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Identification of GUCY2D gene mutations in CORD5 families and evidence of incomplete penetrance

Identification of GUCY2D gene mutations in CORD5 families and evidence of incomplete penetrance
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     MUTATION IN BRIEF   HUMAN MUTATION Mutation in Brief #580 (2003) Online © 2003   WILEY-LISS, INC. DOI: 10.1002/humu.9109  Received 25 October 2002; accepted revised manuscript 25 November 2002. Identification of GUCY2D Gene Mutations in CORD5 Families and Evidence of Incomplete Penetrance Nitin Udar 1 , Svetlana Yelchits 1 , Meenal Chalukya 1 , Vivek Yellore 1 , Steve Nusinowitz 1 , Rosamaria Silva-Garcia 1 , Tamara Vrabec 2 , Irene Hussles Maumenee 3 , Larry Donoso 2 , and Kent W. Small 1* 1  Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA; 2  Henry and Corinne Bower Laboratory, Wills Eye Hospital, Philadelphia, PA; 3  Johns Hopkins University, Dept. of Ophthalmology, Wilmer Eye Institute,  Baltimore, MD *Correspondence to: Kent W. Small, M.D., 200 Stein Plaza, UCLA, Jules Stein Eye Institute, Los Angeles, CA 90095; Tel.: 310 206 7475; Fax: 310 794 2029; E-mail: Grant sponsor: Foundation Fighting Blindness and McCone Foundation Communicated by Mark H. Paalman   Cone rod dystrophy 5 (CORD5) is an autosomal dominant retinal disease that primarily affects cone function. The locus has previously been mapped to human chromosome 17p12-p13 between the markers D17S926/D17S849 and D17S945/D17S804. One of our “unaffected” recombinant individual from family 1175 was subsequently found to cross through this interval. Reexamination revealed that he was in fact mildly affected. This expanded the minimum candidate region. Direct sequencing of the GUCY2D and other candidate genes within this interval was carried out on 2 American families affected with CORD5. There was an R838C missense mutation within the GUCY2D gene in one and a R838H missense mutation in another families. The previously reported mutations for CORD6 are clustered at the same position within the gene. These results indicate that both CORD5 (MIM# 600977) and CORD6 (MIM# 601777) are actually the same disease. We conclude that significant variability in expression and incomplete penetrance exists even within one family.  © 2003 Wiley-Liss, Inc.   KEY WORDS: CORD5, CORD6, autosomal dominant cone rod dystrophy, GUCY2D, RetGC1 INTRODUCTION Small et al. (1996) demonstrated linkage for an autosomal dominant cone dystrophy 5 (CORD5; MIM# 600977) to human chromosome 17p12-13. Balciuniene et al. (1995) also independently showed linkage to the same region with a maximum LOD score of 7.72 at the marker D17S938 in large five generation Swedish CORD5 family. This retinal disorder is characterized by a predominant loss of cone function with relative preservation of rod function, resulting in the degeneration of the central macula. The most common symptoms are photophobia, progressive loss of central vision, and nonspecific color vision defects (Small and Gehrs, 1996). Kelsell et al. (1997) subsequently identified the locus for CORD6 a cone rod dystrophy with a phenotype similar to CORD5 on chromosome 17p12-13. Perlautte et al. (1996) reported mutations in the GUCY2D gene (MIM# 600179) in patients with the recessive form of Leber Congenital Amaurosis (LCA1), the most severe form of inherited  2 Udar et al. retinopathy. Subsequently in 1998, Kelsell et al. (1998) found missense mutation in two neighboring amino acids of this gene at position 837 and 838 to be responsible for autosomal dominant cone rod dystrophy (CORD6; MIM# 601777). In the present study, we have screened candidate genes within the minimal candidate region including GUCY2D in 2 American families with CORD5. METHODS AND MATERIAL We ascertained 2 families consisting of at least three generations each with autosomal dominant CORD5. Family 1175 is a Caucasian family of 95 individuals of which 44 are affected (Small et al. 1996). Family 1177 has a total of 14 individuals of Caucasian (American) srcin with 8 affected individuals. Seventy individuals from family #1175 and 12 individuals from family #1177 were examined by the criteria previously described by Small for CORD5 (Small et al., 1996). Institutional Review Board approval and informed consent was obtained from all participating subjects prior to enrolling them in the study. Blood was obtained by venipuncture for DNA analysis from a total of 83 subjects. The DNA was extracted using the Puregene protocol (Gentra Systems, Minneapolis, USA). Pedigrees - 1175 and - 1177 were drawn using Cyrillic software (Cherwell Scientific