Arch Dermatol Res DOI 10.1007/s00403-013-1436-4
CONCISE COMMUNICATION
Investigation of four novel male androgenetic alopecia susceptibility loci: no association with female pattern hair loss Rima Nuwaihyd • Silke Redler • Stefanie Heilmann • Dmitriy Drichel • Sabrina Wolf • Pattie Birch • Kathy Dobson • Gerhard Lutz • Kathrin A. Giehl • Roland Kruse • Rachid Tazi-Ahnini • Sandra Hanneken Markus Bo¨hm • Anja Miesel • Tobias Fischer • Hans Wolff • Tim Becker • Natalie Garcia-Bartels • Ulrike Blume-Peytavi • Markus M. No¨then • Andrew G. Messenger • Regina C. Betz
•
Received: 15 August 2013 / Revised: 19 November 2013 / Accepted: 6 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Female pattern hair loss (FPHL) is a common hair loss disorder in women and has a complex mode of inheritance. The etiopathogenesis of FPHL is largely unknown; however, it is hypothesized that FPHL and male pattern baldness [androgenetic alopecia (AGA)] share common genetic susceptibility alleles. Our recent findings indicate that the major AGA locus, an X-chromosome region containing the androgen receptor and the ectodysplasin A2 receptor (EDA2R) genes, may represent a common genetic factor underlying both early-onset FPHL and AGA. This gives further support for the widespread assumption of shared susceptibility loci for FPHL and AGA. However, we could not demonstrate association of further AGA risk loci, including 20p11, 1p36.22, 2q37.3,
7p21.1, 7q11.22, 17q21.31, and 18q21.1, with FPHL. Interestingly, a recent study identified four novel AGA risk loci in chromosomal regions 2q35, 3q25.1, 5q33.3, and 12p12.1. In particular, the 2q35 locus and its gene WNT10A point to an as-yet unknown involvement of the WNT signaling pathway in AGA. We hypothesized that the novel loci and thus also the WNT signaling may have a role in the etiopathogenesis of FPHL and therefore examined the role of these novel AGA risk loci in our FPHL samples comprising 440 German and 145 UK affected patients, 500 German unselected controls (blood donors), and 179 UK supercontrols. Patients and controls were genotyped for the top two single nucleotide polymorphisms at each of the four AGA loci. However, none of the genotyped variants displayed any significant association. In conclusion, the
R. Nuwaihyd and S. Redler have contributed equally to this work. R. Nuwaihyd S. Redler (&) S. Heilmann S. Wolf M. M. No¨then R. C. Betz Institute of Human Genetics, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany e-mail: [email protected] R. Nuwaihyd N. Garcia-Bartels U. Blume-Peytavi Department of Dermatology and Allergy, Clinical Research Center for Hair and Skin Science, Charite´-Universita¨tsmedizin Berlin, Charite´platz 1, 10117 Berlin, Germany S. Heilmann M. M. No¨then Department of Genomics, Life and Brain Center, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany D. Drichel T. Becker German Center for Neurodegenerative Diseases (DZNE), Sigmund-Freud-Str. 25, 53127 Bonn, Germany
G. Lutz Dermatological Practice, Hair and Nail, Wesseling, Kronenweg 84, 50389 Wesseling, Germany K. A. Giehl H. Wolff Department of Dermatology, University of Munich, Frauenlobstr. 9-11, 83125 Munich, Germany R. Kruse Dermatological Practice, Paderborn, Alte Brauerei 11, 33098 Paderborn, Germany R. Tazi-Ahnini Department of Infection and Immunity, University of Sheffield, Sheffield S10 2JF, UK S. Hanneken Department of Dermatology, Medical Faculty, University of Du¨sseldorf, Moorenstr. 5, 40225 Du¨sseldorf, Germany
P. Birch K. Dobson A. G. Messenger Department of Dermatology, Royal Hallamshire Hospital, Sheffield S10 2JF, UK
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results of this study provide no support for the hypothesis that the novel AGA loci influence susceptibility to FPHL.
M. Bo¨hm Laboratory for Neuroendocrinology of the Skin and Interdisciplinary Endocrinology, Department of Dermatology, University of Mu¨nster, Von-Esmarch-Str. 58, 48149 Mu¨nster, Germany
AGA loci at 20p11, 1p36.22, 2q37.3, 7p21.1, 7q11.22, 17q21.31, and 18q21.1 [10] in the origin of FPHL could not be shown (Redler et al. [19]). Interestingly, a recent study reported four novel male AGA risk loci 2q35, 5q33.3, 12p12.1, and 3q25.1 [8]. These findings provide exciting new insights into the etiopathogenesis of male AGA, and for the first time clearly point to an androgen-independent biological pathway. The chromosomal region 2q35 encompasses distinct genes of the WNT family, which play a crucial role in the development of a large number of common human disorders [2, 4]. Although the importance of WNT signaling for hair cycling and development has been known for many years [1, 12], a contribution of the WNT pathway to the origin of AGA was not discovered yet. The two top variants identified at 2q35 are both located within intronic regions of WNT genes: rs7349332 within WNT10A (wingless-type MMTV integration site family, member 10A) and rs10193725 within WNT6 (wingless-type MMTV integration site family, member 6). Furthermore, carriers of the risk allele of rs7349332 demonstrated the significantly reduced expression of WNT10A in human hair follicles [8]. As WNT10A is thought to be implicated in anagen induction [16], reduced expression might lead to alteration of the hair cycle by, for example, shortening anagen duration [8]. The recent study therefore provides the first insight into understanding the mechanisms by which WNT signaling may regulate hair cycling in AGA patients [8]. Further support for the involvement of the WNT pathway in AGA is provided by the association at chromosome 12p12.1. The two most significant single-nucleotide polymorphisms (SNPs) at these loci, rs9668810 and rs7975017, are located between the genes SSPN (sarcospan) and ITPR2 (inositol 1,4,5-triphosphate receptor, type 2), both of which are expressed in hair follicles [8]. ITPR2 is thought to function as a receptor for IP3 [25], involved in the WNT pathway. No candidate genes or underlying disease-causing mechanisms have yet been identified for the 3q25.1 and 5q33.3 loci; hence, it is not known if these regions have any functional connection with androgen, WNT or distinct pathways [8]. Based on a hypothesis of a shared genetic background between FPHL and AGA, and a possible involvement of the WNT pathway in disease etiology, we investigated whether these newly identified AGA susceptibility loci are associated with FPHL.
