Diverse mechanisms of human genetic disease: Splice order determination in the COL1A2 gene. Effects that influence splice site mutations in osteogenesis imperfecta and a translocation disrupting SNRPN gene causes Prader-Willi syndrome

The focus of this research involves distinct genetic diseases and underlying molecular etiologies. Osteogenesis Imperfecta (OI) is a disease primarily characterized by varying degrees of bone fragility and deformity due to mutations in one of the two type I collagen genes, COL1A1 or COL1A2. Prader-Willi syndrome (PWS), typically involving obesity and mental retardation, is considered to be a contiguous gene syndrome spanning an imprinted region on chromosome 15q11.2. Affected individuals were used to: (1) investigate the function splice order and sequence content within the COL1A2 gene have on splice site mutation outcomes, and (2) determine the role of a translocation in an individual with PWS.;The introns of the COL1A2 gene were sequenced. Sequence motif identification was accomplished for SR protein RNA recognition sequences (SFS2/ASF, SRp40, and SRp55), polypurine stretches (GAR repeats where R is a purine), and splice site consensus sequences were analyzed. This detailed analysis revealed that for one individual with a 5' splice donor mutation (IVS32G+1 → A) a novel utilization of a cryptic 3' splice acceptor site containing terminal dinucleotides AC occurred transforming the intron into a 5'AT-3'AC splicing system. A characteristic order of splicing was determined for three regions based on individuals that had splice site mutations in these areas. Order of splicing analysis in cells with splice site mutations also revealed that there is a preferred order of splicing and this order was demonstrated to influence the outcome of these splice site mutations. As a consequence of these findings a model of splicing that incorporates the order of intron removal into the scope of factors governing a splicing reaction was developed.;The breakpoint of a translocation (4;15) was determined at the molecular level in an individual with PWS. The translocation was found to disrupt the SNRPN gene between SNRPN exons 2 and 3 on chromosome 15. Expression analysis of various loci imprinted within the 15q11.2 region revealed a disruption in the expression of SNRPN exons 3 and 4 and D15S226E (PAR5). The findings of this study and other reported translocation induced cases further refine the genotype/phenotype correlations in this syndrome.