Four novel variants in MC1R in red-haired South African individuals of European descent: S83P, Y152X, A171D, P256S

Skin, hair and eye pigmentation is a polygenic multifactorial trait determined by the cumulative effects of multiple genetic variants and environmental factors. MC1R is one of the genes involved in pigmentation, and has been implicated in the red hair and pale skin trait in human Caucasoid individuals. This study was undertaken to investigate variants in the MC1R gene in Caucasoid individuals in South Africa, who are of European decent. Seven unrelated individuals were studied, all of whom were found to be either homozygous for a single mutation or compound heterozygous for two different mutations. We report four novel MC1R missense mutations: S83P, Y152X, A171D and P256S. This study supports the view that two mutations are necessary, but not necessarily sufficient, to give rise to the red hair and pale skin phenotype.     

Comprehensive evaluation of allele frequency differences of MC1R variants across populations

The melanocortin 1 receptor (MC1R), a member of the G protein-coupled receptors superfamily, mediates the response to melanocortins and is currently the best-described contributor to normal pigment variation in humans. A remarkably large number of natural polymorphisms, or variants, of the MC1R gene have been identified in different populations. Some of these variants have been associated with specific hair and skin color phenotypes, the presence of freckling, and melanoma and nonmelanoma skin cancer risk. Interestingly, some MC1R variants have been associated with skin cancer beyond their effects on pigmentation. Although the red hair color variants (RHC variants) have been associated with skin cancer risk in the Celtic population, studies in darkly-pigmented Caucasian populations have demonstrated the importance of non-RHC MC1R variants on skin cancer risk as well. We have reviewed and compared allele frequency differences of MC1R variants across geographic regions. We observed large differences in the distribution of variants across populations, with a prominent difference between lightly and darkly-pigmented individuals. Moreover, among Caucasian groups, there were seven variants (p.V60L, p.V92M, p.D84E, p.R151C, p.R160W, p.R163Q, and p.D294H) with significantly different allele frequencies. Exploring differences in allele frequencies of MC1R variants across populations with varying pigmentation and differing skin cancer risk may improve our understanding of the complex relationship between MC1R, pigmentation, and carcinogenesis.     

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).     

Mutation scanning by meltMADGE: validations using BRCA1 and LDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population

We have developed a mutation-scanning approach suitable for whole population screening for unknown mutations. The method, meltMADGE, combines thermal ramp electrophoresis with MADGE to achieve suitable cost efficiency and throughput. The sensitivity was tested in blind trials using 54 amplicons representing the BRCA1 coding region and a panel of 94 unrelated family breast cancer risk consultands previously screened in a clinical diagnostic laboratory. All 10 common polymorphisms, 15/15 previously identified disease-causing mutations, and three previously untested single base changes were identified. Assays of LDLR exons 3 and 8 were validated in 460 familial hypercholesteremics and detected 8/9 known variants. We then applied the exon 3 assay in several DNA banks representing approximately 8000 subjects with known cholesterol values and applied both assays in one DNA bank (n = 3600). In exon 3 we identified one previously reported moderate mutation, P84S (n = 1), also associated with moderate hypercholesteremia in this subject; an unreported silent variant, N76N (n = 1); and known severe hypercholesteremia splice mutation 313+1G-->A (n = 2). Around exon 8 we identified a paucimorphism (n = 35) at the splice site 1061-8T-->C (known to be in complete linkage disequilibrium with T705I) and unreported sequence variants 1186+11G-->A (n = 1) and D335N G-->A (n = 1). The cholesterol value for D335N was on the 96.2 percentile and for T705I, 2/35 carriers were above the 99th percentile. Thus, variants with predicted severe, moderate, and no effect were identified at the population level. In contrast with case collections, CpG mutations predominated. MeltMADGE will enable definition of the full population spectrum of rare, paucimorphic, severe, moderate (forme fruste), and silent mutations and effects.     

