Novel PITX2 Mutations including a Mutation Causing an Unusual Ophthalmic Phenotype of Axenfeld-Rieger Syndrome.

PURPOSE: The aims of this study were to examine novel mutations in PITX2 and FOXC1 in Chinese patients with anterior segment dysgenesis (ASD) and to compare the clinical presentations of these mutations with previously reported associated phenotypes. METHODS: Twenty-six unrelated patients with different forms of ASD were enrolled from our paediatric and genetic eye clinic. The ocular manifestations of both eyes of each patient were recorded. Genomic DNA was prepared from venous leukocytes. All coding exons of PITX2 and FOXC1 were amplified by polymerase chain reaction (PCR) from genomic DNA and subjected to direct DNA sequencing. Analysis of mutations in control subjects was performed by heteroduplex single-strand conformation polymorphism (SSCP) analysis. RESULTS: Sequence analysis of the PITX2 gene revealed four mutations, including c.475_476delCT (P.L159VfsX39), c.64C > T (P.Q22X), c.296delG (P.R99PfsX56), and c.206G > A (P.R69H). The first three mutations were found to be novel. The c.475_476delCT (P.L159VfsX39) mutation, located at the 3' end of the PITX2-coding region, was identified in a Chinese Axenfeld-Rieger syndrome (ARS) patient who presented with an unusual severe phenotype of bilateral aniridia. The clinical characteristics, including the severity and manifestations of the patient's phenotype, were compared with reported PITX2-associated aniridia phenotypes of ARS in the literature. CONCLUSIONS: These results expand the mutation spectrum of the PITX2 gene in patients with ARS. The PITX2 gene may be responsible for a significant portion of ARS with additional systemic defects in the Chinese population. This is the first reported case of a mutation at the 3' end of the PITX2-coding region extending the phenotypic consequences to bilateral aniridia. The traits of ARS could display tremendous variability in severity and manifestations due to the dominant-negative effect of PITX2. Our results further emphasize the importance of careful clinical and genetic analysis in determining mutation-disease associations and may lead to a better understanding of the role of PITX2 in ocular development.

Mutation Survey of Candidate Genes and Genotype-Phenotype Analysis in 20 Southeastern Chinese Patients with Axenfeld-Rieger Syndrome.

Purpose/aim of the study: To conduct a survey spectrum of the PITX2, FOXC1, and PRDM5 genes to reveal genotype-phenotype correlations in a cohort of Southeastern Chinese patients with Axenfeld-Rieger syndrome (ARS). MATERIALS AND METHODS: A total of 20 probands with ARS were recruited in Southeast China. All patients underwent full ocular and systemic examinations. Sanger sequencing was used to analyze all coding regions of and regions adjacent to PITX2, FOXC1, and PRDM5 and 13 upstream regulatory elements of PITX2. Multiplex ligation-dependent probe amplification was performed to detect gross insertions and deletions in PITX2 and FOXC1. Quantitative polymerase chain reaction was used to detect copy number variations in regulatory elements of PITX2. A bioinformatics analysis was conducted to evaluate the pathogenicity of variants. RESULTS: Eleven mutations, including eight novel mutations, were identified in PITX2. Seven of the mutations were truncations. A genotype-phenotype correlation analysis showed that 81.8% (9/11) of patients with mutations in PITX2 developed glaucoma before reaching 10 years old. The proportion of patients without detected mutations was only 33.3% (3/9, P = 0.0399). In patient G1399, ultrasound biomicroscopy revealed that the left eye exhibited a phenotype similar to aniridia with complete angle closure and a remaining stub of iris tissue. CONCLUSION: This is the first genetic study of a cohort of Southeastern Chinese patients with ARS. Eight novel mutations were detected, expanding the mutation spectrum of PITX2. PITX2 may be a major candidate gene for ARS in Southeastern Chinese patients. Truncations may be the primary mutation type in PITX2. Glaucoma onset may be earlier in patients with mutations in PITX2 than in those without mutations in PITX2 and FOXC1. A block of the anterior chamber angle by the end of the iris might represent the main factor influencing the development of glaucoma in ARS patients with an asymmetric aniridia phenotype.