A. Miesel T. Fischer Department of Dermatology, University of Lu¨beck, Ratzeburger Allee 160, 23538 Lu¨beck, Germany
Materials and methods
T. Becker Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany
We selected a total of eight SNPs, consisting of the top two variants of each of the four novel AGA loci, 2q35, 3q25.1, 5q33.3, and 12p12.1 [8].
Keywords Female pattern hair loss Association study Novel AGA susceptibility loci WNT signaling Introduction Androgenetic alopecia (AGA) is the most frequent hair loss disorder in both sexes, termed male pattern baldness (AGA) in men and female pattern hair loss (FPHL) in women. FPHL affects approximately 12 % of women by the age of 30 years and 30–40 % of women by the age of 70 years [3, 6, 13]. The clinical course of FPHL is typically characterized by diffuse and progressive thinning of the hair at the crown, classified according to the scales of Ludwig [11] and Sinclair [22]. Understanding of the etiology of FPHL is incomplete. Recent studies suggest an involvement of the sex steroid hormone receptor estrogen receptor 2 (ESR2) in the development of FPHL [26]. The role of further sexual steroid hormones remains elusive [17, 20, 27]. Another widespread assumption of disease origin is a shared genetic background between FPHL and AGA and common disease-causing mechanisms. This hypothesis is based on the presence of elevated androgen levels and male pattern hair loss in some affected women, the identical histology of FPHL and AGA [7, 11, 21, 22, 24], and the common occurrence of both FPHL and AGA in individual families [9, 14, 23]. At present, several AGA susceptibility loci with genome-wide significance have been identified. Many of these loci are assumed to be involved in androgen metabolism, which implies that an androgen-dependent mechanism is of crucial importance in the development of AGA [5, 10]. Our own recent studies suggest that the X-chromosomal ectodysplasin A2 receptor (EDA2R) gene may be specifically involved in the pathogenesis of early onset FPHL [18]; however, these results stress the need for independent replication in larger samples. An involvement of the male
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The eight selected SNPs were genotyped in the following samples: hair clinic outpatients (440 unrelated German and 145 UK individuals), healthy and unrelated female German blood donors (500), and UK supercontrols (179 women [60 years without FPHL). The inclusion criterion was the presence of FPHL of grade 2–3 according to the Ludwig scale [11], or grade 2–5 FPHL according to the Sinclair scale [22]. Ninety-nine women (German, n = 57; UK, n = 42) presented with a severe form of FPHL (Ludwig grade 3, Sinclair grade 4–5). A total of 290 patients (German, n = 233; UK, n = 57) reported an early-age-of-onset (B40 years). Ethical approval was obtained from the respective Ethics Committee. The study was conducted in concordance with the Declaration of Helsinki Principles. Genotyping was performed in multiplex reactions using the MassARRAY system and a Sequenom Compact MALDI-TOF device (Sequenom Inc., San Diego, CA, USA). For the association analysis, the following SNP quality measures were used as exclusion criteria: minor allele frequency \1 %; P \ 0.05 for Hardy– Weinberg equilibrium in controls; and SNP call rates of \95 %. After applying these criteria, all eight SNPs remained eligible for analysis in both German and UK samples. In the first step, separate analyses were performed for the German and UK samples (Tables 1, 2). In both samples, patients with severe expression of the disease and early-age-of-onset were separately analyzed (Tables 1, 2). In the second step, data from both the samples were included in a meta-analysis. For association testing, the Armitage trend test was used to detect allelic and genotypic effects [15]. Power was calculated using the power.fisher.exact function in R (R version 2.10.1, http://www.rproject.org/).
Results Assuming a risk allele frequency of 0.3, our German and UK samples had 49 and 32 % power, respectively, at the 0.05 level to detect an effect of the magnitude observed for the AGA locus 2q35 [8] for a genotype relative risk (GRR) of 1.34 for the heterozygous and 1.79 for the homozygous risk genotype. In the overall German and UK samples, no significant differences in allele or genotype frequencies were found between patients and controls for any of the investigated variants (Tables 1, 2). The results remained negative in subgroups of patients with severe disease and early-age-ofonset (Tables 1, 2). A meta-analysis of the combined German and UK data also generated no significant results (data not shown).
