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      A novel heterozygous COL4A4 missense mutation in a Chinese family with focal segmental glomerulosclerosis

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          Focal segmental glomerulosclerosis (FSGS) is the most common glomerular histological lesion associated with high‐grade proteinuria and end‐stage renal disease. Histologically, FSGS is characterized by focal segmental sclerosis with foot process effacement. The aim of this study was to identify the disease‐causing mutation in a four‐generation Chinese family with FSGS. A novel missense mutation, c.1856G>A (p.Gly619Asp), in the collagen type IV alpha‐4 gene ( COL4A4) was identified in six patients and it co‐segregated with the disease in this family. The variant is predicted to be disease‐causing and results in collagen IV abnormalities. Our finding broadens mutation spectrum of the COL4A4 gene and extends the phenotypic spectrum of collagen IV nephropathies. Our study suggests that exome sequencing is a cost‐effective and efficient approach for identification of disease‐causing mutations in phenotypically complex or equivocal disorders. Timely screening for COL4A3/COL4A4 mutations in patients with familial FSGS may help both accurately diagnose and treat these patients.

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          Identification of PRRT2 as the causative gene of paroxysmal kinesigenic dyskinesias

          Paroxysmal kinesigenic dyskinesias is a paroxysmal movement disorder characterized by recurrent, brief attacks of abnormal involuntary movements induced by sudden voluntary movements. Although several loci, including the pericentromeric region of chromosome 16, have been linked to paroxysmal kinesigenic dyskinesias, the causative gene has not yet been identified. Here, we identified proline-rich transmembrane protein 2 (PRRT2) as a causative gene of paroxysmal kinesigenic dyskinesias by using a combination of exome sequencing and linkage analysis. Genetic linkage mapping with 11 markers that encompassed the pericentromeric of chromosome 16 was performed in 27 members of two families with autosomal dominant paroxysmal kinesigenic dyskinesias. Then, the whole-exome sequencing was performed in three patients from these two families. By combining the defined linkage region (16p12.1–q12.1) and the results of exome sequencing, we identified an insertion mutation c.649_650InsC (p.P217fsX7) in one family and a nonsense mutation c.487C>T (p.Q163X) in another family. To confirm our findings, we sequenced the exons and flanking introns of PRRT2 in another three families with paroxysmal kinesigenic dyskinesias. The c.649_650InsC (p.P217fsX7) mutation was identified in two of these families, whereas a missense mutation, c.796C>T (R266W), was identified in another family with paroxysmal kinesigenic dyskinesias. All of these mutations completely co-segregated with the phenotype in each family. None of these mutations was identified in 500 normal unaffected individuals of matched geographical ancestry. Thus, we have identified PRRT2 as the first causative gene of paroxysmal kinesigenic dyskinesias, warranting further investigations to understand the pathogenesis of this disorder.
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            Exome Sequencing Identifies ZNF644 Mutations in High Myopia

