Biomedical subjects

R A Sturm

Publications and source records attributed to R A Sturm.

At least 19 recordsLinked to original sources

PubMed2006

A polymorphism in the agouti signalling protein (ASIP) is associated with decreased levels of mRNA.

To date, a role for agouti signalling protein (ASIP) in human pigmentation has not been well characterized. It is known that agouti plays a pivotal role in the pigment switch from the dark eumelanin to the light pheomelanin in the mouse. However, because humans do not have an agouti banded hair pattern, its role in human pigmentation has been questioned. We previously identified a single polymorphism in the 3'-untranslated region (UTR) of ASIP that was found at a higher frequency in African-Americans compared with other population groups. To compare allele frequencies between European-Australians and indigenous Australians, the g.8818A --> G polymorphism was genotyped. Significant differences were seen in allele frequencies between these groups (P < 0.0001) with carriage of the G allele highest in Australian Aborigines. In the Caucasian sample set a strong association was observed between the G allele and dark hair colour (P = 0.004) (odds ratio 4.6; 95% CI 1.4-15.27). The functional consequences of this polymorphism are not known but it was postulated that it might result in message instability and premature degradation of the transcript. To test this hypothesis, ASIP mRNA levels were quantified in melanocytes carrying the variant and non-variant alleles. Using quantitative real-time polymerase chain reaction the mean ASIP mRNA ratio of the AA genotype to the AG genotype was 12 (P < 0.05). This study suggests that the 3'-UTR polymorphism results in decreased levels of ASIP and therefore less pheomelanin production.

Agouti Signaling Protein↗

PubMed2003

Genetic association and cellular function of MC1R variant alleles in human pigmentation.

We have examined MC1R variant allele frequencies in the general population of South East Queensland and in a collection of adolescent dizygotic and monozygotic twins and family members to define statistical associations with hair and skin color, freckling, and mole count. Results of these studies are consistent with a linear recessive allelic model with multiplicative penetrance in the inheritance of red hair. Four alleles, D84E, R151C, R160W, and D294H, are strongly associated with red hair and fair skin with multinomial regression analysis showing odds ratios of 63, 118, 50, and 94, respectively. An additional three low-penetrance alleles V60L, V92M, and R163Q have odds ratios 6, 5, and 2 relative to the wild-type allele. To address the cellular effects of MC1R variant alleles in signal transduction, we expressed these receptors in permanently transfected HEK293 cells. Measurement of receptor activity via induction of a cAMP-responsive luciferase reporter gene found that the R151C and R160W receptors were active in the presence of NDP-MSH ligand, but at much reduced levels compared with that seen with the wild-type receptor. The ability to stimulate phosphorylation of the cAMP response element binding protein (CREB) transcription factor was also apparent in all stimulated MC1R variant allele-expressing HEK293 cell extracts as assessed by immunoblotting. In contrast, human melanoma cell lines showed wide variation in the their ability to undergo cAMP-mediated CREB phosphorylation. Culture of human melanocytes of known MC1R genotype may provide the best experimental approach to examine the functional consequences for each MC1R variant allele. With this objective, we have established more than 300 melanocyte cell strains of defined MC1R genotype.

Alleles↗

PubMed2002

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.

Alleles↗

PubMed2001

The human melanocortin-1 receptor locus: analysis of transcription unit, locus polymorphism and haplotype evolution.

The complete sequence of the MC1R locus has been assembled, the coding region of the gene is intronless and placed within a 12 kb region flanked by the NULP1 and TUBB4 genes. The immediate promoter region has an E-box site with homology to the M-box consensus known to bind the microphthalmia transcription factor (MITF); however, promoter deletion analysis and transactivation studies have failed to show activation through this element by MITF. Polymorphism within the coding region, immediate 5' promoter region and a variable number tandem repeat (VNTR) minisatellite within the locus have been examined in a collection of Caucasian families and African individuals. Haplotype analysis shows linkage disequilibrium between the VNTR and MC1R coding region red hair variant alleles which can be used to estimate the age of these missense changes. Assuming a mean VNTR mutation rate of 1% and a star phylogeny, we estimate the Arg151Cys variant arose 7500 years before the present day, suggesting these variants may have arisen in the Caucasian population more recently than previously thought.