Discussion To our knowledge, this study is the first to investigate the contribution of the novel AGA susceptibility loci to the development of FPHL. We were unable to detect any significant association for the genotyped variants of 2q35, 3q25.1, 5q33.3, and 12p12 in either the overall German and UK samples or the subgroups of severely and early affected individuals. Our findings, therefore, do not support the hypothesis of an association between FPHL and the genotyped variants. The most likely explanation for our negative findings is that the novel AGA loci are not involved in the etiopathogenesis of FPHL and they do not represent overlapping genetic factors between the two common hair loss disorders. Our negative association findings might also be attributable to the relatively small sample sizes used, which may have precluded the detection of small effects. However, our combined sample did have sufficient power to detect larger effects. Population stratification is an unlikely explanation for the negative association results in our present study, as the FPHL sample as well as the male AGA sample was of Central European origin. Our negative findings may also have been due to the limited number of genotyped variants, since we did not attempt to cover the genes completely by a tagging SNP approach. However, we genotyped the top two variants of each of the four novel AGA loci, which were identified in a combined analyses of a replication set of nearly 2,800 AGA patients and a large-scale meta-analysis of seven genome-wide association studies (GWAS) of 3,891 men with AGA. All of the eight variants displayed genome-wide significant association results. Therefore, these variants must be considered as those that are most likely to confer disease risk or be in high linkage disequilibrium with causal variants. Based on the hypothesis of shared genetic risk variants between AGA and FPHL, it is most likely that variants associated with AGA are also associated with FPHL. Nevertheless, we cannot completely exclude that distinct variants of the four loci not genotyped by us are associated with FPHL. The analyses presented here are important as they demonstrate that loci of the WNT signaling pathway are most likely not involved in the pathogenesis of FPHL. Taken together, our findings and the negative association results for the AGA loci 20p11, 1p36.22, 2q37.3, 7p21.1, 7q11.22, 17q21.31, and 18q21.1 (Redler et al. [18, 19]) provide further evidence for the distinct underlying disease-causing mechanisms in FPHL and AGA. Future genetic studies of FPHL should therefore not only be limited to prior assumptions concerning possible pathophysiological pathways, but also include systematic genome-wide approaches. This might lead to the identification
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d
c
b
a
26426420
26428793
rs7975017
158381512
rs1081073
rs9668810
158310631
rs929626
151639765
151653368
rs7648585
rs4679955
a
T/C
T/C
T/A
C/T
T/A
G/A
T/C
C/T
AlleleA/ Allele B
T/C
T/C
T/A
T/A C/T
G/A
T/C
C/T
AlleleA/ Allele B
0.049
0.073
0.22
0.175
0.293
0.195
0
0.024
AA
MAF indicates minor allele frequency
CI confidence interval, OR odds ratio
P values were calculated using the Armitage Trend Test
0.341
0.415
0.414
0.475
0.366
0.415
0.268
0.366
AB
Severe cases
0.05
0.081
0.247
0.187 0.254
0.157
0.017
0.06
AA
National Center for Biotechnology Information (NCBI) build 37.3
12p12.1
5q33.3
3q25.1
219726498
219756383
rs10193725
rs7349332
2q35
Position (bp)
SNP
26428793
rs7975017
Genomic region
26426420
158381512
rs1081073
5q33.3
rs9668810
151653368 158310631
rs4679955 rs929626
12p12.1
151639765
rs7648585
219756383
rs7349332
3q25.1
219726498
rs10193725
2q35
Position (bp)a
SNP
Genomic region
0.61
0.512
0.366
0.35
0.341
0.39
0.732
0.61
BB
0.331
0.367
0.479
0.482 0.448
0.46
0.236
0.318
AB
German controls
b
0.22
0.28
0.427
0.413
0.476
0.04
0.134
0.207
MAF
0.619
0.552
0.274
0.331 0.298
0.383
0.747
0.622
BB
0.928
0.761
0.311
0.279
0.41
0.788
0.984
0.81
P-values
c
0.215 (T)
0.265 (T)
0.486 (T)
0.428 (T) 0.478 (C)
0.387 (G)
0.135 (T)
0.219 (C)
MAF
b
1.03 (0.59–1.77)
1.08 (0.66–1.79)
0.79 (0.5–1.24)
0.77 (0.48–1.22)
1.21 (0.77–1.91)
1.07 (0.67–1.69)
0.99 (0.51–1.92)
0.93 (0.54–1.63)
d
0.346
0.41
0.483
0.478 0.474
0.478
0.207
0.31
AB
OR (95 % CI)
0.057
0.073
0.251
0.176 0.226
0.148
0.016
0.029
AA
All German cases
0.23
0.278
0.492
0.415 0.463
0.387
0.12
0.185
MAFb
0.06
0.082
0.251
0.23
0.169
0.155
0.014
0.018
AA
0.333
0.384
0.493
0.469
0.493
0.466
0.214
0.343
AB
0.607
0.534
0.256
0.301
0.338
0.379
0.772
0.226
0.274
0.498
0.464
0.416
0.388
0.121
0.189
MAFb
0.448
0.525
0.813
0.574 0.539
0.993
0.325
0.169
Pvaluesc
0.639
BB
Early age of onset
0.597
0.517
0.266
0.346 0.3
0.374
0.777
0.661
BB
Table 1 Case–control association analysis for SNPs within 2q35, 3q25.1, 5q33.3 and 12p12.1 in the overall German case–control sample and subgroups
0.654
0.72
0.699
0.646
0.671
0.969
0.468
0.209
P-valuesc
1.07 (0.81–1.4)
1.05 (0.81–1.35)
1.05 (0.83–1.31)
0.95 (0.75–1.9)
0.95 (0.76–1.2)
1 (0.8–1.27)
0.88 (0.63–1.24)
0.83 (0.63–1.12)
OR (95 % CI)d
1.09 (0.87–1.36)
1.07 (0.87–1.31)
1.02 (0.85–1.23)
0.95 (0.79–1.14) 0.94 (0.78–1.13)
1 (0.83–1.21)
0.87 (0.66–1.15)
0.81 (0.64–1.01)
OR (95 % CI)d
Arch Dermatol Res
d
c
b
a
26426420
26428793
rs9668810
158381512
rs7975017
158310631
rs929626
rs1081073
151653368
rs4679955
a
T/C
T/C
T/A
C/T
T/A
G/A
T/C
C/T
MAF indicates minor allele frequency
CI confidence interval, OR odds ratio
P values were calculated using the Armitage Trend Test