            Introduction Myopia is the most common ocular disorder worldwide, with a prevalence of 20–30% in North American, European and Australian populations [1], [2] and as high as 40–70% in the Asian population [3]–[5]. One type of myopia is high myopia, and it is prevalent in 1–2% in the general population [1]–[6]. In high myopia, affected patients' eyes have a spherical equivalent of less than or equal to −6.00 diopter sphere (DS) and an axial length longer than or equal to 26.0 mm. In some cases, high myopia may also show retinal pathological changes with progressive choroidal degeneration in the posterior pole and other complications, potentially resulting in severe vision loss. In such cases, high myopia is referred to as pathological or degenerative myopia, which is one of the leading causes of blindness in the world [1], [7], [8]. The exact pathogenesis of myopia remains unclear. There are indications that environmental factors (such as close working habits, higher education levels and higher socioeconomic class) [9], [10] and genetic predisposition both contribute to the development of myopia [10], [11], especially of high myopia [12]. The evidence that genetic variation plays a crucial role in the occurrence and development of myopia is based on studies showing different frequencies of myopia in different populations [2]–[5], [13], obvious family aggregation trends, twin studies [9], [14], [15], and the identification of 18 linked loci having an association with myopia (OMIM, 160700) [6], [16]–[19]. Myopia can be inherited as a complex trait or in a monogenic form. For the complex form, myopia appears to be the result of an interaction of multiple genes and environmental factors. Recently, several loci have been identified by genome-wide association study (GWAS) as being responsible for complex myopia [17]–[20]. On the other hand, high myopia in a monogenic form may be inherited in an autosomal dominant, autosomal recessive and X-linked recessive manner [15], [21]. In this study, we propose to use exome sequencing to identify a gene responsible for high myopia in a monogenic form in a Han Chinese population. Results Here we describe a Han Chinese family (951) from Chengdu, China, that has monogenic high myopia with a dominant inheritance model. The clinical features of the nineteen of 20 living family members who participated in this study are shown in Table 1. Ten patients within the family 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 −6.27 to −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). 10.1371/journal.pgen.1002084.g001 Figure 1 Pedigree and segregation of the mutation and fundus photograph of a patient from the family. A. Kindred structure and segregation of ZNF664 S672G mutation in the high myopia family. Affected individuals are shown by solid squares (males) or circles (females). Normal individuals are identified by open symbols. Deceased individuals are indicated by a slash (/). M: 672G mutant allele of ZNF644; +: S672 normal allele of ZNF644. B. Fundus photograph of III:2 of the family, showing tigroid or tessellated features and conus pattern of retina. C. Normal fundus appearance of IV:5. 10.1371/journal.pgen.1002084.t001 Table 1 The clinical features of the high myopia family 951 with a ZNF644 gene mutation. Subject No. Age (Yr)* Gender Axial length (mm)** Refractive errors (DS)** Fundus appearance** ZNF644 mutation*** OD OS OD OS OD OS Nucleotide change Amino acid change III:1 71 M 23.8 23.9 0 −0.22 normal normal 2156 A S672 III:2 68 F 28.6 29.5 −11.5 −13.02 tigroid or tesselated tigroid or tesselated 2156 A>G S672G III:3 73 F 30.5 31.1 −16.3 −20.04 tigroid or tesselated tigroid or tesselated 2156 A>G S672G III:4 74 M 24 24.3 0 0 normal normal 2156 A S672 III:5 65 M 27.1 28.2 −9.27 −11.02 tigroid or tesselated tigroid or tesselated 2156 A>G S672G III:7 70 M 23.7 24 −0.5 −0.5 normal normal 2156 A S672 III:9 65 F 23.9 24.1 0.23 0 normal normal 2156 A S672 IV:1 38 M 24.5 24.3 0 0 normal normal 2156 A S672 IV:2 27 F 26.5 27.2 −7.53 −9.49 normal normal 2156 A>G S672G IV:3 25 M 28.9 29.4 −12.11 −12.31 normal normal 2156 A>G S672G IV:4 32 F 24.1 25.2 −0.38 −0.5 normal normal 2156 A S672 IV:5 36 M 24.9 24.8 −0.46 −0.42 normal normal 2156 A S672 IV:6 34 F 24.8 25 −0.48 −0.5 normal normal 2156 A S672 IV:7 31 F 24.8 24.9 −0.32 −0.44 normal normal 2156 A S672 IV:8 34 M 24.3 24.2 −0.2 0.2 normal normal 2156 A S672 IV:9 36 M 23.9 23.8 0.25 0.4 normal normal 2156 A S672 IV:10 30 F 24.9 25.2 −0.1 −0.12 normal normal 2156 A S672 V:1 8 F 26.9 26 −9.54 −6.34 normal normal 2156 A>G S672G V:2 6 M 24.3 24.2 0.22 0.15 normal normal 2156 A S672 *The age when the family member was recruited in 2009. **OS, left eye; OD, right eye; DS, diopter sphere. ***Location: chromosome 1: 9117748 (exon 3 of ZNF644 gene). 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 −9.54 DS (OD) and −6.34 DS (OS) and normal fundus features. III:2, at age 68 in 2009, had refractive errors at −11.49 DS (OD) and −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× 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 transversion 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). 10.1371/journal.pgen.1002084.g002 Figure 2 The distribution of qualities of the sequencing reads for the two analyzed samples. A. The sequencing quality of III:2. B. The sequencing quality of V:1. The X-axis represents the position along each sequence read. In this study we performed 80 bp sequencing, so the label of X-axis is from 0 to 80. The Y-axis is the Phred quality score of each base along sequence reads. The higher the score, the more accurate a base call. The quality scores of 20, 30 and 40 represent 99%, 99.9% and 99.99% accuracy of a base call. The intensity of the blue dot refers to the quantity of sequences with the same quality score. The darker the blue, the more bases/sequences at the dot. The quality scores of first 50 bp of most reads for both samples are higher than those of the last 30 bp, thus the darker blue dots accumulate around Q30 (Phred score of 30) from 0 to 30 bp along reads, while Q5 is observed between 60∼80 bp along reads. In order to get more accurate base calling, we filtered these low quality reads during the data analysis. 10.1371/journal.pgen.1002084.t002 Table 2 Summary statistics for exome sequencing for two individuals with high myopia. Sequencing reads Called coverage Total Unique mapping Overlapping target/unique Nonduplicated Mean coverage Called bases % of CCDS* 23,944,773 22,480,147 8,324,968 7,669,803 96.17% 636,987,416 87.28 28,792,273 27,118,103 10,088,253 9,215,629 97.16% 776,725,130 88.69 *CCDS: consensus coding sequence; %CCDS represents the number of CCDS with over 80% coverage of the total number of CCDS. 10.1371/journal.pgen.1002084.t003 Table 3 Genetic variants identified through exome resequencing. Filter Genetic variants Synonymous SNP Non-synonymous SNP Splice acceptor and donor site Indel Total V:1 5723 4433 447 2370 12973 III:2 5810 4548 501 2642 12701 V:1 and III:2 Shared 2823 2138 243 1406 6610 Not in 30 CHB* data of 1000 Genomes Project or dbSNP131 61 62 5 265 393 *CHB, Han Chinese Beijing. 