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1. Linkage Analysis and Mutation Screening in Triphalangeal Thumb-Associated Limb Malformations 2. Generation of Fgfr3+/P244R Mouse Chimera and Phenotype Analysis of Fgfr2+/P253R Mouse Model

Author: SunZuo
Tutor: ZhangXue;Ethylin Wang Jabs
School: Peking Union Medical College , China
Course: Of Medical Genetics
Keywords: triphalangeal thumb triphalangeal thumb-polysyndactyly syndrome gene mapping linkage analysis point mutation the FGFR genes gene targeting transgenic mouse phenotype
CLC: R682
Type: PhD thesis
Year: 2007
Downloads: 80
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Abstract

Triphalangeal thumb （TPT） is a rare limb malformation with an incidence of 1 in 25,000 births. It can occur as an isolated deformity or associate with other limb malformations. Triphalangeal thumb-polysyndactyly syndrome （TPTPS, MIM 190605） is an autosomal dominant genetic disorder which usually shows a duplicated triphalangeal thumb, normal index finger, and cutaneous syndactyly between fingers 3-5. Using two large Dutch Caucasian kindreds, Heutink and colleagues first mapped the TPT locus to chromosome 7q36, obtaining a maximum Lod score of 12.61 at marker D7S559. This mapping was confirmed by the following linkage analysis in 13 large families of different ethnic origin. These work also suggested that all preaxial polydactyly （PPD） associated with TPT resulted from the same mutant locus at chromosome 7q36. The critical region was further refined to an interval of about 450kb containing the LMBR1/C7ORF2 gene but not the SHH gene. Comparative genomics and functional mapping in transgenic mice showed that there was an approximately 800bp ZPA （zone of polarizing activity） regulatory sequence （ZRS） within the intron 5 of the human LMBR1/C7ORF2 gene. The expression of a LacZ reporter under the control of ZRS is identical to Shh expression in the posterior limb domains where the ZPA located. The role of the ZRS in the pathogenesis of PPD/TPT was further confirmed by identification of point mutations in ZRS, in Hx （hemimelic-extra toes） and M100081 mouse mutants and in 4 patients with TPT. The gene responsible for TPTPS was independently mapped to the same chromosome 7q36 region in 1994. However, the TPTPs locus could not be narrowed down to a smaller region because of the lack of recombination events within the families used for linkage analysis. It has been reported that TPTPS can be occurred with TPT, syndactyly typeⅣ/Haas-type syndactyly （MIM 186200） or THTPTTS （tibial hemimelia-polysyndactyly-triphalangeal thumb syndrome, MIM 188770）, which suggested that TPT, TPTPS, Haas-type syndactyly and THPTTS may be the different variant forms of the same genetic entity. However, it has not been clear if mutations in ZRS can cause TPTPS as well. Therefore, the identification of the TPTPS-causing mutations becomes more important in gaining an insight into the molecular mechanisms underlying the TPT-aasociated limb malformations.We performed linkage analyses and mutation screenings in two Chinese families with typical TPTPS. Linkage and haplotype analyses were carried out using the genetic markers covering the TPT critical region defined by Heus and colleagues in 1999. New markers were selected with the aid of the UCSC Genome Browser on Human May 2004 Assembly. The data we generated in the two Chinese families confirmed the linkage of the disease phenotype to chromosome 7q36, indicating the genetic homogeneity in TPTPS. A recombination event between D7S3037 and a telomeric marker in close proximity excluded the SHH gene in the critical region harboring the mutations for TPTPS. Exons and their flanking intronic sequences of known and predicted genes from the candidate region, C7ORF2/LMBR1, HLXB9, RNF32, NOM1, C7ORF13, LOC389602, LOC393076, LOC393889 and LOC393890, were first PCR-amplified and then subjected to mutation screening by automatic DNA sequencing. No pathogenic mutation was identified. Using the VISTA program, comparative genomic analysis was performed and 10 conserved non-coding sequences including the SHH enhancer ZRS were identified. And direct sequencing of PCR-amplified fragments showed no pathogenic mutation.We also performed sequencing and preliminary functional analyses for the long-range SHH enhancer within intron 5 of the C7ORF2/LMBR1 gene. In a large Mexican family with PPD/TPT, a sequence alteration of C to T at an absolutely conserved base position 402 of the ZRS was found in all three affected individuals available for mutaion analysis. This alteration