Inc, Oxford, UK). Genotyping with microsatellite markers was performed as described by Small et al. 1999. The following markers were genotyped and number in parenthesis represent approximate distance from previous marker in series D17S513, D17S938 (313kb), D17S796 (2kb), D17S906 (445kb), D17S1353 (918kb), D17S1796 (170kb), D17S1805 (793kb), D17S786 (233kb), D17S1858 (56kb), D17S952 (246kb), D17S1791 (42kb), D17S945 (666kb) and D17S804 (42kb). New polymorphic markers were identified by analyzing sequence from clones Genbank ID:- AC005695, AC009335 and AC005747. Flanking primers were designed using Primer 3 ( and analyzed for being polymorphic by genotyping a total of 12 control individuals. The above-mentioned clones were aligned using database at NCBI ( and Celera Discovery System (Celera Genomics, Rockville, US). The new markers we developed are listed below and deposited at the Genome Database ( CORD5NU8 (GDB:11505481), CORD5NU12 (GDB:11505496), CORD5NU17 (GDB:11505497), CORD5NU19 (GDB:11505486), CORD5NU20 (GDB:11505487), CORD5NU26 (GDB:11505488). For DNA sequencing of the candidate genes, primers were synthesized more than 20 bps internal to the intronic region. Primer sequences are available on request from the author (KWS). After PCR amplification using patient and control DNA, the products were separated on a 2% agarose gels. The bands were cut from the gel and purified using Qiagen gel purification kit (Qiagen, Chatsworth, USA). The purified product was used for direct sequencing using Thermo Sequenase radio labeled terminator sequencing kit (USB, Cleveland, USA). The sequencing reactions were separated on a 6% acrylamide, 7M urea sequencing gel in 1X TBE buffer at 80W constant power on a Biorad, DNA sequencing apparatus (Bio-Rad, Hercules, USA). Mutation screening for the R838C and R838H mutations was carried out by amplifying exon 13 from individual DNA samples. The PCR product was digested with HhaI and electrophoresed on a 2% agarose gel. Normal individuals showed a single band after digestion, while the mutants showed one normal band and a larger undigested (mutant) band. Multiple sequence alignment using Clustal X (Thompson et al., 1997, Jeanmougin et al., 1998) was carried out for the following sequences identified as Guanylate cyclase in the NCBI database: Rattus norvegicus NP_077356, Mus musculus NP_032218, Bos P55203, Canis familiaris O19179, Homo sapiens NP_000171, Gallus gallus T42382, Rana pipiens AAF01287 and Hemicentrotus pulcherrimus D21101. Genedoc (Nicholas, 1997) was used to visualize and manually edit the sequence. RESULTS AND DISCUSSION   In order to refine the localization of the CORD5 locus, we genotyped 70 members of a large family (#1175) with autosomal dominant cone rod dystrophy (CORD5). Of these, 38 individuals were affected and 32 unaffected. Haplotype analysis identified 2 critical recombinant individuals, one affected individual #124 and one initially thought to be “unaffected” individual #1031. The nearest telomeric recombinantion was at marker D17S1791. The centromeric recombinant family1175 #124 showed recombinations up to marker D17S804. Since the microsatellite markers in the intervening region (available in the public database at NCBI, GDB, Genethon, CHLC and the Whitehead institute) were not initially dense enough, we created a physical map of this region and  GUCY2D Mutations in Individuals with CORD5 3   identified new markers. The databases at NCBI and Celera Discovery System were utilized to develop a physical map and identify candidate genes. Using a direct sequencing approach we screened 7 genes within the critical interval. Although several base changes were identified none of these segregated with the affected individuals in the family nor were any truncating mutations identified. Figure 1. Microsatellite markers on chromosome 17p13. Blocks indicate region of recombination for the two individuals in family 1175. Individual #124 is affected. Individual #1031 was classified as affected based on ERG findings. Individual #117 has the mutation but does not show the disease phenotype yet.  