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:// 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. 10.1371/journal.pgen.1002084.g003 Figure 3 Genomic structure of the exons encoding the open reading frame of ZNF644 and identified mutations. Five out of six exons are translated (green), and exon 1 and portions of exon 2 and exon 6 are untranslated (red) in the ZNF644 gene (upper panel). Six different mutations in the ZNF644 gene and their sequencing traces are shown at the bottom of the figure (lower panel). 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 −6.0 to −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′ 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′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 2.28×10−6 by Fisher's exact test. This data suggests that there are multiple rare variants in ZNF644 associated with high myopia. 10.1371/journal.pgen.1002084.g004 Figure 4 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). 10.1371/journal.pgen.1002084.t004 Table 4 Characteristics of sporadic cases and controls in the study. Number Age (Yr)* Gender Refractive errors (Diopter)** Axial length (mm)** Male Female Cases 300 33.65±12.66 139 161 −10.54±4.23 (OD), −10.36±4.03 (OS) 28.02±2.02 (OD), 28.00±2.03 (OS) Controls 600 55.85±9.06 287 313 −0.02±0.02 (OD), −0.01±0.01 (OS) 23.91±1.98 (OD), 24.02±2.02 (OS) *The age when the cases and controls were recruited; **OD: right eye; OS: left eye. 10.1371/journal.pgen.1002084.t005 Table 5 The clinical features of affected patients with a ZNF644 gene mutation in the 300 sporadic cases with high myopia. Family Subject No. Age (Yr)* Gender Axial length (mm)** Refractive errors (DS)** Fundus appearance** ZNF644 mutation OD OS OD OS OD OS Nucleotide change Exon Amino acid change Chr.1 location JS007 JS007001 26 M 26.1 26.1 −10.02 −9.47 conus conus 2091 A>G 3 I587V 91177740 JS103 JS103001 55 F 29.1 29.2 −14.47 −15.03 tigroid, CA, Fuchs spot tigroid, CA, Fuchs spot 2091 A>G 3 I587V 91177740 JS104 JS104001 53 M 31.5 29.4 −17.25 −16.49 tigroid, CA tigroid, CA, Fuchs spot 2091 A>G 3 I587V 91177740 JS078 JS078001 28 F 27.8 27.0 −12.03 −9.47 tigroid, macular atrophy tigroid or tesselated 2091 A>G 3 I587V 91177740 JS131 JS131001 31 M 27.2 27.1 −14.67 −15.04 tigroid or tesselated tigroid 2091 A>G 3 I587V 91177740 JS010 JS010001 29 M 26.5 26.1 −10.52 −9.54 conus conus 2180 C>G 3 R680G 91177461 JS064 JS064001 32 F 28.4 28.4 −9.54 −10.97 tigroid, conus tigroid 2238 G>A 3 C699Y 91177403 JS080 JS080001 25 F 26.8 27.0 −11.26 −11.45 tigroid tigroid 2238 G>A 3 C699Y 91177403 JS075 JS075001 19 M 30.5 28.5 −18.02 −15.02 tigroid, conus tigroid 4138 C>G 6 3′UTR+12 C>G 91154931 JS027 JS027001 63 F 26.3 26.5 −8.02 −9.04 tigroid, conus tigroid, conus 4718 G>A 6 3′UTR+592 G>A 91154351 JS047 JS047001 46 M 28.5 28.7 −15.96 −16.02 tesselated, conus, CNV tesselated, conus 4718 G>A 6 3′UTR+592 G>A 91154351 *The age when the patients were recruited. **OD: right eye; OS: left eye; DS, diopter sphere; CA: choriocapillaris atrophy; CNV: choroidal neovascularization. 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). 10.1371/journal.pgen.1002084.g005 Figure 5 Expression of the ZNF644 gene in human tissues. RT-PCR analyses of ZNF644 expression in the human liver, placenta, retina, and retinal pigment epithelium (RPE) with 255 bp of products. GAPDH was used as an internal control for cDNA quantification. Discussion 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 ( 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 whole genome sequencing method will very likely become the most powerful method for disease gene identification as the constant improvements to massively sequencing technologies and the impending massively parallel single-molecule sequencing technologies will reduce method costs and time barriers [38]. Practically, candidate gene approach, positional cloning strategy and exome sequencing or whole genome sequencing methods has been combined to identify the disease-causing genes in humans [39]–[43]. Our data here indicate again that exome sequencing can rapidly identify genes causing dominant Mendelian diseases, which can occur in a heterozygous form. We further were able to identify this gene by sequencing exomes of only two affected patients and using available public databases, such as dbSNP131 and the 1000 Genomes Project. Additionally, the use of second-generation sequencing produces a high level of coverage, with subsequent higher accuracy, and allows more regions of a genome to be sequenced in a very cost effective manner. The 30-fold average coverage we obtained here is a very high sequencing depth. It covered 97% of the target sequence with ∼96% accuracy rate, and thus allowed us to identify variants with high confidence. Using this technique, we successfully identified a gene for high myopia in an affected family. Several lines of evidence provided support for the mutations in ZNF644, and thus the mutated ZNF644 gene, being the cause of high myopia: 1) only the S672G mutation identified in the two affected patients showed complete co-segregation with the disease phenotype in the family studied; 2) our analysis of the ZNF644 gene in 300 unrelated, sporadic high myopia patients identified an additional three missense mutations and two mutations in the 3′ UTR which may affect mRNA stability or microRNA interaction; 3) none of these identified mutations were present in the 30 genomes of Han Chinese Beijing in the 1000 Genomes Project database, the Han Chinese Beijing SNPs in the dbSNP131 database, or 600 normal ethnicity-matched controls; and 4) Comparative analyses of ZNF644 in other species showed that I587 is conserved and S672, R680, C699 are highly conserved among primates, placental animals, and other vertebrate species ( Based on protein structure, ZNF644 is predicted to be a transcription factor (, and given that it has potentially deleterious mutations in patients with high myopia, it may play a role in gene expression regulation in the retina and retinal pigment epithelium (RPE). One important issue in the genetic study of high myopia is the age of disease onset. We would have an informative censoring problem if family members of 951 did not show the disease phenotype because their age was too young. However, the disease phenotype studied in family 951 is very special. The disease onset was at 3–4 years old for all affected patients in the family 951 with high myopia; all affected patients developed high myopia by the age of seven. The