Genetic analyses of oculocutaneous albinism types 1 and 2 with four novel mutations

Background Oculocutaneous albinism (OCA) is a human autosomal-recessive hypopigmentation disorder with hypopigmentation in the skin, hair, and eyes. OCA1 and OCA2 are caused by mutations of the TYR and OCA2 genes, respectively, which are responsible for most oculocutaneous albinism. However, the incidence of oculocutaneous albinism patients in Guangxi remains unclear. Methods To evaluate the molecular basis of oculocutaneous albinism in thirty-six patients in Guangxi, China. Peripheral venous blood samples were collected from these unrelated patients. The TYR and OCA2 genes of all individuals were analyzed by direct DNA sequencing and the sequences compared with are reference database and bioinformatics analysis. Results Among the 36 OCA patients, 8(22.2%) were found mutations on TYR gene, 28 (77.8%) on OCA2. And we identified Twenty-seven different TYR and OCA2 mutations in these patients, including one novel TYR framshift mutation c.561_562insTTATTATGTGTCAAATTATCCCCCA, three novel OCA2 mutations: one nonsense mutation c.2195C > G(p.S732X), one deletation mutation(c.1139-1141delTGG), one missense mutations c.2495A > C(p.H832P). The population screening and the bioinformatic analysis to determined the effects of the mutations, which revealed these four novel mutations were pathogenic. Conclusions This study expands the mutation spectrum of oculocutaneous albinism. Four novel mutational alleles c.1139-1141delTGG, c.1832 T > C and c.2195C > G and of the OCA2 gene and c.561_562insTTATTATGTGTCAAATTATCCCCCA of TYR were associated with OCA. The genotype–phenotype correlations suggest that molecular diagnosis is more accurate and important in OCA. Electronic supplementary material The online version of this article (10.1186/s12881-019-0842-7) contains supplementary material, which is available to authorized users.


Background
Albinism is a group of hereditary disorders caused by a deficit in production of the pigment melanin. It can be classified as oculocutanous albinism (OCA) and ocular albinism (OA) [1]. The most common and visible type of albinism is oculocutaneous albinism, which is a group of autosomal recessive disorders with a reduction or complete absence of melanin in the skin, hair, and eyes and is often associated with ocular changes including photophobia, decreased visual acuity and nystagmus [2]. OCA is subdivided into 7 subtypes (OCA 1-7) based on genes as follows: TYR (OCA1), OCA2 (OCA2), TYRP1 (OCA3), SLC45A2(OCA4), SLC24A5 (OCA6), LRMDA (OCA7) and OCA5 located on chromosome 4q24 and the subtypes can only be accurately diagnosed by genetic gene [3]. OCA affects one in 17,000 individuals worldwide [4]. The prevalence of OCA subtypes differs among ethnic groups. OCA1 has been reported to be the most subtype in Caucasians and accounts for approximately 50% of cases worldwide [5,6]. OCA2 is the most common form of albinism worldwide; in which the prevalence is estimated as 1/30000 due to Caucasians [5], and in the African-American population, in which the prevalence is estimated to be 1:10,000 [6]. OCA3,or rufous OCA (ROCA), it has been reported to affect 1:8500 individuals in Africa, but is virtually unseen in Caucasians and Asiatic populations. OCA3,or rufous OCA (ROCA), it has been reported to affect 1:8500 individuals in Africa, but is virtually unseen in Caucasians and Asiatic populations [7]. OCA4 were reported to explain the disease in approximately 18% of Japanese patients, whereas it is very rare in in Korean, Chinese,Caucasians populations [7,8]. In Han Chinese, the prevalence of OCA is about 1:18000, and OCA1 is the most subtype [9]. OCA1, is caused by mutations of TYR on chromosome 11q14 and it exhibits the most severe phenotype [10]. More than 300 mutations in TYR have been identified in individuals with OCA1 (MIM 203100) phenotype [11]. There are two subtypes of OCA1: OCA1A and OCA1B. OCA1A is caused by a mutation causing a complete lack of tyrosinase activity, which presents with milky skin and white hair throughout life. Whereas type 1B (OCA1B) is caused by the mutations causing reduced activity of tyrosinase, and the white/light yellow hair and white skin of individuals with OCA1B can darken overtime [4,12]. Oculocutaneous albinism type II (OCA2-MIM 203200), an autosomal recessive disorder in which the biosynthesis of melanin pigment is reduced in skin, hair, and eyes, which has been described in all major ethnic groups [13]. OCA2 is caused by mutations of OCA2 (previouslycalled P gene), which is located on chromosome 15q11.2-q12 and consists of 24 exons (23 coding). More than 140 mutations have been identified in its gene body (HGMD Professional http://www.hgmd.cf.ac.uk/ac/all.php). Although the function of OCA2 is not precisely characterized, the p protein consists of 12 transmembranes panning regions and is an integral component of themelanosomal membrane [13]. But it could be involved in the transport of tyrosine, the precursor to melanin synthesis, within the melanocyte. Regulates the pH of melanosome and the melanosome maturation [14][15][16].
As mutations in the TYR and OCA2 genes account for the majority of OCA cases, we have analyzed and examined the TYR and OCA2 genes in thirty-six patients with oculocutaneous albinism in Guangxi Zhuang Autonomous Region of China in the present study to identify the causative mutations for each of them.

