- Email: [email protected]
Implementation of an optimized strategy for genetic testing of the Chinese patients with oculocutaneous albinism
Journal of Dermatological Science 62 (2011) 124–127
Contents lists available at ScienceDirect
Journal of Dermatological Science journal homepage: www.elsevier.com/jds
Implementation of an optimized strategy for genetic testing of the Chinese patients with oculocutaneous albinism Aihua Wei a,b, Xiumin Yang a, Shi Lian c, Wei Li b,* a
Department of Dermatology, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Rd., Chaoyang District, Beijing 100101, China c Department of Dermatology, Xuan Wu Hospital, Capital Medical University, Beijing 100053, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 November 2010 Received in revised form 5 February 2011 Accepted 27 February 2011
Background: Oculocutaneous albinism (OCA) is a relatively common inherited disorder in all populations worldwide. The mutational spectra of OCA are population-specific. Objective: Based on our previous molecular epidemiological studies, we have implemented an optimized strategy for the genetic testing of Chinese OCA patients. Methods: Genomic DNA was extracted from the blood samples of 52 clinically diagnosed OCA patients and 100 unaffected subjects. The amplified DNA segments were screened for mutations of TYR, OCA2, TYRP1, SLC45A2 and HPS1 by direct sequencing. To exclude the previously unidentified alleles (PUAs) from polymorphisms, samples from 100 unaffected controls were sequenced for the same regions of variations. Results: Among the 52 OCA patients, 26 (50.0%) were found mutations on TYR gene, 8 (15.4%) on OCA2, 12 (23.1%) on SLC45A2, 2 (3.8%) on HPS1, and 4 (7.7%) patients uncharacterized. We identified 18 PUAs in these patients, 2 in TYR, 7 in OCA2, 8 in SLC45A2, and 1 in HPS1. Conclusion: The optimized method to screen the OCA mutations is efficiently implemented in the routine genetic testing of Chinese OCA patients accompanied with genetic counseling. ß 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Oculocutaneous albinism Hermansky-Pudlak syndrome Genetic testing Previously unidentified allele
1. Introduction Oculocutaneous albinism (OCA) is an autosomal recessive disorder with a relatively high incidence in Chinese Han population as estimated as 1:18,000 [1]. It manifests as a reduction or complete loss of melanin in the skin, hair, and eyes, often accompanied with eye symptoms such as photophobia, strabismus, moderate to severe visual impairment, and nystagmus. OCA could be caused by mutations in non-syndromic OCA genes (TYR, OCA2, TYRP1 and SLC45A2) or syndromic OCA genes (HPS1, HPS2, HPS3, HPS4, HPS5, HPS6, HPS7, HPS8, LYST, MYO5A, RAB27A and MLPH) [2]. OCA1 is the predominant form which accounts for about 70% of the Chinese OCA patients, while OCA2, OCA4 and HPS1 are less common, reflecting a population-specific distribution of different subtypes of OCA [3]. OCA is clinically characterized as OCA1A, OCA1B or OCA2. OCA1A presents a complete lack of tyrosinase activity and produces a totally depigmented phenotype with affected individuals exhibiting white hair, white skin, and blue, brown or pink iris
throughout life. OCA1B is characterized by reduction of tyrosinase activity. Individuals with OCA1B are born with white hair and then change to blond or yellow with age [4]. OCA2 is characterized by yellow, brown or golden hair at birth with or without darkening of hair color at later age. However, the clinically diagnosed subtypes of OCA could be mixed forms of molecularly diagnosed OCA subtypes, such as clinical OCA1 could be molecularly identified as OCA1, OCA2, OCA4 or HPS1, whereas clinical OCA2 could be OCA1, OCA2, OCA4, or HPS1 [3]. Hermansky-Pudlak syndrome (HPS) is a more severe form of OCA. Patients with HPS often die in their middle ages [5]. Therefore, the genetic testing of OCA is needed for routine diagnosis of OCA to better characterize the prognosis of OCA. Based on our previous genetic epidemiological studies of OCA in Chinese Han population, we have implemented an optimized strategy to molecularly screen the mutations on the known OCA genes. 2. Materials and methods 2.1. Study subjects
* Corresponding author. Tel.: +86 10 6484 8212; fax: +86 10 6484 8212. E-mail address: [email protected] (W. Li).