youngest unaffected member in the family (V:2) is 9 years old now; he does not show any signs of myopia at all. In addition, all affected patients in the family had severe high myopia, which allowed us identify the affected patients easily. Therefore, there is very little chance that an unaffected family member does not show the trait by virtue of being too young. Although it clearly has a ubiquitous level of expression, this is common for other genes involved in eye diseases (for example, the retinitis pigmentosa disease-causing gene PRPC8 is ubiquitously expressed in human tissues [44]), and its expression in eye tissue allows for the ZNF644 gene having activity in the eye. Note that, given that less than 4% of the sporadic high myopia cases had mutations in ZNF644 (we identified 5 different mutations in 11 patients out of 300 cases), the ZNF644 gene is unlikely to play a major role in sporadic high myopia. ZNF644 belongs to the Krüppel C2H2-type zinc-finger protein family, which contains 7 C2H2-type zinc fingers. Among the six identified mutations, four missense mutations were found clustered in exon 3 of the ZNF644 gene, suggesting that this exon may code for important protein domain structures or have regulatory functions. The other two mutations were located in the 3′ UTR of ZNF644 gene, which is a region often important for RNA degradation. The main feature of high myopia is axial elongation of the eye globe. Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, a mutant ZNF644 protein may impact the normal eye development and therefore underlie the axial elongation of the eye globe in high myopia patients. However, the exact mechanism of 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. Materials and Methods Ethics statement 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. Study population 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 ≤−6.0 DS for both eyes and an axial length of the eye globe of ≥26.0 mm for both eyes. For controls, the spherical equivalent for both eyes had to be in the range of −0.50 to +1.0 DS and show no evidence of disease in either eye. Targeted capture and exome sequencing The exome sequencing approach was used to identify the disease-causing genetic variant for the high myopia family in the study (951). Genomic DNA was extracted from peripheral white blood cells, using Gentra Systems PUREGENE DNA purification kit (Minneapolis, MN, USA). Fifteen µg of genomic DNA from each of the two selected individuals (V:1 and III:2, Figure 1A) with high myopia from family 951 were separately sheared into about 200-bp DNA fragments by sonication. Exome capture was performed to collect the protein coding regions of human genome DNA using a NimbleGen 2.1M HD array as described in the manufacturer's instructions (Roche NimbleGen, Inc., Madison, WI, USA). The array was able to capture 18,654 (92%) of the 20,091 genes. The gene sequences for this array are available in the Consensus Coding Sequence Region (CCDS) database ( The exon-enriched DNA libraries were then subjected to a second library construction in preparation for Illumina GA sequencing and were sequenced using the Illumina Genome Analyzer II platform, following the manufacturer's instructions (Illumina, San Diego, USA) [23]. We obtained a mean exome coverage of 30×, which allows each selected region of the genome to be checked, on average, 30 times. Such deep coverage provided sufficient depth to accurately call variants at ∼97% of each targeted exome. Read mapping and variant analysis The human reference genome, together with its gene annotation, was downloaded from the UCSC database (, version hg18 (build36). Alignment of the sequences from the two affected individuals was performed using SOAPaligner after we removed the duplicated reads [32], and SNPs were called using SOAPsnp set with the default parameters [33]. Indels affecting coding sequence or splicing sites were identified as described previously [34]. The thresholds for calling SNPs and short insertions or deletions (indels) included the following: 1) the number of unique mapped reads supporting a SNP had to be ≥4 and ≤100; and 2) the consensus quality score had to be ≥20 (The quality score is a Phred score, generated by the program SOAPsnp 12, quality score 20 represents 99% accuracy of a base call). The shared changes of the two affected individuals were obtained by further comparison of the variants of each of the two affected individuals. All changes were filtered against exome data of 30 genomes from ethnic Han Chinese individuals from Beijing available in the 1000 Genomes Project (February 28, 2011 releases for SNPs and February 16, 2011 releases for indel fttp://, and against the Han Chinese Beijing SNPs in the dbSNP131. Sanger sequencing was then used to validate the identified potential disease-causing variants. SIFT ( was used to predict whether an amino acid substitution affects protein function. Mutation validation All shared variants of the two affected individuals after filtering against the 30 Han Chinese Beijing genomes of 1000 Genomes Project and the Han Chinese Beijing SNPs in the dbSNP131 were then confirmed by direct polymerase chain reaction (PCR)-product sequencing using Bigdye terminator v3.1 cycle sequencing kits (ABI, Foster City, CA, USA) and analyzed on an ABI 3130XL Genetic Analyzer. We used Sanger sequencing to determine whether any of the remaining variants co-segregated with the disease phenotype in family 951 and used MLINK of the LINKAGE program to calculate a two-point LOD score for the detected variants to assess the locus position of the predicted disease gene in family 951 [35]. The primers flanking all exons of ZNF644 were designed using primer 3 ( (Table S1), and all exons of the 300 sporadic patients with high myopia were analyzed using the same method as above. ZNF644 expression We looked at expression of the ZNF644 gene in the human liver, retina, RPE, and placenta. The human liver, retina, and RPE were donated by a deceased 55-year-old Han Chinese male and the human placenta was donated by a 29-year-old Han Chinese female. Total RNA from the human liver, placenta, retina, and RPE was extracted by trizol (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed using a reverse transcription kit (Invitrogen, Carlsbad, CA, USA). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control (Table S2). All RT-PCR products were confirmed by direct sequencing. Supporting Information Table S1 Primers for Sequencing Analysis of ZNF644 Exons. (DOC) Click here for additional data file. Table S2 Primers for gene expression by RT-PCR. (DOC) Click here for additional data file.
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              COL4A3/COL4A4 mutations: from familial hematuria to autosomal-dominant or recessive Alport syndrome.