Amino Acid Sequence↗

PubMed2001

Human pigmentation genes: identification, structure and consequences of polymorphic variation.

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones alpha-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation.

Genes↗

PubMed2001

MC1R genotype modifies risk of melanoma in families segregating CDKN2A mutations.

Mutations in the exons of the cyclin-dependent kinase inhibitor gene CDKN2A are melanoma-predisposition alleles which have high penetrance, although they have low population frequencies. In contrast, variants of the melanocortin-1 receptor gene, MC1R, confer much lower melanoma risk but are common in European populations. Fifteen Australian CDKN2A mutation-carrying melanoma pedigrees were assessed for MC1R genotype, to test for possible modifier effects on melanoma risk. A CDKN2A mutation in the presence of a homozygous consensus MC1R genotype had a raw penetrance of 50%, with a mean age at onset of 58.1 years. When an MC1R variant allele was also present, the raw penetrance of the CDKN2A mutation increased to 84%, with a mean age at onset of 37.8 years (P=.01). The presence of a CDKN2A mutation gave a hazard ratio of 13.35, and the hazard ratio of 3.72 for MC1R variant alleles was also significant. The impact of MC1R variants on risk of melanoma was mediated largely through the action of three common alleles, Arg151Cys, Arg160Trp, and Asp294His, that have previously been associated with red hair, fair skin, and skin sensitivity to ultraviolet light.

Alleles↗

PubMed2001

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma.

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell carcinoma and squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio = 3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information wained through observation of pigmentation phenotype.

Alleles↗

PubMed2000

Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors.

The class III POU gene brn-2, encoding the Brn-2/N-Oct-3 transcription factor, is widely expressed in the developing mammalian central nervous system. Brn-2 has also been found to regulate the melanocytic phenotype with N-Oct-3 DNA binding activity elevated in malignant melanoma, however, its mode of action is yet to be defined. The functional role of the Brn-2 transcription factor has been investigated through the analysis of protein-protein interactions it forms with a number of basal and melanocytic transcriptional regulatory proteins. In vivo interactions were tested by gene-cotransfection using the mammalian GAL4-Herpes Simplex viral protein 16 (VP16) two hybrid formation and direct protein binding by in vitro glutathione S-transferase (GST)-pull down assay. The Brn-2 protein was found to homodimerize in vivo with high affinity, using Brn-2 deletion constructs dimer complex formation was found to be dependent on the presence of both the homeodomain and linker regions of the POU-domain. However, the POU-homoedomain was dispensable for the formation of the dimerization interface in one of the partner molecules but not both, when the POU-linker region was removed the ability to interact was lost irrespective of the presence of the homeodomain. Dimerization of Brn-2/N-Oct-3 was also found to occur in DNA binding assays using melanoma cell line nuclear extracts and a recently reported dimer target sequence probe, which may have significant consequences for gene regulation in melanocytic tumours. Low affinity Brn-2 protein contacts have also been found with the basal transcription complex, including TATA binding protein (TBP) and the transcriptional coactivator p300, and with the Sox-10 and Pax-3 transcription factors that are known to play an important role in melanocyte cell formation.

Binding Sites↗

PubMed2000

Inhibition of melanin synthesis by cystamine in human melanoma cells.