0.332
0.36
0.584
0.579
0.472
0.258 0.472
0.367
AB
0.04
0.04
0.16
0.2
0.16
0.12
0
0
AA
0.28
0.32
0.68
0.6
0.32
0.36
0.24
0.32
AB
Severe cases
0.067
0.084
0.186
0.157
0.135
0.017 0.118
0.034
AA
AlleleA/Allele B
T/C
T/C
T/A
C/T
T/A
T/C G/A
C/T
AlleleA/ Allele B
National Center for Biotechnology Information (NCBI) build 37.3
12p12.1
5q33.3
151639765
219756383
rs7648585
rs7349332
3q25.1
219726498
rs10193725
2q35
Position (bp)
26428793
rs7975017
SNP
26426420
158381512
rs9668810
Genomic region
12p12.1
rs1081073
151653368 158310631
rs4679955
3q25.1
rs929626
rs7349332 rs7648585
5q33.3
219726498 219756383 151639765
rs10193725
2q35
Position (bp)a
SNP
Genomic region
UK controls
0.68
0.64
0.16
0.2
0.52
0.52
0.76
0.68
BB
0.18
0.2
0.5
0.5
0.32
0.3
0.12
0.16
b
0.405
0.335
0.74
0.435
0.486
0.446
0.607
0.316
P-values
0.233 (T)
0.264 (T)
0.478 (T)
0.447 (C)
0.371 (T)
0.146 (T) 0.354 (G)
0.218 (C)
MAF
MAF
0.601
0.556
0.23
0.264
0.393
0.725 0.41
0.599
BB
b
c
0.308
0.345
0.549
0.517
0.482
0.289 0.479
0.357
AB
d
0.72 (0.34–1.55)
0.7 (0.36–1.45)
1.09 (0.61–1.98)
1.24 (0.69–2.24)
0.8 (0.42–1.5)
0.78 (0.41–1.49)
0.8 (0.32–1.97)
0.69 (0.31–1.52)
OR (95 % CI)
0.056
0.07
0.204
0.217
0.149
0.014 0.113
0.028
AA
All UK cases
0.21
0.243
0.479
0.476
0.39
0.158 0.352
0.206
MAFb
0.039
0.053
0.211
0.224
0.145
0.105
0.026
0.026
AA
0.316
0.368
0.526
0.526
0.5
0.5
0.29
0.395
AB
0.645
0.579
0.263
0.25
0.355
0.395
0.684
0.197
0.237
0.474
0.487
0.395
0.355
0.171
0.224
MAFb
0.494
0.556
0.971
0.439
0.616
0.655 0.961
0.718
Pvaluesc
0.579
BB
Early age of onset
0.636
0.585
0.247
0.266
0.369
0.697 0.408
0.615
BB
Table 2 Case–control association analysis for SNPs within 2q35, 3q25.1, 5q33.3 and 12p12.1 in the overall UK case–control sample and subgroups
0.383
0.528
0.932
0.371
0.607
0.977
0.471
0.872
P-valuesc
0.81 (0.51–1.29)
0.87 (0.56–1.35)
0.98 (0.67–1.44)
1.18 (0.8–1.72)
1.11 (0.75–1.63)
1.01 (0.68–1.5)
1.21 (0.72–2.02)
1.04 (0.66–1.64)
OR (95 % CI)d
0.87 (0.6–1.27)
0.89 (0.62–1.28)
1.01 (0.74–1.37)
1.12 (0.82–1.54)
1.09 (0.79–1.5)
1.1 (0.71–1.7) 0.99 (0.72–1.38)
0.94 (0.64–1.37)
OR (95 % CI)d
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of pathways specific to FPHL, which would be a groundbreaking achievement. Acknowledgments The authors thank all patients for their participation in the study. The authors thank the British Skin Foundation (AGM and RTA, 2005) for supporting the FPHL DNA collection. R.C.B. is recipient of a Heisenberg Professorship from the German Research Foundation (DFG). R.C.B. and M.M.N. are members of the DFG-funded Excellence-cluster ImmunoSensation. Conflict of interest The authors have no conflict of interest to declare.
References 1. Andl T, Reddy ST, Gaddapara T, Millar SE (2002) WNT signals are required for the initiation of hair follicle development. Dev Cell 2:643–653 2. Baron R, Kneissel M (2013) WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med 19:179–192 3. Birch MP, Messenger JF, Messenger AG (2001) Hair density, hair diameter and the prevalence of female pattern hair loss. Br J Dermatol 144:297–304 4. Bravo DT, Yang YL, Kuchenbecker K, Hung MS, Xu Z, Jablons DM, You L (2013) Frizzled-8 receptor is activated by the Wnt-2 ligand in non-small cell lung cancer. BMC Cancer 13:316 5. Brockschmidt FF, Hillmer AM, Eigelshoven S et al (2010) Fine mapping of the human AR/EDA2R locus in androgenetic alopecia. Br J Dermatol 162:899–903 6. Gan DC, Sinclair RD (2005) Prevalence of male and female pattern hair loss in Maryborough. J Investig Dermatol Symp Proc 10:184–189 7. Headington JT (1984) Transverse microscopic anatomy of the human scalp. A basis for a morphometric approach to disorders of the hair follicle. Arch Dermatol 120:449–456 8. Heilmann S, Kiefer AK, Fricker N et al (2013) Androgenetic alopecia: identification of four genetic risk loci and evidence for the contribution of WNT signaling to its etiology. J Invest Dermatol 133:1489–1496 9. Ku¨ster W, Happle R (1984) The inheritance of common baldness: two B or not two B? J Am Acad Dermatol 11:921–926 10. Li R, Brockschmidt FF, Kiefer AK et al (2012) Six novel susceptibility loci for early-onset androgenetic alopecia and their unexpected association with common diseases. PLoS Genet 8:e1002746 11. Ludwig E (1977) Classification of the types of androgenetic alopecia (common baldness) occurring in the female sex. Br J Dermatol 97:247–254 12. Millar SE, Willert K, Salinas PC, Roelink H, Nusse R, Sussman DJ, Barsh GS (1999) WNT signaling in the control of hair growth and structure. Dev Biol 207:133–149
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13. Norwood OT (2001) Incidence of female androgenetic alopecia (female pattern alopecia). Dermatol Surg 27:53–54 14. Nyholt DR, Gillespie NA, Heath AC, Martin NG (2003) Genetic basis of male pattern baldness. J Invest Dermatol 121:1561–1564 15. Armitage P (1955) Tests for linear trends in proportions and frequencies. Biometrics 11(3):375–386 16. Reddy S, Andl T, Bagasra A, Lu MM, Epstein DJ, Morrisey EE, Millar SE (2001) Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of sonic hedgehog in hair follicle morphogenesis. Mech Dev 107:69–82 17. Redler S, Birch MP, Drichel D et al (2011) Investigation of variants of the aromatase gene (CYP19A1) in female pattern hair loss. Br J Dermatol 165:703–705 18. Redler S, Brockschmidt FF, Tazi-Ahnini R et al (2012) Investigation of the male pattern baldness major genetic susceptibility loci AR/EDA2R and 20p11 in female pattern hair loss. Br J Dermatol 166:1314–1318 19. Redler