Detection of mutations in MYOC, OPTN, NTF4, WDR36 and CYP1B1 in Chinese juvenile onset open-angle glaucoma using exome sequencing

Juvenile onset open-angle glaucoma (JOAG) affects patients before 40 years of age, causing high intraocular pressure and severe optic nerve damage. To expand the mutation spectrum of the causative genes in JOAG, with a view to identify novel disease-causing mutations, we investigated MYOC, OPTN, NTF4, WDR36 and CYP1B1 in a cohort of 67 unrelated Chinese JOAG patients. Whole exome sequencing was used to identify possible pathogenic mutations, which were further excluded in normal controls. After sequencing and the use of a database pipeline, as well as predictive assessment filtering, we identified a total of six mutations in three genes, MYOC, OPTN and CYP1B1. Among them, 2 heterozygous mutations in MYOC (c. 1109C > T, p. (P370L); c. 1150G > C, p. (D384H)), 2 heterozygous mutations in OPTN (c. 985A > G, p.(R329G); c. 1481T > G, p. (L494W)) and 2 homozygous mutations in CYP1B1 (c. 1412T > G, p.(I471S); c. 1169G > A, p.(R390H)) were identified as potentially causative mutations. No mutation was detected in NTF4 or WDR36. Our results enrich the mutation spectra and frequencies of MYOC, OPTN and CYP1B1 in JOAG among the Chinese population. Further studies are needed to address the pathogenicity of each of the mutations detected in this study.

for complex diseases 18 . In developmental and congenital glaucoma, WES has led to the identification of novel variants in LTBP2 and PXDN 19 . It has also been used to identify mutations in known genes in primary glaucoma effectively and quickly 20 . In the current study, we performed WES on 67 Chinese JOAG patients to detect the full spectra of variants in MYOC, OPTN, NTF4, WDR36 and CYP1B1, with a view to identify novel disease-causing mutations for JOAG.

Results
From the whole exome results of 67 Chinese JOAG patients, totally 79 variants in MYOC, 354 variants in OPTN, 139 variants in WDR36, 45 variants in NTF4 and 199 variants in CYP1B1 were detected. Among them, a total of 6 variants in MYOC, OPTN and CYP1B1 were identified as potential disease-causing mutations in 8 patients (11.94%) after a series of filtering steps (Tables 1 and 2). These variants included 2 variants in MYOC, 2 variants in OPTN and 2 variants in CYP1B1 (Fig. 1). The variants were confirmed by Sanger sequencing. No MYOC or OPTN potential disease causing mutations were detected in 125 controls, while two heterozygous mutations of CYP1B1 (c. 1169G > A p. (R390H)) were found in controls (Table 1).
In MYOC, there were two heterozygous mutations (c. 1109C > T, p. (P370L); c. 1150G > C, p. (D384H)) detected from two cases in this study, among which p.D384H was novel ( Fig. 2A). Substitution of p.D384H was predicted to affect protein function by Polyphen-2, SIFT and Mutation Taster. Additionally, the mutated amino acid is highly conserved among all the tested species (Fig. 3A). p. (P370L) was a reported mutation associated with POAG.
In CYP1B1, 2 homozygous mutations (c.1412T > G, p. (I471S); c.1169G > A, p. (R390H)) were detected in two patients. Both mutations were predicted to be a pathogenic mutation in all three pathogenicity prediction tools used in this study. The I471S mutation was reported in a Chinese primary congenital glaucoma (PCG) study 21 .