We recruited 52 unrelated OCA patients (Table 1) and 100 unaffected subjects from the Chinese Han population. The patients
0923-1811/$36.00 ß 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2011.02.009
A. Wei et al. / Journal of Dermatological Science 62 (2011) 124–127
125
Table 1 Genotypes of 52 Chinese OCA patients. Patient ID TYR 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 OCA2 27 28 29 30 31 32 33 34 SLC45A2 35 36 37 38 39 40 41 42 43 44 45 46 HPS1 47 48 Uncharacterized 49 50 51 52
Sex
Age
Clinical diagnosis
Molecular diagnosis
Mutational allele 1
Mutational allele 2
F M M M F F F M M F F F F M M M M F F M F F F M M M
8y 31y 1m 3m 25y 5y 5y 21y 22y 2y 3y 1m 1m 30y 29y 32y 2m 57y 2y 28y 5y 4m 32 11 1m 20y
OCA1A OCA OCA1 OCA1A OCA1A OCA1A OCA1A OCA1A OCA2 OCA1A OCA1A OCA1 OCA1 OCA1A OCA1A OCA1A OCA1 OCA1A OCA2 OCA1A OCA1B OCA1B OCA1B OCA2 OCA2 OCA1
OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1B OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1B OCA1A OCA1 OCA1B OCA1 OCA1A
c.896G>A (p.R299H) c.929insC c.346C>T (p.R116X) c.896G>A (p.R299H) c.758G>A (p.G253E) c.832C>T (p.R278X) c.832C>T (p.R278X) c.230G>A (p.R77Q) c.832C>T (p.R278X) c.714G>A (p.W238X)* c.832C>T (p.R278X) c.71G>A (p.C24Y) c.929insC c.1168C>G (p.H390D) c.832C>T (p.R278X) c.896G>A (p.R299H) c.895C>A (p.R299S) c.632A>G (p.H211R) c.230G>A (p.R77Q) c.896G>A (p.R299H) c.896G>A (p.R299H) c.896G>A (p.R299H) c.164G>A (p.C55Y) c.1204C>G (p.R402G) c.575C>A (p.S192Y) # rs1042602 c.929insC
c.929insC c.929insC c.896G>A (p.R299H) c.929insC c.896G>A (p.R299H) c.896G>A (p.R299H) c.832C>T (p.R278X) c.896G>A (p.R299H) c.IVS2-7T->A and IVS2 10delTT c.929insC c.896G>A (p.R299H) c.832C>T (p.R278X) c.896G>A (p.R299H) c.1255G>A (p.G419R) c.896G>A (p.R299H) c.929insC c.896G>A (p.R299H) c.232insGGG c.1265T>A (p.M426 K) c.1196delA* c.1265G>A (p.R422Q) c.929insC – – – –
F F F M M F M M
2y 3y 3m 25y 8m 3m 7m 3m
OCA1B OCA2 OCA2 OCA2 OCA2 OCA2 OCA1B OCA2
OCA2 OCA2 OCA2 OCA2 OCA2 OCA2 OCA2 OCA2
c.1560-1562delCCT* c.1255C>T (p.R419W) c.1349C>T (p.T450M) c.1423A>G (p.T475A)* c.1182+1G>A* C.1327G>A (p.V443I) c.1193T>C (p.V398A)* c.406C>T (p.R136X)
c.1610A>T (p.Y537F)* c.2491G>C (p.A831P)* c.1001C>T (p.A334V) – – – – –
F M F F M F F M F M M M
7y 13d 24y 8d 6m 1m 7m 6y 1m 7m 2m 28y
OCA2 OCA1B OCA2 OCA OCA2 OCA2 OCA1 OCA2 OCA1B OCA1 OCA1 OCA2
OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4
c.478G>C (p.D160H) c.663-665delCTC* c.478G>C (p.D160H) c.328G>A (p.G110R) c.143-145delGCT* c.478G>C (p.D160H) c.168-173delGACCCC* c.551C>T (p.A184V)* c.152-153delTG* c.1033-2A>T (IVS4-2A>T)* c.1519G>C (p.V507L) # rs3733808 c.152-153delTG
c.478G>C (p.D160H) c.663-665delCTC c.478G>C (p.D160H) c.1210G>A (p.G404R)* c.478G>C (p.D160H) c.1304C>A (p.S435Y)* – – – – – –
F F
1m 6m
OCA1B OCA2
HPS1 HPS1
c.391C>T (p.R131X) c.1885delC*
c.965insC c.1885delC
M F M M
4y 4m 11m 13
OCA2 OCA2 OCA2 OCA1A
OCA2? ? ? ?
c.1441G>A (p.A481T)# rs74653330 – – –
– – – –
The descriptions in the parentheses denote mutations at protein level. A novel allele appeared the first time in this table is marked with a star (*) symbol. A dash in the mutation column denotes uncharacterized allelic mutation. A question mark represents unconfirmed genotype. A symbol (#) indicates a very rare SNP in this population. In the column of ‘‘Age’’, ‘y’ = year; ‘m’ = month; ‘d’ = day.
were from 19 different provinces of China in the Chinese Albinism Registry [6]. None of these patients have a family history of consanguinity. We followed the criteria for the differentiation of OCA1A, OCA1B and OCA2 as described [3]. Among the 52 OCA patients, thirty were clinically diagnosed as OCA1A or OCA1B, twenty were diagnosed as OCA2 and two OCA patients were not differentially diagnosed due to unclear onset history (Table 1). In all the 52 OCA patients, white skin, blue or brown iris and mild to severe nystagmus were observed. This study was approved by the Internal Review Board of the Bioethics Committee of the Institute of Genetics and Development Biology, Chinese Academy of Sciences. The study was conducted according to Declaration of
Helsinki Principles. Written informed consents were obtained and 8 ml peripheral blood samples were collected from all subjects participating in this study. 2.2. DNA amplifications The optimized strategy for the DNA amplifications of the five OCA genes, TYR, OCA2, TYRP1, SLC45A2 and HPS1, was followed as described [3]. Total genomic DNA was extracted from blood samples by the routine proteinase K/SDS method. Standard PCR amplification procedures were conducted with an annealing temperature of 57–59 8C for all primers. The primer sequences
126
A. Wei et al. / Journal of Dermatological Science 62 (2011) 124–127
are available upon request. The amplifications covered all the exons and exon/intron boundaries of the five OCA genes indicated above. All PCR product sizes were verified by 1.0–1.2% agarose gel electrophoresis. 2.3. PCR product sequencing and identification of novel mutations For direct sequencing, all purified PCR products were sequenced using the ABI PRISM 3700 automated sequencer (Applied Biosystems, Foster City, CA). When a potential novel mutation was considered after careful check with the HGMD (http://www. hgmd.cf.ac.uk/ac/), HPSD (http://liweilab.genetics.ac.cn/HPSD/) and the SNP (http://www.ncbi.nlm.nih.gov/SNP/) databases, direct sequencing of the amplified PCR products from the same region of the 100 unaffected subjects was applied to exclude the possibility of polymorphism. For any possible base pair change in the sequencing results, the MutConv (http://liweilab.genetics.ac.cn/ mutconv/) tool was used to check the possible changes at the protein level [3]. 