儿童青少年肥胖与黑皮素4受体基因多态性的研究进展

肥胖是环境和基因共同作用导致的疾病.黑皮素4受体(melanocortin-4 Feceptor gene,MC4R)是中枢黑皮素系统参与调节摄食行为和能量代谢的枢纽.近年来大量研究发现MC4R基因突变与肥胖发生有关,在儿童青少年肥胖的发生过程也发挥重要作用.本文综述近年来研究较多的MC4R基因多态性与儿童青少年肥胖的相关性.许多人群研究表明MC4R基因多态性与肥胖发生相关,某些作用可能具有种族差异性.MC4R基因多态性可能与其他基因或环境存在交互作用.     

Melanocortin 4 receptor antagonists attenuates morphine antinociceptive tolerance,astroglial activation and cytokines expression in the spinal cord of rat

Chronic use of morphine is accompanied by the development of morphine tolerance, which is one of the major problems associated with opiate treatment. Experimental evidence indicates that melanocortin 4 receptor (MC4R) is involved in development of morphine tolerance. Therefore, we investigated the influence of repeated intrathecal injection of a MC4R antagonist (HS014) on the development of morphine tolerance as measured by hot-plate test. It was also examined whether a single i.t. HS014 administration could counteract the loss of analgesic potency of morphine in morphine tolerant rats. We examined also the influence of i.t. HS014 administration on astrocytes activation and cytokines expression in the spinal cord of rat during morphine tolerance. Morphine treatment (10 mg/kg, i.p. twice daily) over 5 days induced tolerance as reflected by a significant reduction of withdrawal latency from 29.67 ± 1.81 s to 8.67 ± 1.70 s in the hot-plate test. Repeated coadministration of HS014 and morphine, significantly prevented the development of morphine tolerance. A single administration of an MC4R antagonist restored morphine analgesic potency in morphine tolerant rats. Using immunohistochemical staining, we demonstrated the administration of MC4R during the induction of morphine tolerance inhibited the activation of astrocytes; reduced the expression of proinflammatory cytokines interleukin-1β, IL-6, and tumor necrosis factor-α; upregulated the expression of anti-inflammatory cytokines IL-10 at the L5 lumbar spinal cord. These results suggest that MC4R may be involved in the mechanisms of morphine tolerance and antagonists of this receptor may be a possible new target in the search for strategies preventing the development of morphine tolerance.     

MC4R expression in pedunculopontine nucleus involved in the modulation of midbrain dopamine system

Background and objective: Separate studies have implicated the pedunculopontine tegmental nucleus (PPTg) in processing aversive stimuli to dopamine systems, and melanocortin-4 receptor (MC4R) are broadly expressed by the neurons in the PPTg, but the exact neurosubstrate underlying the regulation of dopamine systems by the central melanocortin pathway is poorly understood. Methods: In this study, the PPTg of 6 adult mice expressing green fluorescent protein (GFP) under the control of the MC4R promoter was detected by fluorescence immunohistochemistry. Results: A large number of GFP-positive neurons in the dissipated parts of PPTg (dpPPTg) were found, and approximately 50% of MC4R-GFP- positive neurons in the dpPPTg coexpressed tyrosine hydroxylase, a marker of dopamine neurons, indicating that they were dopaminergic. Conclusions: Our findings support the hypothesis that MC4R signaling in the dpPPTg may involve in the modulation of midbrain dopamine systems.     

Assessment of multiple displacement amplification for polymorphism discovery and haplotype determination at a highly polymorphic locus, MC1R

The identification of common genetic variants such as single nucleotide polymorphisms (SNPs) in the human genome has become central in human population genetics and evolution studies, as well as in the study of the genetic basis of complex traits and diseases. Crucial for the accurate identification of genetic variants is the availability of high quality genomic DNA (gDNA). Since popular sources of gDNA (buccal cells, lymphocytes, hair bulb) often do not yield sufficient quantities of DNA for molecular genetic applications, whole genome amplification methods have recently been introduced to generate a renewable source of double-stranded linear DNA. Here, we assess the fidelity of one method, multiple displacement amplification (MDA), which utilizes bacteriophage Phi29 DNA polymerase to generate amplified DNA from an original source of gDNA, in a representative SNP discovery and genetic association study at the melanocortin 1 receptor (MC1R) locus, a highly polymorphic gene in humans involved in skin and hair pigmentation. We observed that MDA has high fidelity for novel SNP discovery and can be a valuable tool in generating a potentially indefinite source of DNA. However, we observed an allele amplification bias that causes genotype miscalls at heterozygous sites. At loci with multiple polymorphic sites in linkage disequilibrium, such as at MC1R, this bias can create a significant number of heterozygote genotype errors that subsequently misrepresents haplotypes.     