Discussion
In this study, the results of exome sequencing data from 67 JOAG patients identified 6 mutations in the MYOC, OPTN and CYP1B1 genes in 8 unrelated patients. Among them, D384H in MYOC and R329G and L494W in OPTN were novel. These mutations are predicted to affect protein function and were absent in 125 control individuals without glaucoma. MYOC is the first identified glaucoma-causing gene 22 . Over 20 mutations in MYOC have been reported, and the frequency of mutation ranged from 10% to 30% 6,23,24 in study cohorts with the familial trait. In the present study, 5.97% (4/67) of JOAG patients carry a heterozygous mutation in MYOC. Three patients have mutant p. (P370L) in the third exon in MYOC. It has been reported that the turnover rate of mutant p. (P370L) in MYOC fusion proteins was much prolonged compared with wild type 25 . The ubiquitin-proteasome function is compromised and autophagy is induced with mutant p. (P370L) in MYOC 25 . Further study showed a causal association between this p. (P370L) mutation of MYOC and juvenile glaucoma with goniodysgenesis 26 . Of the two mutations detected in the present study, p. (P370L) has been previously reported 26,27 . p. (D384H) is a novel mutation in the The reported role of OPTN in JOAG remains inconsistent. Previous studies have identified several mutations in OPTN associated with POAG 7 , including c.C160G 28 and p.(Lys322Glu) 29 . On the other hand, several studies have shown an absence of OPTN mutations in POAG or JOAG [30][31][32] . L494W has been reported in a Chinese amyotrophic lateral sclerosis study but not in any glaucoma study 33 . The patient who carries p. (L494W) is 33 years old. After a detailed systemic review, we confirmed that she has no manifestation f ALS. The mean age at onset of ALS range from 52.9 to 59.9 years in several reports 33,34 . Therefore, it is necessary to follow that patient at regular intervals. p. (R329G) is a novel mutation in OPTN in our study. Further studies are needed to understand the role of mutations in OPTN in JOAG.
CYP1B1 is associated with PCG, characterized by an autosomal recessive model. CYP1B1 is also involved in the development of JOAG. CYP1B1 (G61E, R368H, R390H, E229K, and 4340delG) may be associated with severe or moderate angle abnormalities and plays an important role in PCG 35 38 . In the current study, a homozygous mutation of R390H in the third exon of CYP1B1 was found in this Chinese JOAG group, consistent with previous reports 38 . A homozygous mutation of I471S in the third exon of CYP1B1 was identified. The locations of Arg390 and I470 at CYP1B1 are in the K helix and L helix, respectively. Both helixes are conserved regions for CYP1B1 and are expected to be involved in proper folding of the molecule. p. (I471S) was first reported to be associated with PCG in a Chinese PCG study 21 , indicating that  [42][43][44] . The mutation frequency of NTF4 is known to be low in the Chinese population 42,44 . Thus, it is not surprising that no mutation was detected in our small cohort. Mutations of WDR36 have been detected in different JOAG populations, including German and Chinese 45,46 . The frequency of mutation of WDR36 is unclear in the Chinese population, and further studies are needed to estimate the WDR36 mutation frequency in this population.
This study is limited by the lack of parents' samples so that we are not able to perform segregation analysis. The mutations carriers' parents were reported to be free of glaucoma. Therefore, whether the mutations detected are de novo mutations cannot be confirmed as for now. Also, there could be other factors that might have affected the penetrance of the mutations so that the patients, even carried the mutations, might not have developed the disease. JOAG may belongs to heterogeneity with different variants in numerous genes, which makes it some familial but most occurring sporadically.
In summary, six variants of MYOC, OPTN and CYP1B1 were found in 11.94% of this Chinese JOAG cohort, including two heterozygous mutations in MYOC in four patients, two heterozygous mutations in OPTN in two patients, and two homozygous mutations in CYP1B1 in two patients. No mutation of NTF4 or WDR36 was detected in this cohort. Although the sample size of this study is small and we have not conducted segregation analysis or functional analyses of the novel mutations, our results provide additional evidence of the mutation spectra and frequencies in Chinese JOAG.

Materials and Methods
Patient recruitment. We recruited unrelated Chinese JOAG patients at the Shantou University/Chinese University of Hong Kong Joint Shantou International Eye Center, Shantou, Guangdong Province, China. Written informed consent was obtained from the participants or their guardians. The study was conducted following the tenets of the Declaration of Helsinki and was approved by the institutional review board and ethics committee of Joint Shantou International Eye Center.
The inclusion criteria for patients with JOAG are based on an age of onset between 3 years of age and early adulthood and the manifestation of highly elevated intraocular pressures without angle abnormalities 3 Table 3. Primers used to amplify the sequences harbouring the variants in this study. Note: PCR conditions: 35 cycles of amplification. Each cycle consists of 30 s denaturation at 94 °C, 60 s annealing ranging from 59.5 °C to 60.9 °C and 1 min extension at 72 °C, with a final extension at 72 °C for 5 min.