S, Dobson K, Drichel D, Heilmann S, wolf S, Brockschmidt FF, Tazi-Ahnini R, Birch P et al (2013) Investigation of six novel loci for male androgenetic alopecia in women with female pattern hair loss.J Dermatol Sci 72(2):186–188 20. Redler S, Tazi-Ahnini R, Drichel D et al (2012) Selected variants of the steroid-5-alpha-reductase isoforms SRD5A1 and SRD5A2 and the sex steroid hormone receptors ESR1, ESR2 and PGR: no association with female pattern hair loss identified. Exp Dermatol 21:390–393 21. Riedel-Baima B, Riedel A (2008) Female pattern hair loss may be triggered by low oestrogen to androgen ratio. Endocr Regul 42:13–16 22. Sinclair R, Jolley D, Mallari R, Magee J (2004) The reliability of horizontally sectioned scalp biopsies in the diagnosis of chronic diffuse telogen hair loss in women. J Am Acad Dermatol 51:189–199 23. Smith MA, Wells RS (1964) Male-type alopecia, alopecia areata, and normal hair in women; family histories. Arch Dermatol 89:95–98 24. Vexiau P, Chaspoux C, Boudou P, Fiet J, Abramovici Y, Rueda MJ, Hardy N, Reygagne P (2000) Role of androgens in femalepattern androgenetic alopecia, either alone or associated with other symptoms of hyperandrogenism. Arch Dermatol Res 292:598–604 25. Wang HY, Malbon CC (2004) Wnt-frizzled signaling to G-protein-coupled effectors. Cell Mol Life Sci 61:69–75 26. Yip L, Zaloumis S, Irwin D, Severi G, Hopper J, Giles G, Harrap S, Sinclair R, Ellis J (2012) Association analysis of oestrogen receptor beta gene (ESR2) polymorphisms with female pattern hair loss. Br J Dermatol 166:1131–1134 27. Yip L, Zaloumis S, Irwin D, Severi G, Hopper J, Giles G, Harrap S, Sinclair R, Ellis J (2009) Gene-wide association study between the aromatase gene (CYP19A1) and female pattern hair loss. Br J Dermatol 161:289–294
CONCISE COMMUNICATION
Investigation of four novel male androgenetic alopecia susceptibility loci: no association with female pattern hair loss Rima Nuwaihyd • Silke Redler • Stefanie Heilmann • Dmitriy Drichel • Sabrina Wolf • Pattie Birch • Kathy Dobson • Gerhard Lutz • Kathrin A. Giehl • Roland Kruse • Rachid Tazi-Ahnini • Sandra Hanneken Markus Bo¨hm • Anja Miesel • Tobias Fischer • Hans Wolff • Tim Becker • Natalie Garcia-Bartels • Ulrike Blume-Peytavi • Markus M. No¨then • Andrew G. Messenger • Regina C. Betz
•
Received: 15 August 2013 / Revised: 19 November 2013 / Accepted: 6 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Female pattern hair loss (FPHL) is a common hair loss disorder in women and has a complex mode of inheritance. The etiopathogenesis of FPHL is largely unknown; however, it is hypothesized that FPHL and male pattern baldness [androgenetic alopecia (AGA)] share common genetic susceptibility alleles. Our recent findings indicate that the major AGA locus, an X-chromosome region containing the androgen receptor and the ectodysplasin A2 receptor (EDA2R) genes, may represent a common genetic factor underlying both early-onset FPHL and AGA. This gives further support for the widespread assumption of shared susceptibility loci for FPHL and AGA. However, we could not demonstrate association of further AGA risk loci, including 20p11, 1p36.22, 2q37.3,
7p21.1, 7q11.22, 17q21.31, and 18q21.1, with FPHL. Interestingly, a recent study identified four novel AGA risk loci in chromosomal regions 2q35, 3q25.1, 5q33.3, and 12p12.1. In particular, the 2q35 locus and its gene WNT10A point to an as-yet unknown involvement of the WNT signaling pathway in AGA. We hypothesized that the novel loci and thus also the WNT signaling may have a role in the etiopathogenesis of FPHL and therefore examined the role of these novel AGA risk loci in our FPHL samples comprising 440 German and 145 UK affected patients, 500 German unselected controls (blood donors), and 179 UK supercontrols. Patients and controls were genotyped for the top two single nucleotide polymorphisms at each of the four AGA loci. However, none of the genotyped variants displayed any significant association. In conclusion, the
R. Nuwaihyd and S. Redler have contributed equally to this work. R. Nuwaihyd S. Redler (&) S. Heilmann S. Wolf M. M. No¨then R. C. Betz Institute of Human Genetics, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany e-mail: [email protected] R. Nuwaihyd N. Garcia-Bartels U. Blume-Peytavi Department of Dermatology and Allergy, Clinical Research Center for Hair and Skin Science, Charite´-Universita¨tsmedizin Berlin, Charite´platz 1, 10117 Berlin, Germany S. Heilmann M. M. No¨then Department of Genomics, Life and Brain Center, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany D. Drichel T. Becker German Center for Neurodegenerative Diseases (DZNE), Sigmund-Freud-Str. 25, 53127 Bonn, Germany
G. Lutz Dermatological Practice, Hair and Nail, Wesseling, Kronenweg 84, 50389 Wesseling, Germany K. A. Giehl H. Wolff Department of Dermatology, University of Munich, Frauenlobstr. 9-11, 83125 Munich, Germany R. Kruse Dermatological Practice, Paderborn, Alte Brauerei 11, 33098 Paderborn, Germany R. Tazi-Ahnini Department of Infection and Immunity, University of Sheffield, Sheffield S10 2JF, UK S. Hanneken Department of Dermatology, Medical Faculty, University of Du¨sseldorf, Moorenstr. 5, 40225 Du¨sseldorf, Germany
P. Birch K. Dobson A. G. Messenger Department of Dermatology, Royal Hallamshire Hospital, Sheffield S10 2JF, UK
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results of this study provide no support for the hypothesis that the novel AGA loci influence susceptibility to FPHL.
