Seventeen families were available for segregation analysis. Square indicates a male individual; circle, a female individual. Shading indicates an affected individual. The proband is indicated by an arrow. F indicates family.
eFigure. Chromatography of Nucleotide Sequencing
Li S, Huang L, Xiao X, Jia X, Guo X, Zhang Q. Identification of CNGA3 Mutations in 46 FamiliesCommon Cause of Achromatopsia and Cone-Rod Dystrophies in Chinese Patients. JAMA Ophthalmol. 2014;132(9):1076-1083. doi:10.1001/jamaophthalmol.2014.1032
Mutations in CNGA3 are the most common cause of achromatopsia and cone-rod dystrophies.
To identify CNGA3 mutations in patients with cone dystrophies or Leber congenital amaurosis.
Design, Setting, and Participants
Clinical data and genomic DNA in 267 Chinese probands from 138 families with cone dystrophies and 129 families with Leber congenital amaurosis collected at the Zhongshan Ophthalmic Center, Guangzhou, China.
Main Outcomes and Measures
Variants in CNGA3 and associated phenotypes, assessed by Sanger sequencing of CNGA3, bioinformatics of variants, and segregation analysis.
Homozygous or compound heterozygous mutations in CNGA3, including 26 novel and 13 known mutations, were identified in 46 probands from 138 families with cone dystrophies, but none were found in any of the probands from 129 families with Leber congenital amaurosis. The 46 probands with CNGA3 mutations could be further classified as likely having achromatopsia (18 probands) and cone-rod dystrophies (28 probands) based on electroretinographic recordings. Analysis of family members in 17 of 46 families demonstrated good segregation of the disease with the CNGA3 mutations.
Conclusions and Relevance
To our knowledge, this study is the first systemic analysis of CNGA3 in Chinese patients and expands the mutational spectrum and associated phenotypes. Our results suggest that CNGA3 mutations are a common cause of cone-rod dystrophies and achromatopsia in the Chinese population. These data indicate that CNGA3-associated cone dystrophies may be a common form of early-onset severe retinal dystrophies. Therapeutic potential such as gene therapy targeting this gene may benefit some children with early-onset severe retinal dystrophies.
Cone dystrophies (CODs) refer to a group of inherited retinal diseases with predominantly cone impairment. CODs are characterized by photophobia, reduced visual acuity, dyschromatopsia, and abnormal cone electroretinographic recordings.1 CODs may be stationary (mostly achromatopsia) or progressive. Progressive CODs are frequently associated with less severe or later rod involvement and are called cone-rod dystrophies (CORDs) if combined with rod dysfunction. CODs occurring in later childhood or in adulthood are usually progressive,1 while CODs manifesting in infancy may be stationary or progressive.
To date, associations with CODs have been reported for mutations in 28 genes according to an online database (RetNet [https://sph.uth.edu/Retnet/]). Previous studies2- 6 used various strategies to evaluate mutations in 25 of 28 genes among a cohort of unrelated Chinese patients with CODs. However, mutations in these genes were identified in only about one-fifth of the patients, suggesting that a genetic cause for most patients remains to be identified.
The cone cyclic nucleotide-gated channel is composed of a heterotetrameric complex of CNGA3 and CNGB3, which are encoded by the CNGA3 gene (OMIM 600053) and the CNGB3 gene (OMIM 605080), respectively.7- 11 Mutations in CNGB3 are the most common cause of achromatopsia7,12 but are a rare cause of CORDs.13,14 On the other hand, mutations in CNGA3 are a common cause of stationary COD (achromatopsia)15,16 but are not generally considered a cause of other retinal dystrophies according to a database (RetNet), although CNGA3 mutations were reported in a patient with Leber congenital amaurosis (LCA)17 and in a few patients with severe progressive COD.13,15,16 Other studies14,18 have shown the occurrence of progressive cone degeneration in achromatopsia. These findings suggest that the CNGA3 mutation is a good candidate for achromatopsia and for other forms of retinal dystrophies. To date, the CNGA3 gene has not been systematically analyzed in Chinese patients with achromatopsia, CORDs, or LCA.
