Martin R. Green

A Genomewide Association Study of Skin Pigmentation in a South Asian Population

We have conducted a multistage genomewide association study, using 1,620,742 single-nucleotide polymorphisms to systematically investigate the genetic factors influencing intrinsic skin pigmentation in a population of South Asian descent. Polymorphisms in three genes--SLC24A5, TYR, and SLC45A2--yielded highly significant replicated associations with skin-reflectance measurements, an indirect measure of melanin content in the skin. The associations detected in these three genes, in an additive manner, collectively account for a large fraction of the natural variation of skin pigmentation in a South Asian population. Our study is the first to interrogate polymorphisms across the genome, to find genetic determinants of the natural variation of skin pigmentation within a human population.

Why Some Women Look Young for Their Age

The desire of many to look young for their age has led to the establishment of a large cosmetics industry. However, the features of appearance that primarily determine how old women look for their age and whether genetic or environmental factors predominately influence such features are largely unknown. We studied the facial appearance of 102 pairs of female Danish twins aged 59 to 81 as well as 162 British females aged 45 to 75. Skin wrinkling, hair graying and lip height were significantly and independently associated with how old the women looked for their age. The appearance of facial sun-damage was also found to be significantly correlated to how old women look for their age and was primarily due to its commonality with the appearance of skin wrinkles. There was also considerable variation in the perceived age data that was unaccounted for. Composite facial images created from women who looked young or old for their age indicated that the structure of subcutaneous tissue was partly responsible. Heritability analyses of the appearance features revealed that perceived age, pigmented age spots, skin wrinkles and the appearance of sun-damage were influenced more or less equally by genetic and environmental factors. Hair graying, recession of hair from the forehead and lip height were influenced mainly by genetic factors whereas environmental factors influenced hair thinning. These findings indicate that women who look young for their age have large lips, avoid sun-exposure and possess genetic factors that protect against the development of gray hair and skin wrinkles. The findings also demonstrate that perceived age is a better biomarker of skin, hair and facial aging than chronological age.

Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth

Epidermal growth factor (EGF), a low-molecular-weight polypeptide (G. Carpenter and S. Cohen, 1979, Annu. Rev. Biochem. 48, 193-216), stimulates the proliferation and keratinisation of cultured embryonic epidermis (S. Cohen, 1965, Dev. Biol. 12, 394-407) and promotes epidermal growth, thickening, and keratinisation when injected into neonatal mice (S. Cohen and G.A. Elliott, 1963, J. Invest. Dermatol, 40, 1-5). We have determined the distribution of the available receptors for epidermal growth factor in rat skin using autoradiography following incubation of explants with 125I-labelled mouse EGF. EGF receptors are detected on the epithelial cells overlying the basement membranes of the epidermis, sebaceous gland, and regions of the hair follicle all of which have proliferative capacity. In marked contrast, tissues which have started to differentiate and lost their growth potential, carry either an undetectable or sharply reduced number of EGF receptors. The EGF receptor number and receptor affinity of epidermal basal cells freshly isolated from rats of increasing age has also been determined. We find that receptor affinity remains unchanged (3.3 nM) but that basal cell surface receptor number decreases markedly with age. This decrease in receptor number is similar in trend to the known drop in basal cell [3H]thymidine labelling index which occurs over the same time period. The data suggest that the distribution of EGF receptors and EGF cell surface receptor number in skin are important in the spatial and temporal control of epithelial proliferation.

SLC24A5 Encodes a trans-Golgi Network Protein with Potassium-dependent Sodium-Calcium Exchange Activity That Regulates Human Epidermal Melanogenesis

A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of ∼43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.

Human hair follicle and epidermal melanocytes exhibit striking differences in their aging profile which involves catalase.

in the DNFB-applied skin. We assume that this distinct migratory activity may be due to the fact that not all T cells in the LNs after DNFB sensitization were DNFB specific. In contrast, almost all TNCB-sensitized T cells actively migrated when DNFB was applied. In addition, we showed reverse motile activities in the TNCB-painted skin. Moreover, skin-infiltrating T cells colocalized with APCs. These findings suggest that skin-infiltrating T cells may actively scan for antigens, and when they meet APCs carrying their specific antigens, they stop migrating and stably interact with APCs. Our results also indicate that in the elicitation phase of CHS, hapten seems to be presented mainly by APCs in the dermis. Additional detailed studies are needed to clarify which subset of dermal APCs is essential for hapten presentation and whether epidermal Langerhans cells contribute.