3. Results 3.1. The distribution of mutational OCA genes was slightly shifted In a total of 52 OCA patients, 93.3% (48/52) of these patients were confirmed by molecular testing, with the remaining 7.7% (4/ 52) uncharacterized after mutational screening of the TYR, OCA2, TYRP1, SLC45A2 and HPS1 gene. Of the 48 molecularly diagnosed patients, 33 patients carried two mutational alleles; 15 carried one mutational allele. Of the identified patients, we found apparent pathologic TYR mutations in 50.0% patients (26/52), OCA2 mutations in 15.4% patients (8/52), SLC45A2 mutations in 23.1% patients (12/52), and 3.8% (2/52) patients with HPS1 mutations (Table 1). No mutation on TYRP1 was found in these patients. Although this distribution is slightly different from the observation in a larger patient screen for 127 OCA patients [3], OCA1 is likewise the predominant type of OCA in these patients. Combined with the two screens, in a total of 179 OCA patients from the Chinese Han population, OCA1, OCA2, OCA4, and HPS1 account for 64.3%, 11.7%, 15.6%, and 2.2% respectively, with the remaining 11 patients (6.2%) of unknown genotypes. 3.2. Eighteen previously unidentified alleles (PUAs) During this screen, we have identified 18 PUAs in these patients, 2 alleles in TYR (p.W238X and c.1196delA), 7 alleles in OCA2 (c.1182+1G>A, p.V398A, p.T475A, c.1560–1562delCCT, p.Y537F, p.A831P, p.V833L), 8 alleles in SLC45A2 (c.143–145delGCT, c.152–153delTG, c.168–173delGACCCC, p.A184V, c.663–665delCT, p.G404R, c.1033-2A>T (IVS4-2A>T), p.S435Y), and 1 allele in HPS1 (c.1885delC). None of these PUAs are found in the 100 unaffected control subjects. 3.3. Common alleles and unusual alleles in this screen In our previous report on the common mutational alleles of TYR gene, p.R299H, c.929insC and p.R278X rank as the top three mutational alleles in Chinese OCA1 patients [3]. There is the same distribution in this study as these three alleles account for 14, 9, 7 alleles respectively among the 48 identified TYR alleles (Table 1). Similarly, the most common mutational allele of SLC45A2 is p.D160H in this study, which is in agreement with our previous report [3]. The p.S192Y allele of TYR gene found in Patient #25 has been regarded as a pathological allele in Chinese OCA patients as we discussed before [3]. Similarly, the p.V507L of the SLC45A2 gene in Patient #45 is regarded as a pathological allele in Asians [8]. We did
not detect the p.Y192 allele or the p.L507 allele in the 100 unaffected controls. Whether the p.A481T allele of OCA2 gene in Patient #49 is a pathologic mutation in the Caucasians [9] or a polymorphism in Japanese [10] is controversial. A transfection study reveals that the p.T481 allele has approximately 70% function of the p.A481 allele in melanogenesis, suggesting it is a relatively mild OCA2 allele [11]. We did not find the p.T481 allele in the 100 unaffected controls. It is possible that the p.T481 allele may contribute to the hypopigmentation in OCA2 patients [12]. 4. Discussion OCA has been widely distributed in different populations. However, the spectra of disease genes and mutational alleles of the known OCA genes vary in different populations. The distributional pattern of TYR, OCA2, SLC45A2 and HPS1 in the Chinese population [3] is apparently different from other populations such as Japanese [13], non-Hispanic Caucasians [9], and Danes [14]. Nevertheless, TYR is the most common OCA gene in all these four populations. In this study, we have revised the spectrum of OCA genes in Chinese Han population by a combination of this screen and our previous screen in 127 OCA patients. That is, OCA1, OCA2, OCA4, and HPS1 account for 64.3%, 11.7%, 15.6%, and 2.2%, respectively. The population-specific distribution of the OCA genes, together with the common mutational alleles or mutational hotspots, is useful for the optimal design of a genetic testing strategy. The optimized method to screen the OCA mutations based on the population studies is efficiently implemented in the routine gene diagnosis of Chinese OCA patients by reaching a detection rate over 90%. The molecular identification of the mutational OCA genes provides useful information for the genetic counseling. Skin, eye and hair color are common pigmentary traits in human beings with apparent variations. A combination of candidate approaches and genome-wide association studies has revealed many candidate genes underlying this variation, including TYR, TYRP1, OCA2, SLC45A2, SLC24A5, MC1R, ASIP, KITLG, SLC24A4, IRF4, TPCN2, SLC7A11 and others [15,16]. Genetic screening of the known OCA genes in different populations has raised several alleles as controversial for a SNP or a pathological mutation. A definition of a SNP or a pathological mutation by the allelic frequency could be population-specific. This is especially in the case of p.S192Y of the TYR gene. It has been reported as a SNP rs1042602 (http://www. ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs1042602) in several populations including Caucasians, Africans, Japanese and South Asian population. However, in Chinese Han population, the allele c.575C frequency is 1.00 in the NCBI SNP database. Likewise, we did not find the c.575A allele in any of the 100 unaffected subjects from the Han population. As the c.575C allele is associated with darken skin [17], it is possible that the c.575C>A mutation would contribute to the development of hypopigmentation in a double heterozygous state with other mutational allele in the Han population. The human color traits on the skin may undergo positive selection in regional populations. For example, population study shows that the p.T481 allele of OCA2 gene is relatively common in the northeastern Asian populations with low ultraviolet radiation [18]. On the other hand, the p.S192 allele of TYR gene is more common in the South Asian population with higher ultraviolet radiation [17]. Thus, the control group from the same population is required during the mutational screen of the OCA genes. More importantly, a functional assay of different alleles for the contribution of hypopigmentation is warranted for resolving the disputes. HPS is a relatively rare form in the Chinese OCA patients. We have so far identified four HPS1 patients in the Chinese Albinism Registry [3,7]. All of them were diagnosed at less than 5 years old. The c.965insC and c.391C>T mutation found in Patient #47 have
A. Wei et al. / Journal of Dermatological Science 62 (2011) 124–127
been reported in other populations as collected in the HPSD database [2]. This patient did not show bruises on the legs as the patient was only one month at diagnosis. Her blood testing showed normal range of platelet counts. The homozygous c.1885delC mutation in Patient #48 leads to the same elongated HPS1 peptide which ends at codon 724 as the result of the homozygous c.1932delC mutation in the first reported Chinese HPS1 patient [7]. Both patients did not have bleeding tendency and showed normal blood counts. It remains to be clarified whether the elongated HPS1 protein is partially functional which causes a mild form of HPS1. Follow-up checks, examination on the platelet dense granules and detailed investigations on the mutational HPS1 protein will be interesting for these four HPS1 patients. In this study, 18 previously unidentified alleles were identified in the TYR, OCA2, SLC45A2 and HPS1 gene. These alleles expand the mutational database in humans. Functional studies for some interesting alleles are undergoing to elucidate the pathogenesis of OCA and the genotype–phenotype relationship. When molecular testing is conducted in a more comprehensive manner, some rare type of OCA or HPS will be expected in the Chinese OCA patients. Acknowledgements This work was supported in part by National Natural Science Foundation of China (No. 30730049), National Basic Research Program of China (No. 2007CB947200), Ministry of Agriculture of China (2009ZX08009-158B) and Natural Science Foundation of Beijing (No. 7092040). References [1] Gong Y, Shao C, Zheng H, Chen B, Guo Y. Study on genetic epidemiology of albinism. Yi Chuan Xue Bao 1994;21:169–72. [2] Li W, He M, Zhou HL, Bourne JW, Liang P. Mutational data integration in geneoriented files of Hermansky-Pudlak syndrome database. Hum Mutat 2006;27: 402–7.
127
[3] Wei A, Wang Y, Long Y, Wang Y, Guo X, Zhou Z, et al. A comprehensive analysis reveals mutational spectra and common alleles in Chinese patients with oculocutaneous albinism. J Invest Dermatol 2010;130: 716–24. [4] King RA, Pietsch J, Fryer JP, Savage S, Brott MJ, Russell-Eggitt I, et al. Tyrosinase gene mutations in oculocutaneous albinism 1 (OCA1): definition of the phenotype. Hum Genet 2003;113:502–13. [5] Huizing M, Gahl WA. Disorders of vesicles of lysosomal lineage: the Hermansky-Pudlak syndromes. Curr Mol Med 2002;2:451–67. [6] He M, Li W. China Genetic Counseling Network (CGCN): a website on genetic counseling and genetic education. Hereditas (Beijing) 2007;29:381–4. [7] Wei A, Lian S, Wang L, Li W. The first case report of a Chinese HermanskyPudlak syndrome patient with a novel mutation on HPS1 gene. J Dermatol Sci 2009;56:130–2. [8] Inagaki K, Suzuki T, Shimizu H, Ishii N, Umezawa Y, Tada J, et al. Oculocutaneous albinism type 4 is one of the most common types of albinism in Japan. Am J Hum Genet 2004;74:466–71. [9] Hutton SM, Spritz RA. Comprehensive analysis of oculocutaneous albinism among non-Hispanic Caucasians shows that OCA1 is the most prevalent OCA type. J Invest Dermatol 2008;128:2442–50. [10] Suzuki T, Miyamura Y, Tomita Y. High frequency of the Ala481Thr mutation of the P gene in the Japanese population. Am J Med Genet 2003;118A: 402–3. [11] Sviderskaya EV, Bennett DC, Ho L, Bailin T, Lee ST, Spritz RA. Complementation of hypopigmentation in p-mutant (pink-eyed dilution) mouse melanocytes by normal human P cDNA, and defective complementation by OCA2 mutant sequences. J Invest Dermatol 1997;108:30–4. [12] Kato A, Fukai K, Oiso N, Hosomi N, Saitoh S, Wada T, et al. A novel P gene missense mutation in a Japanese patient with oculocutaneous albinism type II (OCA2). J Dermatol Sci 2003;31:189–92. [13] Suzuki T, Tomita Y. Recent advances in genetic analyses of oculocutaneous albinism types 2 and 4. J Dermatol Sci 2008;51:1–9. [14] Grønskov K, Ek J, Sand A, Scheller R, Bygum A, Brixen K, et al. Birth prevalence and mutation spectrum in Danish patients with autosomal recessive albinism. Invest Ophthalmol Vis Sci 2009;50:1058–64. [15] Sturm RA. Molecular genetics of human pigmentation diversity. Hum Mol Genet 2009;18:R9–17. [16] Ito S, Wakamatsu K. Human hair melanins: what we have learned and have not learned from mouse coat color pigmentation. Pigment Cell Melanoma Res 2011;24:63–74. [17] Stokowski RP, Pant PV, Dadd T, Fereday A, Hinds DA, Jarman C, et al. A genomewide association study of skin pigmentation in a South Asian population. Am J Hum Genet 2007;81:1119–32. [18] Yuasa I, Umetsu K, Harihara S, Miyoshi A, Saitou N, Park KS, et al. OCA2 481Thr, a hypofunctional allele in pigmentation, is characteristic of northeastern Asian populations. J Hum Genet 2007;52:690–3.
