Mutational analysis of a Chinese family with oculocutaneous albinism type 2

Oculocutaneous albinism (OCA) is an autosomal recessive disorder characterized by hypopigmentation of the skin, hair, and eyes accompanied with ophthalmologic abnormalities. Molecular genetic test can confirm the diagnosis of the four subtypes of OCA (OCA1-4). Herein, we report a Chinese family with two patients affected by OCA. Mutations of TYR, OCA2, TYRP1, and SLC45A2 were examined by using PCR-sequencing. Large deletions or duplications of TYR and OCA2 were examined by Multiplex Ligation-dependent Probe Amplification (MLPA) assay. Compound heterozygous mutations of OCA2, (c.808-3C>G and c.2080-2A>G), were identified in both patients characterized with yellow hair and milky skin, heterochromia iridis, and nystagmus. Several computer-assisted approaches predicted that c.808-3C>G and c.2080-2A>G in OCA2 might potentially be pathogenic splicing mutations. No exon rearrangement (deletion/duplication) of TYR and OCA2 was observed in the patients by MLPA analysis. This study suggests that compound heterozygous mutations, (c.808-3C>G and c.2080-2A>G), in OCA2 may be responsible for partial clinical manifestations of OCA.


INTRODUCTION
Oculocutaneous albinism (OCA) is a heterogeneous and autosomal recessive disorder with an estimated prevalence of 1/17,000 worldwide, and the carrier rate is approximately 1 in 70. OCA is characterized by a reduction or complete loss of pigment in the skin, hair, and eyes accompanied by photophobia, nystagmus, strabismus, and reduced visual acuity due to melanin biosynthesis deficiency [1,2]. OCA is broadly classified as non-syndromic and syndromic OCA based on the presence of other symptoms such as immunodeficiency, bleeding diathesis, or neurological dysfunction [3,4]. Non-syndromic OCA includes four types, OCA1-4, and the clinical diagnosis of OCA subtype is difficult because of its variable clinical phenotype. Emerging evidence shows that molecular and genetic analyses can provide accurate diagnosis and genetic counselling.
The prevalence of different OCA subtypes significantly differs in different ethnic populations. OCA1 and OCA2 are the two most frequent types of OCA, making up 50% and 30% of all OCA cases worldwide, respectively [1,5]. OCA1 is caused by mutations of TYR. OCA2-4, which are somewhat milder, are caused by mutations in OCA2, TYRP1, and SLC45A2, respectively. OCA2 is mainly found in Africa, and the frequencies of OCA3 and OCA4 are approximately 3% and 17% worldwide, respectively [6][7][8].
Genetic tests were carried out to provide an accurate genetic diagnosis and genetic counselling for a Chinese family with two patients affected by OCA characterized by yellow hair, milky skin, photophobia, nystagmus, and reduced visual acuity. Two compound heterozygous mutations (c.808-3C>G and c.2080-2A>G) in OCA2 were identified, which may result in pathogenic splice site mutation and may be responsible for some clinical manifestations of OCA.

Clinical phenotype
The pedigree chart and the clinical features of the male patient (proband) affected by OCA are shown in Figure 1. Both the patients have yellow eyebrows and hair, milky skin, and heterochromia iridis, accompanied with photophobia, impaired visual acuity, and nystagmus. On the other hand, unaffected family members present normal pigmentation.
Several in silico analysis programs were used to predict the effect of the compound heterozygous mutations in OCA2 (c.808-3C>G and c.2080-2A>G) on splicing. The GENIE program performs splice site score calculation. The results indicated that the mutants showed a remarkable decrease of the splice site score ( Table 2). The NetGene2 program showed that both OCA2 c.808-3C>G and c.2080-2A>G mutations abolished a previously predicted splice site ( Figure 4A). The NNSPLICE program showed that OCA c.808-3C>G mutation generated a novel splice site, and the two nucleotides (AG) from the authentic splice site were incorporated into the coding region, creating a frame shift mutation. The NNSPLICE program showed  Figure 4B). These data suggest that both OCA2 c.808-3C>G and c.2080-2A>G mutations may affect OCA2 mRNA splicing, and compound heterozygous mutations (c.808-3C>G and c.2080-2A>G) in OCA2 might be responsible for some clinical manifestations of OCA.
In order to detect rearrangements (deletion/ duplication) of TYR and OCA2, Multiplex Ligationdependent Probe Amplification (MLPA) assay was used and the results showed that no significant exon rearrangement (deletion/duplication) of TYR and OCA2 occurred in the two patients ( Figure 5).