Novel mutations of FRMD7 in Chinese patients with congenital motor nystagmus.

The purpose of the current study was to identify novel mutations in the FRMD7 (FERM domain containing 7) gene and to characterize clinical features in Chinese patients with congenital motor nystagmus. For this purpose, 18 patients with congenital motor nystagmus were selected from the ocular genetic diseases bank of the Pediatric and Genetic Clinic of Zhongshan Ophthalmic Center (Guangdong, China). Direct sequencing was used to analyze the exons and adjacent introns of the FRMD7 gene. The heteroduplex­single strand conformation polypeptide method was used to analyze 96 unrelated normal controls and gene­screening positive patients. Slit lamp photography of the anterior segment, fundus photography, optical coherence tomography and electroretinogram were carried out to identify the clinical features of congenital motor nystagmus. The authors noted that in, 18 patients with congenital motor nystagmus, there were 7FRMD7 gene mutations (six new mutations). The screening rate was 38.89%, including c.41_43delAGA (p.13­15delK); c.473T>A (p.I158N); c.605T>A (p.I202N); c.580G>T (p.A194S); c.811T>A (p.C271S); c.1493insA (p.Y498X); c.57+1G>A (slice mutation). There were no such mutations in the 96 normal controls. These results enriched the gene mutation spectrum of FRMD7. The authors systematically investigated the clinical phenotype of congenital motor nystagmus in a Chinese population. The study provides further evidence for clinical diagnosis and differential diagnosis and genetic counseling.

GPR143 mutations in Chinese patients with ocular albinism type 1.

The aim of the present study was to evaluate mutations of the G protein-coupled receptor 143 (GPR143) gene for ocular albinism type 1 (OA1) in Chinese patients. For the current study, 8 patients with OA1 were selected from the database of ocular genetic diseases. Genomic DNA of OA1 was prepared from venous leukocytes collected from the patients. Cycle sequencing was used to analyze the exons and adjacent introns of GPR143. The variation detected was analyzed by bidirectional DNA sequencing and further evaluated in 96 controls using heteroduplex­single strand conformational polymorphism analysis. Additionally, slit lamp photography of anterior segment, fundus photography and optical coherence tomography (OCT) were performed to identify the clinical features of OA1. In five patients with OA1, 5 GPR143 gene mutations were identified and four of them there were novel mutations. The screening rate is 62.5%, including c.333G>A (p.W111X), c.353G>A (p.G118E) (known mutation), C.658+2T>G (splice mutation), c.215_216insCGCTGC (p.71­72insAA) and c.17T>C (p. L6P). These mutations were absent in the 96 normal controls. Only one patient with OA1 in the present study was female. Patients with OA1 often have congenital nystagmus, refractive error, severe decline of visual acuity (from 0.1 to 0.4) and foveal hypoplasia. Different degrees of pigment loss were evident in the patients' iris and retina, whereas macular structure was not identified in the OCT examination. The findings of the present study expanded the gene mutation spectrum of GPR143 and investigated the clinical phenotype of patients with OA1 in the Chinese population. Additional evidence for clinical diagnosis was provided along with differential diagnosis and genetic counseling.

Genetic and Clinical Analyses of DOA and LHON in 304 Chinese Patients with Suspected Childhood-Onset Hereditary Optic Neuropathy.

