RHC Alleles Research Articles

Sequence diversity of the Rh blood group system in Basques.

Basques show specific cultural, demographic, and genetic characteristics that have placed them as an isolated and unique population within Europe, such as their non-Indo-European language, Euskara. They have historically lived along the Western Pyrenees, between Spain and France, in one of the most important European glacial refugia during the Last Glacial Maximum. The most striking genetic characteristic is their highest frequency of the RhD blood group negative allele, a variant related to the hemolytic disease of the newborn. Both demographic and adaptive processes have been suggested as possible causes of the high frequency of RhD negative in Basques, but neither hypothesis has been clearly demonstrated. While previous studies on the Rh system in Basques have been mostly focused on serological and genotyping diversity, in this work we analyze genotyping and next generation sequencing data in order to provide a general framework of the genetic scenario of the system in Basques. In particular, we genotyped the most relevant variants of the system (D/d, E/e, and C/c), and sequenced three ~6 kb flanking regions surrounding the Rh genes in Basques and also in other populations for comparison. Our results are in agreement with previous studies, with Basques presenting the highest frequency of the RHD deletion (47.2%). Haplotype analyses of D/d, E/e, and C/c variants confirmed an association between the RhC allele, previously suggested to be under positive selection, and the RhD positive variant in non-sub-Saharan populations, including Basques. We also found extreme differentiation for the C/c variant when comparing sub-Saharan to non-sub-Saharan populations.

Abstract 5588: Nevus count and dermoscopic pattern associated with MC1R RHC-variant alleles in a case-control study of melanoma

Abstract The total number of acquired melanocytic nevi (TNC) is the strongest known risk factor for cutaneous malignant melanoma (CMM). An increased risk of CMM is also seen with individuals with red hair, fair skin and poor tanning ability (RHC phenotype); and who carry variant alleles of the MC1R gene designated as R. High freckling scores but reduced nevus counts have been reported in individuals with the RHC phenotype. The relationship between these reciprocal phenotypes of lower vs higher nevus counts, both with increased risk of CMM, is complex and supports hypotheses of different pathways to melanoma formation. We have reported on a case-control study of nevi in volunteers from the South-east Queensland area combining an assessment of pigmentation characteristics, nevus phenotype including dermoscopic nevus subtypes with genotypic comparisons and melanoma risk for 573 individuals. This sample set has been further examined to delineate the nevus characteristics of 46 individuals who are homozygous MC1R RHC allele carriers R/R. We wished to determine if there is any commonality in the pigmentation or melanocytic nevus phenotype including number, size, distribution, profile and colour that may distinguish these patients. Body mass index (BMI), body surface area (BSA), TNC and MC1R genotype were analysed and found that participants with personal melanoma history had higher TNC (P<0.005). We found no association between MC1R genotype and BMI but there exists a weak association between BSA and higher TNC. Within the MC1R R/R cohort, melanoma patients are more likely to have high TNC than controls (P<0.01). The signature dermoscopic pattern for the individuals in the MC1R R/R group is at increased odds of being a non-specific pattern (P<0.01). A significant difference in MC1R R/R TNC dependent on melanoma history exists and there is a predisposition towards a non-specific signature nevus pattern. Citation Format: Phil McClenahan, Kasturee Jagirdar, Katie Lee, Elizabeth McEniery, Sam Beh, Brian Burke, David L. Duffy, H. Peter Soyer, Richard A. Sturm. Nevus count and dermoscopic pattern associated with MC1R RHC-variant alleles in a case-control study of melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5588. doi:10.1158/1538-7445.AM2015-5588

Partial C antigen in sickle cell disease patients: clinical relevance and prevention of alloimmunization

Partial Rh antigens have been widely described in black individuals. Carriers are prone to immunization when exposed to the normal antigens. In sickle cell disease (SCD), patient alloimmunization is a major cause of transfusion failure. The potential of individuals with partial C antigen to make anti-C has not been investigated. We sought partial C status and anti-C production in a cohort of SCD patients with the C+ phenotype, to determine whether exposure to normal C antigen should be avoided. We constituted a cohort of 177 randomly selected SCD patients expressing C antigen. We screened for (C)ce(s) and R(N) haplotypes, presumably associated with partial C antigen in Afro-Caribbeans, and we recorded the number of transfused C+ red blood cell (RBC) units, immunization status, and extended phenotype. Forty-nine patients carried abnormal C antigen, deduced from the presence of (C)ce(s) and/or R(N), not compensated by a normal RHC allele in trans. Among patients with partial C phenotype exposed repeatedly to C+ RBCs, 30% produced anti-C. Two patients experienced hemolysis. In our hospital, with 22% of SCD patients expressing C, prevention of anti-C immunization for all individuals with partial C antigen would require a 7% increase in the use of C- RBC units. These RBCs are already in short supply for SCD patients who are C-. This study demonstrates the need to detect partial C within C+ SCD patients and to prevent immunization. A larger number of Afro-Caribbeans donors is needed to provide these patients with C- RBCs.