Contents lists available at ScienceDirect
Journal of Dermatological Science journal homepage: www.elsevier.com/jds
Implementation of an optimized strategy for genetic testing of the Chinese patients with oculocutaneous albinism Aihua Wei a,b, Xiumin Yang a, Shi Lian c, Wei Li b,* a
Department of Dermatology, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Rd., Chaoyang District, Beijing 100101, China c Department of Dermatology, Xuan Wu Hospital, Capital Medical University, Beijing 100053, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 November 2010 Received in revised form 5 February 2011 Accepted 27 February 2011
Background: Oculocutaneous albinism (OCA) is a relatively common inherited disorder in all populations worldwide. The mutational spectra of OCA are population-specific. Objective: Based on our previous molecular epidemiological studies, we have implemented an optimized strategy for the genetic testing of Chinese OCA patients. Methods: Genomic DNA was extracted from the blood samples of 52 clinically diagnosed OCA patients and 100 unaffected subjects. The amplified DNA segments were screened for mutations of TYR, OCA2, TYRP1, SLC45A2 and HPS1 by direct sequencing. To exclude the previously unidentified alleles (PUAs) from polymorphisms, samples from 100 unaffected controls were sequenced for the same regions of variations. Results: Among the 52 OCA patients, 26 (50.0%) were found mutations on TYR gene, 8 (15.4%) on OCA2, 12 (23.1%) on SLC45A2, 2 (3.8%) on HPS1, and 4 (7.7%) patients uncharacterized. We identified 18 PUAs in these patients, 2 in TYR, 7 in OCA2, 8 in SLC45A2, and 1 in HPS1. Conclusion: The optimized method to screen the OCA mutations is efficiently implemented in the routine genetic testing of Chinese OCA patients accompanied with genetic counseling. ß 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Oculocutaneous albinism Hermansky-Pudlak syndrome Genetic testing Previously unidentified allele
1. Introduction Oculocutaneous albinism (OCA) is an autosomal recessive disorder with a relatively high incidence in Chinese Han population as estimated as 1:18,000 [1]. It manifests as a reduction or complete loss of melanin in the skin, hair, and eyes, often accompanied with eye symptoms such as photophobia, strabismus, moderate to severe visual impairment, and nystagmus. OCA could be caused by mutations in non-syndromic OCA genes (TYR, OCA2, TYRP1 and SLC45A2) or syndromic OCA genes (HPS1, HPS2, HPS3, HPS4, HPS5, HPS6, HPS7, HPS8, LYST, MYO5A, RAB27A and MLPH) [2]. OCA1 is the predominant form which accounts for about 70% of the Chinese OCA patients, while OCA2, OCA4 and HPS1 are less common, reflecting a population-specific distribution of different subtypes of OCA [3]. OCA is clinically characterized as OCA1A, OCA1B or OCA2. OCA1A presents a complete lack of tyrosinase activity and produces a totally depigmented phenotype with affected individuals exhibiting white hair, white skin, and blue, brown or pink iris
throughout life. OCA1B is characterized by reduction of tyrosinase activity. Individuals with OCA1B are born with white hair and then change to blond or yellow with age [4]. OCA2 is characterized by yellow, brown or golden hair at birth with or without darkening of hair color at later age. However, the clinically diagnosed subtypes of OCA could be mixed forms of molecularly diagnosed OCA subtypes, such as clinical OCA1 could be molecularly identified as OCA1, OCA2, OCA4 or HPS1, whereas clinical OCA2 could be OCA1, OCA2, OCA4, or HPS1 [3]. Hermansky-Pudlak syndrome (HPS) is a more severe form of OCA. Patients with HPS often die in their middle ages [5]. Therefore, the genetic testing of OCA is needed for routine diagnosis of OCA to better characterize the prognosis of OCA. Based on our previous genetic epidemiological studies of OCA in Chinese Han population, we have implemented an optimized strategy to molecularly screen the mutations on the known OCA genes. 2. Materials and methods 2.1. Study subjects
* Corresponding author. Tel.: +86 10 6484 8212; fax: +86 10 6484 8212. E-mail address: [email protected] (W. Li).