M. Bo¨hm Laboratory for Neuroendocrinology of the Skin and Interdisciplinary Endocrinology, Department of Dermatology, University of Mu¨nster, Von-Esmarch-Str. 58, 48149 Mu¨nster, Germany
AGA loci at 20p11, 1p36.22, 2q37.3, 7p21.1, 7q11.22, 17q21.31, and 18q21.1 [10] in the origin of FPHL could not be shown (Redler et al. [19]). Interestingly, a recent study reported four novel male AGA risk loci 2q35, 5q33.3, 12p12.1, and 3q25.1 [8]. These findings provide exciting new insights into the etiopathogenesis of male AGA, and for the first time clearly point to an androgen-independent biological pathway. The chromosomal region 2q35 encompasses distinct genes of the WNT family, which play a crucial role in the development of a large number of common human disorders [2, 4]. Although the importance of WNT signaling for hair cycling and development has been known for many years [1, 12], a contribution of the WNT pathway to the origin of AGA was not discovered yet. The two top variants identified at 2q35 are both located within intronic regions of WNT genes: rs7349332 within WNT10A (wingless-type MMTV integration site family, member 10A) and rs10193725 within WNT6 (wingless-type MMTV integration site family, member 6). Furthermore, carriers of the risk allele of rs7349332 demonstrated the significantly reduced expression of WNT10A in human hair follicles [8]. As WNT10A is thought to be implicated in anagen induction [16], reduced expression might lead to alteration of the hair cycle by, for example, shortening anagen duration [8]. The recent study therefore provides the first insight into understanding the mechanisms by which WNT signaling may regulate hair cycling in AGA patients [8]. Further support for the involvement of the WNT pathway in AGA is provided by the association at chromosome 12p12.1. The two most significant single-nucleotide polymorphisms (SNPs) at these loci, rs9668810 and rs7975017, are located between the genes SSPN (sarcospan) and ITPR2 (inositol 1,4,5-triphosphate receptor, type 2), both of which are expressed in hair follicles [8]. ITPR2 is thought to function as a receptor for IP3 [25], involved in the WNT pathway. No candidate genes or underlying disease-causing mechanisms have yet been identified for the 3q25.1 and 5q33.3 loci; hence, it is not known if these regions have any functional connection with androgen, WNT or distinct pathways [8]. Based on a hypothesis of a shared genetic background between FPHL and AGA, and a possible involvement of the WNT pathway in disease etiology, we investigated whether these newly identified AGA susceptibility loci are associated with FPHL.
A. Miesel T. Fischer Department of Dermatology, University of Lu¨beck, Ratzeburger Allee 160, 23538 Lu¨beck, Germany
Materials and methods
T. Becker Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany
We selected a total of eight SNPs, consisting of the top two variants of each of the four novel AGA loci, 2q35, 3q25.1, 5q33.3, and 12p12.1 [8].
Keywords Female pattern hair loss Association study Novel AGA susceptibility loci WNT signaling Introduction Androgenetic alopecia (AGA) is the most frequent hair loss disorder in both sexes, termed male pattern baldness (AGA) in men and female pattern hair loss (FPHL) in women. FPHL affects approximately 12 % of women by the age of 30 years and 30–40 % of women by the age of 70 years [3, 6, 13]. The clinical course of FPHL is typically characterized by diffuse and progressive thinning of the hair at the crown, classified according to the scales of Ludwig [11] and Sinclair [22]. Understanding of the etiology of FPHL is incomplete. Recent studies suggest an involvement of the sex steroid hormone receptor estrogen receptor 2 (ESR2) in the development of FPHL [26]. The role of further sexual steroid hormones remains elusive [17, 20, 27]. Another widespread assumption of disease origin is a shared genetic background between FPHL and AGA and common disease-causing mechanisms. This hypothesis is based on the presence of elevated androgen levels and male pattern hair loss in some affected women, the identical histology of FPHL and AGA [7, 11, 21, 22, 24], and the common occurrence of both FPHL and AGA in individual families [9, 14, 23]. At present, several AGA susceptibility loci with genome-wide significance have been identified. Many of these loci are assumed to be involved in androgen metabolism, which implies that an androgen-dependent mechanism is of crucial importance in the development of AGA [5, 10]. Our own recent studies suggest that the X-chromosomal ectodysplasin A2 receptor (EDA2R) gene may be specifically involved in the pathogenesis of early onset FPHL [18]; however, these results stress the need for independent replication in larger samples. An involvement of the male
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The eight selected SNPs were genotyped in the following samples: hair clinic outpatients (440 unrelated German and 145 UK individuals), healthy and unrelated female German blood donors (500), and UK supercontrols (179 women [60 years without FPHL). The inclusion criterion was the presence of FPHL of grade 2–3 according to the Ludwig scale [11], or grade 2–5 FPHL according to the Sinclair scale [22]. Ninety-nine women (German, n = 57; UK, n = 42) presented with a severe form of FPHL (Ludwig grade 3, Sinclair grade 4–5). A total of 290 patients (German, n = 233; UK, n = 57) reported an early-age-of-onset (B40 years). Ethical approval was obtained from the respective Ethics Committee. The study was conducted in concordance with the Declaration of Helsinki Principles. Genotyping was performed in multiplex reactions using the MassARRAY system and a Sequenom Compact MALDI-TOF device (Sequenom Inc., San Diego, CA, USA). For the association analysis, the following SNP quality measures were used as exclusion criteria: minor allele frequency \1 %; P \ 0.05 for Hardy– Weinberg equilibrium in controls; and SNP call rates of \95 %. After applying these criteria, all eight SNPs remained eligible for analysis in both German and UK samples. In the first step, separate analyses were performed for the German and UK samples (Tables 1, 2). In both samples, patients with severe expression of the disease and early-age-of-onset were separately analyzed (Tables 1, 2). In the second step, data from both the samples were included in a meta-analysis. For association testing, the Armitage trend test was used to detect allelic and genotypic effects [15]. Power was calculated using the power.fisher.exact function in R (R version 2.10.1, http://www.rproject.org/).
