Reciprocal responses of fibroblasts and melanocytes to α-MSH depending on MC1R polymorphisms

Abstract

The melanocortin 1-receptor (MC1R) exhibits several variants in form of single nucleotide polymorphisms (SNPs) which are known to differentially regulate melanocyte function. However, whether and how MC1R polymorphisms also affect fibroblast function, has not been investigated so far. Therefore we measured intracellular cyclic adenosine monophosphate (cAMP) concentrations (cAMP-EIA) and cellular proliferation (CellTiter-Blue) upon stimulation with alpha-melanocyte stimulating hormone (α-MSH) in eight different human fibroblast and melanocyte cell lines with wild type and different MC1R SNPs. We found that fibroblasts, as well as melanocytes, show differences in MC1R function depending on the MC1R genotype. MC1R stimulation with α-MSH in wild type (MC1R_wt) melanocytes results in an increase of intracellular cAMP and cellular proliferation. In contrast, MC1R_wt fibroblasts react with a decrease of intracellular cAMP and proliferation. In MC1R polymorphic fibroblasts (R163Q, R151C and V60L) both effects are significantly alleviated. Similar, but inverse effects could be found in MC1R polymorphic melanocytes (R142H and V92M) with a significantly lower cAMP increase and proliferation rate compared to MC1R_wt melanocytes. Our results indicate that the MC1R displays reciprocal growth responses in melanocytes and fibroblasts, depending on the MC1R genotype. Thus, the MC1R seems to be not solely important for the skin pigmentary system, but also for the fibroblast function, and might influence different processes of the dermal compartment like wound healing, fibrosis and keloid formation.

Introduction

The Proopiomelanocortin (POMC) system of the human skin consists of peptide hormones (MSH, Adenocorticotropic Hormone (ACTH), Endorphins, etc.,) that originate from the precursor protein POMC, and the corresponding receptors (Melanocortin receptors, Endorphin receptors). The Melanocortin 1 receptor (MC1R) represents one out of a group of five G-protein coupled receptors (MC1R-MC5R) ubiquitously expressed in all cells of the skin (keratinocytes, melanocytes, fibroblasts, cells of the immune system). MC1R consists of seven transmembrane domains and has a high affinity for POMC derived α-MSH. The activation of the MC1R leads to an increase of intracellular cAMP levels which trigger the activation of protein kinase A (PKA). PKA phosphorylates the cAMP responsive binding protein (CREB), which binds to the cAMP responsive element (CRE) in the promoter region of the microphthalmia transcription factor (MITF). For full activation of the MITF-promoter, CREB needs to bind the CREB binding protein (CBP).1 MITF is a member of the MYC-superfamily of basic-helix-loop-helix (bHLH) zipper-transcription factors and activates genes involved in both skin pigmentation and collagen metabolism.1–3

The MC1R gene is localised on the long arm of Chromosom 16 (16q24.3).4,5 The gene consists of an intronless coding region and is highly polymorphic.6–8 Robbins et al. described the association of some MC1R polymorphisms and receptor functionality modulating the fur colour of mice.9 Based on this discovery, several investigations of the functionality of the MC1R variants were carried out in humans. Population studies showed that several MC1R single nucleotide polymorphisms (SNPs) (D84E, R151C, R160W, D294H, R142H, I155T, V60L, V92M, R163Q) are associated with a poor tanning ability and an augmented risk to develop skin cancer, designating the MC1R a melanoma susceptibility gene (red hair and fair skin (RHC) phenotype).10–16

Interestingly, the alleles differ in their penetrance concerning the RHC phenotype.17,18 The strong RHC alleles comprise the common polymorphisms R151C, R160W and D294H and rare polymorphisms D84E and R142H, which increase odds ratios for red hair 50–120 fold. The weak RHC alleles V60L, V92M and R163Q have odds ratios for red hair between 2 and 6. Polymorphisms R142H, R151C, R160W, D294H and V60L are found in 30% of the individuals of north-european populations and are responsible for 30% of all red haired phenotypes.19

On the molecular level, it has been shown that the aforementioned RHC alleles R142H, R151C, R160W and D294H affect MC1R function.20,21 However, the molecular mechanisms of these effects are still not clarified. One possibility is, according to some recent studies, that the intracellular retention of the receptor and the receptor density on the plasma membrane is different depending on different MC1R variants.20,21 On the other hand the fact that the MC1R-variants differ in their ability to generate cAMP implicates varying degrees in the activation of the involved G-proteins. As heterozygote variants reveal an intermediate functional state between wt and homozygote variants a gene-dose-effect can be assumed.22,23

