AOphthalmology and Medical Genetics, BOphthalmology, CMedical Genetics, 1University of Alberta, Edmonton, Alberta, Canada
2Developmental Biology Unit, UCL Institute of Child Health, London, United Kingdom
Commercial Relationships: B. Chanda, None; M. Asai-Coakwell, None; J.C. Sowden, None; M.A. Walter, None; O.J. Lehmann, None.
Support: CIHR, AHFMR, CRC
Purpose:Determining the causation of chromosome 6p25 copy number variations (CNVs) that underlie Axenfeld Rieger Syndrome (ARS), is expected to elucidate the genesis of an important pediatric glaucoma subtype, and facilitate molecular diagnostic testing.
Methods:Array comparative genome hybridization was undertaken to localize the breakpoints in the largest ARS pedigree collection worldwide with 6p25 CNVs, prior to long range PCR. Some 240 primer pairs were used to amplify breakpoint-spanning junctional fragments with sequencing and in silico analyses employed to determine the genomic architecture of the regions flanking the breakpoints.
Results:The 6p25 CNVs extent were determined at the base pair level in all pedigrees [duplication: # 1-2: 492kb, # 3-6: 480kb, #7: 512kb; deletion: #8: 1216kb and #9: 30kb; ring chromosome #10: 2257kb deletion]. Haplotype analysis confirmed the existence of founder effects with just five unique segmental rearrangements (3 duplications, 2 deletions) identified. In all segmental duplications, a head to tail orientation of duplicons with insertion or deletion of nucleotides were found. In segmental deletion pedigree #8, an unusually large (367bp) novel insert was identified with no significant homology to any known genomic sequences. The forkhead box transcription factor, FOXC1, was the only gene to be encompassed in all 6p25 CNVs. A mechanistic spectrum involving non-allelic homologous recombination (NAHR) and non-homologous end joining (NHEJ), was observed in all pedigrees, with evidence of a novel mechanism in one pedigree.
Conclusions:Characterization of the 6p25 CNVs revealed constant involvement of FOXC1. Their non-recurrent nature, with differing degrees of NAHR & NHEJ in an autosome, extends the range and complexity associated with chromosomal rearrangements. The novel genomic architecture observed in pedigree #8 is compatible with a DNA replication based mechanism that broadens the means by which CNVs are known to occur. From the clinical perspective, identification of a small number of common rearrangements that underlie a substantial portion of ARS, facilitates molecular diagnostic testing with scope for genotype-phenotype correlation and predicting clinical outcome.
Keywords: gene microarray transcription factors genetics
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