Results Assuming a risk allele frequency of 0.3, our German and UK samples had 49 and 32 % power, respectively, at the 0.05 level to detect an effect of the magnitude observed for the AGA locus 2q35 [8] for a genotype relative risk (GRR) of 1.34 for the heterozygous and 1.79 for the homozygous risk genotype. In the overall German and UK samples, no significant differences in allele or genotype frequencies were found between patients and controls for any of the investigated variants (Tables 1, 2). The results remained negative in subgroups of patients with severe disease and early-age-ofonset (Tables 1, 2). A meta-analysis of the combined German and UK data also generated no significant results (data not shown).
Discussion To our knowledge, this study is the first to investigate the contribution of the novel AGA susceptibility loci to the development of FPHL. We were unable to detect any significant association for the genotyped variants of 2q35, 3q25.1, 5q33.3, and 12p12 in either the overall German and UK samples or the subgroups of severely and early affected individuals. Our findings, therefore, do not support the hypothesis of an association between FPHL and the genotyped variants. The most likely explanation for our negative findings is that the novel AGA loci are not involved in the etiopathogenesis of FPHL and they do not represent overlapping genetic factors between the two common hair loss disorders. Our negative association findings might also be attributable to the relatively small sample sizes used, which may have precluded the detection of small effects. However, our combined sample did have sufficient power to detect larger effects. Population stratification is an unlikely explanation for the negative association results in our present study, as the FPHL sample as well as the male AGA sample was of Central European origin. Our negative findings may also have been due to the limited number of genotyped variants, since we did not attempt to cover the genes completely by a tagging SNP approach. However, we genotyped the top two variants of each of the four novel AGA loci, which were identified in a combined analyses of a replication set of nearly 2,800 AGA patients and a large-scale meta-analysis of seven genome-wide association studies (GWAS) of 3,891 men with AGA. All of the eight variants displayed genome-wide significant association results. Therefore, these variants must be considered as those that are most likely to confer disease risk or be in high linkage disequilibrium with causal variants. Based on the hypothesis of shared genetic risk variants between AGA and FPHL, it is most likely that variants associated with AGA are also associated with FPHL. Nevertheless, we cannot completely exclude that distinct variants of the four loci not genotyped by us are associated with FPHL. The analyses presented here are important as they demonstrate that loci of the WNT signaling pathway are most likely not involved in the pathogenesis of FPHL. Taken together, our findings and the negative association results for the AGA loci 20p11, 1p36.22, 2q37.3, 7p21.1, 7q11.22, 17q21.31, and 18q21.1 (Redler et al. [18, 19]) provide further evidence for the distinct underlying disease-causing mechanisms in FPHL and AGA. Future genetic studies of FPHL should therefore not only be limited to prior assumptions concerning possible pathophysiological pathways, but also include systematic genome-wide approaches. This might lead to the identification
123
123
d
c
b
a
26426420
26428793
rs7975017
158381512
rs1081073
rs9668810
158310631
rs929626
151639765
151653368
rs7648585
rs4679955
a
T/C
T/C
T/A
C/T
T/A
G/A
T/C
C/T
AlleleA/ Allele B
T/C
T/C
T/A
T/A C/T
G/A
T/C
C/T
AlleleA/ Allele B
0.049
0.073
0.22
0.175
0.293
0.195
0
0.024
AA
MAF indicates minor allele frequency
CI confidence interval, OR odds ratio
P values were calculated using the Armitage Trend Test
0.341
0.415
0.414
0.475
0.366
0.415
0.268
0.366
AB
Severe cases
0.05
0.081
0.247
0.187 0.254
0.157
0.017
0.06
AA
National Center for Biotechnology Information (NCBI) build 37.3
12p12.1
5q33.3
3q25.1
219726498
219756383
rs10193725
rs7349332
2q35
Position (bp)
SNP
26428793
rs7975017
Genomic region
26426420
158381512
rs1081073
5q33.3
rs9668810
151653368 158310631
rs4679955 rs929626
12p12.1
151639765
rs7648585
219756383
rs7349332
3q25.1
219726498
rs10193725
2q35
Position (bp)a
SNP
Genomic region
0.61
0.512
0.366
0.35
0.341
0.39
0.732
