Identification of candidate regions for a novel Usher syndrome type II locus - PubMed (original) (raw)

. 2008 Sep 19:14:1719-26.

Zeineb Benzina, Houria Dhouib, Imen Hadjamor, Mustapha Amyere, Leila Ayadi, Khalil Turki, Bouthaina Hammami, Noureddine Kmiha, Hassen Kammoun, Bochra Hakim, Ilhem Charfedine, Miikka Vikkula, Abdelmonem Ghorbel, Hammadi Ayadi, Saber Masmoudi

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Identification of candidate regions for a novel Usher syndrome type II locus

Imen Ben Rebeh et al. Mol Vis. 2008.

Abstract

Purpose: Chronic diseases affecting the inner ear and the retina cause severe impairments to our communication systems. In more than half of the cases, Usher syndrome (USH) is the origin of these double defects. Patients with USH type II (USH2) have retinitis pigmentosa (RP) that develops during puberty, moderate to severe hearing impairment with downsloping pure-tone audiogram, and normal vestibular function. Four loci and three genes are known for USH2. In this study, we proposed to localize the gene responsible for USH2 in a consanguineous family of Tunisian origin.

Methods: Affected members underwent detailed ocular and audiologic characterization. One Tunisian family with USH2 and 45 healthy controls unrelated to the family were recruited. Two affected and six unaffected family members attended our study. DNA samples of eight family members were genotyped with polymorphic markers. Two-point and multipoint LOD scores were calculated using Genehunter software v2.1. Sequencing was used to investigate candidate genes.

Results: Haplotype analysis showed no significant linkage to any known USH gene or locus. A genome-wide screen, using microsatellite markers, was performed, allowing the identification of three homozygous regions in chromosomes 2, 4, and 15. We further confirmed and refined these three regions using microsatellite and single-nucleotide polymorphisms. With recessive mode of inheritance, the highest multipoint LOD score of 1.765 was identified for the candidate regions on chromosomes 4 and 15. The chromosome 15 locus is large (55 Mb), underscoring the limited number of meioses in the consanguineous pedigree. Moreover, the linked, homozygous chromosome 15q alleles, unlike those of the chromosome 2 and 4 loci, are infrequent in the local population. Thus, the data strongly suggest that the novel locus for USH2 is likely to reside on 15q.

Conclusions: Our data provide a basis for the localization and the identification of a novel gene implicated in USH2, most likely localized on 15q.

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Figures

Figure 1

Figure 1

Pedigree, haplotype and statistical data for a Tunisian family segregating Usher type 2 syndrome. A-C: In pedigree, the square symbol indicates male, the circle symbol denotes female and black symbols represent affected individuals. Haplotypes for polymorphic markers in three candidate regions on Chromosome 2, 4, and 15 are shown. The disease-linked haplotype is indicated by black bar for markers listed while other haplotypes in gray and white. The critical linkage interval of each candidate region was indicated by box on haplotypes. Analysis of these markers allowed us to refine the boundary of the critical linkage intervals to 14.7 Mb, 25.5 Mb, and 55 Mb respectively. Among interesting candidate genes on chromosome 2 and 15 region two CERKL and MYO1E were selected for mutation screening. D-F: Multipoint lod scores for markers on three candidate regions on Chromosome 2, 4, and 15. Lod scores for the different markers studied were computed using Genehunter software. Maximum lod score of 1.765 was identified for the candidate regions on chromosome 4 between D4S2989 and D4S402 and chromosome 15 between

D15S978

and D15S1036. A maximum lod score of 1.51 was found on chromosome 2 between rs155100 and rs1157595. The following abbreviation was used: Mega base (Mb).

Figure 2

Figure 2

Serial audiograms of two affected members (BT188 and BT189) shown for the right (R) and left (L) ears separately. Pure tone air-conduction threshold (y-axis) is expressed in decibels (dB). The blue one represents the audiogram from 18-year-old BT188. Both the red and the green represent the audiograms for the patient BT189. The red audiogram was made when he was 24 whereas the second was made at the age of 28. BT188 was not available for audiometric test at the beginning of the study. Audiometric test showed a moderate sloping bilateral sensorineural HI in these two usher patients. The green and the red audiograms for the patient BT189 showed that there is no progression of hearing loss at an interval of four years.

Figure 3

Figure 3

Visual field test results obtained on the right (RE) and the left eye (LE) of the two patients BT188 and BT189. A: Result of measuring the visual fields on BT189 at 28 years of age. B: Result of measuring the visual fields on BT188 at the age of 18 years. A series of random lights of different intensities are flashed in the peripheral field of vision of both patients. When they perceive the computer-generated light suddenly appearing in their field of view they press a button to indicate their responses, then we see this spot (Dot see). If the patient is unable to see the light in an appropriate portion of his field of view, then we see on the computer a spot (Dot don’t see) indicating vision loss. Visual field loss was more severe in the older brother BT189. But in both patients, the nasal and temporal fields were not preserved, and only the central field was maintained.

Figure 4

Figure 4

Ganzfeld-Electroretinogram of the right and left eyes of the patient BT189. The following abbreviations were used: Left eye (LE), right eye (RE), electroretinogram (ERG), visual-evoked potentials (VEP), positive peak (P1 and P2), negative peak (N1 and N2). The ERG and the VEP tests the function of the visual pathway from the retina (ERG) to the occipital cortex (VEP). These tests were conducted by placing a standard ERG device attached to the skin on 2 mm above the orbit. VEPs were recorded simultaneously from electrode attached to the occipital scalp 2 mm above the region on the midsaggital plane. An electrode placed on the fore head provided a ground. The results can be directly related to the part of a visual field that might be defective. This is based on the anatomical relationship of the retinal images and the visual field. After dark adaptation for 30 min, the doctor will place anesthetic drops in the patient's eye and place a contact lens on the surface of the eye. Once the contact lens is in place, a series of blue, red and white lights will be shown to the patient. The VEP is an evoked electrophysiological potential that can be extracted, using signal averaging, from the electroencephalographic activity recorded at the scalp. Both ERG and VEP were differentially amplified band pass filtred (0,1,30 Hz), recorded over 300 ms epochs, and signal average. 2 trials were given. The visual evoked potential to flash stimulation consists of a series of negative and positive waves. The earliest detectable response has a peak latency of approximately 30ms post-stimulus. For the flash VEP, the most robust components are the N2 and P2 peaks. Measurements of P2 amplitude should be made from the positive P2 peak at around 207.3 ms. The ERG recorded in BT189 showed an absence of responses. While the VEP showed a normal responses in both eyes. These traces confirm the evidence of a significant bilateral global retinal degeneration. Only cone flicker responses of less than 15% of the normal mean were recordable under photopic conditions while all other responses were below noise level, a typical finding for patients with retinitis pigmentosa.

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