In studies to determine whether pigmentation can be regulated physiologically by thiols, human melanoma cells (MM418c5) and melanocytes were found to become depigmented when cultured continuously in 50 microM cystamine. Cystamine was depleted from the culture medium and the treatment was nontoxic and reversible. Cysteamine, dithiothreitol, and phenylthiourea were less effective, and glutathione, cysteine, and cystine were inactive. Tyrosinase (dopa oxidase) activity was not greatly affected except for induction of a lag period. In contrast, tyrosinase activity in an amelanotic melanoma cell line (MM96L) was rapidly inhibited without consumption of cystamine/cysteamine, in association with the generation of free thiol in the culture medium, and could be enhanced by the cystine transport inhibitor, glutamate. Tyrosinase expressed by a recombinant vaccinia virus was inhibited by cystamine treatment of MM96L and HeLa cells. Cystamine treatment lowered the degree of cross-linking of the pigmentation antigen gp75/TRP-1 in MM418c5 cells. Tyrosinase protein and mRNA levels in MM418c5 cells were not affected by cystamine. The results show that cystamine at a concentration close to physiologic levels has multiple effects on the melanogenic pathway. In amelanotic cells, tyrosinase has a short half-life and is readily inhibited by cystamine/cysteamine whereas tyrosinase in the more mature melanosomes of the pigmented cell appears to be less accessible to proteolytic and thiol attack. Inhibition of melanin synthesis in the latter cell type may arise to a significant degree from reduction of cystamine to cysteamine, which sequesters quinones.

Cystamine↗

PubMed2000

Melanocortin-1 receptor polymorphisms and risk of melanoma: is the association explained solely by pigmentation phenotype?

Risk of cutaneous malignant melanoma (CMM) is increased in sun-exposed whites, particularly those with a pale complexion. This study was designed to investigate the relationship of the melanocortin-1 receptor (MC1R) genotype to CMM risk, controlled for pigmentation phenotype. We report the occurrence of five common MC1R variants in an Australian population-based sample of 460 individuals with familial and sporadic CMM and 399 control individuals-and their relationship to such other risk factors as skin, hair, and eye color; freckling; and nevus count. There was a strong relationship between MC1R variants and hair color and skin type. Moreover, MC1R variants were found in 72% of the individuals with CMM, whereas only 56% of the control individuals carried at least one variant (P<.001), a finding independent of strength of family history of melanoma. Three active alleles (Arg151Cys, Arg160Trp, and Asp294His), previously associated with red hair, doubled CMM risk for each additional allele carried (odds ratio 2.0; 95% confidence interval 1. 6-2.6). No such independent association could be demonstrated with the Val60Leu and Asp84Glu variants. Among pale-skinned individuals alone, this association between CMM and MC1R variants was absent, but it persisted among those reporting a medium or olive/dark complexion. We conclude that the effect that MC1R variant alleles have on CMM is partly mediated via determination of pigmentation phenotype and that these alleles may also negate the protection normally afforded by darker skin coloring in some members of this white population.

Data Interpretation, Statistical↗

PubMed1998

UVB-specific regulation of gene expression in human melanocytic cells: cell cycle effects and implication in the generation of melanoma.

Analysis of the expression of a number of known genes in cultured human cells has revealed UVB-induced changes that may be specific for melanocytic cells. The response of c-fos, p53 and HIV-LTR reporter constructs to UVB and UVC was reduced in MM96L melanoma cells compared to HeLa. Cell cycle arrest produced by UVA, gamma radiation, cisplatin or the antimetabolite deoxyinosine differed from that of UVB. Cell cycle analysis after multiple doses of UVB raised the possibility that UVB-induced pRb depletion could result in increased mutation and thus enhanced tumourigenesis of irradiated melanocytes in skin subjected to a defined pattern of UVB exposure. To extend the analysis of gene expression in cultured melanocytic cells to uncharacterised genes, promoter trap cell clones containing unknown genes 'tagged' by a beta-galactosidase reporter construct were generated from MM96L cells. Altered gene expression in clones treated with a panel of DNA-damaging agents was quantitated by measurement of beta-galactosidase activity. Of the clones containing 'tagged' endogenous promoters induced by UVB, 52% were induced only by UVB and not by other DNA-damaging agents (cisplatin, N-methyl-N-nitro-nitrsoguanidine, fotemustine). One third of the clones were also activated by TPA suggesting that general DNA damage responses involving PKC are activated less frequently than unique pathways of gene activation. Overall, 60% of the 50 clones that responded to the panel of agents were induced by only one of the agents, indicating that a high proportion of genes are induced by agent-specific mechanisms. In the long term, promoter trapping may allow the full repertoire of UVB-inducible genes to be characterised.

