Genetic Studies of TYRP1 and SLC45A2 in Pakistani Patients with Nonsyndromic Oculocutaneous Albinism

T Kausar et al. Mutations of OCA Genes in a Pakistani Population

ACKNOWLEDGMENTS We are thankful to Brian Diffey, Thomas Ruenger, Irene Kochevar, Eri Verter, and the Wellman Center for Photomedicine (Boston, MA). The experiment was carried out and drafted in Boston, MA, and Providence, RI, respectively.

Alina Markova1,2,3 and Martin A. Weinstock2,3 1

Department of Internal Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA; Department of Dermatology, Alpert Medical School of Brown University, Providence, Rhode

2

Island, USA and 3Dermatoepidemiology Unit-111D, Veterans Affairs Medical Center, Providence, Rhode Island, USA E-mail: [email protected]

REFERENCES Nails Magazine (2012) 2011–2012 Industry Statistics. ohttp://www.nailsmag.com/page/70218/ market-research4 de Gruijl FR, Van der Leun JC (1994) Estimate of the wavelength dependency of ultraviolet carcinogenesis in humans and its relevance

to the risk assessment of a stratospheric ozone depletion. Health Phys 67:319–25 Diffey BL, Farr PM (2007) The challenge of followup in narrowband ultraviolet B phototherapy. Br J Dermatol 157:344–9 MacFarlane DF, Alonso CA (2009) Occurrence of nonmelanoma skin cancers on the hands after UV nail light exposure. Arch Dermatol 145: 447–9 Schoon D, Bryson P, McConnell J (2010) Do UV nail lamps emit unsafe levels of ultraviolet light? http://www.schoonscientific.com/ downloads/UV-Nail-Lamp-Facts.pdf

Genetic Studies of TYRP1 and SLC45A2 in Pakistani Patients with Nonsyndromic Oculocutaneous Albinism Journal of Investigative Dermatology (2013) 133, 1099–1102; doi:10.1038/jid.2012.432; published online 29 November 2012

TO THE EDITOR Autosomal recessive oculocutaneous albinism (OCA) is a genetic disorder characterized by the partial or complete loss of pigmentation in the skin, hair, and iris (Tomita and Suzuki, 2004). OCA is associated with a number of vision problems and can severely affect an individual’s quality of life (Oetting and King, 1999; Tomita and Suzuki, 2004; Chaki et al., 2011). Many of the molecular components that are necessary for the development, trafficking, and maintenance of melanin pigment have been discovered by identifying the genes for the nonsyndromic and syndromic forms of OCA in humans. Identifying the genetic causes of OCA, conducting genetic testing, detecting carriers, and improving patients’ understanding of the implications and consequences of the disorder through genetic counseling are essential, especially in populations that commonly practice inbreeding (Hussain and Bittles, 1998). The Pakistani population is one such example; however, genetic information regarding OCA in this population is limited (Jaworek et al., 2012). To the best of our knowledge, only one allele of OCA3 (TYRP1) and no mutations in OCA4 (SLC45A2) have

Abbreviation: OCA, oculocutaneous albinism

been documented in the Pakistani population (Forshew et al., 2005; Jaworek et al., 2012). Here, we report the identification of four Pakistani albinism mutations, including three SLC45A2 alleles and one 22-nucleotide deletion in TYRP1, segregating in four large families (Figure 1). After obtaining institutional review board approval and written informed consent from all participating individuals and the parents of minor subjects, consistent with the tenets of the Declaration of Helsinki, we enrolled 75 large, multigeneration families segregating nonsyndromic OCA (i.e., without any other apparent clinical phenotypes) from various regions of Pakistan (see Supplementary Materials online) and conducted a comprehensive molecular analysis of TYRP1 and SLC45A2. Mutations in TYRP1 rarely cause OCA, and, to date, only nine alleles have been detected in individuals of Southern African, African-American, Caucasian, Indian, or Pakistani descent (Boissy et al., 1996; Manga et al., 1997; Forshew et al., 2005; Rooryck et al., 2006; Hutton and Spritz, 2008; Rooryck et al., 2008; Chiang et al., 2009). Mutations in TYRP1 typically result in reddish brown or golden hair with

