4+/-2.7 kg/m2) was also screened. Three novel mutations in the MC3R gene (A293T, I335S and X361S) were identified among the obese patients. The mutations segregated with obesity in the members of the families studied. In vitro expression studies of each mutation demonstrated a loss of function of the I335S-mutated receptor. These findings suggest that, in humans, MC3R mutations may be a cause of a dominantly inherited form of obesity. However, this association as well as the specific phenotypic characteristics resulting from these mutations need to be further evaluated in larger series of obese subjects.     

Association of polymorphisms in the melanocortin receptor type 2 (MC2R, ACTH receptor) gene with heroin addiction

The melanocortin receptor type 2 (MC2R or adrenocorticotropic hormone, ACTH receptor) gene (MC2R) encodes a protein involved in regulation of adrenal cortisol secretion, important in the physiological response to stressors. A variant of MC2R, -179A>G, results in reduction of promoter activity and less adrenal action. We hypothesize that altered stress responsivity plays a key role in the initiation of substance abuse. By direct resequencing of the promoter region and exons 1 and 2 of the MC2R gene in 272 subjects including Caucasians, Hispanics and African Americans with approximately equal numbers of former heroin addicts and normal volunteers, we identified five novel variants each with allele frequency <2%. Previously reported polymorphisms -184G>A (rs2186944), -179A>G, 833A>C (rs28926182), 952T>C (rs4797825), 1005C>T (rs4797824) and 1579T>C (rs4308014) were each in allelic frequency >/=2% in one or more ethnic groups. These polymorphisms were genotyped in 632 subjects (260 Caucasians, 168 Hispanics, 183 African Americans and 21 Asians) using TaqMan assays. Significant differences in genotype frequency among ethnic groups studied were found for each of the six variants analyzed. We found a significant association (p=0.0004, experiment-wise p=0.0072) of the allele -184A with a protective effect from heroin addiction in Hispanics. Also, in Hispanics only we found the haplotype GACT consisting of four variants (-184G>A, -179A>G, 833A>C and 1005C>T) to be significantly associated with heroin addiction (p=0.0014, experiment-wise p=0.0168), whereas another haplotype, AACT, consisting of the same variants, was associated with a protective effect from heroin addiction (p=0.0039, experiment-wise p=0.0468).     

Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations

A subtype of phenylalanine hydroxylase (PAH) deficiency that responds to cofactor (tetrahydrobiopterin, BH4) supplementation has been associated with phenylketonuria (PKU) mutations. The underlying molecular mechanism of this responsiveness is as yet unknown and requires a detailed in vitro expression analysis of the associated mutations. With this aim, we optimized the analysis of the kinetic and cofactor binding properties in recombinant human PAH and in seven mild PKU mutations, i.e., c.194T>C (p.I65T), c.204A>T (p.R68S), c.731C>T (p.P244L), c.782G>A (p.R261Q), c.926C>T (p.A309V), c.1162G>A (p.V388M), and c.1162G>A (p.Y414C) expressed in E. coli. For p.I65T, p.R68S, and p.R261Q, we could in addition study the equilibrium binding of BH4 to the tetrameric forms by isothermal titration calorimetry (ITC). All the mutations resulted in catalytic defects, and p.I65T, p.R68S, p.P244L, and most probably p.A309V, showed reduced binding affinity for BH4. The possible stabilizing effect of the cofactor was explored using a cell-free in vitro synthesis assay combined with pulse-chase methodology. BH4 prevents the degradation of the proteins of folding variants p.A309V, p.V388M, and p.Y414C, acting as a chemical chaperone. In addition, for wild-type PAH and all mild PKU mutants analyzed in this study, BH4 increases the PAH activity of the synthesized protein and protects from the rapid inactivation observed in vitro. Catalase and superoxide dismutase partially mimic this protection. All together, our results indicate that the response to BH4 substitution therapy by PKU mutations may have a multifactorial basis. Both effects of BH4 on PAH, i.e., the chemical chaperone effect preventing protein misfolding and the protection from inactivation, may be relevant mechanisms of the responsive phenotype.     

Identification of mutations in the GNPTA (MGC4170) gene coding for GlcNAc-phosphotransferase alpha/beta subunits in Korean patients with mucolipidosis type II or type IIIA

Mucolipidosis types II and III are autosomal recessive inherited diseases caused by a deficiency in the lysosomal enzyme N-acetylglucosamine-1 phosphotransferase (GlcNAc-phosphotransferase), which adds phosphate to function as a recognition marker for the uptake and transport of lysosomal enzymes. We investigated mutations in the GNPTA (MGC4170) gene, which codes for the alpha/beta subunits of phosphotransferase, and in the GNPTAG gene, which codes for its gamma subunits in five Korean patients with mucolipidosis type II or IIIA. We identified seven mutations in the GNPTA gene, but none in GNPTAG. The mutations in type II patients included p.Q104X (c.310C>T), p.R1189X (c.3565C>T), p.S1058X (c.3173C>G), p.W894X (c.2681G>A), and p.H1158fsX15 (c.3474_3475delTA), all of which are nonsense or frameshift mutations. However, a splicing site mutation, IVS13+1G>A (c.2715+1G>A) was detected along with a nonsense or a frameshift mutation (p.R1189X or p.E858fsX3 (c.2574_2575delGA)) in two mucolipidosis type IIIA patients. This report shows that mutations in the GNPTA gene coding for the alpha/beta subunits of phosphotransferase, and not mutations in the GNPTAG gene, account for most of the genetic mutations found in Korean patients with mucolipidosis type II or IIIA.     