Leber hereditary optic neuropathy (LHON) and dominant optic atrophy (DOA), the most common forms of hereditary optic neuropathy, are easily confused, and it is difficult to distinguish one from the other in the clinic, especially in young children. The present study was designed to survey the mutation spectrum of common pathogenic genes (OPA1, OPA3 and mtDNA genes) and to analyze the genotype-phenotype characteristics of Chinese patients with suspected childhood-onset hereditary optic neuropathy. Genomic DNA and clinical data were collected from 304 unrelated Chinese probands with suspected hereditary optic neuropathy with an age of onset below 14 years. Sanger sequencing was used to screen variants in the coding and adjacent regions of OPA1, OPA3 and the three primary LHON-related mutation sites in mitochondrial DNA (mtDNA) (m.3460G>A, m.11778G>A and m.14484T>C). All patients underwent a complete ophthalmic examination and were compared with age-matched controls. We identified 89/304 (29.3%) primary mtDNA mutations related to LHON in 304 probands, including 76 mutations at m.11778 (76/89, 85.4% of all mtDNA mutations), four at m.3460 (4/89, 4.5%) and nine at m.14484 (9/89, 10.1%). This result was similar to the mutation frequency among Chinese patients with LHON of any age. Screening of OPA1 revealed 23 pathogenic variants, including 11 novel and 12 known pathogenic mutations. This study expanded the OPA1 mutation spectrum, and our results showed that OPA1 mutation is another common cause of childhood-onset hereditary optic neuropathy in Chinese pediatric patients, especially those with disease onset during preschool age.

X-linked heterozygous mutations in ARR3 cause female-limited early onset high myopia.

PURPOSE: To identify genetic mutations in three families with early onset high myopia (eoHM) limited to female members. METHODS: Genomic DNA was collected from participating members of families XF1, XF2, and XF3. Genome-wide linkage scans were performed on the largest family (XF1). Whole exome sequencing was performed on seven samples, including five samples (four affected and one unaffected) from family XF1, as well as the two probands from family XF2 and XF3. Variants were analyzed with multistep bioinformatics analyses. Sanger-dideoxy sequencing was used to verify candidate variations in families and controls. RESULTS: The genome-wide linkage scans performed on family XF1 detected a candidate locus on chromosome Xp11.1-Xq13.3 with a maximum logarithm of the odds (LOD) score of 2.48 and 3.01 for markers DXS991 and DXS986, respectively. Parallel whole exome sequencing identified a novel c.893C>A (p.Ala298Asp) mutation in ARR3 located on Xq13.1 in family XF1, which was shared by all four affected individuals but not the unaffected individual. Two other novel mutations in ARR3, c.298C>T (p.Arg100*) and c.239T>C (p.Leu80Pro), were detected in families XF2 and XF3, respectively. These mutations were predicted to be damaging and were not present in the normal controls and existing databases. All three mutations cosegregated with eoHM in each of the three families, in which all heterozygous female members are affected whereas all hemizygous male family members are not affected. Transmission of the mutations and eoHM in the three families demonstrates an unusual pattern of X-linked female-limited inheritance. CONCLUSIONS: These data suggest that heterozygous mutations in ARR3 might be responsible for X-linked female-limited eoHM in the three families, a pattern contrary to the standard X-linked recessive trait. To our knowledge, eoHM is the first human disease associated with mutations in ARR3 and the second X-linked female-limited disease identified thus far. Identification of ARR3 associated with X-linked female-limited trait provides not only additional evidence of this unusual hereditary pattern but also an additional model for investigating the molecular mechanism responsible for female-limited phenotypes.

RGR variants in different forms of retinal diseases: The undetermined role of truncation mutations.

It has been previously reported that mutations in retinal G protein coupled receptor (RGR) are associated with retinitis pigmentosa. The present study aims to systemically analyze the potential role of variants of RGR in retinal diseases. Variants in coding regions and splice sites of RGR were selected from a whole exome sequencing dataset of 820 probands with various forms of genetic ocular diseases. Potential variants of RGR were further confirmed by Sanger sequencing and analyzed in available family members. Clinical data was reviewed for patients with RGR variants. As a result, a total of five variants in RGR were detected in six probands with different types of ocular diseases. Of the five variants, two were novel heterozygous truncation variations, c.266C>A (p.S89*) and c.722_723delCC (p.S241Yfs*29), identified in two probands with high myopia and confirmed by Sanger sequencing. Segregation analysis on available family members demonstrated p.S89* and p.S241Yfs*29 were also present in unaffected relatives. The other three variants of RGR were heterozygous missense variants randomly occurring in patients with different genetic ocular diseases. No homozygous or compound heterozygous variants were detected. The results of the present study suggested that the heterozygous truncation variants in RGR were less likely to be pathogenic. Further studies are expected to evaluate the pathogenicity of variants in RGR.