orange highlights (Boissy et al., 1996; Manga et al., 1997; Forshew et al., 2005; Rooryck et al., 2006; Hutton and Spritz, 2008; Rooryck et al., 2008). A sequence analysis of TYRP1 in our cohort revealed a 22-base-pair deletion (c.647_668del) in the coding region in family PKAB131 (Figure 1 and Supplementary Figure S1 online). Deletion of these nucleotides is predicted to cause a frameshift in the reading frame that will result in a truncated (p.Glu216GlyfsX42) TYRP1 protein (Table 1). The affected individuals of family PKAB131 had golden hair with orange highlights, brown eyes with foveal hypoplasia, and nystagmus (Figure 1 and Table 1). All of the affected individuals had white skin at birth that gradually darkened on the body parts exposed to sunlight, such as the face, arms, and hands. However, pigment deficiency was still observable on the body parts not directly exposed to sunlight. To date, this is the second mutation of TYRP1 that causes an OCA3 phenotype in a family of Pakistani origin. A sequence analysis of SLC45A2 revealed three variants that are likely to be pathogenic in three families (Figure 1, Table 1 and Supplementary Figure S2 online). Two missense substitutions were identified: p.Leu84Pro (c.251T4C) and p.Ala511Val (c.1532C4T) in families PKAB053 and PKAB051, respectively (Figure 1). Both missense mutations www.jidonline.org 1099

T Kausar et al. Mutations of OCA Genes in a Pakistani Population

OCA3 family PKAB131

c.647_668del; p.Glu216GlyfsX42

2 3

1

2

2

1

OCA4 families PKAB051

October 2009

2

May 2012

1

c.251T>C p.Leu84Pro

PKAB053

3

2

PKAB059

c.889-6T>G

6

3

c.1532C>T; p.Ala511Val

1

1

2

5

November 2009

4 3

3

January 2010

4

May 2012

5

6

May 2012

6

Figure 1. Mutant alleles of TYRP1and SLC45A2 segregating in four Pakistani families. Pedigrees of four multigenerational Pakistani families cosegregating recessive nonsyndromic oculocutaneous albinism (OCA) and mutant alleles of TYRP1 or SLC45A2. The filled and clear symbols represent affected and unaffected individuals, respectively. The double lines indicate consanguineous marriages. TYRP1 or SLC45A2 mutations segregating in these families are also indicated. Photographs of multiple affected individuals from each family are also shown. The numbers on the family pedigrees indicate individuals shown. All the individuals have given written consent to show their images. For several of the affected individuals, multiple photographs taken at different ages revealed no significant changes in the pigmentation of the hair, iris, or skin. Some of the affected individuals have used hair dyes.

affected amino-acid residues that are conserved among the SLC45A2 orthologs, and these mutations are predicted to be deleterious (Table 1 and Supplementary Figure S2 online). Molecular modeling of wild-type and mutant SLC45A2 proteins suggested that the p.Leu84Pro mutation alters the hydrogen bonding pattern in the alpha helix; specifically, the replacement of leucine with proline eliminates three potential

hydrogen bonds between neighboring amino acids that are critical for the stabilization of the alpha helix (Supplementary Figure S3 online). Proline is considered to be a more rigid amino acid and can create a kink in the chain; however, this effect may not be noticeable in the model, because other chains may help maintain the overall structure. The alanine residue at position 511 forms three hydrogen bonds

1100 Journal of Investigative Dermatology (2013), Volume 133

within a distance of 2.5 A˚ (Supplementary Figure S3 online). Replacement of this alanine with valine creates an additional cryptic hydrogen bond (Supplementary Figure S3 online). Furthermore, valine is bulkier than alanine and may cause steric strain against the neighboring alpha helix. An alanine to glutamate mutation at amino acid position 511 (p.Ala511Glu) has been previously documented (Rooryck et al.,

T Kausar et al. Mutations of OCA Genes in a Pakistani Population

Table 1. Genetic and clinical assessment of the affected individuals with mutations in TYRP1 and SLC45A2 Gene

TYRP1

Family

SLC45A2

PKAB131

PKAB053

PKAB051

PKAB059

Ethnicity

Ladd

Lakhat

Kingray

Rehmani

Consanguinity

Yes

Yes

Yes

Yes

Nucleotide change

c.647_668del

c.251T4C

c.1532C4T

c.889-6T4G

Amino acid change

p.Glu216GlyfsX42

p.Leu84Pro

p.Ala511Val

Splicing error

0/374

0/374

0/378

0/378

MutationTaster

Pathogenic

Pathogenic

Pathogenic

NA

Polyphen-2

Damaging

Damaging

Damaging

NA

SNPs3D

Damaging

Damaging

Damaging

NA

Allele frequency1 In silico analysis

Sex

F

F

F

F

F

M

F

Age (years)