Detection of 37bp insert in RHD allele and 109bp insert in RHC allele

Objective To investigate polymorphism of 37 bp insert in RHD allele and 109 bp insert in RHC allele.Methods 37 bp insert of RHD allele and 109 bp insert of RHC allele from 82 cases of Hans with RhD(+),as well as from 206 cases of Hans and 70 cases of Uigurs with RhD(-)were detected with PCR-SSP.Results 37 bp insert of all samples above can't be detected,namely there don't exist RHD ψ in all the samples.of 76 cases of Hans from 82 cases with RhD(+)carring RhC antigen,3 cases(3.95%)lose 109 bp insert of RHC.Of 75 cases of Hans from 206 cases with RhD(-)carring RhC antigen,4 cases(5.33%)inexist 109 bp insert of RHC.There don't exist RhCC phenotype in 70 cases of Uigurs with RhD(-),and of which 3 cases with RhCc phenotype 109bp insert can be detected.109 bp insert can't be detected in other 204 cases with Rhcc phenotype.Conclusions 109 bp insert of RHC allele exist polymorphism in Chinese,but it can't be affirmed if there don't exist RHD ψ in Chinese. Key words: RHD allele; RHC allele; Insert; Polymorphism; PCR-Sequence specific primer

MC1R variation and melanoma risk in the Swedish population in relation to clinical and pathological parameters

The genetic background of cutaneous malignant melanoma (CMM) includes both germ line aberrations in high-penetrance genes, like CDKN2A, and allelic variation in low-penetrance genes like the melanocortin-1 receptor gene, MC1R. Red-hair colour associated MC1R alleles (RHC) have been associated with red hair, fair skin and risk of CMM. We investigated MC1R and CDKN2A variation in relation to phenotype, clinical factors and CMM risk in the Swedish population. The study cohort consisted of sporadic primary melanoma patients, familial melanoma patients and a control group. An allele-dose dependent increase in melanoma risk for carriers of variant MC1R alleles (after adjusting for phenotype), with an elevated risk among familial CMM patients, was observed. This elevated risk was found to be significantly associated with an increased frequency of dysplastic nevi (DN) among familial patients compared to sporadic patients. MC1R variation was found to be less frequent among acral lentiginous melanomas (ALM) and dependent on tumour localisation. No association was found between CDKN2A gene variants and general melanoma risk. Two new variants in the POMC gene were identified in red haired individuals without RHC alleles.

The Dc(G48)es haplotype is frequent among the Dce haplotypes within a white population

The absence of hybrid Rhesus boxes denotes an RHD homozygous status and helps to detect the presence of Dce haplotypes instead of dce. RHCE exon 1 C48, characteristic of RHC alleles, and RHCE exon 5 G733, responsible for VS antigenicity, have been noted in many RHce alleles but it was not clearly established whether they occurred in the same allele and/or cosegregate together with RHD. Samples from 148 white trios (father, mother, and child) were studied. Rh phenotype was performed by hemagglutination. Hybrid Rhesus box, RHCE exon 1 G48C, RHCE exon 5 C733G, and RHC intron 2 polymorphisms were analyzed by polymerase chain reaction. Haplotypes were determined considering serologic, molecular, and segregation data. RHCE exon 1 C48 and RHCE exon 5 G733 were present in RHce alleles that cosegregated with RHD forming Dce haplotypes. Both transversions were not frequently found in the same RHce allele. Of the 33 Dce haplotypes, 16 (48.5%) had a C at position 48 [Dc(C48)e], 11 (33.3%) had a G at position 48 with a G at position 733 [Dc(G48)e(s)], 5 (15.2%) had a G at position 48 [Dc(G48)e], and 1 (3.0%) had a C at position 48 with a G at position 733 [Dc(C48)e(s)]. The results show four molecular backgrounds for the Dce haplotype and reflect the contribution of African alleles to the genetic pool of the population under study. The molecular characterization of Dce and its frequency distribution may develop a better understanding of the phylogeny of Rh haplotypes.

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells

α-Melanocyte-stimulating hormone (α-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected HEK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to α-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent.

Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.

Skin colour and skin cancer - MC1R, the genetic link.