Molecular genetics of Leber congenital amaurosis in Chinese: New data from 66 probands and mutation overview of 159 probands.

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy. We have previously performed a mutational analysis of the known LCA-associated genes in probands with LCA by both Sanger and whole exome sequencing. In this study, whole exome sequencing was carried out on 66 new probabds with LCA. In conjunction with these data, the present study provides a comprehensive analysis of the spectrum and frequency of all known genes associated with retinal dystrophy in a total of 159 Chinese probands with LCA. The known genes responsible for all forms hereditary retinal dystrophy were included based on information from RetNet. The candidate variants were filtered by bioinformatics analysis and confirmed by Sanger sequencing. Potentially causative mutations were further validated in available family members. Overall, a total of 118 putative pathogenic mutations from 23 genes were identified in 56.6% (90/159) of probands. These mutations were harbored in 13 LCA-associated genes and in ten genes related to other forms of retinal dystrophy. The most frequently mutated gene in probands with LCA was GUCY2D (10.7%, 17/159). A series of mutational analyses suggests that all known genes associated with retinal dystrophy account for 56.6% of Chinese patients with LCA. A comprehensive molecular genetic analysis of Chinese patients with LCA provides an overview of the spectrum and frequency of ethno-specific mutations of all known genes, as well as indications about other unknown genes in the remaining probands who lacked identified mutations.

Mutation survey and genotype-phenotype analysis of COL2A1 and COL11A1 genes in 16 Chinese patients with Stickler syndrome.

PURPOSE: To identify mutations in COL2A1 and COL11A1 genes and to examine the genotype-phenotype correlation in a cohort of Chinese patients with Stickler syndrome. METHODS: A total of 16 Chinese probands with Stickler syndrome were recruited, including nine with a family history of an autosomal dominant pattern and seven sporadic cases. All patients underwent full ocular and systemic examinations. Sanger sequencing was used to analyze all coding and adjacent regions of the COL2A1 and COL11A1 genes. Multiplex ligation-dependent probe amplification was performed to detect the gross indels of COL2A1 and COL11A1. Bioinformatics analysis was performed to evaluate the pathogenicity of the variants. RESULTS: Five mutations in COL2A1 were identified in six of 16 probands, including three novel (c.85C>T, c.3356delG, c.3401delG) mutations and two known mutations (c.1693C>T, c.2710C>T). Of the five mutations, three were truncated mutations, and the other two were missense mutations. Putative pathogenic mutations of the COL11A1 gene were absent in this cohort of patients. Gross indels were not found in COL2A1 or COL11A1 in any of the probands. High myopia was the most frequent initial ocular phenotype of Stickler syndrome. In this study, 12 Chinese probands lacked obvious systemic phenotypes. CONCLUSIONS: In this study, three novel and two known mutations in the COL2A1 gene were identified in six of 16 Chinese patients with Stickler syndrome. This is the first study in a cohort of Chinese patients with Stickler syndrome, and the results expand the mutation spectrum of the COL2A1 gene. Analysis of the genotype-phenotype correlation showed that the early onset of high myopia with vitreous abnormalities may serve as a key indicator of Stickler syndrome, while the existence of mandibular protrusion in pediatric patients may be an efficient indicator for the absence of mutations in COL2A1 and COL11A1.

Exome sequencing identified null mutations in LOXL3 associated with early-onset high myopia.