              COL4A3/COL4A4 mutations: From familial hematuria to autosomal-dominant or recessive Alport syndrome. Mutations of the type IV collagen COL4A5 gene cause X-linked Alport syndrome (ATS). Mutations of COL4A3 and COL4A4 have been reported both in autosomal-recessive and autosomal-dominant ATS, as well as in benign familial hematuria (BFH). In the latter conditions, however, clinical features are less defined, few mutations have been reported, and other genes and non-genetic factors may be involved. We analyzed 36 ATS patients for COL4A3 and COL4A4 mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and direct sequencing. Sporadic patients who had tested negative for COL4A5 mutations were included with typical cases of autosomal recessive ATS to secure a better definition of the phenotype spectrum. We identified seven previously undescribed COL4A3 mutations: in two genetic compounds and three heterozygotes, and one in COL4A4. In agreement with the literature, some of the mutations of compound heterozygotes were associated with microhematuria in healthy heterozygous relatives. The mutations of heterozygous patients are likely dominant, since no change was identified in the second allele even by sequencing, and they are predicted to result in shortened or abnormal chains with a possible dominant-negative effect. In addition, both genes showed rare variants of unclear pathogenicity, and common polymorphisms that are shared in part with other populations. This study extends the mutation spectrum of COL4A3 and COL4A4 genes, and suggests a possible relationship between production of abnormal COL IV chains and dominant expression of a continuous spectrum of phenotypes, from ATS to BFH.