Novel and recurrent spastin mutations in a large series of SPG4 Italian families

Background

Hereditary spastic paraplegias (HSP) are heterogeneous neurodegenerative disorders, genetically classified according to the identified disease gene or locus. Clinically, HSP are distinguished in pure and complicated forms. Mutations in the spastin gene (SPAST) are responsible for SPG4 and account approximately for 50% of the dominantly inherited paraplegias with a pure HSP phenotype.

Methods

Molecular screening of the SPAST gene allowed the identification of 31 Italian mutation carriers, from 19 unrelated families. Genetic testing was performed by direct sequencing and multiplex ligation-dependent probe amplification. Subjects carrying SPAST mutations were retrospectively evaluated for clinical phenotype and disability score assessment.

Results

We found 12 recurrent mutations, and 7 novel SPAST mutations. Twenty-eight patients exhibited a pure spastic paraplegia phenotype, while 3 subjects were asymptomatic mutation carriers. Four patients were sporadic cases. Age at onset ranged from 10 to 61 years. Disability score increased with age at examination and disease duration. Patients with onset >38 years presented a faster disease progression, and a higher disability functional index, than the patients with earlier onset (p < 0.04).

Conclusions

Our study enlarges the number of pathogenic SPAST mutations, and confirms the association with a pure spastic paraplegia phenotype. Age at onset was highly variable and correlates with the rate of disease progression. Future longitudinal clinical studies are needed to confirm these observations.     

Impact of Single Nucleotide Polymorphism in IL-4, IL-4R Genes and Systemic Concentration of IL-4 on the Incidence of Glioma in Iraqi Patients

Glioma is the most common and believed to be one of the most aggressive tumors of the central nervous system (CNS) in humans. Very little information is available on the etiology and pathogenesis of these tumors to date. A significant gap remains in our current understanding of the molecular pathways involved in the genesis, progression and clinical behavior of these tumors. Recently, several single nucleotide polymorphisms (SNPs) have been identified in cytokine gene sequences, particularly within the promoter region of these genes, and have been shown to be associated with the development of different types of brain tumors. The present study investigates the association of C-33T SNP in the interleukin-4 (IL-4) gene with systemic IL-4 level and the S503P SNP in the IL-4R gene with the incidence of glioma.Blood samples were collected from 100 histologically confirmed adult patients with glioma, and 30 apparently healthy individuals from the same area. DNA was extracted from each blood sample, and the IL-4 and IL-4R genes were amplified using polymerase chain reaction (PCR) with gene-specific primers. Systemic IL-4 concentration was assessed in serum samples from each participant by enzyme-linked immunosorbent assay (ELISA). We observed a negative association between the homozygous genotype (CC) of the SNP C-33T of the IL-4 gene with the incidence of glioma (OR=0.19, 95% CI=0.035-1.02), while the T allele of the SNP demonstrated a significant protective association against glioma. Similarly, the heterozygous (CT) and homozygous mutant (CC) of the SNP S503P of the IL-4R gene demonstrated a significant association with glioma development (OR=0.405, 95% CI=0.17-0.969 and OR=0.147, 95% CI=0.036-0.6 respectively), while the C allele exhibited a highly significant association with protection from glioma formation.These findings suggest that the T allele of the SNP C-33T in the IL-4 gene and the C allele of the SNP S503P in IL-4R may have a protective role against glioma development.     

CFH基因R1210C突变与中国人老年黄斑变性的相关性研究

目的 补体因子H(complement factor H,CFH)基因R1210C突变与高加索人老年黄斑变性(age-related macular degeneration,AMD)显著相关.我们通过对中国大陆汉族人群进行病例-对照研究,评估CFH基因R1210C突变与中国人老年黄斑变性的相关性.方法 收集258例中国汉族人湿性AMD患者作为病例组,同时收集来自同一地区426名正常中国汉族人作为对照,应用直接测序法检测CFH基因中R1210C的突变情况,并比较病例组和对照组的差异.结果 258例中国人AMD患者和426名正常中国人的CFH基因中未发现R1210C突变.结论 中国人老年黄斑变性与CFH基因R1210C突变可能无关.     