Because of the important role of MC1R in the pigmentary system of the skin, most investigations of its polymorphisms on a cellular level were done in melanocytes so far.3,22 These studies could demonstrate that MC1R polymorphisms compared to MC1R_wt not only impair melanogenesis, but also affect the UV-response of melanocytes, leading to an augmented growth arrest and apoptosis by ineffective DNA-repair. These findings explain the role or MC1R polymorphisms as a major melanoma risk factor.24–31

In fibroblasts, the MC1R has been shown to regulate fibroblast function by affecting the synthesis and degradation of collagen.32,33 In contrast to melanocytes, the impact of MC1R polymorphisms on receptor function in fibroblasts has not been investigated so far.

The aim of this study was therefore to investigate the functional relevance of MC1R _wt compared with MC1R polymorphisms in dermal fibroblasts, and to relate the findings to melanocytes, which are well characterized concerning MC1R, as positive control.

Results

Characterization of MC1R gene in melanocyte and fibroblast cell lines.

MC1R gene was characterised by sequencing several melanocyte and fibroblast cell lines. We found two MC1R wild-type (MC1R_wt) and two MC1R polymorphic melanocyte cell lines (homozygous V92M and heterozygous R142H), and one MC1R_wt and three MC1R polymorphic fibroblast cell lines (heterozygous R163Q, heterozygous V60L and heterozygous R151C) respectively (Table 1).

Reciprocal cAMP induction and growth responses in MC1R_wt melanocytes and fibroblasts after α-MSH stimulation.

First, we compared the α-MSH effect on cAMP production and cell proliferation in melanocytes and fibroblasts with MC1R_wt (Fig. 1). MC1R_wt melanocytes showed a significant increase in cellular cAMP concentration of 50% (p = 0.003) following α-MSH (1 µM) treatment (Fig. 1A), whereas the cAMP of fibroblasts showed a decrease of 60% (p = 0.007) (Fig. 1C).

The proliferation measurements of MC1R_wt melanocytes and MC1R_wt fibroblasts with and without α-MSH stimulation are depicted in Figure 1B and D. Measuring time points were: day 0 (the day when α-MSH was added, see methods), day 2 and in the case of fibroblasts additionally day 4. The additional measuring point (day 4) was added as the effect of α-MSH on fibroblast growth was less pronounced.

Untreated melanocytes showed a proliferation of 46% from day 0 to day 2 whereas the α-MSH stimulated melanocytes revealed a proliferation of 75% for the same time period. The difference of 29% between the non-stimulated and stimulated MC1R_wt melanocytes was highly significant (p < 0.001) (Fig. 1B).

The untreated MC1R_wt fibroblasts showed an increase in proliferation from day 0 to day 4 of 484%, whereas the fibroblasts, treated with α-MSH, showed a lower increase of 434%. The growth reduction in fibroblast was significant: 50% from day 0 to day 4 (p < 0.001) (Fig. 1D).

In summary, the results in Figure 1 show that α-MSH induces a significant augmentation of both cAMP and cell proliferation in MC1R_wt melanocytes. On the contrary, in MC1R_wt fibroblasts, both cAMP levels and cell growth were significantly reduced.

Functional implications of MC1R polymorphisms on cAMP induction and proliferation.

Since we could demonstrate that α-MSH has inverse biologic effects on MC1R_wt fibroblasts and melanocytes, we intended to examine, if and how MC1R polymorphisms would affect the observed responses to α-MSH.

In Figure 2A and C cAMP measurements in MC1R polymorphic melanocytes treated or untreated with α-MSH, are shown. The increase in cAMP for R142H and V92M cell lines was 2.5% (p = 0.326) and 10.5% (p = 0.32) respectively. The proliferation measurements from the same cell lines stimulated with α-MSH are presented in Figure 2B and D. Hormone stimulation induced an increase in proliferation on day 2 of 23% (p < 0.001) and 12% (p < 0.001) for R142H and V92M cell lines respectively.

The polymorphic fibroblast cell lines underwent similar treatment as the melanocytes. Measurements of intracellular cAMP following α-MSH stimulation in R163Q, R151C and V60L are shown in Figure 3A, C and E respectively. The cell lines displaying MC1R polymorphisms showed no significant activation of the MC1R. We could observe a reduction in cAMP of 26% (p = 0.1) or increase of 0.6% (p = 0.49) and 26% (p = 0.26) for R163Q, R151C and V60L cell lines respectively, which all failed to reach significance.

