Identification of Two Novel Mutations in the CHM Gene Causing Choroideremia

To investigate the molecular cause of choroideremia in two unrelated patients with no detectable mutations in the CHM gene. Two unrelated patients were examined by an ophthalmologist to obtain a clinical diagnosis. Patient and control cells were cultured and used as a source of DNA, RNA, and protein for analysis. Exonic regions of CHM were Sanger sequenced and copy number analysis was performed by multiplex ligation-dependent probe amplification. mRNA transcripts were analyzed by Sanger sequencing from synthesized cDNA. Protein expression was probed with western blot analysis. Suspecting an inversion, PCR in one case and inverse PCR in the second case were employed to locate the precise DNA breakpoints. Patient 1 and 2 were clinically diagnosed with choroideremia by experienced ophthalmologists. In both cases, sequencing of the promoter and coding regions of the CHM gene revealed no mutation and copy number analysis of the gene did not detect the presence of any large deletions or duplications. RNA obtained from the patient cells showed only a partial transcript in patient 1, and an abnormal CHM splice isoform in patient 2. The results from RNA and DNA analyses suggested that both patients' genomic DNA might contain an inversion mutation. In patient 1, a long-range PCR product amplified over two breakpoints confirmed an inversion event. This 6 kb inversion spanned from the 5'UTR to the first intron (NM_000390.4(CHM): c.[-836_-826del; 49 + 5526_49 + 5856del; -825_49 + 5525inv; insCGTCT].). In patient 2, inverse PCR revealed a different novel inversion of approximately 85 kb, spanning from intron 2 to intron 8 (NM_000390.4(CHM):c.116 + 6659_1166 + 20670inv). To detect these two inversions in future choroideremia samples, multiplex PCR assays were developed that produce distinct banding patterns that are diagnostic for these two mutations. We have identified inversion mutations in the CHM gene resulting in choroideremia. Though uncommon, inversions should be investigated as a possible cause of the disease in CHM patients, especially for those in whom no point mutations or copy number variants are found.