Molecular Characterization of Choroideremia-Associated Deletions Reveals an Unexpected Regulation of CHM Gene Transcription

Abstract

Choroideremia (CHM) is a X-linked recessive chorioretinal dystrophy due to deficiency of the CHM gene product, i.e., Rab escort protein isoform 1 (REP1). To date, gene therapy for CHM has shown variable effectiveness, likely because the underlying pathogenic mechanisms as well as genotype-phenotype correlation are not yet fully known. Small nucleotide variants leading to premature termination codons (PTCs) are a major cause of CHM, but about 20% of patients has CHM gene deletions. To improve understanding of the disease mechanisms, we analyzed molecular features of seven deletions involving the CHM gene sequence. We mapped the deletion breakpoints by using polymerase chain reaction, sequencing and array comparative genomic hybridization; to identify rearrangement-promoting DNA sequences, we analyzed genomic architecture surrounding the breakpoint regions. Moreover, in some CHM patients with different mutation types, we measured transcript level of CHM and of CHML, encoding the REP2 isoform. Scattered along the whole CHM gene and in close proximity to the deletion breakpoints we found numerous repeat elements that generate a locus-specific rearrangement hot spot. Unexpectedly, patients with non-PTC variants had increased expression of the aberrant CHM mRNA; CHML expression was higher than normal in a patient lacking CHM and its putative regulatory sequences. This latest evidence suggests that mechanisms regulating CHM and CHML gene expression are worthy of further study, because their full knowledge could be also useful for developing effective therapies for this hitherto untreatable inherited retinal degeneration.

Genetic epidemiology of inherited retinal diseases in a large patient cohort followed at a single center in Italy

“…Sixty patients harboring structural variants (i.e. extended deletions or duplications) in CHM , NMNAT1 , NPHP1 , PCDH15 , PRPF31 , RAX2 , RP2 , RPGR , USH1G , USH2A and WFS1 were solved by combining the above-mentioned approaches with multiplex ligation-dependent probe amplification (MLPA), array comparative genomic hybridization (aCGH) 41 , Sanger sequencing or in silico tools for CNV detection (e.g. CONTRA 42 , Vargenius 43 ).…”

“…For some cases that remained unsolved after an NGS-based analysis, we implemented complementary approaches to identify disease-causing mutations (e.g. Sanger-based analysis of RPGR ORF15 23 , search for rare structural variants/larger copy number variations (CNVs) by experimental and in silico approaches 41 – 43 ). Because whole-exome and clinical exome approaches do not efficiently detect all known deep-intronic variants associated with IRDs, we screened by Sanger sequencing known intronic variants (e.g.…”

Comprehensive Molecular Analysis of Disease-Related Genes as First-Tier Test for Early Diagnosis, Classification, and Management of Patients Affected by Nonsyndromic Ichthyosis

“…The relatively high number of XLI in our patients' cohort has undoubtedly contributed to increasing the diagnostic yield of our procedure. Similar to other X-linked disorders, XLI is mostly caused by deletions (80-90%), but about 10-20% of patients have pathogenic SNVs [28][29][30]. Therefore, multiple ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH) are the methods of choice for XLI diagnosis, even though they cannot detect pathogenic SNVs [30,31].…”

De Novo Large Deletions in the PHEX Gene Caused X-Linked Hypophosphataemic Rickets in Two Italian Female Infants Successfully Treated with Burosumab

“…Chromosomal deletions of variable sizes that remove single exons or eliminate a whole gene and, sometimes, various contiguous genes are frequent causes of numerous X-linked genetic diseases [29,30]. The relatively high incidence of deletions indicates that chromosome X is predisposed to genomic rearrangements; indeed, X-linked diseases often appear sporadically in a family as consequence of de novo mutational events, including large deletions [29].…”