Proliferation measurements of each particular fibroblast cell line are shown in Figure 3B, D and F. R163Q cells showed a minor decrease of cell growth after treatment with α-MSH of 8% (p = 0.06) from day 0 to day 4 (Fig. 3B). The proliferation measurements for the other two polymorphic fibroblast cell lines R151C and V60L showed similar minimal decreases of 7% (p = 0.07) and 6% (p = 0.06) from day 0 to day 4.

The results presented in Figure 3 show that fibroblasts with MC1R polymorphisms, consistent with an impaired receptor function, do not react with a significant decrease of cAMP levels and do not show a significant reduction of growth.

cAMP production and proliferation differ significantly between MC1R_wt and MC1R polymorphic melanocytes and fibroblasts.

Regarding the results presented in Figures 1–3 we were interested to see, if the differences between wt and polymorphic cell lines were significant in both cell types. For that purpose we compared the pooled data for cAMP levels in MC1R_wt and MC1R polymorphic melanocytes and fibroblasts stimulated with α-MSH (Fig. 4). Our statistical analysis showed that cAMP levels in MC1R_wt melanocytes are significantly higher than in the polymorphic cell lines (p = 0.013) (Fig. 4A), whereas cAMP levels in MC1R_wt fibroblasts are significantly lower than in the fibroblasts with MC1R polymorphisms (p = 0.036) (Fig. 4C). The cAMP concentrations in untreated wild-type versus polymorphic cells were not significantly different (p = 0.266 and p = 0.312).

An analogous examination was also performed for the effects of α-MSH on proliferation. In MC1R_wt melanocytes we found a significantly stronger increase in proliferation than in MC1R polymorphic melanocytes (p = 0.008) (Fig. 4B), whereas MC1R_wt fibroblasts showed a significantly stronger inhibition of proliferation compared to the MC1R polymorphic fibroblasts (p = 0.003) (Fig. 4D).

These findings emphasise the reciprocal response to MC1R stimulation in melanocytes and fibroblasts, and underline the significant inverse effects of MC1R polymorphisms on the receptor function in melanocytes as well as in fibroblasts.

Discussion

As already well known, stimulation of MC1R_wt melanocytes with α-MSH leads to a G-protein coupled induction of cAMP driving the CRE-MITF pathway and promoting melanin synthesis and cell proliferation.1–3 MC1R polymorphisms contribute to a different receptor function, which additionally depends upon whether one or both alleles are affected: gain of function, no change, reduction or loss of function.22 The most important finding of our study is that MC1R_wt melanocytes and MC1R_wt fibroblasts display a reciprocal MC1R function concerning cAMP induction and cell growth. Furthermore, we could show that polymorphisms of MC1R also affect the receptor function in dermal fibroblasts, what has only been shown for human melanocytes so far.

As expected, cAMP did not increase significantly in both polymorphic melanocyte cell lines after α-MSH stimulation and were significantly lower compared to MC1R_wt melanocytes. We were surprised to observe that proliferation in MC1R polymorphic melanocytes seems not to be compromised, as significant differences could be found after stimulation with α-MSH versus unstimulated melanocytes. Given that cAMP levels correlate with the receptor functionality of MC1R, our results suggest that either even minimal MC1R activation suffice to stimulate cellular proliferation in melanocytes irrespective of the polymorphisms investigated here or α-MSH exhibits MC1R-independent yet unknown effects in melanocytes. However, comparing proliferation rates of MC1R_wt and polymorphic MC1R melanocytes the cell growth after stimulation with α-MSH was significantly more pronounced in MC1R_wt melanocytes. These findings are in agreement with previously published reports in reference 22, 25 and 26.

In contrast to melanocytes, MC1R_wt fibroblasts revealed significantly decreased cAMP levels and decelerated cell growth following α-MSH stimulation. Heterozygous MC1R polymorphisms R163Q, V60L, R151C found in our fibroblast cell lines accordingly abolished these inhibitory effects of α-MSH: cAMP levels and fibroblast proliferation were not significantly affected compared to untreated cells, suggesting an impaired receptor function. These observations are additionally supported by the finding that cAMP levels and growth rate after stimulation with α-MSH are significantly lower in fibroblast with MC1R_wt compared to MC1R polymorphic fibroblasts.