Combined effects of MC4R and FTO common genetic variants on obesity in European general populations

Genome-wide association scans recently identified common polymorphisms, in intron 1 of FTO and 188 kb downstream MC4R, that modulate body mass index (BMI) and associate with increased risk of obesity. Although their individual contribution to obesity phenotype is modest, their combined effects and their interactions with environmental factors remained to be evaluated in large general populations from birth to adulthood. In the present study, we analyzed independent and combined effects of the FTO rs1421085 and MC4R rs17782313 risk alleles on BMI, fat mass, prevalence and incidence of obesity and subsequent type 2 diabetes (T2D) as well as their interactions with physical activity levels and gender in two European prospective population-based cohorts of 4,762 Finnish adolescents (NFBC 1986) and 3,167 French adults (D.E.S.I.R.). Compared to participants carrying neither FTO nor MC4R risk allele (20–24% of the populations), subjects with three or four risk alleles (7–10% of the populations) had a 3-fold increased susceptibility of developing obesity during childhood. In adults, their combined effects were more modest (~1.8-fold increased risk) and associated with a 1.27% increase in fat mass (P?=?0.001). Prospectively, we demonstrated that each FTO and MC4R risk allele increased obesity and T2D incidences by 24% (P?=?0.02) and 21% (P?=?0.02), respectively. However, the effect on T2D disappeared after adjustment for BMI. The Z-BMI and ponderal index of newborns homozygous for the rs1421085 C allele were 0.1 units (P?=?0.02) and 0.27 g/cm³ (P?=?0.005) higher, respectively, than in those without FTO risk allele. The MC4R rs17782313 C allele was more associated with obesity and fat mass deposition in males than in females (P?=?0.003 and P?=?0.03, respectively) and low physical activity accentuated the effect of the FTO polymorphism on BMI increase and obesity prevalence (P?=?0.008 and P?=?0.01, respectively). In European general populations, the combined effects of common polymorphisms in FTO and MC4R are therefore additive, predictive of obesity and T2D, and may be influenced by interactions with physical activity levels and gender, respectively.     

EIF4G1 R1205H and VPS35 D620N mutations are rare in Parkinson's disease from South Africa

The R1205H mutation in the eukaryotic translation initiation factor 4G1 (EIF4G1) gene and the D620N mutation in the vacuolar protein sorting 35 (VPS35) gene were recently found in patients with autosomal dominant or sporadic forms of Parkinson's disease (PD). In the present study, 418 South African PD patients and 528 control subjects of diverse ethnicities were screened using the KASP (Kompetitive Allele Specific PCR) genotyping assay. The mutations were not found in our study, suggesting that they are not a common cause of PD in South African patients. Further studies are needed on the frequency of these 2 mutations in other sub-Saharan African populations.     

哮喘患者外周血淋巴细胞IL-4Rα链的表达及其意义

为了解过敏性哮喘患者外周血淋巴细胞IL 4受体 (IL 4R )的变化规律及其影响因素。本工作将研究对象分为正常对照组 (n =12 )、过敏性哮喘患者缓解期组 (n =14 )和急性发作期组 (n =16 ) ,分离外周血单个核细胞 (PBMC ) ,取 1× 10 6个细胞加入或不加入 10ng/mlIL 3、IL 5及GM CSF培育 2 4h ,利用流式细胞仪测量PBMC中淋巴细胞亚群膜IL 4Rα的表达。结果显示 :哮喘患者缓解期B细胞IL 4Rα表达阳性率明显低于正常人及哮喘急性发作期 ,而哮喘患者急性发作期CD3+ 细胞、CD4 + 细胞和CD8+ 细胞IL 4Rα表达阳性率则较哮喘缓解期和正常人明显升高 ,差异有显著性 (P均 <0 0 5 )。经IL 3、IL 5及GM CSF刺激后 ,正常组仅见B细胞携带IL 4Rα的比例显著下降 (P <0 0 5 ) ,T细胞携带IL 4Rα的比例则无变化 ;而哮喘患者缓解期T、Th细胞携带IL 4Rα的比例有明显上升 (P均 <0 0 5 ) ,B细胞携带IL 4Rα的比例则无变化 ;哮喘患者急性发作期T细胞和B细胞携带IL 4Rα的比例均无变化。这提示CD4 + 细胞携带IL 4R比例的升高可能在过敏性哮喘发病过程中起重要作用 ,而IL 3、IL 5及GM CSF则有助于Th细胞携带IL 4R比例的上调     

