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CLINICAL GENETICS ISSN lm9-9163

X-linked exudative vitreoretinopathy caused by an arginine to leucine substitution (R121L) in the Norrie disease protein

Johnson K, Mintz-Hittner, HA, Conley YP, Ferrell RE. X-linked exudative vitreoretinopathy caused by an arginine to leucine substitution (R121L) in the Nome disease protein. Clin Genet 1996: 5 0 113-1 1 5 . 0 Munksgaard, 1996

Wg report the cosegregation of an arginine to leucine substitution at position 121 of the Nome disease protein in a large kindred where exudative vitre- oretinopathy segregates as an X-linked recessive trait. The clinical phenotype and rate of disease progression were extremely variable, with progression to total retinal detachment from less than age 2 years to more than 21 years. To date, all mutations in X-linked vitreoretinopathy have been missense muta- tions, presumably not affecting the three-dimensional structure of the NDP gene product, and clustered around residues 121-126 of the Nome protein. This contrasts with the diversity of mutations seen in the more severe, allelic Norrie disease.

Exudative vitreoretinopathy (EVR) is characterized by abno'rmal vascularization of the peripheral retina. The clinical characteristics of EVR are extremely variable, ranging from macular ectopia and a mod- erate decrease in visual acuity (see Fig. 2) to retinal detachment with vitreous hemorrhage and total blindness. While EVR is most often transmitted as an autosomal dominant trait (McKusick 133780), Plager et al. (1992) reported a family in which EVR segregated as an X-linked recessive trait (EVRX; McKusick 305390) and pointed out earlier pedi- grees consistent with an X-linked mode of inherit- ance (Criswick & Schepens, 1969, Dudgeon 1979). Fullwood et al. (1993) reported possible linkage be- tween EVRX and markers near the Nome disease (NDP) gene locus, and suggested the possibility that EVRX and NDP are allelic disorders. NDP (McKu- sick 3 1060) is an X-linked recessive neurodevelop- mental disorder characterized by bilateral retinal

Kimberly Johnson, Helen A. Mink-Hither', Yvette P. Conley and Robert E. Ferrell Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh PA and 'Department of Ophthalmology and Visual Science, University of Texas, School of Medicine, Houston, TX, USA

Key words: exudative vitreoretinopathy - Norrie disease - retinopathy Robert E. Ferrell. Ph.0. Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA. Tel: (412) 624-3018, Fax: (412) 624-3020 Received in revised version 15 April, accepted for publication 15 April 1996

dysplasia and retinal detachment with vitreous hem- orrhage and total blindness. A variable degree of mental retardation and hearing loss are frequently present.

Chen et al. (1993) provided strong support for an allelic relationship between EVRX and ND, with the discovery of a missense mutation in exon 3 lead- ing to a leucine to phenylalanine substitution at po- sition 124 of the NDP protein in the family reported by Fullwood et al. (1993). Subsequently, Fuentes et al. (1993) reported a G+A transition at nucleotide 778 leading to an arginine+glutamine substitution at position 121 of the NDP protein in a Spanish fam- ily segregating for EVRX. To further define allelic heterogeneity at the NDP locus, we analyzed the NDP gene in a five-generation family in which EVRX segregates as an X-linked recessive trait. A preliminary report of this family has been presented (Johnson et al. 1994).

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Fig. I. Pedigree of a five-generation EVRX family. Carrier sta- tus confirmed by genotyping is indicated by 0; affected males are indicated by filled squares and phenotypically normal indi- viduals by open symbols.

Materials and methods

Thirty-one members of a single kindred (Fig. 1) were examined for evidence of EVR by fluorescein angi- ography and electrophysiology. Informed consent to participate in the genetic study was obtained, and an EDTA anticoagulated whole blood sample was col- lected from each examined family member. High molecular weight DNA was isolated by the method of Miller et al. ( 1988). Oligonucleotide primers flanking exons 1-3 of the NDP gene were used to amplify exons and splice junctions from genomic DNA (Meindl et al. 1992), using the polymerase chain reac- tion (PCR; Saiki et al. 1988). Single-strand confor- mational analysis was carried out by the method of Orita et al. (1989). PCR products were directly sequenced using dye-labeled dideoxy chain termina- tion chemistry and cycle sequencing. Sequencing reaction products were analyzed using the Applied Biosystems automatic sequencer.