Pigmentary traits such as red hair, fair skin, lack of tanning ability and propensity to freckle (the RHC phenotype) have been identified as genetic risk factors for both melanoma and non-melanocytic skin cancers when combined with the environmental risk factor of high ultraviolet light exposure. The human melanocortin-1 receptor (MC1R) is a key determinant of the pigmentation process and can account in large part for the diverse range of variation in human pigmentation phenotypes and skin phototypes. The coding sequence is highly polymorphic in human populations, with several of these variant forms of the receptor now known to be associated with the RHC phenotype. We have examined variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins with defined pigmentation characteristics. Variant allele frequencies have also been determined in several case-control studies of sporadic melanoma, basal cell carcinoma and squamous cell carcinoma, and in familial melanoma kindreds collected within Australia. These studies have shown that three RHC alleles - Arg151Cys, Arg160Trp and Asp294His - were associated with increased risk in all forms of skin cancer and with penetrance and age of onset in familial melanoma in mutation carriers. There is a significant RHC allele heterozygote carrier effect on skin phototype and skin cancer risk, which indicates that variant alleles do not behave in a strictly recessive manner. Ultimately, the genetic and chemical assessment of melanin synthesis rather than skin colour will be the best indicator for skin cancer risk, and such genetic association studies combined with functional analysis of variant alleles should provide the link to understanding skin phototypes.

Rh genotyping: avoiding false-negative and false-positive results among individuals of African ancestry.

High homology, variant alleles, and silent alleles have made the development of completely reliable genotyping assays for the RHD and RHC alleles difficult. An RHD pseudogene (RHDPsi) possessing a 37-bp insertion within exon 4 is common among serologically RhD-negative individuals of African descent and generates false-positive results in previously reported RhD genotyping assays. Genotyping RhC is problematic due to exon 2 homology between RHD and RHC; however, an RHC-specific 109-bp insertion within intron 2 has been reported useful for genotyping. Primers flanking the exon 4 insertion point were used for detection of RHD and RHDPsi among a total of 231 serotyped individuals: 134 African American, 85 Caucasian, and 12 RhD serotype-negative/genotype-positive, D-sensitized women. Primers flanking the RHC-specific intron 2 insertion were used to genotype 282 serotyped individuals (128 African American, 154 Caucasian) and were compared to RHC genotyping using the exon 1 RhC-specific nt48 cytosine polymorphism. Complete correlation was observed between genotyping with the RHDPsi primer pair and serotyping among 219 individuals and 10/12 previous RHD false-positive genotyping results were resolved. RHDPsi was detected in 19% (n = 4/21) of RhD seronegative African Americans and 4.4% (n = 5/113) of RhD seropositive African Americans. When using the 109-bp intron 2 insertion for genotyping of RHC, a 23.9% (n = 11/46) false-negative rate was observed among African American RhCc serotyped heterozygotes. Utilization of the exon 1 nt48 cytosine for indirect genotyping of RHC yielded a 7.2% (n = 4/55) and 56.3% (n = 45/80) false-positive rate among Rhcc Caucasians and African Americans, respectively. We conclude that these additional reactions, though not sufficient alone, can be useful supplements to existing Rh genotyping assays.

RHC/c genotyping based on polymorphism in the promoter region of the RHCE gene

Designing of PCR tests for the RHC allele is difficult because of the high DNA sequence homology between RHC and RHD genes, which differ by only a one-nucleotide substitution at position 48 in exon 1 of the RHCE gene. We sequenced the promoter region of the RHCE gene, and compared our results with the reported sequence. Genomic DNA was prepared from blood samples collected from 656 Japanese donors. The DNA segment encompassing the promoter region and exon 1 of the RHCE gene from 30 donors was amplified by PCR and analyzed by DNA sequencing. Four nucleotide differences between RHC/c and RHD were found at positions −468, −304, −58, and −46. On the basis of the nucleotide differences at positions −468 ( RHCE vs. RHD) and −292 ( RHC vs. RHc), we then developed a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for RHC/c genotyping. Analysis of the genomic DNA from the 656 donors revealed that this method could discriminate RHC from RHc, irrespective of the RHD genotype, with only a few exceptions. The combination of our system and the intron 2-based PCR-RFLP method previously reported may prove to be more accurate than either of the methods alone, and therefore, useful and valuable for RHC/c genotyping.

Partial expression of RHc on the RHD polypeptide.

In the human Rh blood group system, c is, after D, the most immunogenic antigen. The background of a new partial c phenotype (D(c)), identified on the RBCs of two unrelated white persons, was studied. This was done by analyzing the reactivity of the RBCs from the donors with anti-c reagents, by performing sequence analysis, and by carrying out transduction studies. Serologic results suggested the existence of a new partial c phenotype. Genomic DNA and cDNA analysis revealed a normal RHCe allele, a normal RHD allele, and an RHD allele that carried two point mutations: 307T>C and 329T>C (the latter known to be associated with the DVII, Tar-positive phenotype). No normal RHc allele was found. Thus, it was most likely that c is encoded by the mutated RHD allele (phenotype DD(c)CCee). Indeed, subsequent transduction of K562 erythroleukemic cells with an RHD cDNA carrying the 307T>C point mutation (leading to S103P) resulted in the expression of c. In the human Rh system, P103 is involved in the expression of c. Moreover, c can be expressed in vivo on the D polypeptide.