                Author and article information

                J Cell Mol Med
                J. Cell. Mol. Med
                Journal of Cellular and Molecular Medicine
                John Wiley and Sons Inc. (Hoboken )
                29 July 2016
                December 2016
                : 20
                : 12 ( doiID: 10.1111/jcmm.2016.20.issue-12 )
                : 2328-2332
                [ 1 ] Center for Experimental MedicineThe Third Xiangya Hospital Central South University ChangshaChina
                [ 2 ] Department of NeurologyThe Third Xiangya Hospital Central South University ChangshaChina
                [ 3 ] Department of Clinical LaboratoryThe Third Xiangya Hospital Central South University ChangshaChina
                [ 4 ] Department of RadiologyThe Third Xiangya Hospital Central South University ChangshaChina
                [ 5 ] Department of NephrologyThe Third Xiangya Hospital Central South University ChangshaChina
                [ 6 ] Cancer Research InstituteXiangya School of Medicine Central South University ChangshaChina
                Author notes
                [* ] Correspondence to: Hao DENG, M.D., Ph.D.

                E‐mail: hdeng008@


                These authors contributed equally to this paper.

                © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 2, Tables: 1, Pages: 5, Words: 4052
                Funded by: New Xiangya Talent Project, China
                Original Article
                Original Articles
                Custom metadata
                December 2016
                Converter:WILEY_ML3GV2_TO_NLMPMC version:4.9.8 mode:remove_FC converted:28.11.2016


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