PURPOSE: To identify null mutations in novel genes associated with early-onset high myopia using whole exome sequencing. METHODS: Null mutations, including homozygous and compound heterozygous truncations, were selected from whole exome sequencing data for 298 probands with early-onset high myopia. These data were compared with those of 507 probands with other forms of eye diseases. Null mutations specific to early-onset high myopia were considered potential candidates. Candidate mutations were confirmed with Sanger sequencing and were subsequently evaluated in available family members and 480 healthy controls. RESULTS: A homozygous frameshift mutation (c.39dup; p.L14Afs*21) and a compound heterozygous frameshift mutation (c.39dup; p.L14Afs*21 and c.594delG; p.Q199Kfs*35) in LOXL3 were separately identified in two of the 298 probands with early-onset high myopia. These mutations were confirmed with Sanger sequencing and were not detected in 1,974 alleles of the controls from the same region (507 individuals with other conditions and 480 healthy control individuals). These two probands were singleton cases, and their parents had only heterozygous mutations. A homozygous missense mutation in LOXL3 was recently reported in a consanguineous family with Stickler syndrome. CONCLUSIONS: Our results suggest that null mutations in LOXL3 are likely associated with autosomal recessive early-onset high myopia. LOXL3 is a potential candidate gene for high myopia, but this possibility should be confirmed in additional studies. LOXL3 null mutations in human beings are not lethal, providing a phenotype contrary to that in mice.

Molecular genetics of cone-rod dystrophy in Chinese patients: New data from 61 probands and mutation overview of 163 probands.

Cone-rod dystrophy (CORD) is a common form of inherited retinal degeneration. Previously, we have conducted serial mutational analysis in probands with CORD either by Sanger sequencing or whole exome sequencing (WES). In the current study, variants in all genes from RetNet were selected from the whole exome sequencing data of 108 CORD probands (including 61 probands reported here for the first time) and were analyzed by multistep bioinformatics analysis, followed by Sanger sequencing and segregation validation. Data from the previous studies and new data from this study (163 probands in total) were summarized to provide an overview of the molecular genetics of CORD. The following potentially pathogenic mutations were identified in 93 of the 163 (57.1%) probands: CNGA3 (32.5%), ABCA4 (3.8%), ALMS1 (3.1%), GUCY2D (3.1%), CACNA1F (2.5%), CRX (1.8%), PDE6C (1.8%), CNGB3 (1.8%), GUCA1A (1.2%), UNC119 (0.6%), RPGRIP1 (1.2%), RDH12 (0.6%), KCNV2 (0.6%), C21orf2 (0.6%), CEP290 (0.6%), USH2A (0.6%) and SNRNP200 (0.6%). The 17 genes with mutations included 12 known CORD genes and five genes (ALMS1, RDH12, CEP290, USH2A, and SNRNP200) associated with other forms of retinal degeneration. Mutations in CNGA3 is most common in this cohort. This is a systematic molecular genetic analysis of Chinese patients with CORD.

Identification of MFRP Mutations in Chinese Families with High Hyperopia.

PURPOSE: Mutations in MFRP have been reported to cause autosomal recessive posterior microphthalmia, nanophthalmos, and an ophthalmic syndrome characterized by posterior microphthalmia, high hyperopia, retinitis pigmentosa, foveoschisis, and optic disc drusen. High hyperopia is a consistent sign of this syndrome. The purpose of this study was to detect MFRP mutations in 46 unrelated Chinese probands with high hyperopia. METHODS: Clinical data and genomic DNA were collected from 46 Chinese probands diagnosed as having high hyperopia. Genomic DNA from 42 probands was initially analyzed by whole exome sequencing. MFRP variants were confirmed by Sanger sequencing. The coding sequence of MFRP for four additional probands was also analyzed by Sanger sequencing. Candidate MFRP variants were further validated in available family members and 192 normal individuals. RESULTS: Potential pathogenic compound heterozygous mutations, including c.287_291del (p.P96Lfs*6), c.1615C>T (p.R539C), c.664C>A (p.P222T), c.1150dup (p.H384Pfs*8), and c.1549C>T (p.R517W), were detected in three of the 46 probands included in this study. The clinical data revealed that all patients in this study had high hyperopia of +13.50D or higher and an eye axial length of 16.78 mm or less. Electroretinography showed normal responses in a patient with missense mutations and reduced rod responses in another patient with missense and truncation mutations in whom optical coherence tomography showed developmental cystoid macular degeneration in both eyes. CONCLUSIONS: The current study expands our knowledge of the mutation spectrum of MFRP and its associated phenotypes. To our knowledge, this is the first report of MFRP mutations in a Chinese cohort.

KIF11 mutations are a common cause of autosomal dominant familial exudative vitreoretinopathy.