was not detected in 118 chromosomes from unrelated Mexican controls by restriction analysis using enzyme Rsa I, suggesting that the alteration might be a pathogenic point mutation. In a Chinese family with PPD/TPT, we did not find any mutation within the ZRS. Using the enhancer-GFP-Tol system, we set up the transgenic zebrafish models containing the ZRS-402 mutation and normal ZRS, respectively. The GFP expression within the heart was detected around 30 hours post-fertilization （hpf） and the expression within the fins was found around 100 hpf. No obvious difference in GFP expression between mutant and normal control has been observed so far and further works remained to be done to validate the mutation in vivo.In summary, we have confirmed the genetic mapping of TPTPS in Chinese families and excluded the SHH gene in the critical region; showed that TPTPS and PPD/TPT might result from different mutations, and identified a novel point mutation of ZRS in a Mexican family with PPD/TPT. The fibroblast growth factor receptor （FGFR） family consists of four single-pass transmembrane receptor tyrosine kinases, FGFR1-4. Identical gain-of-function mutations in the FGFR genes, P252R in FGFR1, P253R in FGFR2 and P250R in FGFR3, have been identified to result in type I Pfeiffer, Apert and Muenke craniosynostosis syndromes, respectively. Apert syndrome （OMIM 101200）, an autosomal dominant disorder characterized by severe skull malformations and syndactyly of the hands and feet, is caused by the gain-of-function mutations in FGFR2. Approximately 2/3 of patients with Apert syndrome have a FGFR2 S252W mutation while 1/3 have a FGFR2 P253R mutation. It has been hypothesized that the P253R mutation is associated with milder craniofacial abnormalities and more severe syndactyly than the S252W mutation. Two Fgfr2-S252W knock-in mouse models have been independently created by two different research groups. All the mice carried S252W mutation exhibited the phenotypes similiar to human Apert syndrome. However, none of them showed syndactyly. The generation and analysis of a knock-in mouse model with the P253R mutation will be very useful to study the genotype-phenotype correlations between the two FGFR2 mutations and the pathogenesis of Apert syndrome.We generated and characterized Fgfr2+/P253R transgenic mice. Necropsy, histological analysis, bone and cartilage staining were conducted on heterozygous mutant mice at postnatal day 0 （P0）, P5 and P10. Various abnormalities of skeleton, such as skull, long bone and stemebra were found. Incompletely fused palate was present in all the mutant mice from embryonic 15.5 （E15.5） to P10. None of the mutants exhibited syndactyly. Most of these observations were similar to those found in the Fgfr2+/S252W mice except that the Fgfr2+/P253R mice could survive longer and might have milder phenotypes. These data suggested that the Fgfr2+/P253R transgenic mouse could be a model for human Apert syndrome and further molecular analysis will shed new light on the mechanism underlying the disease.FGFR3, as a negative regulator of bone growth, plays a very important role during the development. The P250R mutation in the FGFR3 gene has been shown to associate with several genetic entities including Muenke, Saethre-Chotzen, Crouzon, Pfeiffer and Beare-Stevenson syndromes, and sometimes hearing loss. The variable phenotypes caused by the same P250R mutation in FGFR3 suggested the importance of its interaction with the genetic background during the formation of phenotypes.We cloned a 12.0kb genomic DNA fragment containing exons 3-13 of the mouse Fgfr3 gene into the pBluescript SK plasmid. A 726₇31CC＞AG substitution, resulting in a P244R mutation at the residue homologous to human FGFR3 amino acid 250, was introduced into exon 7 using site-directed mutagenesis. We inserted a TK cassette at the 5’ SpeⅠsite upstream of the targeting DNA fragment and a neo cassette with flanking loxP recognition sites into intron 7, oriented in an opposite transcriptional direction to the target Fgfr3. The final targeting vector of 17.0 kb was confirmed by sequencing, linearized by SacⅡdigestion, and introduced into G4 ES cells by electroporation. ES cells and the clones with the targeted mutant allele were identified by Southern blotting. After aggregation and embryo implantation, chimeric Fgfr3+/P244R mice were generated. Further phenotype analysis of this mouse model will give an insight into the genetic interactions between the mutation and the modifiers in the pathogenesis.

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