We recruited 52 unrelated OCA patients (Table 1) and 100 unaffected subjects from the Chinese Han population. The patients
0923-1811/$36.00 ß 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2011.02.009
A. Wei et al. / Journal of Dermatological Science 62 (2011) 124–127
125
Table 1 Genotypes of 52 Chinese OCA patients. Patient ID TYR 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 OCA2 27 28 29 30 31 32 33 34 SLC45A2 35 36 37 38 39 40 41 42 43 44 45 46 HPS1 47 48 Uncharacterized 49 50 51 52
Sex
Age
Clinical diagnosis
Molecular diagnosis
Mutational allele 1
Mutational allele 2
F M M M F F F M M F F F F M M M M F F M F F F M M M
8y 31y 1m 3m 25y 5y 5y 21y 22y 2y 3y 1m 1m 30y 29y 32y 2m 57y 2y 28y 5y 4m 32 11 1m 20y
OCA1A OCA OCA1 OCA1A OCA1A OCA1A OCA1A OCA1A OCA2 OCA1A OCA1A OCA1 OCA1 OCA1A OCA1A OCA1A OCA1 OCA1A OCA2 OCA1A OCA1B OCA1B OCA1B OCA2 OCA2 OCA1
OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1B OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1A OCA1B OCA1A OCA1 OCA1B OCA1 OCA1A
c.896G>A (p.R299H) c.929insC c.346C>T (p.R116X) c.896G>A (p.R299H) c.758G>A (p.G253E) c.832C>T (p.R278X) c.832C>T (p.R278X) c.230G>A (p.R77Q) c.832C>T (p.R278X) c.714G>A (p.W238X)* c.832C>T (p.R278X) c.71G>A (p.C24Y) c.929insC c.1168C>G (p.H390D) c.832C>T (p.R278X) c.896G>A (p.R299H) c.895C>A (p.R299S) c.632A>G (p.H211R) c.230G>A (p.R77Q) c.896G>A (p.R299H) c.896G>A (p.R299H) c.896G>A (p.R299H) c.164G>A (p.C55Y) c.1204C>G (p.R402G) c.575C>A (p.S192Y) # rs1042602 c.929insC
c.929insC c.929insC c.896G>A (p.R299H) c.929insC c.896G>A (p.R299H) c.896G>A (p.R299H) c.832C>T (p.R278X) c.896G>A (p.R299H) c.IVS2-7T->A and IVS2 10delTT c.929insC c.896G>A (p.R299H) c.832C>T (p.R278X) c.896G>A (p.R299H) c.1255G>A (p.G419R) c.896G>A (p.R299H) c.929insC c.896G>A (p.R299H) c.232insGGG c.1265T>A (p.M426 K) c.1196delA* c.1265G>A (p.R422Q) c.929insC – – – –
F F F M M F M M
2y 3y 3m 25y 8m 3m 7m 3m
OCA1B OCA2 OCA2 OCA2 OCA2 OCA2 OCA1B OCA2
OCA2 OCA2 OCA2 OCA2 OCA2 OCA2 OCA2 OCA2
c.1560-1562delCCT* c.1255C>T (p.R419W) c.1349C>T (p.T450M) c.1423A>G (p.T475A)* c.1182+1G>A* C.1327G>A (p.V443I) c.1193T>C (p.V398A)* c.406C>T (p.R136X)
c.1610A>T (p.Y537F)* c.2491G>C (p.A831P)* c.1001C>T (p.A334V) – – – – –
F M F F M F F M F M M M
7y 13d 24y 8d 6m 1m 7m 6y 1m 7m 2m 28y
OCA2 OCA1B OCA2 OCA OCA2 OCA2 OCA1 OCA2 OCA1B OCA1 OCA1 OCA2
OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4 OCA4
c.478G>C (p.D160H) c.663-665delCTC* c.478G>C (p.D160H) c.328G>A (p.G110R) c.143-145delGCT* c.478G>C (p.D160H) c.168-173delGACCCC* c.551C>T (p.A184V)* c.152-153delTG* c.1033-2A>T (IVS4-2A>T)* c.1519G>C (p.V507L) # rs3733808 c.152-153delTG
c.478G>C (p.D160H) c.663-665delCTC c.478G>C (p.D160H) c.1210G>A (p.G404R)* c.478G>C (p.D160H) c.1304C>A (p.S435Y)* – – – – – –
F F
1m 6m
OCA1B OCA2
HPS1 HPS1
c.391C>T (p.R131X) c.1885delC*
c.965insC c.1885delC
M F M M
4y 4m 11m 13
OCA2 OCA2 OCA2 OCA1A
OCA2? ? ? ?
c.1441G>A (p.A481T)# rs74653330 – – –
– – – –
The descriptions in the parentheses denote mutations at protein level. A novel allele appeared the first time in this table is marked with a star (*) symbol. A dash in the mutation column denotes uncharacterized allelic mutation. A question mark represents unconfirmed genotype. A symbol (#) indicates a very rare SNP in this population. In the column of ‘‘Age’’, ‘y’ = year; ‘m’ = month; ‘d’ = day.
were from 19 different provinces of China in the Chinese Albinism Registry [6]. None of these patients have a family history of consanguinity. We followed the criteria for the differentiation of OCA1A, OCA1B and OCA2 as described [3]. Among the 52 OCA patients, thirty were clinically diagnosed as OCA1A or OCA1B, twenty were diagnosed as OCA2 and two OCA patients were not differentially diagnosed due to unclear onset history (Table 1). In all the 52 OCA patients, white skin, blue or brown iris and mild to severe nystagmus were observed. This study was approved by the Internal Review Board of the Bioethics Committee of the Institute of Genetics and Development Biology, Chinese Academy of Sciences. The study was conducted according to Declaration of
Helsinki Principles. Written informed consents were obtained and 8 ml peripheral blood samples were collected from all subjects participating in this study. 2.2. DNA amplifications The optimized strategy for the DNA amplifications of the five OCA genes, TYR, OCA2, TYRP1, SLC45A2 and HPS1, was followed as described [3]. Total genomic DNA was extracted from blood samples by the routine proteinase K/SDS method. Standard PCR amplification procedures were conducted with an annealing temperature of 57–59 8C for all primers. The primer sequences
126
A. Wei et al. / Journal of Dermatological Science 62 (2011) 124–127
are available upon request. The amplifications covered all the exons and exon/intron boundaries of the five OCA genes indicated above. All PCR product sizes were verified by 1.0–1.2% agarose gel electrophoresis. 2.3. PCR product sequencing and identification of novel mutations For direct sequencing, all purified PCR products were sequenced using the ABI PRISM 3700 automated sequencer (Applied Biosystems, Foster City, CA). When a potential novel mutation was considered after careful check with the HGMD (http://www. hgmd.cf.ac.uk/ac/), HPSD (http://liweilab.genetics.ac.cn/HPSD/) and the SNP (http://www.ncbi.nlm.nih.gov/SNP/) databases, direct sequencing of the amplified PCR products from the same region of the 100 unaffected subjects was applied to exclude the possibility of polymorphism. For any possible base pair change in the sequencing results, the MutConv (http://liweilab.genetics.ac.cn/ mutconv/) tool was used to check the possible changes at the protein level [3]. 