More Complicated Than The (toe) Deformity Genetic Mapping And Disease-causing Mutation Analysis Of The Retinoblastoma Rb1 Gene In Pathogenic Mutation Detection

Paper 1:Genetic mapping and identification of pathogenic mutations in complex polysyndactylyCongenital limb malformations occur at a live birth rate of 0.5‰-1‰,characterized by alteration of identity and number of digits.Limb malformations occur as an isolated malformation of the hands or feet or as a part of a syndrome.Polydactyly,sometimes accompanied by syndactyly,is one of the most frequently observed congenital hand malformations.Triphalangeal thumb-polysyndactyly syndrome(TPTPS;MIM 174500) is an autosomal dominant disorder comprising triphalangeal thimb,polydactyly and syndactyly of finger 3-5 or milder syndactyly of finger 4-5.The malformations are usually bilateral and asymmetric.Triphalangeal thumb is a constant finding and finger 2 is almost always normal.Feet are usually less severely affected.Nonsyndromic syndactyly is a common limb malformation in human beings,which is also inherited in an autosomal dominant way.According to Temtamy and McKusicks',nonsyndromic syndactyly is classified into five types.Syndactyly typeⅣ(SD4;MIM 186200),also known as Haas syndactyly,is characterized by bilateral cutanious syndactyly of all fingers,and polydactyly with 6 metacarpals and 6 digits without bone fusion.Flexion of the fingers often gives the hands a cup-shaped form.Feet are mostly less severely affected and tibial aplasia is observed occasionally.The zone of polarizing activity(ZPA) plays a central role in limb development and morphogenesis.The Sonic hedgehog protein produced and secreted by the mecenchymal cells within the ZPA determines the identitiy and number of digits.The human SHH gene locates in chromosome 7q36,a region related with several limb development disorders, including preaxial polydactyly/triphalangeal thumb(PPD/TPT;MIM 174500),TPTPS, SD4,tibial hemimelia-polysyndactyly-triphalangeal thumbs syndrome(THPTTS;MIM 188770),acheiropodia(ACHR;MIM 200500)and acropectoral syndrome(ACRPS;MIM 605967).Triphanlangeal thumb is a shared phenotype among PPD/TPT,TPTPS and THPTTS.Interestingly,in 1999,Heus et al.refined PPD/TPT locus to a critical region of 450kb,excluding SHH.Lettice and colleages later identified a long range limb specific Shh enhancer,termed ZRS(ZPA regulatory sequence).The ZRS is highly conserved from mammalian to fish,capable of driving restricted expression of a reporter gene in ZPA located in the posterial limb bud.The mouse and human ZRS enhancers are both within intron five of the Lmbr1/LMBR1 genes,848kb and 979kb centromeric to Shh/SHH loci,respectively.The direct link between ZRS and the PPD phenotype was established by the identification of different point mutations within the ZRS,including eight in human PPD,three in mouse models for PPD,and three in polydactylous cats.A Tai man with TPTPS was reported having a more severely affected daughter consistent with THPTTS.The fact that TPTPS,SD4 and THPTTS present in different family members within a single family indicates a shared disease causing mechanism.Copy number variation(CNV) refers to structural variants involving DNA copy number changes more than 1kb,usually insertion,deletion or duplicstion.CNV is estimated to cover 12%of human genome and involves larger spread of DNA sequence compared with SNP regardless of whose relative high frequency of 1 per 300 nucleutides. CNV may cause human diseases termed as genomic disorders as far as deletion, duplication or structural disruption of dosage sentitive genes is concerned.In the 6 Han Chinese TPTPS/SD4 kindreds we collected,typical TPTPS is the phenotype of almost all the patients in family 1,and both phenotype of TPTPS and SD4 are observed in patients of family 2,3,4 and 6.Patients of family 5 are characterized by typical SD4.We performed two points linkage analysis in four families.At a recombination rate ofθ=0, the added up LOD score reached 17.32 at the TG marker within the LMBR1 gene,with 9.52,3.72,2.65,1.43 for family 1 to family 4,respectively.The significant positive LOD score affirms the linkage of TPTPS/SD4 to chromesome 7q36.Moreover,by the subsequent haplotype analysis,we refined TPTPS/SD4 critical region to an interval telomeric to SHH between markers AGAA and AATT,representing a genomic extent of about 647kb encompassing the PPD locus.Previously,we sequenced all exons of the four known genes and the ZRS within the critical region and no pathogenic mutation was identified.Interestingly,the sequence chromatogram of a known SNP(rs10254391) in ZRS showed a remarkable allele imbalance in all the analyzed affected