Antineoplastic Agents↗

PubMed1998

Human pigmentation genes and their response to solar UV radiation.

Identification and characterisation of the genes involved in melanin pigment formation, together with the study of how their action is influenced by exposure to UV radiation, is providing a molecular understanding of the process of skin photoprotection through tanning. The mechanisms underlying this change in epidermal melanin involve both a transcriptional response of the pigmentation genes and post-translational control of the melanin biosynthetic pathway. UV rays are known to interact with numerous molecules within cells, and among these the photochemical reactions involving lipids and DNA are implicated in modulating melanogenesis. The combination of DNA damage, the formation of diacylglycerol, and the action of the melanocyte stimulating hormone receptor are all likely to be involved in UV-induced tanning.

Animals↗

PubMed1998

Human pigmentation genetics: the difference is only skin deep.

There is no doubt that visual impressions of body form and color are important in the interactions within and between human communities. Remarkably, it is the levels of just one chemically inert and stable visual pigment known as melanin that is responsible for producing all shades of humankind. Major human genes involved in its formation have been identified largely using a comparative genomics approach and through the molecular analysis of the pigmentary process that occurs within the melanocyte. Three classes of genes have been examined for their contribution to normal human color variation through the production of hypopigmented phenotypes or by genetic association with skin type and hair color. The MSH cell surface receptor and the melanosomal P-protein are the two most obvious candidate genes influencing variation in pigmentation phenotype, and may do so by regulating the levels and activities of the melanogenic enzymes tyrosinase, TRP-1 and TRP-2.

Albinism↗

PubMed1998

Complete sequence and polymorphism study of the human TYRP1 gene encoding tyrosinase-related protein 1.

The complete 24,667 nucleotide sequence spanning the human TYRP1 gene has been determined from the inserts of two overlapping lambda clones. A LINE-1 repeat element is immediately adjacent to and may demarcate the immediate 5' promoter region of the gene. A search for polymorphism within the seven TYRP1 coding exons has been performed by an RNase mismatch detection procedure. Analysis of the TYRP1 gene in 100 Caucasian individuals of varying hair color has found no amino acid sequence variation nor revealed any hemizygous mutant allele in the hypopigmented phenotype of two 9p- syndrome patients.

Base Sequence↗

PubMed1998

Redox regulation of Brn-2/N-Oct-3 POU domain DNA binding activity and proteolytic formation of N-Oct-5 during melanoma cell nuclear extraction.

Reversible oxidation sensitivity of N-Oct-3 DNA binding activity was seen when melanoma extracts and recombinant Brn-2 protein were treated with a variety of metals, hydrogen peroxide and the cysteine disulphide bond forming agent diamide. Western blot analysis of diamide-oxidized N-Oct-3 protein indicated that this was likely to be due to intramolecular disulphide bonding. The potential role of oxidative loss of N-Oct-3 DNA binding activity is discussed in relation to redox changes that may occur during the early phase of apoptosis in neuronal cell lines and tissues. Brn-2 C-terminal antibody Western blot analysis of melanoma cell line nuclear extracts prepared using a combination of sodium dodecyl sulphate and NP-40 detergent cell lysis procedures demonstrated the formation of N-Oct-5 DNA binding activity via N-terminal proteolytic clipping of Brn-2/N-Oct-3.

Animals↗

PubMed1997

Tumor selectivity and transcriptional activation by azelaic bishydroxamic acid in human melanocytic cells.

