Clinical and genetic studies in a family with a new splice-site mutation in the choroideremia gene

Abstract

Purpose: To describe the clinical and molecular findings of an Italian family with a new mutation in the choroideremia (CHM) gene.

Methods: We performed a comprehensive ophthalmologic examination, fundus photography, macular optical coherence tomography, perimetry, electroretinography, and fluorescein angiography in an Italian family. The clinical diagnosis was supported by western blot analysis of lymphoblastoid cell lines from patients with CHM and carriers, using a monoclonal antibody against the 415 C-terminal amino acids of Rab escort protein-1 (REP-1). Sequencing of the CHM gene was undertaken on genomic DNA from affected men and carriers; the RNA transcript was analyzed with reverse transcriptase-PCR.

Results: The affected men showed a variability in the rate of visual change and in the degree of clinical and functional ophthalmologic involvement, mainly age-related, while the women displayed aspecific areas of chorioretinal degeneration. Western blot did not show a detectable amount of normal REP-1 protein in affected men who were hemizygous for a novel mutation, c.819+2T>A at the donor splicing site of intron 6 of the CHM gene; the mutation was confirmed in heterozygosity in the carriers.

Conclusions: Western blot of the REP-1 protein confirmed the clinical diagnosis, and molecular analysis showed the new in-frame mutation, c.819+2T>A, leading to loss of function of the REP-1 protein. These results emphasize the value of a diagnostic approach that correlates genetic and ophthalmologic data for identifying carriers in families with CHM. An early diagnosis might be crucial for genetic counseling of this type of progressive and still untreatable disease.

Figure 1

Pedigree of an Italian family with choroideremia. Squares and circles indicate males and females, respectively, and the darkened symbols represent the affected members. The patient above the arrow is the proband. Under the symbol (square or circle) of each subject are two rows of information: individual identifier (I:1, I:2, etc.) and genotype for the CHM mutation.

Figure 2

Optical coherence tomography, visual field, and fluorescein angiography of the proband and his brother. Widespread chorioretinal atrophy with relative sparing of the macular region and visual field constriction are found. Note the greater severity of the anatomic and functional findings of the proband II:4 (A, B, C) compared to his older brother II:3 (D, E, F). RE,right eye; LE, left eye.

Figure 3

Comparison of electroretinography findings of family members. Note the undetectable full-field electroretinography of all responses in the proband (II:4). The dark adapted responses were unrecordable in II:3 and markedly impaired in III:1 and III:2, while the light adapted responses were less compromised. Electrofunctional responses in female carriers (II:2 and I:2) were slightly impaired. RE, right eye; LE, left eye.

Figure 4

Fundus photography and perimetry of the two children. Retinal pigment epithelium mid-periphery pigment changes are already visible. Perimetry shows concentric reduction in the visual field in patient III:1 (A, B) and patient III:2 (C, D). RE, right eye; LE, left eye.

Figure 5

Immunoblot analysis of the Rab escort protein-1 protein from patients II:4 (2, 9), II:3 (3, 10), III:1 e III:2 (5, 6); from the female carrier II:2 (4, 8); from the normal controls: male (1) and female (7); lane 11: ladder in the range of 20–220 kDa (MagicMark XP Western Protein Standard) and female (7, 11).

Figure 6

A novel splice-site mutation of CHM gene. A: Mutation analysis of CHM gene performed by direct sequencing on genomic DNA from patient II:4 showed a new mutation c.819+2T>A. B: The female carrier II:2 resulted heterozygous for the mutation c.819+2T>A. C: Mutation analysis of CHM gene performed by RT-PCR in mRNA from the proband (line 3), female carrier II:2 (line 4), wild type male (line 2), Ladder 100 bp (line 1) and negative reaction control (line 5). D: Sequencing analysis of the 394 bp cDNA reverse transcripted from mRNA patient II:4 show that the whole exon 6 is deleted as result of splicing mutation c.819+2T>A. E: Consequence of CHM gene splicing mutation c.819+2T>A on mRNA.