Fundus photographs and optical coherence tomography (OCT) of high myopia patients from the sporadic cases. A. The fundus of the patient JS047001 (Table 5) showing tigroid or tessellated features, conus, and CNV (choroid neovascularization). Optical Coherence Tomography (OCT) examination of this patient showed continuity of retinal pigment epithelial layer and broken photoreceptor layer (E). B. The fundus of the patient JS103001 (Table 5) showing tigroid or tessellated features, numerous areas of atrophy of the pigment epithelium, and choriocapillaries extending into the macular region and Fuchs spot. OCT examination of this patient showed discontinuity and irregular apophysis of the reflective pigment epithelial layer (F). C. The fundus of the patient JS104001 (Table 5) showing tigroid or tessellated features, numerous areas of atrophy of the pigment epithelium, and choriocapillaris. OCT examination of this patient showed foveal thinning and atrophies of the retinal neuroepithelial layer (G). D. Normal fundus photograph and OCT examination of a normal Control (H). doi:10.1371/journal.pgen.1002084.g004 

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... ZNF644 action and its role in high myopia pathogenesis remains unclear, and future functional studies will be important. To date, there have been no documented studies on the ZNF644 gene, and the data here indicating its involvement in a devastating eye disease provide excellent motivation for future investigation of the ZNF644 gene, which in turn should enable dissection of its relationship with high myopia pathogenesis. All procedures used in this study conformed to the tenets of the Declaration of Helsinki. The Institutional Review Board and the Ethics Committee of Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital approved the protocols used. Informed consent was obtained from all participants. We undertook exome sequencing and validation studies between September 1, 2009 and December 2, 2009. This study included a Han Chinese family (designated as 951) with high myopia that had 30 (20 living) family members (Figure 1, Table 1); 300 sporadic patients with high myopia; and 600 matched, normal controls. The 300 sporadic patients and the 600 controls were non-related, were of Han Chinese ethnicity (Figure 4, Table 4), came from the Chengdu region of Sichuan Province, China, and were recruited at the ophthalmic clinic at Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, China. All participants underwent an extensive, standardized examination by ophthalmologists, including visual acuity (VA) testing, a detailed clinical examination, optical coherence tomography (OCT), and ocular imaging prior to genetic testing. Refractive error and the radius of corneal curvature in the horizontal and vertical meridian were measured using an autorefractor (KR8800, Topcon, Tokyo, Japan). Final refractive error status was established with subjective visual acuity testing by trained and certified optometrists. The diagnosis for high myopia in this study required a spherical equivalent of #2 6.0 DS for both eyes and an axial length of the eye globe of $ 26.0 mm for both eyes. For controls, the ...

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... were diagnosed with high myopia; six were alive and available for this study. The four deceased patients were diagnosed based on available medical records (Figure 1A). The six living had refractive errors ranging from 2 6.27 to 2 20.00 diopter sphere (DS) for the left eye (OS) and from 7.51 to 11.49 DS for the right eye (OD), and eye globe axial length ranging from 26.0 to 31.1 mm for OS and 26.9 to 30.5 mm for OD. They also had pre-school age of onset. Three elderly patients showed typical fundus features of high myopia: a thinning of the RPE and the choriocapillaris, which gives what is described as a ‘tigroid’ or ‘tessellated’ fundus appearance (Figure 1B, Table 1). For this analysis, we selected two affected family members (V:1 and III:2) (Figure 1A, Table 1). V:1, the proband, at age 8 in 2009, had refractive errors at 2 9.54 DS (OD) and 2 6.34 DS (OS) and normal fundus features. III:2, at age 68 in 2009, had refractive errors at 2 11.49 DS (OD) and 2 13.02 DS (OS) and tessellated or tigroid features (Table 1, Figure 1B). We used exome sequencing to identify potential variants responsible for high myopia in this family. We generated an average of 2.4 Gb of sequence with 30 6 average coverage for each individual as single-end, 80-bp reads, and about 97% ( , 32.98 Mb in length) of the targeted bases were covered sufficiently to pass our thresholds for calling SNPs and short insertions or deletions (indels) (Table 2). The bases with quality scores above 20 (99% accuracy of a base call) represent over 75% of total sequence data, while the error rate is below 3% (Figure 2). Table 3 presents the exome genetic variants identified from the exome sequencing analysis. The numbers in Table 3 are comparable to what was reported in two previously published results [4,20]. The transition versus transver- sion ratio is 2.95 and 2.69 for the two samples respectively. The rate of heterozygous versus homozygous variants is 1.33 and 1.38 for the two samples respectively. For patients V:1 and III:2, respectively, we identified 10,156 and 10,358 SNPs (synonymous and non- synonymous) in coding regions; 447 and 501 variants (SNPs and indels) in introns that may affect splicing (within 5 bp of the intron/ exon junction); and 2,370 and 2,642 indels in coding regions or introns. Given that these patients are related and they are expected to share the causal variant for high myopia, we filtered all the detected variations in these patients against each other and found that they shared 6,610 variants (SNPs and indels) (Table 3). Because high myopia is a rare disorder but has a clear phenotype, there is a very low likelihood of the causal mutation in these patients being shared with a wider healthy population. We therefore compared the shared variants in these patients with the Han Chinese Beijing SNPs from dbSNP131 and the data from 30 genomes of Han Chinese Beijing recently available from the 1000 Genome Project (February 28, 2011 releases for SNPs and February 16, 2011 releases for indel