3

6

19

3

16

18

22

Hair color

Golden

Golden

Brown

White

Brown

Brown

Brown

Skin tone

White

White

Pinkish white

Pinkish white

White

White

White

Iris color

Brown

Brown

Gray/brown

Gray/blue

Gray/blue

Gray/brown

Gray/blue

Visual acuity Right eye

6/60

6/60

5/60

6/36

6/36

6/36

6/248

Left eye

6/60

6/60

5/60

6/36

6/36

6/36

6/248

Hypometric astigmatism

Hypometric astigmatism

Compound astigmatism

Hypometric astigmatism

Hypometric astigmatism

Type of refraction error Fundus Foveal hypoplasia Photophobia Nystagmus

Hypometropia Hypometropia Albiniotic

Albiniotic

Albiniotic

Albiniotic

Albiniotic

Albiniotic

Albiniotic

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Present

Present

Present

Present

Present

Present

Present

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Abbreviations: F, female; M, male; NA, not applicable. 1 Frequencies were determined by sequencing at least 374 chromosomes from geographically and ethnically matched subjects without any history of ocular disease. No carrier for these four alleles was found in the 1000 Genomes database, or in 5,400 individuals listed in the NHLBI-ESP variant database (see URLs in Supplementary Material online).

2008). Our molecular modeling predicted that glutamate at position 511 could potentially create a hydrogen bond (in the same manner as valine) within a ˚ (Supplementary Figure distance of 2.5 A S3 online), which would affect the folding and secondary structure of mutant SLC45A2. A sequence analysis of SLC45A2 also revealed a putative splice-site mutation in intron 3 (c.889-6T4G) segregating with nonsyndromic OCA in family PKAB059 (Figure 1). To determine whether c.889-6T4G alters the normal splicing of SLC45A2 mRNA, we performed an exon-trapping assay. In this assay, the wild-type construct produced a PCR product of 321 bp (pSPL3 þ exon 4) when amplified with vector primers (Supplementary Figure S2 online). The

construct with a mutated 50 splice site (c.889-6T4G) produced a band of similar size; however, sequencing analysis revealed an insertion of 5 bp from intron 3 directly upstream of exon 4 (Supplementary Figure S2 online). The results of the exon-trapping assay demonstrate that the c.889-6T4G mutation created a cryptic splice acceptor site, leading to aberrant splicing and the insertion of a 5-bp intron sequence into the spliced mRNA product. If a similar splicing effect occurs in affected individuals homozygous for the c.8896T4G mutation, this will lead to a shift in the reading frame and a predicted stop codon in exon 4 (p.Thr297PhefsX6), resulting in an mRNA that presumably undergoes nonsense-mediated decay (Maquat, 2004). However, the effect of

the c.889-6T4G mutation on in vivo splicing of SLC45A2 pre-mRNA is not known. All of the affected individuals from the OCA4 families exhibited a loss of pigmentation in their skin, hair, and eyes, regardless of sex or age (Figure 1 and Table 1). Intra-familial variation in hair color was noted among individuals and ranged from white to honey blonde or brown (Table 1). All of the affected individuals were photophobic, exhibited nystagmus and foveal hypoplasia, and their visual acuity was affected to varying degrees (Table 1). In summary, SLC45A2 mutations accounted for 4% (3 of 75 families; 95% confidence interval (CI), 1.45– 11.1%) of OCA in our Pakistani cohort study, whereas only one pathogenic www.jidonline.org 1101

A-C Lauer et al. Vitamin C as a Radical Scavenger

mutation in TYRP1 (1.3%; 95% CI, 0.32–7.1%) was found. This knowledge, in combination with data from previous studies, will improve our understanding of the molecular epidemiology of OCA in Pakistan. The results of our study will be important for future OCA diagnoses, genetic counseling, and functional studies of the TYRP1 and SLC45A2 proteins.

1

Division of Pediatric Ophthalmology, Cincinnati Children’s Hospital Research Foundation, Cincinnati, Ohio, USA and 2 Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan E-mail: [email protected] or [email protected] 3 These authors contributed equally to this work. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank the families for their participation and cooperation in our study. We also thank Dr S. Riazuddin for a critique of the manuscript. The work in Pakistan was supported by a research grant (HEC-1262) from the Higher Education Commission, to R.S.S. This work was also supported by intramural research funds granted by the Cincinnati Children’s Hospital Research Foundation (CCHMC) to Z.M.A. and by a Career Development Award from the Research to Prevent Blindness Foundation.