Patients
August 2017 to May 2019, thirty-six unrelated OCA patients (33 minors and 3 adults) and their parents and 300 unaffected subjects were recruited in our study. All the patients were from the provinces of Guangxi, China. The families were referred to the Guangxi Maternal and Child Health Hospital for advice about diagnosis or highly suspected in patients with non-syndromic ocular albinism and Genetic testing demands of the patient. We followed the criteria for the differentiation of OCA1A, OCA1B and OCA2 as described [16]. Among the 36 OCA patients, sixteen were clinically diagnosed as OCA1A or OCA1B, twenty were diagnosed as OCA2 (Table 1). In all the 36 OCA patients, varying colors of the skin and hair, and abnormal ophthalmological findings including photophobia, nystagmus and reduced visual acuity were observed. The 300 normal controls matched by gender and ethnic origin were selected and recruited from healthy individuals from the Center of Genetic Metabolism Genetic Metabolism at Guangxi Maternaland Child Health Hospital. All participants agreed to carry out a genetic analysis and singed written informed consent for the study approved by the Genetic and Metabolic Central Laboratory of Guangxi Zhuang Autonomous Region Women and Children Care Hospital (Nanning, China).

Mutation identification and analysis
Sanger sequencing analysis of the relevant PCR fragments in exons of the TYR and OCA2 genes revealed

In silico analysis novel variants
Our studies revealed four novel variants, which located in TYR and OCA2, respectively (Table 1 and Fig. 1). The functional sites of the SIFT, PolyPhen2.0 and Mutation   (Table 5). According to the ACMG standards and guidelines for the interpretation of sequence variants, these novel mutations is likely pathogenic or pathogenic (Table 5).