Concerning the inhibitory effects of α-MSH on MC1R_wt fibroblasts our results are contrary to recently published data by Böhm and colleagues, who found an increase of intracellular cAMP in fibroblasts after α-MSH stimulation.33 The fibroblasts investigated in that study were however not further characterized concerning their MC1R gene sequence. Thus, MC1R polymorphisms could be the explanation for this controversy, as we could demonstrate positive, albeit not significant effects of α-MSH on intracellular cAMP in MC1R_V60L fibroblasts. The inhibitory effects of α-MSH on MC1R_wt fibroblasts may provide a link to the antifibrotic properties of α-MSH described in the literature.33,34

The different downstream mechanisms after MC1R activation inducing opposite effects of α-MSH in melanocytes and fibroblasts are not defined yet. Possibly, the intrinsic receptor activity and the regulation of MC1R (phosphorylation, internalisation, degradation and recycling) play a role.35,36 However, divergent signalling pathways or different sets of target genes depending on epigenetic mechanisms in different cell types are further considerations. Nevertheless the contrary effects of α-MSH on melanocytes and fibroblasts could be a hint to a key role of the cutaneous POMC system as a complex player in the endocrine adaptation and coordination of skin functions.

Our findings suggest that besides the importance for individual pigmentation,8 MC1R polymorphisms can regulate other physiological processes in the skin. Regarding the fibroblasts, α-MSH delivers antifibrotic effects through modulation of collagen metabolism33,34 and, as shown in this study, by a MC1R mediated inhibition of proliferation of MC1R_wt fibroblasts. This could have significant clinical importance. For instance, in a mouse model of scleroderma α-MSH reduced the bleomycin induced collagen-synthesis and tissue fibrosis.34 Furthermore, there is evidence for a concomitant cytoprotective and anti-apoptotic effect of α-MSH in dermal fibroblasts.37 In addition, certain MC1R polymorphisms have been associated with an increased risk for severe photoaging of facial skin38 while patients having MC1R_R160W responded with a significantly increased risk for severe acute radiosensitivity after radiotherapy.39

In conclusion, our work may spark investigating the role of MC1R polymorphisms in skin aging as well as in a variety of fibrosing diseases such as keloids, systemic sclerosis, arterial fibrosclerosis and nephrosclerosis, which are found more often in pigmented races compared to Caucasians.40–43 These studies are ongoing in our laboratory.

Materials and Methods

Cell culture.

Primary human melanocyte and fibroblast cultures were established from neonatal foreskins of healthy individuals with different pigmentation obtained from the Department of Urology, University Hospital of the Saarland. The cells were incubated at 37°C, 5% CO₂ and 95% humidity. The next day medium was changed to discard cell debris. To obtain a monoculture of primary human dermal fibroblasts from the isolated cell mix the medium was changed to DMEM (Invitrogen) containing 10% FCS (Promocell) and 1% Penicillin and Streptomycin (Sigma). To establish a mono-culture of primary human dermal melanocytes the isolated cell mix was treated with 400 ng/ml G418. Viability of the fibroblasts was controlled microscopically for three days and medium was changed daily. If on day six vital fibroblasts were still visible the procedure was repeated. The obtained melanocyte monoculture was cultivated in melanocyte growth medium with supplement mix (Promocell) containing 0.4% bovine pituary extract (BPE), 1 ng/ml basic fibroblast growth factor, 5 µg/ml Insulin, 0.5 µg/ml hydrocortisone, 1% streptomycin, penicillin and amphotericin B.

Sequencing of the MC1R gene.

Prior to sequencing the MC1R DNA was amplified by PCR as described: The genomic DNA of the MC1R gene can be amplified directly as the MC1R gene is intronless and polymorphisms are detectable on the gene level.