This study used Sanger sequencing to analyze the coding exons and their adjacent intronic regions of CNGA3 in 267 Chinese unrelated probands with CODs or LCA.
This study was approved by the institutional review board of the Zhongshan Ophthalmic Center, Guangzhou, China. Written informed consent was obtained from all participants or their guardians before the study. In total, 267 Chinese unrelated probands, including 138 families with CODs and 129 families with LCA, were selected from the genomic DNA repository established by our team at the Zhongshan Ophthalmic Center in 1996. Using a method described previously,19 genomic DNA was prepared from venous leukocytes from the proband of each family and from some family members, as well as 96 unrelated healthy control subjects from a previous study.20
Nine pairs of primers were used to amplify 7 coding exons and adjacent regions of CNGA3; all were designed using Primer3 online tools (http://frodo.wi.mit.edu/)21 (Table 1) (the reference sequences from the National Center for Biotechnology Information were NC_000002.11 for genomic DNA, NM_001298.2 for messenger RNA, and NP_001289.1 for protein). A touchdown polymerase chain reaction was used to amplify the sequences as previously described.22 Amplicons were purified and then analyzed with a sequencing kit (ABI BigDye Terminator Cycle version 3.1 using an ABI 3130 Genetic Analyzer; Applied Biosystems). The sequences of the probands were compared with the CNGA3 consensus sequences from the National Center for Biotechnology Information database using a program to search for variants (SeqMan II; DNASTAR, Inc). Each variant was bidirectionally sequenced and then verified in available family members and 96 controls. Variants were described in accord with the nomenclature suggested by the Human Genome Variation Society (http://www.hgvs.org/mutnomen/).23 The effect of a missense variant on the encoded protein was predicted using online tools (PolyPhen-2 [http://genetics.bwh.harvard.edu/pph2/]24 and the Sorting Tolerant From Intolerant algorithm [http://sift.jcvi.org/]25). Potential splicing change was predicted using another online tool (Berkeley Drosophila Genome Project [http://www.fruitfly.org/]).26
Complete sequencing analysis of CNGA3 in 267 probands revealed 39 variants (Table 2 and the eFigure in the Supplement) in 55 probands, including 26 novel and 13 known mutations.15,16,27- 29 The 39 mutations are composed of 24 missense, 8 nonsense, 1 indel, 2 insertion, 2 deletion, and 2 splice site variations. In 4 probands, sequencing detected the common heterozygous polymorphism rs2271041, c.592G>A (p.Glu198Lys).
The 55 probands with CNGA3 variants included 46 with homozygous or compound heterozygous mutations and 9 with heterozygous mutations. Only homozygous or compound heterozygous CNGA3 mutations were considered causative because mutations in CNGA3 have been associated with only autosomal recessive retinal diseases.13,15- 17 All 46 probands with homozygous or compound heterozygous mutations were identified from 138 probands with CODs (Table 3). Family members were available for 17 of 46 probands with CNGA3 mutations. Analysis of the family members of 17 probands demonstrated that the mutations were well segregated with the disease in the 17 families (Figure). None of 129 probands with LCA had homozygous or compound heterozygous mutations in CNGA3. No additional analysis was conducted for 9 probands with heterozygous variation alone because none of the probands’ families demonstrated a pedigree pattern of autosomal dominant inheritance.
Available clinical data for 46 probands with CNGA3 mutations are summarized in Table 3. All probands with CNGA3 mutations had photophobia or poor vision in early childhood, and all but 2 underwent their first ophthalmologic examination before age 10 years. The 46 probands with CODs and CNGA3 mutations can be classified into 2 subgroups based on electroretinographic recordings: 18 probands probably had achromatopsia because they had different degrees of reduced cone responses, and 28 probands likely had CORDs owing to reduced cone responses and less severe reduced rod responses.