Protection by Food-derived Antioxidants from UV-A1–Induced Photodamage, Measured Using Living Skin Equivalents¶

Abstract In a study of biomarkers of ultraviolet-A1 radiation (UV-A1)-induced skin damage, living skin equivalent cultures (LSE) were treated with the antioxidants hesperetin and quercetin-3-glucoside and irradiated with 25 or 50 J/cm2 UV-A1. Changes in the following biomarkers were measured; Interleukin 1-alpha (IL-1α), Heme Oxygenase-1 (HO-1), TdT-mediated dUTP nick end labeling (TUNEL) and 8-hydroxy-2′-deoxyguanosine (8-OHdG). IL-1α and HO-1 were analyzed by real-time PCR, Western blot, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. TUNEL and 8-OHdG were determined by (immuno)histochemical techniques. Sections were stained with hematoxylin and eosin (H&E). UV-A1 induced keratinocyte and fibroblast vacuolation and nuclear pyknosis, intense TUNEL staining of fibroblasts and increased staining of cells and nuclei for 8-OHdG. Lesser or marginal increases in intensity followed staining for HO-1 and IL-1α. The IL-1α increase was confirmed by ELISA assays of the medium supernatants. Hesperetin and quercetin-3-glucoside reduced changes in H&E, 8-OHdG, TUNEL and IL-1α. Quercetin-3-glucoside reduced the amount of IL-1α in LSE media. These observations support the use of the selected biomarkers to monitor UV-A1 damage and provide evidence that dietary ingredients could reduce ultraviolet-A radiation-induced damage.

Nutrigenetics: where next for the foods industry?

In spite of concerns about affluencerelated conditions such as obesity and diabetes, the Western world has enjoyed extraordinary improvements in health and well-being over the past century. During the last 150 years, the average female life expectancy has increased by 3 months per year and now exceeds 85 years in Japanese women. The main causes of this remarkable phenomenon are improvements in nutrition, sanitation, hygiene and infection control. Individuals are ever more captivated by opportunities for improved personal health and appearance; therefore, it is not surprising that consumer studies suggest preferences for food products having positive health benefits. At the same time, consumers believe that they are all ‘different’ so it is reasonable that food choices should reflect this diversity. As we all want to see healthier populations, it is increasingly topical to ask how food companies can further contribute to health and longevity, and how new concepts and techniques from the genomics era can aid the industry to improve healthy living. Indeed, many population studies have concluded that the micronutrients from high fruit and vegetable diets improve health and longevity; so how might the food industry further contribute to improvements in human health and what are the pitfalls? An unconventional vision for the foods industry is (i) that major benefits for society will arise in the future from modest, individual improvements in health and well-being generating a cumulative, significantly improved pattern of health and well-being across entire populations; and (ii) that these accumulated benefits from now on will far outweigh those achieved by new medical treatments. In essence, population benefits bring improved health for all, while medicines, though of irreplaceable and immense value to individuals, only immediately benefit the current proportion of the population with treatable conditions (Figure 1). Indeed, improvements in population health can lead to many people not developing a specific disease at all. The population argument can be illustrated by the role of fluoride in improving dental health. Dental decay affected virtually everyone in developed countries prior to the widespread introduction of fluoride in the early 1960s. The anticaries property of fluoride was discovered in the 19th century and through epidemiological observations in the USA, but the major benefits for mankind and marked reduction in caries incidence in the Western world were mainly achieved by fluoride delivery to the general public in cheap and/or consumer pleasing forms. This was implemented in part through publicly funded research, water fluoridation and education, but mainly by commercial product sales to consumers who appreciated and enjoyed the immediate benefits of the product forms. Extrapolating from the fluoride paradigm, an ideal vision for health care is illustrated in the figure: the ambition for all in society to achieve a fully acceptable lifestyle for the whole of their life aided by consumer desirable foods that promote health. In this respect, public health organisations and the commercial health care and foods industries share a mutual interest and motivation in providing better health and well-being solutions for society. Turning to some of the issues, there are abundant examples of drugs caus-

Identifying genetic risk variants for coronary heart disease in familial hypercholesterolemia: an extreme genetics approach.

Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), a disorder characterized by coronary heart disease (CHD) at young age. We aimed to apply an extreme sampling method to enhance the statistical power to identify novel genetic risk variants for CHD in individuals with FH. We selected cases and controls with an extreme contrast in CHD risk from 17 000 FH patients from the Netherlands, whose functional LDLR mutation was unequivocally established. The genome-wide association (GWA) study was performed on 249 very young FH cases with CHD and 217 old FH controls without CHD (above 65 years for males and 70 years of age for females) using the Illumina HumanHap550K chip. In the next stage, two independent samples (one from the Netherlands and one from Italy, Norway, Spain, and the United Kingdom) of FH patients were used as replication samples. In the initial GWA analysis, we identified 29 independent single nucleotide polymorphisms (SNPs) with suggestive associations with premature CHD (P<1 × 10−4). We examined the association of these SNPs with CHD risk in the replication samples. After Bonferroni correction, none of the SNPs either replicated or reached genome-wide significance after combining the discovery and replication samples. Therefore, we conclude that the genetics of CHD risk in FH is complex and even applying an ‘extreme genetics’ approach we did not identify new genetic risk variants. Most likely, this method is not as effective in leveraging effect size as anticipated, and may, therefore, not lead to significant gains in statistical power.

The biology and genetics of curly hair

Hair fibres show wide diversity across and within all human populations, suggesting that hair fibre form and colour have been subject to much adaptive pressure over thousands of years. All human hair fibres typically have the same basic structure. However, the three‐dimensional shape of the entire fibre varies considerably depending on ethnicity and geography, with examples from very straight hair with no rotational turn about the long axis, to the tightly sprung coils of African races. The creation of the highly complex biomaterials in hair follicle and how these confer mechanical functions on the fibre so formed is a topic that remains relatively unexplained thus far. We review the current understanding on how hair fibres are formed into a nonlinear coiled form and which genetic and biological factors are thought to be responsible for hair shape. We report on a new GWAS comparing low and high curl individuals in South Africa, revealing strong links to polymorphic variation in trichohyalin, a copper transporter protein CUTC and the inner root sheath component keratin 74. This builds onto the growing knowledge base describing the control of curly hair formation.

NCKX5, a natural regulator of human skin colour variation, regulates the expression of key pigment genes MC1R and alpha-MSH and alters cholesterol homeostasis in normal human melanocytes.

Natural human skin colour is determined both by environmental exposure to ultraviolet light and through inherited genetic variation in a very limited number of genes. Variation of a non-synonymous single-nucleotide polymorphism (nsSNP; rs1426654) in the gene (SLC24A5) encoding the NCKX5 protein is associated with differences in constitutive skin colour in South Asians. The nsSNP encodes the substitution of alanine for threonine at residue 111 (A111T) near a transmembrane region required for exchanger activity, a region which is highly conserved across different species and between NCKX family members. We have shown that NCKX5 is located at the trans-Golgi network of melanocytes and functions as a potassium-dependent sodium-calcium exchanger. When heterologously expressed, the 111T variant of NCKX5 shows significantly lower exchanger activity than the A111 variant. We have postulated that lower exchanger activity causes the reduced melanogenesis and lighter skin in Thr111-positive individuals. We used gene expression microarrays with qPCR replication and validation to assess the impact of siRNA-mediated knockdown of SLC24A5 on the transcriptome of cultured normal human melanocytes (NHM). Very few genes associated with melanogenesis were altered at the transcript level except for MC1R, suggesting that SLC24A5 interacts with at least one well-characterized melanogenic signalling pathway. More surprisingly, the expression of a number of cholesterol homeostatic genes was altered after SLC24A5 knockdown, and the total cholesterol content of NHM was increased. Cholesterol has previously been identified as a potential melanogenic regulator, and our data imply that NCKX5 exchanger function influences natural variation in skin pigmentation via a novel, unknown mechanism affecting cellular sterol levels.