0.61
BB
0.331
0.367
0.479
0.482 0.448
0.46
0.236
0.318
AB
German controls
b
0.22
0.28
0.427
0.413
0.476
0.04
0.134
0.207
MAF
0.619
0.552
0.274
0.331 0.298
0.383
0.747
0.622
BB
0.928
0.761
0.311
0.279
0.41
0.788
0.984
0.81
P-values
c
0.215 (T)
0.265 (T)
0.486 (T)
0.428 (T) 0.478 (C)
0.387 (G)
0.135 (T)
0.219 (C)
MAF
b
1.03 (0.59–1.77)
1.08 (0.66–1.79)
0.79 (0.5–1.24)
0.77 (0.48–1.22)
1.21 (0.77–1.91)
1.07 (0.67–1.69)
0.99 (0.51–1.92)
0.93 (0.54–1.63)
d
0.346
0.41
0.483
0.478 0.474
0.478
0.207
0.31
AB
OR (95 % CI)
0.057
0.073
0.251
0.176 0.226
0.148
0.016
0.029
AA
All German cases
0.23
0.278
0.492
0.415 0.463
0.387
0.12
0.185
MAFb
0.06
0.082
0.251
0.23
0.169
0.155
0.014
0.018
AA
0.333
0.384
0.493
0.469
0.493
0.466
0.214
0.343
AB
0.607
0.534
0.256
0.301
0.338
0.379
0.772
0.226
0.274
0.498
0.464
0.416
0.388
0.121
0.189
MAFb
0.448
0.525
0.813
0.574 0.539
0.993
0.325
0.169
Pvaluesc
0.639
BB
Early age of onset
0.597
0.517
0.266
0.346 0.3
0.374
0.777
0.661
BB
Table 1 Case–control association analysis for SNPs within 2q35, 3q25.1, 5q33.3 and 12p12.1 in the overall German case–control sample and subgroups
0.654
0.72
0.699
0.646
0.671
0.969
0.468
0.209
P-valuesc
1.07 (0.81–1.4)
1.05 (0.81–1.35)
1.05 (0.83–1.31)
0.95 (0.75–1.9)
0.95 (0.76–1.2)
1 (0.8–1.27)
0.88 (0.63–1.24)
0.83 (0.63–1.12)
OR (95 % CI)d
1.09 (0.87–1.36)
1.07 (0.87–1.31)
1.02 (0.85–1.23)
0.95 (0.79–1.14) 0.94 (0.78–1.13)
1 (0.83–1.21)
0.87 (0.66–1.15)
0.81 (0.64–1.01)
OR (95 % CI)d
Arch Dermatol Res
d
c
b
a
26426420
26428793
rs9668810
158381512
rs7975017
158310631
rs929626
rs1081073
151653368
rs4679955
a
T/C
T/C
T/A
C/T
T/A
G/A
T/C
C/T
MAF indicates minor allele frequency
CI confidence interval, OR odds ratio
P values were calculated using the Armitage Trend Test
0.332
0.36
0.584
0.579
0.472
0.258 0.472
0.367
AB
0.04
0.04
0.16
0.2
0.16
0.12
0
0
AA
0.28
0.32
0.68
0.6
0.32
0.36
0.24
0.32
AB
Severe cases
0.067
0.084
0.186
0.157
0.135
0.017 0.118
0.034
AA
AlleleA/Allele B
T/C
T/C
T/A
C/T
T/A
T/C G/A
C/T
AlleleA/ Allele B
National Center for Biotechnology Information (NCBI) build 37.3
12p12.1
5q33.3
151639765
219756383
rs7648585
rs7349332
3q25.1
219726498
rs10193725
2q35
Position (bp)
26428793
rs7975017
SNP
26426420
158381512
rs9668810
Genomic region
12p12.1
rs1081073
151653368 158310631
rs4679955
3q25.1
rs929626
rs7349332 rs7648585
5q33.3
219726498 219756383 151639765
rs10193725
2q35
Position (bp)a
SNP
Genomic region
UK controls
0.68
0.64
0.16
0.2
0.52
0.52
0.76
0.68
BB
0.18
0.2
0.5
0.5
0.32
0.3
0.12
0.16
b
0.405
0.335
0.74
0.435
0.486
0.446
0.607
0.316
P-values
0.233 (T)
0.264 (T)
0.478 (T)
0.447 (C)
0.371 (T)
0.146 (T) 0.354 (G)
0.218 (C)
MAF
MAF
0.601
0.556
0.23
0.264
0.393
0.725 0.41
0.599
BB
b
c
0.308
0.345
0.549
0.517
0.482
0.289 0.479
0.357
AB
d
0.72 (0.34–1.55)
0.7 (0.36–1.45)
1.09 (0.61–1.98)
1.24 (0.69–2.24)
0.8 (0.42–1.5)
0.78 (0.41–1.49)
0.8 (0.32–1.97)
0.69 (0.31–1.52)
OR (95 % CI)
0.056
0.07
0.204
0.217
0.149
0.014 0.113
0.028
AA
All UK cases
0.21
0.243
0.479
0.476
0.39
0.158 0.352
0.206
MAFb
0.039
0.053
0.211
0.224
0.145
0.105
0.026
0.026
AA
0.316
0.368
0.526
0.526
0.5
0.5
0.29
0.395
AB
0.645
0.579
0.263
0.25
0.355
0.395
0.684
0.197
0.237
0.474
0.487
0.395
0.355
0.171
0.224
MAFb
0.494
0.556
0.971
0.439
0.616
0.655 0.961
0.718
Pvaluesc
0.579
BB
Early age of onset
0.636
0.585
0.247
0.266
0.369
0.697 0.408
0.615
BB
Table 2 Case–control association analysis for SNPs within 2q35, 3q25.1, 5q33.3 and 12p12.1 in the overall UK case–control sample and subgroups
0.383
0.528
0.932
0.371
0.607
0.977
0.471
0.872
P-valuesc
0.81 (0.51–1.29)
0.87 (0.56–1.35)
0.98 (0.67–1.44)
1.18 (0.8–1.72)
1.11 (0.75–1.63)
1.01 (0.68–1.5)
1.21 (0.72–2.02)
1.04 (0.66–1.64)
OR (95 % CI)d
0.87 (0.6–1.27)
0.89 (0.62–1.28)
1.01 (0.74–1.37)
1.12 (0.82–1.54)
1.09 (0.79–1.5)
1.1 (0.71–1.7) 0.99 (0.72–1.38)
0.94 (0.64–1.37)
OR (95 % CI)d
Arch Dermatol Res
123
Arch Dermatol Res
of pathways specific to FPHL, which would be a groundbreaking achievement. Acknowledgments The authors thank all patients for their participation in the study. The authors thank the British Skin Foundation (AGM and RTA, 2005) for supporting the FPHL DNA collection. R.C.B. is recipient of a Heisenberg Professorship from the German Research Foundation (DFG). R.C.B. and M.M.N. are members of the DFG-funded Excellence-cluster ImmunoSensation. Conflict of interest The authors have no conflict of interest to declare.
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