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A recurrent mutation in GUCY2D associated with autosomal dominant cone dystrophy in a Chinese family.

Purpose To identify the genetic locus and mutation responsible for autosomal dominant cone dystrophy (adCOD) in a large Chinese family and to describe the phenotypes of the patients. Methods Genomic DNA and clinical data were collected from the family. Genome-wide linkage analysis was performed to map the disease locus, and Sanger dideoxy sequencing was used to detect the mutation in a candidate gene. Results Initially, genome-wide linkage analysis mapped the disease to 17p13.1 between D17S831 and D17S799, with a maximum lod score of 2.71 for D17S938 and D17S1852 at theta=0. Sequence analysis of the guanylate cyclase 2D gene (GUCY2D) in the linkage interval detected a recurrent heterozygous mutation, c.2513G>A (p.Arg838His). This mutation was present in all eight patients with adCOD, but neither in any of the six unaffected family members nor in 192 control chromosomes. Conclusions adCOD in this family is caused by a recurrent mutation in GUCY2D. adCOD can be detected in the first few years after birth in the family by fundus observation and electroretinogram recordings.

In the present study, progressive COD was found in a three-generation Chinese family with eight affected individuals. A genome-wide linkage study mapped the COD locus to 17p13.1. Sequencing the candidate gene in the linkage interval identified a recurrent c.2513G>A (p.Arg838His) mutation in GUCY2D (OMIM 600179).

METHODS
Family with cone dystrophy: adCOD was identified in a threegeneration family living in a small town in Guandong province, China. Eight patients and six unaffected individuals in the family participated in this study. Written informed consent was obtained from the participating individuals or their guardians before the collection of clinical data and genomic samples. This study was approved by the Internal Review Board of the Zhongshan Ophthalmic Center, Sun Yatsen University, Guangzhou, China and followed the tenets of the Declaration of Helsinki and the Guidance of Sample Collection of Human Genetic Diseases (863-Plan) of the Ministry of Public Health of China. Genomic DNA was prepared from venous leukocytes. Genotyping and linkage analysis: Genotyping for 14 family members was performed using 5′-fluorescently labeled  . Genotyping data were analyzed using the Gene Mapper version 3.5 software package (Applied Biosystems). Two-point linkage analysis was performed by using the MLINK program of the FASTLINK implementation of the LINKAGE program package [12,13]. COD in the family was analyzed as an autosomal dominant trait with full penetrance and with a disease-gene allele frequency of 0.0001. Haplotypes were generated using the Cyrillic 2.1 program (Cyrillic Software, Wallingford, UK) and confirmed by inspection.
Mutation identification in GUCY2D: PCR was used to amplify the genomic fragments of GUCY2D. Primers employed to amplify the 19 coding exons and their adjacent intronic region of GUCY2D were the same as those previously reported [14].   (Figure 1). The disease in the family had been passed in three generations. Eight individuals in the family were considered to be affected with adCOD based on clinical information (Table 1; Figure 2, and Figure 3).

Fourteen individuals in the family participated in this study
Two young affected girls (III:1 and III:2) in the family, aged at two and a half years and nine months, respectively, had no visual symptom at the time of examination ( Figure 1 and Table 1). However, fundus observation revealed carpetlike changes with multiple fine yellowish-white spots in the macular region, as well as pallor in the temporal optic discs of these two girls ( Figure 2). Electroretinography (ERG) recordings demonstrated reduced cone responses that confirmed the clinical findings ( Figure 3).
For the other six adult patients, all had photophobia and blurred vision noticed at about 6 to 8 years old, with progressively decreased visual acuity thereafter. Night vision in these patients was well preserved. Examination of Ishihara color plates on 5 patients suggested color vision defects. Macular atrophy and temporal pallor of the optic disc were present in all six adult patients ( Figure 2). Cone responses were significantly reduced in three adult patients who received ERG examination (Figure 3). Rod responses were normal in all five patients (three adults and two young girls), as measured by ERG recordings (Figure 3). The genome-wide linkage scan excluded linkage to other regions and mapped the adCOD to 17p13.1 between D17S831 and D17S799, with a maximum lod score of 2.71 at theta=0 (Figure 1, Table 2). One gene known to cause CORD, GUCY2D, was present in the linkage interval. Subsequently, sequencing analysis of GUCY2D detected a recurrent heterozygous mutation, c.2513G>A (p.Arg838His). This mutation cosegregated with adCOD in the family with a maximum lod score of 2.71, but was not present in six unaffected family members or in 192 control chromosomes.

DISCUSSION
We identified a Chinese family with eight patients showing signs of adCOD that was transmitted as autosomal dominant trait in the family. A genome-wide linkage analysis mapped the disease to 17p13.1 between D17S831 and D17S799. Subsequent mutational screening of a candidate gene in the linkage interval identified a recurrent heterozygous c. 2513G>A (p.Arg838His) mutation in GUCY2D. This mutation was present in all eight patients in the family, but was absent in unaffected family members and normal controls. All of these lines of evidence support the view that a mutation in GUCY2D is responsible for the adCOD in the family.
To date, at least 125 mutations in GUCY2D have been identified to be responsible for retinal degeneration, including Leber congenital amaurosis, CORD, COD, and retinitis pigmentosa, based on HGMD® Professional 2011 accessed as of June 24, 2011. A review of the original reports revealed that 10 of the 125 mutations were associated with COD or CORD in 33 families (Table 3). For the 10 mutations in the 33 families, codon 838 accounted for six mutations (66.7%) in 29 families (90.6%). Up to the present, all patients with GUCY2D mutations at codon 838 exhibited COD or CORD except for one patient, who had c.2513G>C (p.Arg838Pro) and Leber congenital amaurosis, but no clinical details were present [15]. It is unclear if there is any functional difference among these different mutations involving codon 838.
Of the 33 families with GUCY2D mutations, 22 had adCORD, 1 had arCORD, and 10 had adCOD. For the 10 adCOD families, 5 had the c.2512C>T (p.Arg838Cys) mutation [9], 4 had the c.2513G>A (p.Arg838His) mutation [9,16], and 1 had p.P575L mutation [17]. The phenotypes of the patients were essentially similar but the ages at onset varied significantly. In the Chinese family, photophobia and  poor vision was the initial symptom presented at age around 6 to 8 years old, which appeared earlier than those patients with the p.Arg838His mutations in previously reported families. In addition, the visual acuity for the Chinese patients was generally worse than that for patients in previous reports. The rod function in the Chinese patients was well preserved. Our study provided clinical phenotypes for the two youngest patients at ages less than two and half years old, while previous studies only provided phenotypes for patients over 10 years old. Except for the common signs and symptoms for COD or CORD, myopia has been reported as a common sign in several families, such as myopia greater than 5D in at least one eye in 18 of 29 patients (62%) in a family with the p.Arg838Cys mutation [18], myopia greater than 6D in all 10 patients of two families with the p.Arg838His or Arg838Cys mutation [19], and moderate myopia in patients of a family with the p.
[Glu837Asp, Arg838Ser] mutation [20]. In this study, high myopia was present in five of the eight patients.
In summary, adCOD was identified in a Chinese family and was caused by a recurrent mutation in GUCY2D. This is the first report of GUCY2D mutation-associated adCOD in the Chinese population. Fundus and ERG changes in the two patients less than three years old provide the earliest signs for adCOD, which would be valuable in clinical diagnosis.