Previously, CNGA3 mutations had been reported in 81 families, including 76 families with total color blindness (or achromatopsia),13,15,16,27- 43 three families with progressive COD,13,16 a family with LCA,17 and a family with oligocone trichromacy44 according to information from an online database (HGMD Professional [https://portal.biobase-international.com/hgmd/pro/gene.php?gene=CNGA3]). One study16 also indicated that CNGA3 mutations detected in achromatopsia were also present in patients with CORDs. In the present study, CNGA3 mutations were identified in 46 of 138 families (33.3%) with CODs, including 28 families with CORDs and 18 families with achromatopsia. The finding that most of the 46 families with CNGA3 mutations had CORDs was unexpected because mutations in CNGA3 are generally considered a common cause of achromatopsia, in which usually only cone photoreceptors are affected by the mutations. The detection of mutations in about one-fifth of Chinese probands with CORDs by systematic analysis of 25 CORD-associated genes2- 6 revealed that the finding of 28 probands with CORDs and CNGA3 mutations was greater than the number of patients with mutations detected among all 25 CORD-associated genes. This large number might increase with age because some of the achromatopsia probands with CNGA3 mutations may have progressed to having CORDs. These data indicate that CNGA3 mutations may be the most common cause of CORDs among Chinese patients. In addition, when comparing genes associated with retinal diseases (eg, the CYP4V2 gene mutation in Bietti crystalline corneoretinal dystrophy45 and the rhodopsin gene mutation in retinitis pigmentosa46), the large number of mutations identified in CNGA3 suggests that CNGA3 mutations are by far the most common cause of hereditary retinal degeneration in the Chinese population.
In summary, an online database (HGMD Professional) reports 81 CNGA3 mutations, including 69 missense,13,15- 17,27- 44 eight nonsense,16,27,29 three small insertion,16,27 and a small deletion.16 In the present study, 39 CNGA3 variants were detected, including 26 novel and 13 known mutations. The 39 variants could be classified as missense (24 variants), nonsense (8 variants), indel (1 variant), insertion (2 variants), deletion (2 variants), and splice site variations (2 variants). Most CNGA3 mutations (29 of 39 [74.4%]) occurred in exon 7. None of these variants were present in 192 control chromosomes. The types of variations identified in the present study are comparable with previous findings except that splice site changes and indels have not been reported previously, to our knowledge. The 26 novel mutations identified in this study expand the mutation spectrum of CNGA3. No specific genotype-phenotype correlation has been identified between different CNGA3 mutations and phenotypic variations (achromatopsia and CORDs).
Accepted for Publication: February 14, 2014.
Corresponding Author: Qingjiong Zhang, MD, PhD, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, 54 Xianlie Rd, Guangzhou 510060, China (firstname.lastname@example.org).
Published Online: June 5, 2014. doi:10.1001/jamaophthalmol.2014.1032.
Author Contributions: Dr Zhang had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Drs Li and Huang contributed equally to this work.
Study concept and design: Huang, Zhang.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Li, Huang, Xiao, Jia.
Critical revision of the manuscript for important intellectual content: Guo, Zhang.
Statistical analysis: Li, Huang, Xiao, Jia.
Obtained funding: Zhang.
Administrative, technical, or material support: Li, Huang, Xiao, Jia, Guo.
Study supervision: Zhang.
Conflict of Interest Disclosures: None reported.
Funding/Support: The work was supported by grants 81170881 and U1201221 from the National Natural Science Foundation of China, by grant 2010CB529904 from the national 973 Program, by Project 985 of Sun Yat-Sen University, and by fundamental research funds of the State Key Laboratory of Ophthalmology (Dr Zhang).
Role of the Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Additional Contributions: We thank the patients and their family members for their participation.