Discussion
Oculocutaneous albinism is a recessive hereditary group of diseasesis characterized by reduce or completely absent melanin synthesis despite adequate numbers of structurally normal melanocytes in the skin, hair, and eyes [16]. The common disease-causing genes of OCA are TYR and OCA2 gene. Tyrosinase is a coppercontaining glycoprotein and a oxidase and involved in the formation of pigments, such as melanins and other polyphenolic compounds [11]. The mutations of TYR causing the OCA1 are delineated by five key functional sites of the enzyme; two locations copper-binding sites while others are located at the 3′-end of the copper B-binding region near the amino terminus of the protein, and between the CuA and CuB domains [18]. OCA2 is caused by mutations in the OCA2 gene, resulting in alterationsin the p protein, and which might in turn affect melanin biosynthesis. OCA2 is a transmembrane protein found in the melanosomal membrane. Numerousstudies suggest that this protein could be involved in the transport of tyrosine, the precursor to melanin synthesis within the melanocyte and regulates the pH of melanosome and the melanosome maturation [19][20][21].
In this study, we identified compound heterozygous and homozygous mutations in TYR or OCA2 in 36 Guangxi Chinese individuals by direct sequencing. The distribution of mutational OCA genes was slightly shifted, OCA2 is the most common type in our oculocutaneous albinism population in Guangxi, China. In a total of 36 OCA patients, 8 were clinically diagnosed with OCA1, 28 were diagnosed with OCA2 (Table 1). Of  (Table 4).
Among patients with OCA1, most of patients genotypically have "tyrosinase-negative" OCA1A and patient 4 have OCA1B, associated with low residual tyrosinase catalytic activity. The mutation c.896G > A(p.R299H) has already been reported [22] which is located in the central portion of tyrosinase and causing the enzyme activity deficient, therefore, the patient was classified as OCA1A. Tomita Yoshioka et al. [23] demonstrated that the frameshift mutation c.929dupC(p.R311Kfs) resulting in a truncated inactive tyrosinase. The heterozygous mutations c.230G > A(p.R77Q) and c.1199G > T(p.W400 L) in the TYR gene identified in patient 2 were firstly reported in Japanese and Taiwan patients [24]. The mutation of c.230G > A(p.R77Q) is the major ones in Japanese patients with OCA1A [25]; Chang-Hai Tsai et al. [26] demonstrated that the mutation of c.1199G > T(p.W400 L) might disrupt the second copper binding site of this polypeptide. Therefore, the patients(1-3,5-8) were classified as OCA1A. Patient 4 was compound heterozygous for c.996G > A (p.M332I) and c.1265G > A (p.R422Q) changes in the TYR gene. Grønskov K et al. identified the M332I allele in Denmark for the first time [27], and in 1991, Giebel LB et al. demonstrated the substitution of R422Q results in a tyrosinase polypeptide that is temperature-sensitive [11], the patient was classified as OCA1B. According to the study of Richards S et al., c.346C > T (p.R116X) was a pathegentic mutation, which resulted in premature termination codon downstream [17]. To the best of our knowledge, c.561_562insTTATTATGT GTCAAATTATCCCCCA(G190Cfs*12) was a novel mutation, which was not present in the HGMD Professional Database, dbSNP, or the 1000 Genomes database. The variant caused a frameshift alteration after codon 190 leading to a premature termination codon (PTC) which located at codon 202 and resulting in a inactive tyrosinase .
There, we analyzed the genetic defects underlying OCA2 in 28 albino patients (patients 9-36, Table 1), and identified sixteen known known mutations, three novel mutations. Except the novel mutations, other 16 mutations have been reported [16,[27][28][29][30][31][32][33][34][35], (https://www.scholarmate.com/S/ ivheaf]. Pei-Wen Chiang et al. [24] identified the c.808-3C > G allele in Hispanic for the first time, in their studies, c.808-3C > G would affect the splicing of OCA2 and induce abnormal mRNA splicing. In our study, Seven Patients were inherited this missense substitutions and one patient was homozygous for c.808-3C > G, this is the first time report in Chinese. The c.808-3C > G splicing site mutation represents a significant proportion of the P gene mutations (16.1%) in our oculocutaneous albinism population in Guangxi, China. The site of c.1832 T > C has been reported by Chunyue Miao as a pathogenic mutation  As the study has reported that most missense mutations occur in the loops between the transmembrane domains [37], the mutations of P211L, A334V, V443I, T475P, R455G, P198L, T450 M, A481T, R572C, S788 L, and A7 87T were close to the interface between the transmembrane domains and the intra-or extracellular regions. Of the 19 distinct mutations in the OCA2 gene, three novel mutations have not been presented in dbSNP (http://www.ncbi.nlm.nih.gov/dbvar), the 1000 Genomes Database (http://browser.1000genomes.org/index.html), the HGMD Professional Database (http://www.hgmd.cf. ac.uk/ac/all.php), or the Albinism Datebase (http://www. ifpcs.org/albinism/oca2mut.html). To the best of our knowledge, c.1139-1141delTGG in the OCA2 gene was a novel mutation, the mutation occur within dinucleotide repeats and may have arisen because of slipped mispairing, and it located at extracellular regions. The mutation of c.2195C > G(p.S732X) change from TAC to TAG which is a novel mutation and located in the structure of transmembrane domains resulted in premature termination codon downstream. The truncated protein lacked transmembrane domains, which might have caused the transport of tyrosine dysfunction and the precursor to melanin synthesis dysfunction and resulted in location of the protein in the nucleus. The c.2495A > C(p.H832P) mutation which located in the structure of transmembrane domains and the hydrophilic histidine becomes hydrophobic proline which may change the transmembrane structure of OCA2, and further affect the tyrosinase activity.
In our study, the clinical phenotypes of 26 OCA2 patients identified showed a wide variety OCA. The Sviderskaya studies [34] shows that the A481T allele had approximately 70% functional activity in melanogenesis compared with that of the wild-type human P cDNA, furthermore, p.481Thr was reported to be an Asian-specific hypopigmentation allele [38]. Jeppe D. Andersen's date [35] showed that p.481Thr and p.443Ile was sufficient to lower the pigmentation levels in healthy individuals. These findings suggesting that the novel alleles of c.1139-1141delTGG, c.2195C > G(p.S732X) and c.2495A > C(p.H832P),might have no or very low functional activity in melanogenesis. The missense, splicing and deletion mutations destroyed the spatial structure and the integrity of the OCA2 protein, which would delayed the activity of the OCA2 protein different degrees in melanogenesis. Functional studies of the variants is particularly important on the relationships of phenotypes and genotypes of the P gene, and the mechanism for this need futhuer study.