BACKGROUND/AIMS: To identify KIF11 mutations in patients with familial exudative vitreoretinopathy (FEVR) and to describe the associated phenotypes. METHODS: Mutation analysis in a cohort of patients in a single institute was conducted. Bioinformatics was performed for whole exome sequencing, and the variants were confirmed by Sanger sequencing. Clinical data and DNA samples were collected from 814 unrelated Chinese probands, including 34 with FEVR, at the Pediatric and Genetic Eye Clinic, Zhongshan Ophthalmic Centre, Guangzhou, China. RESULTS: Four novel heterozygous truncation mutations in KIF11, including c.131_132dupAT (p.P45Ifs*92), c.2230C>T (p.Q744*), c.2863C>T (p.Q955*) and c.2952_2955delGCAG (p.G985Ifs*6), were detected in four of 34 probands with FEVR. Combined with our previously identified mutations in FEVR cases (n=14), KIF11 mutations were identified in 8.3% (4/48) of all probands with FEVR. Ocular phenotypes documented in patients with KIF11 mutations showed a significant great variability of FEVR from the avascular zone in the peripheral retina to bilateral complete retinal detachment. Analysis of available family members in family QT1314 and QT937 showed segregation of KIF11 mutations with the phenotype of FEVR as expected. The family QT964 with two affected siblings and unaffected parents demonstrated a peculiar somatic mosaicism in the mother who had a low copy number variant (about 7% in her leucocyte DNA). CONCLUSIONS: Identification of mutations in 8.3% patients suggests KIF11 mutations as a common cause of FEVR. Patients with KIF11 mutations showed typical, but variable, signs of FEVR with or without microcephaly, lymphoedema and mental retardation.

Mutation analysis of the genes associated with anterior segment dysgenesis, microcornea and microphthalmia in 257 patients with glaucoma.

Genetic factors have an important role in the development of glaucoma; however, the exact genetic defects remain to be identified in the majority of patients. Glaucoma is frequently observed in patients with anterior segment dysgenesis (ASD), microcornea or microphthalmia. The present study aimed to detect the potential mutations in the genes associated with ASD, microcornea and microphthalmia in 257 patients with glaucoma. Variants in 43 of the 46 genes, which are associated with ASD, microcornea or microphthalmia, were available in whole­exome sequencing. Candidate variants in the 43 genes were selected following multi­step bioinformatic analysis and were subsequently confirmed by Sanger sequencing. Confirmed variants were further validated by segregation analysis and analysis of controls. Overall, 70 candidate variants were selected from whole­exome sequencing, of which 53 (75.7%) were confirmed by Sanger sequencing. In total, 27 of the 53 were considered potentially pathogenic based on bioinformatic analysis and analysis of controls. Of the 27, 6 were identified in BEST1, 4 in EYA1, 3 in GDF6, 2 in BMP4, 2 in CRYBA4, 2 in HCCS, and 1 in each of CRYAA, CRYGC, CRYGD, COL4A1, FOXC1, GJA8, PITX2 and SHH. The 27 variants were detected in 28 of 257 (10.9%) patients, including 11 of 125 patients with primary open­angle glaucoma and 17 of 132 patients with primary angle­closure glaucoma. Variants in these genes may be a potential risk factor for primary glaucoma. Careful clinical observation and analysis of additional patients in different populations are expected to further these findings.

Identification of Genetic Defects in 33 Probands with Stargardt Disease by WES-Based Bioinformatics Gene Panel Analysis.