Azelaic bishydroxamic acid (ABHA), a potent differentiating agent for lymphoid cells, was selectively toxic for 5 human tumor cell lines and transformed human melanocytes and keratinocytes (dose for 37% survival, D37, 30-100 microg/mL) compared with normal cells (melanocytes, fibroblasts; D37 > 300 microg/mL). Dendritic morphology was the only indicator found for increased differentiation, markers for the pigmentation pathway being unchanged or inhibited by ABHA. In contrast to hexamethylene bisacetamide and azelaic acid, ABHA significantly increased the HIV LTR, SV40 and c-fos promoter activities during a 24 hr treatment. Metallothionein promoter activity was enhanced by 5 hr treatment with ABHA in a sensitive melanoma cell line (MM96L) but was inhibited in a more resistant line (HeLa); c-fos promoter activity was inhibited in HeLa during this time. Transcription from a p53 binding response element was inhibited in MM96L by a 24 hr ABHA treatment but enhanced in HeLa. ABHA may represent a structural prototype for designing more potent and selective anti-melanoma agents.

Acetamides↗

PubMed1997

Rufous oculocutaneous albinism in southern African Blacks is caused by mutations in the TYRP1 gene.

Oculocutaneous albinism (OCA) is the most common autosomal recessive disorder among southern African Blacks. There are three forms that account for almost all OCA types in this region. Tyrosinase-positive OCA (OCA2), which is the most common, affects approximately 1/3,900 newborns and has a carrier frequency of approximately 1/33. It is caused by mutations in the P gene on chromosome 15. Brown OCA (BOCA) and rufous OCA (ROCA) account for the majority of the remaining phenotypes. The prevalence of BOCA is unknown, but for ROCA it is approximately 1/8,500. Linkage analysis performed on nine ROCA families showed that ROCA was linked to an intragenic marker at the TYRP1 locus (maximum LOD score = 3.80 at straight theta=.00). Mutation analysis of 19 unrelated ROCA individuals revealed a nonsense mutation at codon 166 (S166X) in 17 (45%) of 38 ROCA chromosomes, and a second mutation (368delA) was found in an additional 19 (50%) of 38 chromosomes; mutations were not identified in the remaining 2 ROCA chromosomes. In one family, two siblings with a phenotypically unclassified form of albinism were found to be compound heterozygotes for mutations (S166X/368delA) at the TYRP1 locus and were heterozygous for a common 2.7-kb deletion in the P gene. These findings have highlighted the influence of genetic background on phenotype, in which the genotype at one locus can be influenced by the genotype at a second locus, leading to a modified phenotype. ROCA, which in southern African Blacks is caused by mutations in the TYRP1 gene, therefore should be referred to as "OCA3," since this is the third locus that has been shown to cause an OCA phenotype in humans.

Africa, Southern↗

PubMed1997

Characterization of melanocyte stimulating hormone receptor variant alleles in twins with red hair.

The association between MSHR coding region variation and hair colour in humans has been examined by genotyping 25 red haired and 62 non-red Caucasians, all of whom were 12 years of age and members of a twin pair study. Twelve amino acid substitutions were seen at 11 different sites, nine of these being newly described MSHR variants. The previously reported Val92Met allele shows no association with hair colour, but the three alleles Arg151Cys, Arg160Trp and Asp294His were associated with red hair and one Val60Leu variant was most frequent in fair/blonde and light brown hair colours. Variant MSHR genotypes are associated with lighter skin types and red hair (P < 0.001). However, comparison of the MSHR genotypes in dizygotic twin pairs discordant for red hair colour indicates that the MSHR gene cannot be solely responsible for the red hair phenotype, since five of 13 pairs tested had both haplotypes identical by state (with three of the five having both identical by descent). Rather, it is likely that additional modifier genes exist, making variance in the MSHR gene necessary but not always sufficient, for red hair production.

Alleles↗