3. Results 3.1. The distribution of mutational OCA genes was slightly shifted In a total of 52 OCA patients, 93.3% (48/52) of these patients were confirmed by molecular testing, with the remaining 7.7% (4/ 52) uncharacterized after mutational screening of the TYR, OCA2, TYRP1, SLC45A2 and HPS1 gene. Of the 48 molecularly diagnosed patients, 33 patients carried two mutational alleles; 15 carried one mutational allele. Of the identified patients, we found apparent pathologic TYR mutations in 50.0% patients (26/52), OCA2 mutations in 15.4% patients (8/52), SLC45A2 mutations in 23.1% patients (12/52), and 3.8% (2/52) patients with HPS1 mutations (Table 1). No mutation on TYRP1 was found in these patients. Although this distribution is slightly different from the observation in a larger patient screen for 127 OCA patients [3], OCA1 is likewise the predominant type of OCA in these patients. Combined with the two screens, in a total of 179 OCA patients from the Chinese Han population, OCA1, OCA2, OCA4, and HPS1 account for 64.3%, 11.7%, 15.6%, and 2.2% respectively, with the remaining 11 patients (6.2%) of unknown genotypes. 3.2. Eighteen previously unidentified alleles (PUAs) During this screen, we have identified 18 PUAs in these patients, 2 alleles in TYR (p.W238X and c.1196delA), 7 alleles in OCA2 (c.1182+1G>A, p.V398A, p.T475A, c.1560–1562delCCT, p.Y537F, p.A831P, p.V833L), 8 alleles in SLC45A2 (c.143–145delGCT, c.152–153delTG, c.168–173delGACCCC, p.A184V, c.663–665delCT, p.G404R, c.1033-2A>T (IVS4-2A>T), p.S435Y), and 1 allele in HPS1 (c.1885delC). None of these PUAs are found in the 100 unaffected control subjects. 3.3. Common alleles and unusual alleles in this screen In our previous report on the common mutational alleles of TYR gene, p.R299H, c.929insC and p.R278X rank as the top three mutational alleles in Chinese OCA1 patients [3]. There is the same distribution in this study as these three alleles account for 14, 9, 7 alleles respectively among the 48 identified TYR alleles (Table 1). Similarly, the most common mutational allele of SLC45A2 is p.D160H in this study, which is in agreement with our previous report [3]. The p.S192Y allele of TYR gene found in Patient #25 has been regarded as a pathological allele in Chinese OCA patients as we discussed before [3]. Similarly, the p.V507L of the SLC45A2 gene in Patient #45 is regarded as a pathological allele in Asians [8]. We did
not detect the p.Y192 allele or the p.L507 allele in the 100 unaffected controls. Whether the p.A481T allele of OCA2 gene in Patient #49 is a pathologic mutation in the Caucasians [9] or a polymorphism in Japanese [10] is controversial. A transfection study reveals that the p.T481 allele has approximately 70% function of the p.A481 allele in melanogenesis, suggesting it is a relatively mild OCA2 allele [11]. We did not find the p.T481 allele in the 100 unaffected controls. It is possible that the p.T481 allele may contribute to the hypopigmentation in OCA2 patients [12]. 4. Discussion OCA has been widely distributed in different populations. However, the spectra of disease genes and mutational alleles of the known OCA genes vary in different populations. The distributional pattern of TYR, OCA2, SLC45A2 and HPS1 in the Chinese population [3] is apparently different from other populations such as Japanese [13], non-Hispanic Caucasians [9], and Danes [14]. Nevertheless, TYR is the most common OCA gene in all these four populations. In this study, we have revised the spectrum of OCA genes in Chinese Han population by a combination of this screen and our previous screen in 127 OCA patients. That is, OCA1, OCA2, OCA4, and HPS1 account for 64.3%, 11.7%, 15.6%, and 2.2%, respectively. The population-specific distribution of the OCA genes, together with the common mutational alleles or mutational hotspots, is useful for the optimal design of a genetic testing strategy. The optimized method to screen the OCA mutations based on the population studies is efficiently implemented in the routine gene diagnosis of Chinese OCA patients by reaching a detection rate over 90%. The molecular identification of the mutational OCA genes provides useful information for the genetic counseling. Skin, eye and hair color are common pigmentary traits in human beings with apparent variations. A combination of candidate approaches and genome-wide association studies has revealed many candidate genes underlying this variation, including TYR, TYRP1, OCA2, SLC45A2, SLC24A5, MC1R, ASIP, KITLG, SLC24A4, IRF4, TPCN2, SLC7A11 and others [15,16]. Genetic screening of the known OCA genes in different populations has raised several alleles as controversial for a SNP or a pathological mutation. A definition of a SNP or a pathological mutation by the allelic frequency could be population-specific. This is especially in the case of p.S192Y of the TYR gene. It has been reported as a SNP rs1042602 (http://www. ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs1042602) in several populations including Caucasians, Africans, Japanese and South Asian population. However, in Chinese Han population, the allele c.575C frequency is 1.00 in the NCBI SNP database. Likewise, we did not find the c.575A allele in any of the 100 unaffected subjects from the Han population. As the c.575C allele is associated with darken skin [17], it is possible that the c.575C>A mutation would contribute to the development of hypopigmentation in a double heterozygous state with other mutational allele in the Han population. The human color traits on the skin may undergo positive selection in regional populations. For example, population study shows that the p.T481 allele of OCA2 gene is relatively common in the northeastern Asian populations with low ultraviolet radiation [18]. On the other hand, the p.S192 allele of TYR gene is more common in the South Asian population with higher ultraviolet radiation [17]. Thus, the control group from the same population is required during the mutational screen of the OCA genes. More importantly, a functional assay of different alleles for the contribution of hypopigmentation is warranted for resolving the disputes. HPS is a relatively rare form in the Chinese OCA patients. We have so far identified four HPS1 patients in the Chinese Albinism Registry [3,7]. All of them were diagnosed at less than 5 years old. The c.965insC and c.391C>T mutation found in Patient #47 have
A. Wei et al. / Journal of Dermatological Science 62 (2011) 124–127
been reported in other populations as collected in the HPSD database [2]. This patient did not show bruises on the legs as the patient was only one month at diagnosis. Her blood testing showed normal range of platelet counts. The homozygous c.1885delC mutation in Patient #48 leads to the same elongated HPS1 peptide which ends at codon 724 as the result of the homozygous c.1932delC mutation in the first reported Chinese HPS1 patient [7]. Both patients did not have bleeding tendency and showed normal blood counts. It remains to be clarified whether the elongated HPS1 protein is partially functional which causes a mild form of HPS1. Follow-up checks, examination on the platelet dense granules and detailed investigations on the mutational HPS1 protein will be interesting for these four HPS1 patients. In this study, 18 previously unidentified alleles were identified in the TYR, OCA2, SLC45A2 and HPS1 gene. These alleles expand the mutational database in humans. Functional studies for some interesting alleles are undergoing to elucidate the pathogenesis of OCA and the genotype–phenotype relationship. When molecular testing is conducted in a more comprehensive manner, some rare type of OCA or HPS will be expected in the Chinese OCA patients. Acknowledgements This work was supported in part by National Natural Science Foundation of China (No. 30730049), National Basic Research Program of China (No. 2007CB947200), Ministry of Agriculture of China (2009ZX08009-158B) and Natural Science Foundation of Beijing (No. 7092040). References [1] Gong Y, Shao C, Zheng H, Chen B, Guo Y. Study on genetic epidemiology of albinism. Yi Chuan Xue Bao 1994;21:169–72. [2] Li W, He M, Zhou HL, Bourne JW, Liang P. Mutational data integration in geneoriented files of Hermansky-Pudlak syndrome database. Hum Mutat 2006;27: 402–7.
127
[3] Wei A, Wang Y, Long Y, Wang Y, Guo X, Zhou Z, et al. A comprehensive analysis reveals mutational spectra and common alleles in Chinese patients with oculocutaneous albinism. J Invest Dermatol 2010;130: 716–24. [4] King RA, Pietsch J, Fryer JP, Savage S, Brott MJ, Russell-Eggitt I, et al. Tyrosinase gene mutations in oculocutaneous albinism 1 (OCA1): definition of the phenotype. Hum Genet 2003;113:502–13. [5] Huizing M, Gahl WA. Disorders of vesicles of lysosomal lineage: the Hermansky-Pudlak syndromes. Curr Mol Med 2002;2:451–67. [6] He M, Li W. China Genetic Counseling Network (CGCN): a website on genetic counseling and genetic education. Hereditas (Beijing) 2007;29:381–4. [7] Wei A, Lian S, Wang L, Li W. The first case report of a Chinese HermanskyPudlak syndrome patient with a novel mutation on HPS1 gene. J Dermatol Sci 2009;56:130–2. [8] Inagaki K, Suzuki T, Shimizu H, Ishii N, Umezawa Y, Tada J, et al. Oculocutaneous albinism type 4 is one of the most common types of albinism in Japan. Am J Hum Genet 2004;74:466–71. [9] Hutton SM, Spritz RA. Comprehensive analysis of oculocutaneous albinism among non-Hispanic Caucasians shows that OCA1 is the most prevalent OCA type. J Invest Dermatol 2008;128:2442–50. [10] Suzuki T, Miyamura Y, Tomita Y. High frequency of the Ala481Thr mutation of the P gene in the Japanese population. Am J Med Genet 2003;118A: 402–3. [11] Sviderskaya EV, Bennett DC, Ho L, Bailin T, Lee ST, Spritz RA. Complementation of hypopigmentation in p-mutant (pink-eyed dilution) mouse melanocytes by normal human P cDNA, and defective complementation by OCA2 mutant sequences. J Invest Dermatol 1997;108:30–4. [12] Kato A, Fukai K, Oiso N, Hosomi N, Saitoh S, Wada T, et al. A novel P gene missense mutation in a Japanese patient with oculocutaneous albinism type II (OCA2). J Dermatol Sci 2003;31:189–92. [13] Suzuki T, Tomita Y. Recent advances in genetic analyses of oculocutaneous albinism types 2 and 4. J Dermatol Sci 2008;51:1–9. [14] Grønskov K, Ek J, Sand A, Scheller R, Bygum A, Brixen K, et al. Birth prevalence and mutation spectrum in Danish patients with autosomal recessive albinism. Invest Ophthalmol Vis Sci 2009;50:1058–64. [15] Sturm RA. Molecular genetics of human pigmentation diversity. Hum Mol Genet 2009;18:R9–17. [16] Ito S, Wakamatsu K. Human hair melanins: what we have learned and have not learned from mouse coat color pigmentation. Pigment Cell Melanoma Res 2011;24:63–74. [17] Stokowski RP, Pant PV, Dadd T, Fereday A, Hinds DA, Jarman C, et al. A genomewide association study of skin pigmentation in a South Asian population. Am J Hum Genet 2007;81:1119–32. [18] Yuasa I, Umetsu K, Harihara S, Miyoshi A, Saitou N, Park KS, et al. OCA2 481Thr, a hypofunctional allele in pigmentation, is characteristic of northeastern Asian populations. J Hum Genet 2007;52:690–3.