Tasleem Kausar1,2,3, Thomas J. Jaworek1,3, Nabeela Tariq2, Sobia Sadia2, Muhammad Ali2, Rehan S. Shaikh2 and Zubair M. Ahmed1,2

REFERENCES Boissy RE, Zhao H, Oetting WS et al. (1996) Mutation in and lack of expression of tyrosinase-related protein-1 (TRP-1) in melanocytes from an individual with brown oculocutaneous albinism: a new subtype of albinism classified as ‘‘OCA3’’. Am J Hum Genet 58:1145–56 Chaki M, Sengupta M, Mondal M et al. (2011) Molecular and functional studies of tyrosinase variants among Indian oculocutaneous albinism type 1 patients. J Invest Dermatol 131:260–2

Hussain R, Bittles AH (1998) The prevalence and demographic characteristics of consanguineous marriages in Pakistan. J Biosoc Sci 30:261–75 Hutton SM, Spritz RA (2008) A comprehensive genetic study of autosomal recessive ocular albinism in Caucasian patients. Invest Ophthalmol Vis Sci 49:868–72 Jaworek TJ, Kausar T, Bell SM et al. (2012) Molecular genetic studies and delineation of the oculocutaneous albinism phenotype in the Pakistani population. Orphanet J Rare Dis 7:44 Manga P, Kromberg JG, Box NF et al. (1997) Rufous oculocutaneous albinism in southern African Blacks is caused by mutations in the TYRP1 gene. Am J Hum Genet 61:1095–101 Maquat LE (2004) Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat Rev Mol Cell Biol 5:89–99 Oetting WS, King RA (1999) Molecular basis of albinism: mutations and polymorphisms of pigmentation genes associated with albinism. Hum Mutat 13:99–115 Rooryck C, Morice-Picard F, Elcioglu NH et al. (2008) Molecular diagnosis of oculocutaneous albinism: new mutations in the OCA1-4 genes and practical aspects. Pigment Cell Melanoma Res 21:583–7

Chiang PW, Spector E, Scheuerle A (2009) A case of Asian Indian OCA3 patient. Am J Med Genet A 149A:1578–80

Rooryck C, Roudaut C, Robine E et al. (2006) Oculocutaneous albinism with TYRP1 gene mutations in a Caucasian patient. Pigment Cell Res 19:239–42

Forshew T, Khaliq S, Tee L et al. (2005) Identification of novel TYR and TYRP1 mutations in oculocutaneous albinism. Clin Genet 68:182–4

Tomita Y, Suzuki T (2004) Genetics of pigmentary disorders. Am J Med Genet C Semin Med Genet 131C:75–81

Radical Scavenging Capacity in Human Skin before and after Vitamin C Uptake: An In Vivo Feasibility Study Using Electron Paramagnetic Resonance Spectroscopy Journal of Investigative Dermatology (2013) 133, 1102–1104; doi:10.1038/jid.2012.420; published online 29 November 2012

TO THE EDITOR Vitamin C (VitC) is a potent radical scavenger (Buettner and Jurkiewicz, 1996) and is a component of the antioxidant system in human skin. The skin is constantly confronted with reactive oxygen species and other free radicals as a result of environmental inflictions (Thiele et al., 2001), as well as the endogenous formation of radicals (Berger, 2005). The skin comprises a variety of antioxidants, e.g., VitC,

which must be taken in with food as human cells lack the enzymes for synthesis. In healthy volunteers, oral and topical application of VitC reduces skin photo damage (Dayan et al., 2011). Supplementation of VitC might be controversial, but it is commonly implemented. The aim of this investigation was to conduct a feasibility study to measure changes in the radical scavenging capacity of human skin in vivo in response to the supplementa-

Abbreviations: EPR, electron paramagnetic resonance; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy; VitC, vitamin C

1102 Journal of Investigative Dermatology (2013), Volume 133

tion with moderate VitC doses. Furthermore, we were interested in learning about the kinetics of VitC uptake. It was investigated whether the uptake is dose dependent. A total of 33 healthy volunteers (aged between 22 and 57 years, 16 men and 17 women) were supplemented with dose 1 of VitC (100 mg calcium ascorbate per day), dose 2 of VitC (180 mg calcium ascorbate per day), or a placebo for 4 weeks. The study was approved by the Ethics Committee of the Charite´–Universita¨tsmedizin Berlin and conducted in accordance with the Declaration of Helsinki Principles. Participants had given their

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