For DNA purification we used the High Pure PCR Preparation Kit (Roche), DNA concentrations were measured photometrically at 260 nm. For PCR 2 µl DNA (50 ng/µl), 1 µl forward and reverse primer (10 pM), 1 µl dNTPs (10 mM), 0.5 µl TaqPolymerase (5 U/µl), 5 µl 10x Buffer were used per 50 µl reaction. The primer sequences were as follows: primer1forward (P1F) 5′-GTG ACC GGA CAG ACT GG-3′, primer1reverse (P1R) 5′-GCC TGC CTC CTT CCA TCT-3′, P2F 5′-GGT AGA TGC CAG GAG GTG TC-3′, P2R 5′-ACC AGC AGG TCC GAC AAG-3′, P3F 5′-TGC ACT CAC CCA TGT ACT GC-3′, P3R 5′-CCA GCA GAG GAA GAA AAT GC-3′, P4F 5′-CGT GGT CTT CTT CCT GGC TA-3′, P4R 5′-GGA CCA GGG AGG TAA GGA AC-3′. The MC1R DNA was amplified for 35 cycles (primers 1 and 2 forward and reverse 1 min 95°C, 30 sec 94°C, 30 sec 55°C, 45 sec 72°C, 7 min 72°C, primers 3 and 4 forward and reverse 2 min 95°C, 20 sec 94°C, 20 sec 65°C, 45 sec 72°C, 7 min 72°C) in an GeneAmp PCR System 240C (Perkin Elmer). 5 µl of the PCR product was electrophoresed on a 2% agarose Gel. The remaining PCR product was purified either using the Quiaquick PCR Purification Kit or the Quiaquick Gel extraction kit depending on the purity of the obtained PCR product.

For sequencing 1 µl of the purified PCR product (10 ng/µl), 3.2 µl Primer (forward or reverse) (1 pM), and 2 µl of ready reaction mix (Sigma) and H₂O (HPLC-cleaned) were used per 20 µl reaction. 25 cycles were carried out (5 sec 94°C, 10 sec 96°C, 5 sec 50°C, 4 min 60°C). The product obtained from the sequencing PCR was precipitated with 2 µl NaOAc (3 M, pH 4.6) and dried at 90°C. The precipitated probes were measured in an automatic sequencing machine (ABI Prism 310 Genetic analyser, Applied Biosystems) in the Institute of Human Genetics, University Hospital of the Saarland.

The obtained data were analysed with ChromalsPro software.

Measurements of intracellular cAMP concentration.

Biotrac enzyme immunoassay (EIA) (Amersham Biosciences) was used to determine intracellular cAMP levels. The cells were seeded in 96-well-flatbottom plates and incubated for two days at 37°C. For the melanocytes the medium was changed after 24 h to BPE-free melanocyte medium. The melanocytes were seeded at a density of 5 × 10⁵, the fibroblasts at a density of 2 × 10⁴ per well in 100 µl medium. On day three the cells were stimulated for 20 minutes with α-MSH (1 µM) and lysed over night according to the manufacturer's protocol.

Proliferation measurements.

CellTiter-Blue cell viability assay (Promega) was used to measure cellular proliferation. The measurements were carried out in a fluorescent microplate reader (Genius Pro, Tecan).

Initially the melanocytes were seeded in BPE-free medium. The cells were transferred into 12-well plates at a density of 1,000 cells/ml (Fibroblasts) or 1.6 × 10⁴ cells/ml (Melanocytes) in 1 ml medium and were stimulated with 1 µM α-MSH. Untreated melanocytes and fibroblasts served as controls. The day of stimulation was defined as day zero. Further measurements were carried out on day 2 after stimulation for the melanocytes and on days 2 and 4 for the fibroblasts. At the defined time points the proliferation measurement was carried out as follows: After reducing the volume of culture medium from 1 ml to 500 µl per well the cells were incubated for one hour with 100 µl of CellTiter-Blue at standard conditions. 200 µl of the supernatants were transferred into a black 96-well-plate with transparent bottom and the fluorescence was measured at 560 nm excitation and 590 nm emission. The measurements were quantified as RFU (relative fluorescent unit).

Statistics.

The cAMP and RFU values of unstimulated control cells of each experiment were set as 100% and the values of the α-MSH stimulated cells were accordingly related, so that the resulting data could be evaluated independently from the absolute cell counts. Differences between stimulated and unstimulated cells were evaluated using paired student's t-test.

The comparison between MC1R_wt and MC1R polymorphic melanocytes and fibroblasts concerning intracellular cAMP concentrations and proliferation after α-MSH stimulation has been performed by Mann-Whitney-U test. For the proliferation analysis the absolute RFU differences of α-MSH stimulated minus unstimulated cells (d2 for melanocytes and d4 for fibroblasts) were used. For this analysis the data of wild-type and polymorphic MC1R were pooled for melanocytes and fibroblasts respectively. The value distributions are presented as box plots.