fttp://www.1000genome.org). This left a total of 393 variants that were shared between these two patients. Of these, 332 genetic variants (including 62 non-synonymous SNPs, 5 splice acceptor and donor sites, and 265 indels) were predicted to potentially have a functional impact on the gene (Table 3). We carried out Sanger sequencing validation on these 332 variants, and obtained accuracy of 98% (66/67) for called SNPs and 96% (254/265) for indels, indicating the high quality of our variant calling method. We then performed segregation analysis by Sanger sequencing on the 66 validated SNPs and 254 indels, using the available 19 members of family 951 (Figure 1). Only one variant co-segregated with the disease phenotype in this family: an A to G change in exon 3 (2156A . G), resulting in an S672G amino acid change, in the zinc finger protein 644 gene isoform 1 ( ZNF644 , located at 1p22.2) (Table 1, Figure 1, Figure 3). We obtained a LOD score of 3.19 at theta = 0 given an autosomal dominant mode of inheritance with full penetrance and 0.0001 for the disease allele frequency. The power to obtain a LOD score greater than 3 was 88% when tested by SLINK, providing further support for this mutation being the disease-causing change for family 951. We then assessed the presence of the co-segregating mutation in the 600 matched normal controls using direct PCR sequencing of the ZNF644 exon 3, and did not find it in the 600 controls. We further carried out direct PCR sequencing of the ZNF644 exons in an additional 300 unrelated (based on their self-identified geographical ancestry), sporadic high myopia patients. The 300 patients had refractive errors ranging from 2 6.0 to 2 29.0 DS for both eyes and an axial eye globe length from 26 to 33 mm for both eyes (Table 4). Some of these individuals also showed severe retinal pathological changes in the fundus appearance, an abnormal RPE, and photoreceptor layer alterations at the time of the OCT examination (Figure 4, Table 5). In the 300 sporadic patients, we identified a total of 8 variants when we sequenced the ZNF644 exons, and five out of these 8 variants (present in 11 unrelated individuals) were absent in all the 600 controls. Among these five mutations identified from the sporadic cases, three were in exon 3 (I587V, R680G and C699Y) and two were in the 3 9 untranslated region (UTR) ( + 12 C . G and + 592 G . A) (Figure 3, Table 5). The remaining three out of the 8 variants were found in both cases and controls, two (T404T and V444V) were synonymous changes which may not affect the biological function of ZNF644 and one was located in the 3 9 UTR ( + 1015 C . G). The P -value for the 17 potentially functional variants in 301 patients with high myopia (One mutation identified in one member of the high myopia family 951 plus the 6 mutations identified in the 16 unrelated patients from the 300 sporadic cases) compared with these variants being seen in 600 controls (3 in 600 controls) was 2.28 6 10 2 6 by Fisher’s exact test. This data suggests that there are multiple rare variants in ZNF644 associated with high myopia. To make sure that the ZNF644 gene is expressed in the eye, we examined ZNF644 expression in different human tissues using reverse transcript polymerase chain reaction (RT-PCR). The ZNF644 gene was expressed in the human retina and RPE as well as in the liver and placenta (Figure 5). For the past several decades, standard methods for identifying genes underlying disease in a monogenic form have primarily been through selecting candidate genes for testing or by using positional cloning. The candidate gene approach requires less work and costs less because only the candidate gene needs to be sequenced, but this method requires prior knowledge of the pathogenesis of a disease for gene selection. This fundamentally impedes the disease gene identification speed because the pathogeneses of many diseases have not yet been unmasked. Without pathogenesis information of a disease, the traditional positional cloning strategy can be used first to map the disease gene in the chromosome and then to identify the disease-causing gene within a specific interval. Thus the pathogenesis of a disease can be explored based on the identified disease gene. However, the positional cloning method requires marked locus heterogeneity and the availability of a large family. Focusing on the exome can be especially fruitful in disease gene identification given that previous studies have indicated that approximately 85% of causal mutations for human diseases are located within the coding region and canonical splice acceptor and donor sites (. ac.uk/ac/index.php). Therefore, through sequencing and comparing the coding region of affected and unaffected individuals within a family and filtering the benign changes using a public database, such as 1000 Genomes Project and dbSNP databases, the mutation in the coding region can be identified even within small families and without knowing the pathway of a disease and marked locus heterogeneity. Currently, the cost of the exome sequencing method is even less than that of the positional cloning strategy. So, this method will not only speed up disease gene identification but will enable us to systematically tackle previously intractable monogenic disorders. In fact, exome sequencing approaches have been successfully used to identify disease genes for Mendelian disorders in recent studies [21– 36]. Unfortunately, compared to the positional cloning strategy, the exome sequencing method may not identify the mutations in non- coding regions. This limitation promotes the use of the whole sequencing method to identify disease genes [26,37]. Theoretically the whole gene sequencing will eventually become the best method of disease gene identification, because this method has the advantages of both positional cloning and exome sequencing methods. It can also identify disease genes caused by a large indel, inversion, translocation, and other chromosome structure aberrant. However, at the current stage, whole genome sequencing costs more and needs a lot of bioinformatics work, and this restricts its use in disease gene identification. Presently exome sequencing is a powerful tool with low cost for identifying genes that underlie disease. The ...