Mutation identification and analysis of SLC45A2
Compound heterozygous mutations in SLC45A2 (c.814G>A and c.890C>T, which result in p.Glu272Lys and p.Thr297Ile, respectively) have been identified in both the male patient and a healthy unaffected girl in the  family. These data suggest that compound heterozygous mutations in SLC45A2 (c.814G>A and c.890C>T) may not be associated with OCA in this family.

DISCUSSION
OCA2 accounts for 30% of all OCA cases worldwide with an estimated prevalence of 1/38,000-1/40,000. It is the most frequent form of OCA in the African population with a higher prevalence of 1/3,900-1/1,500 and in the African-American population, the prevalence is estimated to be 1/10,000 [11,12]. OCA2 is characterized by variable hypopigmentation of the skin and hair, which may range from minimal to near normal, accompanied with ocular changes. Nystagmus is present before 3-4 months of age. Strabismus and visual inattention may occur in the first six  OCA2 c.808-3C>G wild type (acctagaccgagcagtgccagatcccagatggtgtctcaggtgaaaagcctcaccataacttatgctttggcttgtaCaggtcactcacaactggacggtgt atttaaatccgaggagaagcgagcactcagtgatgagcaggacctttgaggtactgaccaggtgagttctcagtgagtgaggtgttggggcaggctct) and the mutant (acctagaccgagca gtgccagatcccagatggtgtctcaggtgaaaagcctcaccataacttatgctttggcttgtaGaggtcactcacaactggacggtgtatttaaatccgaggagaagcgagcact cagtgatgagcaggacctttgaggtactgaccaggtgagttctcagtgagtgaggtgttggggcaggctct) were used for sequence input. OCA2 c.2080-2A>G wild type (tcattttcaagactttttttttaaatcttgcatatattttcggttctaaactgattctcaccacacatcctttcttctAggcattggcacatctccacttaatagaatatgttggagaacaaactgctttgctaataaaggtaa aataaatgctata atagaaggcactccagccactgttctttgattttgtgaaaaaa) and the mutant (tcattttcaagactttttttttaaatcttgcatatattttcggttctaaactg attctcaccacacatccttt cttctGggcattggcacatctccacttaatagaatatgttggagaacaaactgctttgctaataaaggtaaaataaatgctataatagaaggcactccagccactgttctttgattttgtg aaaaaa) were used for sequence input. The capital words indicate the mutation site. months of age. Iris color ranges from blue to brown. Hair color may darken over time, although the hair color ranges from light yellow to light brown in newborns [13,14]. OCA2 is caused by mutations in OCA2, a human homologue of mouse pink-eye dilution gene located on chromosome 15q11.2-q12 containing 24 exons (23 coding domains). The encoded protein, known as the P protein, is an integral membrane protein composed of 838 amino acid residues that consists of 12 transmembrane spanning regions and is involved in tyrosine transport, which is a precursor to melanin synthesis and pigmentation in the skin, hair, and eye [15,16]. P protein is involved in the regulation of the pH of melanosomes or served as a melanosomal tyrosine transporter [17]. The most common mutation in OCA2 is a 2.7-kb deletion, which removes exon 7 and results in a frame shift mutation in the first luminal loop of OCA2 protein, producing a truncated and non-functional protein. This mutation is detected in Africans, sub-Saharan African heritage, African-Americans, and Haitian, suggesting a founder effect [18,19]. Rooryck et al. stated that rearrangements of OCA2 might be present in more than 20% of patients with OCA2 [20]. P protein may disturb the pigmentation characteristics by altering the melanosomal tyrosine or tyrosinase function due to OCA2 mutations, but melanocytes of patients with OCA2 still produce small amounts of melanin. OCA2 is caused by homozygous or compound heterozygous OCA2 mutation, and recessive compound heterozygous mutation indicated that the mutant alleles of both copy are at different locations on the same gene. Patients in the compound heterozygous state may present with a less severe phenotype compared with those presenting with the homozygous form [13]. In the HGMD database, 154 mutations were included, and missense mutations account for 60%, while splicing mutations account for 11%.
No TYR or TYRP1 mutation was identified in the two patients. Two splice site mutations in OCA2 (c.808-3C>G and c.2080-2A>G) have been identified in both patients, while the healthy family members presented with only one of the two mutations. OCA2 c.808-3C>G was first identified in a Hispanic family with OCA, and this is the first report in Chinese population. OCA2 c.2080-2A>G has not been reported in any ethnic population yet. GENIE, NNSPLICE, and NetGene2 programs have been used to predict the effect of the two splice site mutations on OCA2 mRNA splicing. All three programs showed that both OCA2 c.808-3C>G and c.2080-2A>G mutations may affect OCA2 mRNA splicing by abolishing previous splice sites or generating a new splice site. The expression of OCA2 is very low in leukocytes, and we failed to amplify OCA2 mRNA. In vitro experiments may further confirm these effects.
To exclude the large deletion or duplication of exons in TYR and OCA2, MLPA was also performed to detect  exon rearrangements, and the results showed that no deletion or duplication of exons was found in the patients.
In the current study, compound heterozygous mutations in SLC45A2 (c.814G>A and c.890C>T) were identified in both a male patient and an unaffected girl in the family. Therefore, compound heterozygous mutations in SLC45A2 may not be the causative mutation for OCA in this family.
In conclusion, this study expands the mutation spectrum of OCA. Compound heterozygous mutations (c.808-3C>G and c.2080-2A>G) in OCA2 might be responsible for some clinical manifestations of OCA.

