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M. N. Preising, C. F. Friedburg, M. Jäger, B. Lorenz; Molecular Genetic Evidence That Nonsense Mediated Decay (NMD) is the Mechanism Underlying Autosomal Recessive Bestrophinopathy (ARB). Invest. Ophthalmol. Vis. Sci. 2010;51(13):3089.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the hypothesis that autosomal recessive bestrophinopathy (ARB) is the NULL-phenotype of hBEST1 mutations.
Index patient was a 6 y old boy showing typical ophthalmological features of ARB, and presenting with a heterozygous single nucleotide deletion (c.867delC) in the hBEST1 gene. Peripheral venous blood was taken from the patient, his unaffected father, mother, and paternal grandfather to evaluate the segregation of mutation. Whole RNA was isolated from lymphoblastoid cells from peripheral venous blood using the PAXgene Blood RNA System.Two fragments covering the 5’ and 3’ end of hBEST1 were amplified by quantitative reverse transcription PCR (qRT-PCR) from isolated lymphoblastoid cell RNA using the VersoTM 1-Step qRT-PCR Kit and normalized against GAPDH as a housekeeping gene. RNA was applied by 1 µg / 25 µl PCR set-up and amplification was performed for 50 cycles. qRT-was performed in triplicate.
The deletion was identified in the patient's father and sister in the heterozygous state. The maternal mutation could not yet be determined in the coding parts of the gene by direct sequencing of the patient's DNA.Both hBEST1 qRT-PCR-products were amplified from the RNA samples of the relatives carrying the mutation but not from the RNA sample of the patient indicating complete nonsense mediated decay of the message.
We provided molecular genetic evidence supporting the hypothesis that ARB is the NULL phenotype of hBEST1 mutations by introducing qRT-PCR of lymphoblastoid cell RNA as a diagnostic test for hBEST1 mutations causing preterm translation stops.
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