RHD/CE typing by polymerase chain reaction using sequence-specific primers.

Current DNA-based Rh system typing strategies may detect the two RH genes and their prevalent alleles, but they are known to fail sometimes, when rare RH alleles (e.g., D category phenotypes) are encountered. It is almost impossible to find a single DNA-based method that can accommodate the great heterogeneity within the human Rh system. An easy-to-perform DNA-based method for the detection of the two RH genes and their alleles, including variant RHD alleles, was developed. By the use of one RHD/C-, seven RHD-, and four RHCE-specific polymerase chain reactions, all triggered to work at identical thermocycling conditions, the DNA of 77 blood donors carrying weak D and that of 200 random donors with common D phenotype was investigated. In addition, 77 selected samples of ccDee and rare Rh system phenotypes were examined. Among 77 samples of weak D, one Rh33 and six DVI categories were detected, one of which showed new RHD-specific nucleotide patterns. In DFR and CCee samples, novel variant RHD alleles were found. RHD DNA types of 200 random donors were found to be concordant with their D phenotype. For RHE and RHe genotyping, a full correlation with serologic phenotypes was found. Our method for genotyping RHC and RHc failed in some cases, because of an already published RHc allelic variation, which we have called RHc(cyt48). An estimate of the frequency of this RHc(cyt48) allele in a white population was made. The presented exon-scanning RHD/CE polymerase chain reaction using sequence-specific primers complements current DNA-based Rh system typing strategies and is superior in the detection of variant RHD alleles.

Involvement of Ser103 of the Rh polypeptides in G epitope formation

Almost all red cells that carry D and/or C antigens also express the G antigen (Rh12). A study was conducted on the molecular background of the G epitope. Two unrelated donors with the rare ccDEe, G- phenotype and one donor with the ccEe, G+ phenotype were studied. Genomic DNA and cDNA of these donors were studied with polymerase chain reaction, Southern blot, and sequence analysis, with special focus on exon 2, because it is only in this exon that there are supposed to be similarities between RHD and the RHC allele, but not between RHD and the RHc allele. In both ccDEe, G- donors, a nucleotide substitution was found in exon 2 of RHD; T307 was replaced by C307, which predicted a Ser->Pro substitution at amino acid position 103 of the D polypeptide. The ccEe, G+ donor carried the complete exon 2 of RHD. Moreover, despite the absence of all known D epitopes, this donor also carried RHD characteristics in exons 1 to 3 and exon 9 and further downstream. Ser103, encoded by exon 2 of the RH genes, is involved in G epitope formation.

PCR-based determination of Rhc and RhE status of fetuses at risk of Rhc and RhE haemolytic disease.

After anti-RhD, anti-Rhc is the most important red cell alloantibody which can cause haemolytic disease of the newborn (HDN) when the mother is Rhc-negative and the fetus Rhc-positive. We report here the development of polymerase chain reaction (PCR) assays which detect the presence of the Rhc alleles in amniotic cells by the use of allele-specific primers (ASP). It is expected that such determination will help in the management of pregnancies at risk of Rhc haemolytic disease. In the course of this study we have similarly performed PCR-ASP experiments to detect fetal RHE alleles since, in rare cases, anti-RhE can also cause HDN.

Genetic-environmental interactions in the variation of blood pressure in Tecumseh, Michigan

Emigrants and those who die were found to have higher age adjusted systolic BP than non-migrants and emigrants showed higher BP than non-migrants. For two single-locus cases, and their combined two-locus marker case, the BP differences among marker phenotypes were not homogeneous between non-migrants and those who die and between emigrants and those who die. The BP of emigrant marker phenotypes for the non-migrant-emigrant contrast varied in the same direction as the BP of marker phenotypes of those who died in the non-migrant-die contrast, but in the former contrast the marker phenotype-subsample interaction was not judged statistically significant by the available samples. This may result from the lack of specificity in the emigrant sub-sample for comparison which is presumed to have experienced high levels of stress. Our data suggests that life crises which generate stress exaggerate differences in BP between genotypes of loci which affect the level or rate of rise of BP. The average effects of both Rh-C and Kell alleles for emigrants resembled that of those who die more closely than that of non-migrants. These results are consistent with an explanation which attributes to stress encountered in life crises a major role in accounting for phenotypic differences in BP. Its effect is to raise BP in certain predisposed genotypes which are marked by dosages of the c and k alleles.