The diagnosis of congenital endothelial corneal dystrophy was made clinically in all patients by an experienced ophthalmologist. Early‐onset perceptive hearing loss was observed in all affected members in six of the seven families (table 1).12,13
In family C1 no hearing loss was reported, but the only affected child was too young (2 years old) for this feature to be informative. The parents were asymptomatic in all families. Families H3 and H5 have been described previously.12,13
In addition, we studied a subject (not shown in table 1) of the same ethnicity as family H6 (Sephardi Jews from Morroco and Turkey, not consanguineous) who developed sensorineural hearing loss at age 7, and consulted at age 21 with visual symptoms caused by corneal oedema associated with endothelial dystrophy. At age 28, he presented recurrent syncopes associated with a prolonged PR interval, a bifascicular block, a transient atrial flutter and significant cardiac pauses, and a cardiac pacemaker was implanted.
Table 1Clinical findings and SLC4A11 mutations in probands and families
Peripheral blood was sampled with informed consent for DNA analysis in all nuclear families, four of which were consanguineous. In family H2, only DNA of the proband was available. DNA was extracted by the standard phenol–chloroform method.14
In the four consanguineous families, linkage analysis was carried out with microsatellite markers of the 20p13 region to which CDPD1 was mapped.12
The following markers were studied: D20S864, D20S103, D20S117, D20S199, D20S179, D20S113, D20S198, D20S842, D20S181, D20S473, D20S867, D20S889, D20S116, D20S482, D20S437, D20S95, D20S905, D20S194, D20S156 and D20S851. Marker order was obtained from the Marshfield Comprehensive Human Genetic Maps (http://research.marshfieldclinic.org/genetics/GeneticResearch/compMaps.asp
) and the UCSC Genome Browser (http://genome.ucsc.edu/cgi‐bin/hgGateway
). DNA was amplified by PCR using 15 ng of DNA from each individual in a final volume of 15 μl, followed by polyacrylamide gel electrophoresis and silver staining.
We used the following primers for amplification and sequencing of the 19 coding exons and adjacent intronic sequences of SLC4A11: SLC4A11EX1D, CCTGCTTCCCTTTCTCCC; SLC4A11EX1R, GTAGGCTATGCACCCTGGAG; SLC4A11EX2–3D, GAGCCCCTCCTTCCTGTG; SLC4A11EX2–3R, AGGGAAGCCATCACCTCAG; SLC4A11EX4–5D, ACCAGGCAGTGACAGCATC; SLC4A11EX4–5R, ATGGGACACCCAGTTCCAC; SLC4A11EX6D, CTAGCAGAGGTCGCCAGG; SLC4A11EX6R, AAGCAGAGGGCGGGTAAC; SLC4A11EX7–8D, ATGGGGAGAGCACCTTCAC; SLC4A11EX7–8R, GATGCAGGACAGGCACAC; SLC4A11EX9–10D, CTTCACTGATGGTACGTGGC; SLC4A11EX9–10R, GACACGAATCACTGCAGGC; SLC4A11EX11–12D, GAGATGGTGCCTGAGACCC; SLC4A11EX11–12R, AGTGCAGAACCTCCCATCTC; SLC4A11EX13–14D, CCTTTCTCCCTGAGATCCCC; SLC4A11EX13–14R, GGTTGTAGCGGAACTTGCTC; SLC4A11EX15–16D, GTGGGTGACGTGGGGTAG; SLC4A11EX15–16R, ATGTGGCCAGAGGCTCC; SLC4A11EX17–18D, CTGGCCACATGGGACATAG; SLC4A11EX17–18R, GCCCATTCTCCACACCTAGAC; SLC4A11EX19D, GGTGTCCACTGCCTTCTCTC; SLC4A11EX19R, TACACCTCCCCTCACAGCTC.
PCR products were purified using ExoSAP‐IT For PCR Product Clean‐Up (USB Corporation, Cleveland, Ohio, USA), sequenced using the Big Dye Terminator cycle sequencing kit v2 (Applied Biosystems, Foster City, California, USA) and analysed on a 3130 Genetic Analyzer sequencing machine (Applied Biosystems). Sequences were inspected in silico for mutations using the SeqScape software V.2.0 (Applied Biosystems).
Control DNA samples were obtained with informed consent from a cohort of volunteers including subjects of European, Moroccan, Indian, South American and non‐Ashkenasi Jewish origin. We controlled the absence of each missense mutation observed in this study in a sample of at least 100 unrelated control subjects, including at least 35 ethnically matched subjects, by PCR amplification followed by denaturing high‐performance liquid chromatography (dHPLC) analysis and, when indicated, direct sequencing of amplimers, which produced variant dHPLC electrophoregrams.
This study was approved by the ethics committee of University Hospital Erasme – ULB, Brussels, Belgium.