Genetic analysis and prenatal diagnosis of 20 Chinese families with oculocutaneous albinism

Abstract Background Oculocutaneous albinism (OCA) is a group of heterogeneous genetic disorders characterized by abnormal melanin synthesis in the hair, skin, and eyes. OCA exhibits obvious genetic and phenotypic heterogeneity. Molecular diagnosis of causal genes can be of help in the classification of OCA subtypes and the study of OCA pathogenesis． Methods In this study, Sanger sequencing and whole exome sequencing were used to genetically diagnose 20 nonconsanguineous Chinese OCA patients. In addition, prenatal diagnosis was provided to six OCA families. Results Variants of TYR, OCA2, and HPS1 were detected in 85%, 10%, and 5% of affected patients, respectively. A total of 21 distinct variants of these three genes were identified. Exons 1 and 2 were the hotspot regions of the TYR variants, and c.895C > A and c.896G > A were the hotspot variants. We also found seven novel variants: c.731G > A, c.741C > A, c.867C > A, and c.1037‐2A > T in TYR, c.695dupT and c.1054A > G in OCA2, and c.9C > A in HPS1. Genetic tests on six fetuses revealed three carrier fetuses, two normal fetuses, and one affected fetus. The follow‐up results after birth were consistent with the results of prenatal diagnosis (one fetus terminated during pregnancy was not followed up). Conclusions This study expands our understanding of the genotypic spectrum of the Chinese OCA population. The findings indicate that prenatal diagnosis can provide important information for genetic counseling.

in visual acuity, nystagmus, strabismus, and photophobia. OCA is the most common form of albinism, affecting about 1 in 20 000 individuals worldwide. 1 The prevalence is higher (about 1 in 18 000) in the Chinese population. 2 Based on the complexity of the system involved, OCA can be further classified as either nonsyndromic OCA or syndromic OCA. Due to the genetic heterogeneity of OCA and the overlapping phenotypes of different OCA subtypes, it is difficult to distinguish between different OCA subtypes and to identify causal genes. As such, molecular genetic testing has become the only reliable method for OCA classification. 8 In this study, Sanger sequencing and whole exome sequencing (WES) were used for pathogenic variant screening of OCA-causing genes in 20 Chinese families with albinism. In addition, prenatal diagnosis and informative genetic counseling were provided to six OCA families.

| Patients
The present study was approved by the institutional research ethics committee of Wenzhou Central Hospital, Zhejiang, China, and the study protocol conforms to the ethical guidelines of the Declaration of Helsinki. Written informed consent was obtained from the patient or the guardians of the patient. A total of 20 OCA families from East China were screened between 2013 and 2019, including six families who underwent prenatal diagnosis. All patients denied any family history of consanguinity. The following clinical data were collected: skin, hair, and iris color; ophthalmological findings (photophobia, nystagmus, and eyesight); and other phenotypes (eg, abnormalities of the immune system, hemorrhagic diathesis).

| Sanger sequencing
The genomic DNA (gDNA) was extracted from amniocytes or peripheral blood using a Qiagen DNA Blood Midi/Mini kit (Qiagen), following the manufacturer's protocol. Variant screening of the TYR gene

| Whole exome sequencing
WES was performed in the other 10 families (family [11][12][13][14][15][16][17][18][19][20]; this included two families (family 19 and 20) in which direct Sanger sequencing failed to identify any pathogenic variants. Exon-containing fragments were enriched by SureSelect Human All Exon V6 (Agilent, USA), and a HiSeq2500 sequencer was used for sample sequencing (Illumina). Paired-end sequencing was performed for each sample, and more than 95% of bases in the targeted regions were covered by at least 20 reads. Raw data generated by WES were filtered to obtain high-quality clean reads and were further aligned to the NCBI Human Reference Genome (hg19/GRCh37) using the Burrows-Wheeler Aligner (BWA). Then, SAMtools and Genome Analysis ToolKit (GATK) were used to detect single nucleotide polymorphisms or sequence variants in BAM files. Variants located in coding regions or exon-intron junctions of 33 candidate genes (Table S2) responsible for albinism or diseases whose phenotypes partially overlap with albinism were analyzed emphatically.

| Clinical phenotype
In this study, all patients had typical OCA symptoms of the skin, hair, and eyes. Clinical features of these 20 patients are described in Table 1.
Among these patients, there were several special cases. Specifically, patient 10 died 15 days after birth due to congenital diaphragmatic hernia. In addition, patients 9, 19, and 20 had pigmented nevi on their bodies.

| Identification of sequence variants
In the current study, (likely) pathogenic variants in the TYR (GenBank:    in African and African American populations. 10 In contrast, TYRP1

| D ISCUSS I ON
variants and HPS1 variants are prevalent in Southern Africa and Northwestern Puerto Rico, respectively. 11,12 In this study of Chinese OCA patients, variants in TYR, OCA2, and HPS1 were found in 85%, 10%, and 5%, of patients, respectively, which is similar to the report of Wei et al 13 In contrast, SLC45A2 variants were totally absent here but have been reported to account for 10%-20% of Chinese OCA cases. 13,14 This highlights the genetic heterogeneity of OCA even in patients from the same ethnic group. In addition, differences in the included number of cases may be another explanation. caused arginine to be substituted by glycine at codon 352 and was predicted to be deleterious by all in silico tools. Patient 19 also carried another known pathogenic variant, c.1426A > G (p.N476D). 21 We further determined that the c.1054A > G variant was in trans (different copy of OCA2 gene) with the known variant by testing the next generation of the patient.
HPS is a rare genetic disorder characterized by oculocutaneous albinism, bleeding tendency and, in some cases, lysosomal storage disease. 22  In summary, this study successfully detected causal variants in 20 OCA families. A total of 21 distinct variants of the TYR, OCA2, and HPS1 genes were identified by direct Sanger sequence and WES, of which, seven variants were novel. In addition, effective prenatal diagnosis and genetic counseling were provided to six OCA families to assist them to avoid the birth of affected children. This study expands our understanding of the genotypic spectrum of the Chinese OCA population and highlights that the development of new genetic testing technologies can contribute to more accurate and efficient clinical diagnoses.

ACK N OWLED G M ENTS
We would like to thank all the patients and family members who participated in this work for their cooperation and patience. This study was supported by Wenzhou Central Hospital. We appreciate the financial support of the Medical and Health Science and Technology Project of Zhejiang Province support plan (2020KY921); Science and Technology Planning Project of Wenzhou (ZS2017004). Also, the authors wish to acknowledge the support of clinical geneticists and certified genetic counselors.

AUTH O R S ' CO NTR I B UTI O N S
All authors contributed to the study conception and design.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that provided the evidence for the study are available from the corresponding author upon reasonable request.