Mapping Of Pathogenic Gene In Three DGI-Ⅱ Families

Dentinogenesis imperfecta (DGI) is an autosomal dominant dental disease that occurs with an incidence of approximately 1:8,000 live births. It was classified into three subgroups: Type I, type II and type III. DGI type I is associated with osteogenesis imperfecta resulted from mutations in COL1A1. DGI-II, also known as hereditary opalescent dentin, is associated with dentin formation defect with a very high penetrance approximately 100%. DGI-III is found in a southern Maryland tri-racial inbred population known as the "Brandywine isolation".Kong Xiangyin et al disclosed mutations in DSP gene in three out of six Chinese DGI-II families they collected in 2001, however, there remained three families with no mutation in DSP. In this study, highly polymorphic STRPs in 4q21 were employed for linkage analysis on the three DGI-II families, at the same time, certain candidate genes were screened by PCR and DNA sequencing. 1. Linkage analysis of three Dentinogenesis Imperfecta Type II familiesTwo point linkage analysis on DGI-II family from Yueyang using six highly polymorphic STRPs in 4q21 suggested an linkage with D4S451,D4S2622, DMP1 and D4S1563(Lod score>1). Multipoint linkage analysis supported an linkage with DSPP although the Lod score of this point is lower than 1 in two point analysis. Recombination was observed in D4S451, D4S2622, DMP1 and D4S1563 during haplotype establishment. Especially, a crossing-over of DSPP was observed in family members IV:39 and V:19,which suggested a different location of DGI-II gene from that reported by Kong et al in 2001. To further confirm this speculation, highly polymorphic STRPs with thicker hereditary interval is needed for linkage analysis.Two point linkage analysis on DGI-II family from Ningnan using six highly polymorphic STRPs in 4q21 suggested an linkage with DSPP,SPP1,DMP1 and D4S1563(Lod score>l). hi multipoint analysis, the maximum Lod score was obtained between DSPP and DMP1. Recombination was observed in D4S451, D4S2690, SPP1 and D4S1563 during haplotype establishment. Therefore the locus was mapped between D4S2690 and DMP1. This locus was similar to reported data before.Two point linkage analysis on DGI-II family from Dalian using four highly polymorphic STRPs in 4q21 could not establish any linkage with D4S451, DSPP, SPP1 or D4S1563, for the Lod scores of these points were always negative when 9 varied from 0 to 0.4. The failure of establishing the haplotype in this locus also suggested no linkage with 4q21 in this family. Given the relatively small pidgree, more families are needed for further analysis. 2. Mutation screening of DGI-II candidate genesIn the critical region, there distribute in order of hereditary distance a group of bone-tooth mineral matrix phospho-glycoproteins bonemorphogenetic protein3 (BMP3), dentin sialo phosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein II (BSP), Matrix extracellular phosphoglycoprotein (MEPE) , and osteopontin (OPN; HGMW-approved symbol SPP1); Furthermore, there are many locus candidate genes such as NUDT9 (nucleoside diphosphate linked moiety X-type motif 9), KLHL8 (kelch-like 8), LOC2207693(similar to heat shock protein 90-beta) etc.To screen for DGI-II gene, we sequenced all of the genes mentioned. Here we report the screening results of DSPP promoter, DPP, MEPE and BSP:No mutation was identified in DPP coding sequence and DSPP promoter. Together with the negative results in screening of DSP before, the difference between our results and that of Kong suggested a possible heterogeneity.Screening of MEPE revealed a c.1027G>A transition. This variation introduced an amino acid substitution of V330I; A c.797G>A transition was identified in BSP and resulted in a substitution of G219R. No complete segregation with phenotype was observed on these two transition. Therefore MEPE and BSP were excluded as the pathogenic gene.