Subjects and clinical evaluation
This study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All procedures were carried out in accordance with the approved guidelines. One patient was a 25-year-old male, and another patient was a 37-year-old female. Both of them presented the same clinical ophthalmologic characteristics, including heterochromia iridis, milky skin, yellow hair, photophobia, nystagmus, and reduced visual acuity. Family history and pedigree chart were drawn to evaluate the inheritance model. Written informed consent was obtained from all participants and authorization to publish personal photographs was obtained from the male patient only. However, he only allowed us to publish photographs of his hair and eyebrows, but not of his eyes.

Strategy for mutational screening
Mutational screening of TYR was prioritized for patients with OCA. OCA2, SLC45A2, and TYRP1 were sequentially screened for mutations when no mutation was found in TYR.

DNA extraction and mutational analysis
DNA extraction and PCR-based Sanger sequencing were performed as previously described [21]. Briefly, each 50-μL PCR mixture contained 100 ng of genomic DNA, 2 μL of 10 μM forward and reverse primers (with a final concentration of 400 nM), and 25 μL of 2× Taq PCR MasterMix (Takara, Dalian, China). PCR was carried out in Veriti thermocycler (Applied Biosystems, Foster City, CA, USA) using the following protocol: 95°C for 3 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s; and a final extension at 72°C for 7 min. The amplified products were purified with a cycle-pure kit (Axygen, Wujiang, China) and sequenced using an ABI 3500 Dx sequencer (Applied Biosystems). In order to detect exon rearrangements (deletion/duplication) of TYR and OCA2 and to increase the mutation rate, MLPA assay Kit (P325-OCA2) from MRC-Holland (Amsterdam, Netherlands) was used and the procedure was performed according to manufacturer's instructions. The mutation was named according to the recommendation of sequence variants by Human Genomic Variation Society (HGVS: http://www.hgvs.org/). The interpretation of sequence variants was made according to the recommendation of the American College of Medical Genetics (ACMG) and Genomics and the Association for Molecular Pathology (AMP) [22]. All primer sequences are listed in Table 3.