All statistical analyses were performed with statistical software PASW Statistics 18 (SPSS, Inc., Chicago, IL); p-values of < 0.05 were considered as significant.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures and Tables

Figure 1 cAMP production and proliferation of MC1R_wt melanocytes and MC1R_wt fibroblasts following α-MSH stimulation. Intracellular cAMP concentration was measured in MC1R_wt melanocytes (A, n = 9) and fibroblasts (C, n = 5) untreated (black bars) or treated with 1 µM α-MSH for 20 min (red). Proliferation of MC1R_wt melanocytes (B, n = 24) and MC1R_wt fibroblasts (D, n = 12) untreated (black) or treated with 1 µM α-MSH (red). Measurements were carried out three hours (day 0), 48 hours (day 2) and 96 hours (day 4) following stimulation with 1 µM α-MSH. Error bars represent standard error of mean (SEM).

Figure 2 cAMP production and proliferation of melanocytes with MC1R polymorphisms following α-MSH stimulation. Intracellular cAMP concentration was measured in melanocytes with MC1R polymorphisms (A, n = 5 and C, n = 5) untreated (black bars) or treated with 1 µM α-MSH for 20 min (red). Proliferation of melanocytes with MC1R polymorphisms (B, n = 12 and D, n = 12) untreated (black) or treated with 1 µM α-MSH (red). Measurements were carried out three hours (day 0) and 48 hours (day 2) following stimulation with 1 µM α-MSH. Error bars represent SEM. The MC1R polymorphisms are described in Table 1.

Figure 3 cAMP production and proliferation of fibroblasts with MC1R polymorphisms following α-MSH stimulation. Intracellular cAMP concentration was measured in fibroblasts with MC1R polymorphisms (A, n = 3, C, n = 3 and E, n = 3) untreated (black bars) or treated with 1 µM α-MSH for 20 min (red). Proliferation of fibroblasts with MC1R polymorphisms (B, n = 12, D, n = 12 and F, n = 12) untreated (black) or treated with 1 µM α-MSH (red). Measurements were carried out three hours (day 0) and 48 hours (day 2) following stimulation with 1 µM α-MSH. Error bars represent SEM. The polymorphisms are described in Table 1.

Figure 4 Comparison of cAMP levels and proliferation rate in MC1R_wt and MC1R polymorphic melanocytes and fibroblasts. Intracellular cAMP levels after stimulation with 1 µM α-MSH were compared in MC1R_wt (grey) and MC1R polymorphic (MC1R_SNP) (blue) melanocytes (A) and fibroblasts (C). The differences in cell proliferation with and without α-MSH treatment (RFU after α-MSH stimulation—RFU of untreated cells) were compared in MC1R_wt and MC1R_SNP melanocytes (B) and fibroblasts (D). The value distributions are presented as box plots.

Table 1 Characterisation of the MC1R variants in melanocyte and fibroblast cell lines

Melanocyte cell line	Base-exchange non coding region	Base-exchange coding region	Term for the polymorphism
B-011	Base 152 c → c/t Base 286 a → g	Base 1583 aca → ac(g/a) As 314 Thr → mute	wt
I-014	none	Base 915 gtg → atg AS 92 Val → Met	V92M (homozygous)
M-012	Base 152 c → c/t Base 197 g → a/g Base 416 a → a/t	Base 1583 aca → ac(g/a) As 314 Thr → mute	wt
E-013	none	Base 1066 cgc → c(a/g)c AS 142 Arg → His Base 1583 aca → acg As 314 Thr → mute	R142H (heterozygous)

Fibroblast cell line	Base-exchange non coding region	Base-exchange coding region	Term for the polymorphism
H-211	Base 152 c → c/t Base 197 g → a/g Base 416 a → a/t	none	wt
C-212	Base 152 c → c/t Base 197 g → a/g Base 416 a → a/t	Base 1129 cga → c(a/g)a AS 163 Arg → Gln	R163Q (heterozygous)
K-214	Base 152 c → c/t Base 197 g → a/g Base 416 a → a/t	Base 819 gtg → (t/g)tg AS 60 Val → Leu	V60L (heterozygous)
D-213	none	Base 1092 cgc → (t/c)gc AS 151 Arg → Cys	R151C (heterozygous)

Acknowledgments

We are grateful to Prof. Dr. M. Hoth, Drs. A.S. Wenning, E. Schwarz, I. Bogeski and C. Kummerow for their help and suggestions. We thank A. Stark, A. Weinhold and H. Palm for their technical assistance.