Abstract
Abstract: :
Purpose:We have previously identified the molecular boundaries of a deletion involving chromosome 12q21 in a patient with clinical features of cornea plana. Cornea plana can be inherited as an autosomal recessive (AR) or autosomal dominant trait (AD), and the genetic loci for both the AD and AR forms have been mapped to 12q21. Mutations in keratocan are responsible for an AR form of cornea plana. Keratocan is one of four small leucine rich repeat proteins (SLRP) located in a cluster on chromosome 12q21. Each of these genes has significant expression in the cornea and sclera, making them excellent candidate genes for the AD form of cornea plana. To confirm that these genes are included in this patient’s deletion, we have sequenced all the coding sequence and flanking intron sequence for these four genes. Methods:Genomic DNA was prepared from peripheral blood samples obtained from the patient and appropriate family members. For all four of the SLRP genes (keratocan, lumican, DSPG3 and decorin), all exons and flanking intron sequence were amplified and sequenced using ABI chemistries and an automated ABI 3100 sequencer. Results:Using microsatellite repeat markers we have previously shown that the patient’s deletion spans a region of 25 megabases with the proximal breakpoint located at 70 mB and the distal breakpoint at 95 mB on chromosome 12. Included within this region are the four SLRP genes evaluated in this study: DSPG3 (89.8 mB), Keratocan (89.9 mB), Lumican (90.0 mB) and Decorin (90.1 mB). DNA sequence analysis revealed 21 polymorphic sites distributed throughout the SLRP cluster: 4 cSNPs, 3 insertion/deletion polymorphisms and 14 SNPs. The mother of the child carrying the deletion was heterozygous for 10 of the 21 polymorphic sites, while the deletion patient had only one allele at each site. DNA sequence variants within the coding sequence or flanking intron sequence were not found in any of the four SLRP genes in the deletion patient. Conclusions:The AD and AR recessive forms of cornea plana both map to the small region containing the SLRP gene cluster. However, mutations have only been found in pedigrees affected by the AR form of the disease in the gene for keratocan. The clinical features of the patient we have identified are most consistent with the AD form of the disease, indicating that only one copy of the responsible gene has been altered. Our results show that, in addition to the keratocan gene, the genes for the other three SLRPs on chromosome 12 are also included in this patient’s deletion, identifying them as excellent candidate genes for the autosomal dominant form of cornea plana.
Keywords: gene mapping • cornea: basic science • gene screening