individuals who were heterozygous for this SNP, and this imbalance was not detectable in unaffected family members or in unrelated normal controls.The allele imbalance at rs10254391 implies the presence of a copy number difference between the affected and unaffected family members.Detection of ZRS copy number amplification was performed by real time quantitive PCR in all 6 families,and the duplication found was also directly observed in interphase neuclei of a patient from family 2 by interphase fluoresence in situ hybridization.The pathogenity of ZRS duplication could be further supported by observing a 24kb DNA duplication in the transgenic insertion site of Ssq mouse model.Combination of real time quantative PCR and Affymetrix SNP array 6.0 enable the finemapping of DNA duplications and cloning of break junction fragments.Break junctions of family 1,3 and 5 were cloned,with a final determination of DNA invtervals as 144,859bp,216,761bp,324,189bp in family 1,3 and 5,respectively.The minimum DNA duplication was refined to 379kb,294kb and 398kb for family 2,family 4 and family 6,respectively.According to the sequence features and genome contents in the break ends of the three break junction fragments cloned,we infer that nonallelic homologous recombination (NAHR) and nonhomologous endpoint joining(NHEJ) or fork stalling and template switching(FosTes) may take part in the formation of the DNA duplication of patients in family 1,while nonhomologous endpoint joining(NHEJ) may account for those in family 3 and family 5.In summary,we refined TPTPS/SD4 critical region to a DNA interval of 647kb in chromosome 7q36 and identified the responsible pathogenic mutation as DNA duplications involving ZRS.We also defined the extents of the duplications which were not directly related with the severity of the phenotype.Based on the phenotype resemblance and genetic homogeneity in the patients with TPTPS/SD4 in our research and those with related limb malformation reported by others,we speculate that PPD/TPT, TPTPS,SD4 and THPTTS constitute a phenotype continuum.Paper 2:Identification of Germline RB1 Mutations in RetinoblastomaConstitutional RB1 gene mutations are well accepted as the initiating genetic lesions during development of retinoblastoma.Patients presenting with RB1 germline mutations are recognized as hereditary retinoblastoma,with those without RB1 germline mutations as non-heridatory form.Almost all bilateral and familial cases are hereditary form.Only 15%of unilateral cases are hereditary form,while the remaining ones are non-hereditory sporadic form.Testing for RB1 mutations in peripheral blood facilitates genetic counseling by identification of patients with hereditary retinoblastoma and determination of carriers among relatives at risk.To date,nearly 600 distinct RB1 mutations from various ethic backgrounds have been recorded,however,based on a meta-analysis report on RB1 mutation,Chinese population only contributes 22 distinct mutaions.We conducted genetic screening of 31 Han Chinese patients with retinoblastoma,using Denaturing High-Performance Liquid Chromatography(DHPLC) or High Resolution Melting(HRM) analysis,and then direct PCR sequencing.The patients with negative results after the above screening for small alterations were subjected to Multiple Ligation Probe Amplification(MLPA) analysis.In addition,transcript analysis was performed to detect abnormal splicing due to splice site mutations or DNA rearrangement deep in the introns of RB1 gene which may not be detected by the methods mentioned above.In total,we found 18 distinct mutations in 19 hereditary cases,among which 10 are novel(10/19,52.6%),comprising 6 frameshift mutations(p.Leu647HisfsX10, p.Asn598LysfsX13,p.Gly310ValfsX6,p.Glu746GlyfsX3,p.Ser393LysfsX2, p.Val314PhefsX2),1 splice site mutation(c.2663+1G＞A) resulting in premature stop codon(p.Thr841X) by exon skipping,2 large deletions(including whole RB1 gene with the range of 3Mb and 9Mb,respectively) and 1 exon skipping(p.Glu464_Ser565del) detected on RNA level with the mutation on DNA level unidentified yet.Moreover,we have successfully provided prenatal gene testing and proper genetic counseling for 12 pregnancies by detecting RB1 mutations with samples from cordonal blood,chorionic villus or amniotic fluid.Our study extends the spectrum of RB1 gene mutations in Chinese population.It shows that screening of RB1 germline mutations with combinational methods is sensitive and efficient,with a positive detection rate of 100%in patients with hereditary retinoblastoma,therefore confirms the feasibility of RB1 mutation testing being an integral part of current management of patients with retinoblastoma.