Results and discussion

Visual acuity in affected males ranged from 20/100 to no light perception. The rate of progression of peripheral retinal pathology varied greatly in this family, with total retinal detachment occurring at ages of less than 2 years to more than 21 years. Ret- inal findings in this kindred are illustrated in Fig. 2. Systematic single-strand conformational analysis of amplified exonic and splice junction sequences revealed an unusual conformer that was present in hemizygous affected males, absent in phenotypi- cally normal males, and present in a heterozygous pattern in carrier females. Direct sequence analysis of the normal and mutant conformer revealed a sin- gle difference, a G+T transversion that is predicted to lead to the substitution of a hydrophobic leucine residue for the positively charged arginine residue normally present at position 121 of the NDP gene product. This mutation destroys a MspI restriction

site normally present at residues 776-779 of the Norrie cDNA (Genbank X65724). Exon 3 was amplified from genomic DNA of 31 family mem- bers, digested with MspI and subjected to agarose gel electrophoresis. All clinically normal males and unrelated spouses gave a constant fragment of about 170 bp and doublet of 72 bp and 54 bp fragments. Affected males gave the 170 bp constant fragment and a 126 bp fragment due to loss of the MspI site; carrier females have four fragments of 170, 126, 72 and 54 bp. In this family, the absence of the MspI

Fig. 2. Top: Fundus photographs of the left eye of pedigree member IV-3 (Fig. 1). Visual acuity is 20/80 with -6.00 correc- tion. Macular ectopia is present (macula is circled). There is pe- ripheral extra retinal fibrovascular proliferation with a periph- eral tractional retinal detachment (in the inferotemporal quad- rant). Bottom: Fluorescein angiograms of the same area, demonstrating an abnormal foveal avascular zone (macula is circled). The peripheral vascular pattern is abnormal and the in- ner retinal vessels do not extend to the ora sernta.

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site segregated with disease in affected males and known carrier females, and was not observed in nor- mal males, unrelated spouses or in a sample of 100 randomly selected females from the general popula- tion. The occurrence of a missense mutation which would replace a positively charged arginine residue by a hydrophobic leucine, the cosegregation of this substitution with disease in a large kindred, and the absence of this substitution in unaffected individuals and in the general population support our conclu- sion that FEVR in this family is caused by a muta- tion in exon 3 of the Norrie gene.

Meindl et al. (1992) noted significant homology between the C-terminal cysteine-rich region of the Norrie gene product and the cysteine-rich domains of the mucin family of extracellular proteins. These homologies all involve extracellular proteins with a proposed role in intracellular signaling. They pro- posed that the Nome gene product is secreted in the developing optic cup where it functions as an organ- izational signal. In a more detailed analysis, Meitinger et al. (1 993) used the results of sequence pattern searches and three-dimensional modeling to suggest that the NPD protein tertiary structure was similar to that of transforming growth factor 13, and was a member of a family of growth factors charac- terized by a “cystine knot” motif. All members of this gene family affect their target cell through inter- action with cell-surface receptors. The correlation between Norrie gene mutations and clinical pheno- types is incomplete, mutations causing the more se- vere Nome phenotype are quite diverse (Berger et al. 1992, Chen et al. 1993, Meindl et al. 1992, 1995, Zhu & Maumenee 1994), while characterized muta- tions causing EVRX are confined to residues 121- 126 of the NPD gene product. In addition to the R121L mutation reported here, these include a L124F mutation reported by Chen et al. (1993), re- current R12Q mutations reported by Fuentes et al. (1993) and Meindl et al. (1995), and a recurrent R121W mutation reported by Fuchs et al. (1995) and Shastry et al. (1995). This suggests a highly specific disruption of NPD function in the milder EVRX cases.

Acknowledgements

This work supported in part by an unrestricted Departmental Grant from Research to Prevent Blindness (H.A.M.-H.), the Hermann Eye Fund (H.A.M.-H.) and N.I.H. Grant EY09859 (K. J . and R. E. F.)

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