| Smith-Fineman-Myers syndrome (SFMS,MIM309580) is a syndromic X-linked mental retardation first described in two brothers. The phenotype comprises unusual facial appearance, short stature, microcephaly,dolichocephaly, micrognathia, prominent upper central incisors, bridged palmar creases, foot deformities, early hypotonia, hyperreflexia, behavior problems, and mental retardation. In 1999,Gong et al. studied a large Chinese family with Smith-Fineman-Myers syndrome and mapped the SFMS locus on chromosome Xq25 between markers DXS8064 and DXS8050 and the physical distance is 19.59Mb. Our further study showed that SFMS exhibited locus heterogeneity. In the presence of genetic heterogeneity, it is very difficult to narrow down the critical region by collecting more SFMS families. To facilitate identifying the SFMS candidate gene, the critical region was narrow down to a distance of 10.18Mb by using the newly selected STRs. From this interval, six candidate genes were analyzed and excluded as pathogenic genes for SFMS. Analysis of five STR loci newly selected from X chromosome Our previous research has placed the gene for SFMS in Xq24-26.3 and this region covers about 19.59Mb and contains a minimum of 70 distinct genes. The following study told us that SFMS is a genetic heterogeneous condition. It is not an easy thing to identify the disease gene from such a large region by positional candidate cloning. In the presence of the genetic heterogeneity in SFMS, it is impossible to fulfill the aim through collecting more SFMS families.To further refine the disease locus, and facilitate positional candidate cloning of the disease gene, eight STR (short tandem repeat. STR) markers in the critical region were selected from two genomic clones on X chromosome by using BCM search Launcher. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. Among these eight STRs, Five were polymorphic and named XSTR1, XSTR2, XSTR3, XSTR4, and XSTR5 respectively. Chi-Square test indicated the distribution of genotypes agreed with Hardy-Weinberg equilibrium (P>0.05).? Refined mapping of Smith-Fineman-Myers syndrome by using new STR loci To narrow down the SFMS candidate region, eight new STRs CEN-DXS6854-DXS6855-DXS8068-DXS1187-DXS8071-DXS6748-DXS8041-DXS691-TEL and five newly selected STRs has been analyzed in SFMS family by using PCR-PAGE. Crossover has been observed on XSTR3 and XSTR4, so the locus for Smith-Fineman-Myers syndrome could be mapped between XSTR3 and XSTR4. Due to the Physical distance between XSTR3 and previous upper side is 1.59Mb, between XSTR4 and lower side is 7.82Mb. Therefore, the mapping interval could be narrowed to a 10.18Mb region. In present, it is the minmum interval that can be difined because there are no other informative markers in this region. The candidate interval was reduced about 50%.More than ten genes were excluded such as ARHGEF6, a previously recognized MRX gene. The reduced region will speed the identification of true SFMS gene.? Determination of genomic structure of SMARCA1 and MST4 gene. SMARCA1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin,subfamily A, memberl, SMARCA1) belongs to SWI/SNF2 family.While XNP,One gene in this family is an important X-linked mental retardation gene.So it is considered as a candidate gene to study. Also, according to the location and putative function of MST4 ,it is an important candidate gene .To facilitate mutation detection and function analysis, genomic structure of SMARCA1 and MST4 was determined By comparing the cDNA sequence ofSMARCA1 with the genomic sequences, and confirmed by amplifying and sequencing the sequences of exons and splicing junction. The results show that SMARCA1 gene contains 24 exons and 23 introns, MST4 gene e contains 12 exons and 11 introns.All the exon/intron boundaries follow the GT-AG rule and are in good agreement with the exon/intron consensus sequence. The results also show that MST4 gene has two splice variants. The characterization of genomic structure of SMARCA1 and MST4 gene allows us to detect disease -causing mutation within the gene and further study its biological function.? Mutation analysis of six candidate genes in SFMS family.From the SFMS candidate region,six genes have been chosen to perform mutation analysis based on their location and putative function.The GLUD2 gene, also known as Gludpl, belongs to a complex gene family including GLUDl and four other pseudogenes (Gludp2, GludpS, Gludp4, and Gludp5). The products of GLUDl and GLUD2 are 96% identical at the amino acid level. Recombinant GLUD2 protein is very similar to the soluble GDH in human brain and is capable of catalyzing the oxidative deamination of glutamate. According to its function and location, we hypothesized that a defect in the X-linked neuron-specific GLUD2 gene may be involved in the primary pathogenesis of SFMS.GPC3 and GPC4 encode a heparan sulfate proteoglycan of the glypican family. Heparan sulfate proteoglycans are proteins that are substituted with heparan sulfate. Heparan sulfate is a complex polysaccharide that interacts with heparin-binding growth factors and influences the signaling activities of these factors. The glypicans compose a family of cell surface heparan sulfate proteoglycans that are linked to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. So far, six members of the glypican family are known in vertebrates. All glypicans share a characteristic cysteine motif, which is thought to result in a unique tertiary structure, and have consensus sequences for glycosaminoglycan attachment close to their C-termini. Glypicans playa role in the regulation of morphogen signaling. Mutations in dally, a glypican homologue in Drosophila, result in a disturbance of cell cycling and produce morphological defects in several adult tissues, including the eyes, antennae, wings and genitalia. In 1996,Pilia found that GPC3 mutated in an X-linked mental retardation, Simpson-Golabi-Behmel syndrome.To date, many XLMR genes such as GDI 1. 0PHN1, PAK3,FGD1, RPS6KA3(RSK2), ARHGEF6 were found involved in the pathway of signal transduction.Given that MST4 is biologically active in the activation of MEK/ERK pathway and in mediating cell growth and transformation, we chose it as a candidate gene and performed mutation analysis. Unfortunately, we didn't find any change in coding region. Also because of above reason,GPCR2 has been investigated and excluded as SFMS genes by using same method. Also, SMARCA1 was perform mutation detection in SFMS family.Though no variant was found in these six genes in our SFMS family, they are important candidate genes needed to be scrutinized in other XLMR mapped in this region in the future.In summary, we have refined the SFMS locus to a 10.18Mb interval and this work will facilitate candidate gene analysis in the critical interval. Among of these, SMARCA1,GPC3,GPC4,GPCR2, MST4 and GLUD2 were evaluated as the candidates for SFMS by mutation analysis and no intragenic mutation was found. However, any mutations in promoter or regulatory elements located outside of the amplified regions would not have been detected. Such mutations could modify gene expression at the transcriptional or translational level in SFMS individuals.
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