Stargardt disease (STGD) is the most common hereditary macular degeneration in juveniles, with loss of central vision occurring in the first or second decade of life. The aim of this study is to identify the genetic defects in 33 probands with Stargardt disease. Clinical data and genomic DNA were collected from 33 probands from unrelated families with STGD. Variants in coding genes were initially screened by whole exome sequencing. Candidate variants were selected from all known genes associated with hereditary retinal dystrophy and then confirmed by Sanger sequencing. Putative pathogenic variants were further validated in available family members and controls. Potential pathogenic mutations were identified in 19 of the 33 probands (57.6%). These mutations were all present in ABCA4, but not in the other four STGD-associated genes or in genes responsible for other retinal dystrophies. Of the 19 probands, ABCA4 mutations were homozygous in one proband and compound heterozygous in 18 probands, involving 28 variants (13 novel and 15 known). Analysis of normal controls and available family members in 12 of the 19 families further support the pathogenicity of these variants. Clinical manifestation of all probands met the diagnostic criteria of STGD. This study provides an overview of a genetic basis for STGD in Chinese patients. Mutations in ABCA4 are the most common cause of STGD in this cohort. Genetic defects in approximately 42.4% of STGD patients await identification in future studies.

Unique Variants in OPN1LW Cause Both Syndromic and Nonsyndromic X-Linked High Myopia Mapped to MYP1.

PURPOSE: MYP1 is a locus for X-linked syndromic and nonsyndromic high myopia. Recently, unique haplotypes in OPN1LW were found to be responsible for X-linked syndromic high myopia mapped to MYP1. The current study is to test if such variants in OPN1LW are also responsible for X-linked nonsyndromic high myopia mapped to MYP1. METHODS: The proband of the family previously mapped to MYP1 was initially analyzed using whole-exome sequencing and whole-genome sequencing. Additional probands with early-onset high myopia were analyzed using whole-exome sequencing. Variants in OPN1LW were selected and confirmed by Sanger sequencing. Long-range and second PCR were used to determine the haplotype and the first gene of the red-green gene array. Candidate variants were further validated in family members and controls. RESULTS: The unique LVAVA haplotype in OPN1LW was detected in the family with X-linked nonsyndromic high myopia mapped to MYP1. In addition, this haplotype and a novel frameshift mutation (c.617_620dup, p.Phe208Argfs*51) in OPN1LW were detected in two other families with X-linked high myopia. The unique haplotype cosegregated with high myopia in the two families, with a maximum LOD score of 3.34 and 2.31 at θ = 0. OPN1LW with the variants in these families was the first gene in the red-green gene array and was not present in 247 male controls. Reevaluation of the clinical data in both families with the unique haplotype suggested nonsyndromic high myopia. CONCLUSIONS: Our study confirms the findings that unique variants in OPN1LW are responsible for both syndromic and nonsyndromic X-linked high myopia mapped to MYP1.

Mutation analysis in 129 genes associated with other forms of retinal dystrophy in 157 families with retinitis pigmentosa based on exome sequencing.

PURPOSE: Mutations in 60 known genes were previously identified by exome sequencing in 79 of 157 families with retinitis pigmentosa (RP). This study analyzed variants in 129 genes associated with other forms of hereditary retinal dystrophy in the same cohort. METHODS: Apart from the 73 genes previously analyzed, a further 129 genes responsible for other forms of hereditary retinal dystrophy were selected based on RetNet. Variants in the 129 genes determined by whole exome sequencing were selected and filtered by bioinformatics analysis. Candidate variants were confirmed by Sanger sequencing and validated by analysis of available family members and controls. RESULTS: A total of 90 candidate variants were present in the 129 genes. Sanger sequencing confirmed 83 of the 90 variants. Analysis of family members and controls excluded 76 of these 83 variants. The remaining seven variants were considered to be potential pathogenic mutations; these were c.899A>G, c.1814C>G, and c.2107C>T in BBS2; c.1073C>T and c.1669C>T in INPP5E; and c.3582C>G and c.5704-5C>G in CACNA1F. Six of these seven mutations were novel. The mutations were detected in five unrelated patients without a family history, including three patients with homozygous or compound heterozygous mutations in BBS2 and INPP5E, and two patients with hemizygous mutations in CACNA1F. None of the patients had mutations in the genes associated with autosome dominant retinal dystrophy. CONCLUSIONS: Only a small portion of patients with RP, about 3% (5/157), had causative mutations in the 129 genes associated with other forms of hereditary retinal dystrophy.

Evaluation of 12 myopia-associated genes in Chinese patients with high myopia.