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a trial of enzyme replacement therapy

Mucopolysaccharidosis type VI (MPS VI), or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Seven MPS VI patients were chosen for the initial clinical trial of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Nine substitutions (c.289C>T [p.Q97X], c.629A>G [p.Y210C], c.707T>C [p.L236P], c.936G>T [p.W312C], c.944G>A [p.R315Q], c.962T>C [p.L321P], c.979C>T [p.R327X], c.1151G>A [p.S384N], and c.1450A>G [p.R484G]), two deletions (c.356_358delTAC [p.Y86del] and c.427delG), and one intronic mutation (c.1336+2T>G) were identified. A total of 7 out of the 12 mutations identified were novel (p.Y86del, p.Q97X, p.W312C, p.R327X, c.427delG, p.R484G, and c.1336+2T>G). Two of these novel mutations (p.Y86del and p.W312C) were expressed in Chinese hamster ovary cells and analyzed for residual ARSB activity and mutant ARSB protein. The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified among the patients, along with the silent mutation c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient, and, together with genotype information, were used to predict the expected clinical severity of each MPS VI patient.     

New founder germline mutations of CDKN2A in melanoma-prone families and multiple primary melanoma development in a patient receiving levodopa treatment

Germline mutations in the CDKN2A gene have been shown to predispose individuals to cutaneous malignant melanoma. Here, we describe three melanoma-prone families and one isolated patient affected by multiple melanoma who carried a tandem germline mutation of CDKN2A at the nucleotide level, [c.339G>C;c.340C>T], [p.Leu113Leu;p.Pro114Ser]. We also describe three other melanoma-prone families that carried a missense germline CDKN2A mutation, c.167G>T, p.Ser56Ile. All these families and patients resided in southeast France. We analyzed six 9p21 markers where the CDKN2A gene is located and found that carrier haplotypes for both mutations were consistent with two respective common founder ancestors. In one family, we identified two fourth-degree relatives homozygous for the Ser56Ile mutation, indicating a possible consanguinity. Furthermore, we observed that a carrier of the founder CDKN2A [p.Leu113Leu;p.Pro114Ser] mutation as well as two MC1R moderate-risk variants, [p.Arg151Cys(+)p.Arg163Gln] developed 22 primary melanomas in the three years that followed initiation of levodopa therapy for Parkinson's disease. This observation suggests that there is a need for reconsideration of the hypothesis that levodopa may play a role in melanoma development, at least when in the context of a high-risk genetic background.     

Family history of von Hippel-Lindau disease was uncommon in Chinese patients: suggesting the higher frequency of de novo mutations in VHL gene in these patients

Von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome caused by germline mutations in VHL tumor suppressor gene. It is characterized by hemangioblastoma in central nervous system and retina, renal cell carcinoma or cyst, pheochromocytoma, pancreatic cyst and tumor, endolymphatic-sac tumor, and papillary cystadenoma in epididymis and broad ligament. Here, we used PCR-direct sequencing and universal primer quantitative fluorescent multiplex PCR (UPQFM-PCR) to detect VHL mutations in 16 patients clinically diagnosed with VHL disease. PCR-direct sequencing detected 12 germline mutations (75%, 12/16), in which a novel mutation of c.451A>T/p.Ile151Phe found in one proband had not been reported previously. UPQFM-PCR found two large deletions (12.5%, 2/16). The two remaining patients carried non-typical disease-causing mutations, including one silent mutation (c.481C>A/p.Arg161Arg) and one mutation in 3'-UTR (c.642+70C>A). Remarkably, 56.3% (9/16) probands did not have family history of VHL disease, suggesting the higher frequency of de novo mutations in Chinese patients. We also summarized Chinese VHL disease patients with VHL mutation findings published in the literature to provide information about the spectrum of VHL mutations in Chinese VHL disease patients.     

Molecular characterization of familial hypercholesterolemia in Spain: identification of 39 novel and 77 recurrent mutations in LDLR

Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), an autosomal dominant inherited disorder associated with an increased risk of premature atherosclerosis. The aim of this study was to characterize the LDLR mutations in a group of 476 apparently non-related Spanish FH patients. The promoter region and the 18 exons with their flanking intron sequences of the LDLR gene were screened by PCR-SSCP analysis and DNA sequencing. In addition, we tested for the presence of the mutation p.R3500Q in the gene coding for apolipoprotein B-100 (apo B-100). We found 77 mutations previously described, and 39 novel mutations affecting the LDLR gene: 8 missense, 5 nonsense, 15 frameshift, 5 splicing, 4 in frame, one nucleotide change in the non-coding sequence of exon 1, and one silent variant. We have identified al least one of these LDLR gene mutations in 329 subjects (69%). Four patients were homozygous, 4 patients were compound heterozygous, 48 patients were found to carry two different sequence variants in the same allele and 4 patients carried three different sequence variants in the same allele. Additionally, 4 subjects were carriers of the p.R3500Q mutation in the apo B gene. All of these findings indicate that there is a broad spectrum of mutations and sequence variants in the LDLR gene causing FH in Spain.