We identified and generated 11 new polymorphic markers (marked CORD5NU8-26). These new markers were genotyped for our key recombinant individuals and their immediate relatives i.e. #2023, #1033, #1032, #1066, #1031, #125, #124, #117. After segregation and haplotype analysis, we found that the two individuals crossed through the region at markers D17S945 through CORD5NU26 as shown in Figure 1. This indicated that there could be an error in diagnosis of one or both of these individuals. Therefore, these individuals were subjected to a repeat funduscopic examination and electrophysiological studies. Individual #124 had revealed pigment clumping and macular granularity at age 15. Reexamination at age 30 confirmed these observations based on an abnormal ERG. Individual #1031 had a normal fundus and ERG at age 47. On reexamination at age 56 he revealed a “borderline” abnormal ERG and therefore diagnosed as very mildly affected. The change in affectation status of individual #1031, a previously unaffected individual was not completely unexpected. Small and Gehrs (1996) pointed out that the age of onset for CORD5 can vary between 3-51 years. A high quality full field and multifocal ERG appears to be the only reliable test to aid in making the correct diagnosis in subtle cases. Incomplete  GUCY2D Mutations in Individuals with CORD5 5   Individual #117 is the son of #1031 who had a late age of onset and might show the same variability. Using the HhaI digestion assay, which recognizes both the mutations described above, we screened 200 chromosomes from normal individuals and did not find the change in any one of them. The same assay was used by Kelsell et al. (1998) and Payne et al. (2001) to screen 600 and 100 normal chromosomes respectively. Within family 1175, the disease (CORD5) has an age dependent penetrance (Small et al. 1996) 60% had age of onset in the first decade of life, 80% had onset within the first 2 decades, afterwards the penetrance was estimated to be 95%. Because a few of the individuals are mildly affected as reflected in their low disease scores, a small uncertainty (<10%) existed regarding their affectation status. Variable expressivity is reflected in the extent of macular degeneration observed in individuals from the same family. The relative difference in functional activity observed in different mutants at Arg838 (Wilkie et al., 2000) is likely to be the reason for the difference in disease manifestation. Other cellular and environmental factors might also influence the differences in phenotype. Using the public sequence database at NCBI (, we have identified the homologues for GUCY2D from various species including Rattus, Mus, Bos, Canis, Human, Chicken, Rana, Hemicentrotus, Sea Urchin and Brissus. Orthologues are difficult to identify when additional flanking sequence and syntenic regions are not comparable. We performed a ClustalX alignment of these sequences. Even though the evolutionary distance between the species used in this data set is immense, we found that at codon 837 and 838, the amino acids were highly conserved within all the species in our dataset Figure 3. These results give additional support to the fact that codon 838 is important for the photoreceptor functioning of GUCY2D gene and the phototransduction cycle. 810 820 830 840 850 860 Rattus NP_077356 : KGR KT NIIDS MLR  MLEQYSS NLEDLIR ERTEELEQEKQKTDR LLTQ MLPPS  Mus NP_032218 : KGR KT NIIDS MLR  MLEQYSS NLEDLIR ERTEELEQEKQKTDR LLTQ MLPPS  Bos P55203 : KGR K M  NIIDS MLR  MLEQYSS NLEDLIR ERTEELELEKQKTDR LLTQ MLPPS  Canis O10179 : KGR KT NIIDS MLR  MLEQYSS NLEDLIR ERTEELELEKQKTDR LLTQ MLPPS  Human NP_000171 : KGR KT NIIDS MLR  MLEQYSS NLEDLIR ERTEELELEKQKTDR LLTQ MLPPS  Gallus T42382 : KGR KT NIIDS MLR  MLEQYSS NLEDLIR ERTEELEIEKQKTDKLLTQ MLPPS  Rana AAF01287 : KGKKT NIIDS MLR  MLEQYSS NLEDLIR ERTEELE V EKQKTEKLSFQ MLPPS  Hemicentrotus D21101: KGLKP NILD N MI A IMER YT N NLEELV DERTQELQKEK A KTEQLLHR  MLPPS   Figure 3.   Clustal X alignment of amino acid sequence of guanylate cyclase homologoues from human and other species. The arrow indicates codon 838, which is the mutation site.   ACKNOWLEDGMENTS Foundation Fighting Blindness and McCone Foundation. REFERENCES Balciuniene J, Johansson K, Sandgren O, Wachtmeister L, Holmgren G, Forsman K. 1995. A gene for autosomal dominant progressive cone dystrophy (CORD5) maps to chromosome 17p12-p13. Genomics 30:281-286. Cremers FP, Van Den Hurk JA, Den Hollander AI. 2002. Molecular genetics of Leber congenital amaurosis. Hum Mol Genet. 11:1169-1176. Dharmaraj SR, Silva ER, Pina AL, Li YY, Yang JM, Carter CR, Loyer MK, El-Hilali HK, Traboulsi EK, Sundin OK, Zhu DK, Koenekoop RK, Maumenee IH. Mutational analysis and clinical correlation in Leber congenital amaurosis. 2000. Ophthalmic Genet. 21:135-150. Downes SM, Payne AM, Kelsell RE, Fitzke FW, Holder GE, Hunt DM, Moore AT, Bird AC. 2001. Autosomal dominant cone-rod dystrophy with mutations in the guanylate cyclase 2D gene encoding retinal guanylate cyclase-1. Arch Ophthalmol. 119:1667-1673. Jeanmougin F, Thompson J, Gouy M, Higgins D and Gibson T. 1998. Multiple sequence alignment with Clustal X. TIBS 23:403-405.
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