Conclusion
In summary, we reported thirty six OCA patients and the molecular basis of their disease were identified by PCRsequencing of all exons of the TYR and OCA2 genes. This study expands the mutational database in humans. It is the first report of the c.1714C > T(p.R572C), c.1139-1141d elTGG and c.2195C > G(p.S732X) mutations in the P gene and c.561_562insTTATTATGTGTCAAATTATCC CCCA(G190Cfs*12) in TYR gene. The study shows that OCA2 may be the most common type and the alleles of c.1832 T > C(p.L611P), c.808-3C > G may be the hotspots of OCA2 in oculocutaneous albinism population in Guangxi, China. Molecular genetic testing of TYR, OCA2 is a useful tool for clinical diagnosis and genetic counseling of OCA.

Additional file
Additional file 1: Figure S1. DNA sequencing result from TYR (Patient1-8) and OCA2 (Patient9-36) gene, changes also seen in the father and mother. (DOCX 999 kb) Abbreviations OCA: Oculocutaneous albinism; OCA2: Oculocutaneous albinism II gene; SLC45A2: Solute carrier family 45, member 2 gene; TYR: Tyrosinase gene; TYRP1: Tyrosinase related protein 1 gene data;XF and YQ participated in the study coordination and revised the manuscript. All authors read and approved the final version of the manuscript.

Funding
This work was supported by the Project of Yu-Miao (Grant No. GXWCH-YMJH-2017006), which has been used for the patient recruitment and determining genetic variants. This study was also supported by the Open Project Program of the Shanghai Key Laboratory of Birth Defect (grant no. 16DZKF1014), which has been used for validation of the pathogenicity of identified variants. The funders are not involved in the study design, data collection and analysis, performing experiments and in writing the manuscript.
Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files.
Ethics approval and consent to participate All procedures in this study were approved by the Institutional Review Boards and Ethics Committees of Guangxi Maternal and Child Health Hospital. Detailed written informed consent was obtained from all participants. For the patients who are underage, written informed consent for participation in this study was obtained from the patients' parents or guardians.

Consent for publication
Not applicable.