PURPOSE: Two recent large-scale genome-wide association studies identified significant associations between myopia and single nucleotide polymorphisms (SNPs) near the PRSS56, BMP3, KCNQ5, LAMA2, TOX, TJP2, RDH5, ZIC2, RASGRF1, GJD2, RBFOX1, and SHISA6 genes. Our study is to examine whether rare variants in these genes contribute to high myopia. METHODS: Whole-exome sequencing was performed on samples of 298 probands with early-onset high myopia (eoHM; spherical refraction in each meridian ≤ -6.00 [diopters] D in both eyes; age of onset < 7 years) and 195 controls (different forms of retinal degeneration including Leber congenital amaurosis, cone-rod dystrophy, and familial exudative vitroretinopathy). Potential variations in these genes were selected for further validation and comparison to the controls. Moreover, Sanger sequencing was used to evaluate the coding regions and the upstream 800 bps of GJD2 in 395 additional subjects with late-onset moderate to high myopia (loMHM; spherical refraction in each meridian ≤ -4.00 D; age of onset ≥ 7 years) and 403 healthy controls (-0.50 D ± 1.00 D). RESULTS: Exome sequencing of the 298 probands with eoHM identified 25 rare variants that were predicted to affect coding residues. The segregation analysis and the distribution of rare variants between patients and controls did not provide evidence to support their involvement in myopia. Sanger sequencing of GJD2 in an additional 395 subjects with loMHM and 403 healthy controls did not identify myopia-associated variants. CONCLUSIONS: We did not find evidence to support the association of myopia with rare variants in these genes, probably due to our limited sample size. Additional studies are expected to validate these results.

Mutation analysis of Leber congenital amaurosis­associated genes in patients with retinitis pigmentosa.

The genetic defects underlying approximately half of all retinitis pigmentosa (RP) cases are unknown. A number of genes responsible for Leber congenital amaurosis (LCA) may also cause RP when they are mutated. Our previous study revealed that variants in the most frequently mutated nine exons accounted for approximately half of the mutations detected in a cohort of patients with LCA. The aim of the present study was to detect mutations in LCA-associated genes in patients with RP using two different strategies. Sanger sequencing was used to screen mutations in the nine exons in 293 patients with RP and exome sequencing was used to detect variants in 12 LCA-associated genes in 157 of the 293 patients with RP and then to validate the variants by Sanger sequencing. Potential pathogenic mutations were identified in four patients with early onset RP, including homozygous CRB1 mutations in two patients, compound heterozygous CRB1 mutations in one patient and compound heterozygous CEP290 mutations in one patient. The present study indicated that mutations in CEP290 may also be associated with RP but not with LCA. With the exception of CEP290, the remaining 11 genes known to be associated with LCA but not with RP are unlikely to be a common cause of RP.

Exome Sequencing on 298 Probands With Early-Onset High Myopia: Approximately One-Fourth Show Potential Pathogenic Mutations in RetNet Genes.

PURPOSE: To investigate mutations in 234 genes associated with retinal dystrophies in a cohort of 298 probands with early-onset high myopia using whole exome sequencing. METHODS: Genomic DNA from 298 probands with early-onset high myopia was analyzed by whole exome sequencing. Variants from 234 genes were selected and analyzed by multistep bioinformatics analyses. RESULTS: Systematic analysis of variants in the 234 genes identified potential pathogenic mutations in 34 of 234 genes in 71 of 298 (23.8%) probands. Of the 71 probands, 44 (62.0%) had mutations in 11 genes responsible for ocular diseases accompanied by high myopia, including COL2A1, COL11A1, PRPH2, FBN1, GNAT1, OPA1, PAX2, GUCY2D, TSPAN12, CACNA1F, and RPGR. Initial clinical records of the 71 patients with mutations did not show recognizable signs of original diseases other than high myopia. CONCLUSIONS: Mutations in genes known to be responsible for retinal diseases were found in approximately one-fourth of the probands with early-onset high myopia. The high mutation frequency of RetNet genes in these patients can provide clues for genetic screening and further specific clinical examinations of high myopia to promote long-term follow-up assessment and prompt treatment of some diseases.