Genetical,clinical, and functional analysis of a large international cohort of patients with autosomal recessive congenital ichthyosis due to mutations in NIPAL4

Autosomal recessive congenital ichthyosis (ARCI) belongs to a heterogeneous group of disorders of keratinization. To date, 10 genes have been identified to be causative for ARCI. NIPAL4 (Nipa‐Like Domain‐Containing 4) is the second most commonly mutated gene in ARCI. In this study, we present a large cohort of 101 families affected with ARCI carrying mutations in NIPAL4. We identified 16 novel mutations and increase the total number of pathogenic mutations in NIPAL4 to 34. Ultrastructural analysis of biopsies from six patients showed morphological abnormalities consistent with an ARCI EM type III. One patient with a homozygous splice site mutation, which leads to a loss of NIPAL4 mRNA, showed additional ultrastructural aberrations together with a more severe clinical phenotype. Our study gives insights into the frequency of mutations, a potential hot spot for mutations, and genotype–phenotype correlations.     

Modulation of sensory inhibition in the ventrobasal thalamus via activation of group II metabotropic glutamate receptors by 2R,4R-aminopyrrolidine-2,4-dicarboxylate

Recordings were made from single neurones responsive to somatosensory input in the ventrobasal thalamus of the anaesthetised rat. GABAergic afferent inhibition arising from the thalamic reticular nucleus was evoked using a condition-test vibrissal stimulation paradigm. Local iontophoretic application of the group II metabotropic glutamate receptor (mGluR) agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) in the vicinity of the recorded neurones produced a reduction of the afferent inhibition (from 78±3.0% to 25±5.3%), presumably via a presynaptic mechanism. This effect could be antagonised by LY307452, a known group II mGluR antagonist. In contrast, two selective group I mGluR agonists, (S)-3,5-dihydroxyphenylglycine (DHPG) and trans-azetidine-2,4-dicarboxylate (tADA), were without effect on the GABAergic inhibition. These data show that group II but not group I mGluRs can have a significant role in the modulation of GABAergic afferent inhibition in the ventrobasal thalamus. This could be of importance in the control of sensory discriminative processes and functions of sleep, arousal and seizure generation. Received: 6 January 1998 / Accepted: 23 February 1998     

Membrane CD45R isoform exchange on CD4 T cells is rapid,frequent and dynamic in vivo

CD4 T cells bearing high (240–190 kDa) and low (180 kDa) molecular mass isoforms of the leukocyte common antigen CD45 define functionally distinct subsets which have been equated with naive and memory T cells. In the rat, CD4 T cells expressing a high molecular mass isoform [identified by monoclonal antibody MRC-OX22 (anti-CD45RC)] exchange this for the 180 kDa molecule (CD45RC^?) when stimulated by antigen. Here we show, by transferring mature allotype-marked CD45RC^? CD4 T cells (depleted of immature Thy-1⁺ CD45RC^? recent thymic emigrants) into normal euthymic recipients, that many T cells re-express the high molecular mass isoform in less than 6 h. By 24 h, 30–60% of CD45RC^? CD4 T cells became CD45RC⁺; within a week the entire cohort appeared to exchange the low for the high molecular mass isoform. Isoform exchange was dynamic and many CD4 T cells returned once again to the CD45RC^? state. CD45RC^? CD4 T cells declined in number more rapidly than the CD45RC⁺ subset after transfer. The results suggest that CD45R isoforms distinguish between resting T cells (CD45RC⁺) and those which have encountered antigen in the recent past. CD45R isoforms would appear to be unsuitable markers of naive and memory T cells.