Study Of APC Gene Mutation And Linked STR Markers In A Family With Familial Adenomatous Polyposis

Familial adenomatous polyposis(FAP)is one of late-onset autosomal dominant disorders with 80～100%penetrance by age of 25 years old.The incidence of FAP is approximately 7.4 per 100,000 individuals.FAP is characterized by the development of multiple adenomatous polyps in colorectum and the extremely high potential to progress to colorectal cancer(CRC).It was estimated that the malignant incidence of FAP is almost 100%if a total colectomy and ileorectal anastomosis is not performed in the last teens.Because of its carcinogenicity and penetrance,the gene diagnosis of FAP is very important for the members in FAP family.It can make FAP diagnosis before the symptoms appear,direct the marriage and prenatal counseling.The birth of FAP patients can be prevented by gene diagnosis through chorionic villus sampling,amniocentesis and cordocentesis.With the assisted reproductive techniques,the FAP pregnancy could be avoided by preimplantation genetic diagnosis.FAP is caused by a functional mutation in adenomatous polyposis coli(APC)gene. The APC gene is located at 5q21-q22 with open reading frame(8.5kp)containing with 15 exons.Exon 15(6.5Kbp)encompasses about 75%of coding sequence of APC gene and has a corresponding mRNA of approximately 10 kb.APC gene encodes 2843 amino acids and the gene products are critical to Wnt pathway,and participate in the procedures of cell migration,adhesion,proliferation,apoptosis,differentiation and so on.APC gene is known as a tumor suppressor.Inactivation of APC protein resulted from alteration of APC gene is the cause of FAP.Mutation of APC gene leads to over-activation of Wnt/β-catenin,misdirection of microtubule binding site,imbalance of cellular proliferation and differentiation and development of tumors.APC gene mutations include germline mutation and somatic mutation.60%-65%of all somatic mutations are clustered in the sequence between codon 1286 and 1513.Therefore,this region is termed as the mutation cluster region(MCR).Somatic mutation is the common causes of sporadic colorectal cancer,which does generally not transfer to the offsprings.FAP is resulted from germline mutation of APC gene and can be transmitted through generations.So far,more than 826 different kinds of germline mutations of APC gene have been reportedin FAP families.60%of the mutations are located in the coding region near to 5' of the exon 15.Over 98%of those mutations are nonsense(30%)and frameshift mutations(68%)and the sequence from codon 1061 to 1309 is the sites frequently involved.Mutations could result in the truncation of APC protein and tumorigenesis.The functions of some point mutations of APC gene,such as codon 1309, 1061,1317 and 1307,have been definitely identified.However,the influences on tumorigenesis of some other point mutations,such as 1822,1125,1126 and 2502,are still uncertain.So far,APC gene mutations have been identified in 60-80%of all FAP families and remained unknown in 20-30%.Too large deletions to detect by conventional gene screening methods,mutations located in introns or regulatory regions of the APC gene that is escaped from the detection and un-identified gene related with the functions of APC gene are the possible explanations.Applying polymorphic markers linked to APC gene is another approach for the gene diagnoses of those FAP families.Short-tandem repeats(STR),interspersed in eukaryote genomes,is highly polymorphic among individuals due to wide variations in the number of their two to five CA base pair long repeat units.The information provided by polymorphic marker can be markedly improved by constructing haplotypes consisting of a defined pattern of alleles at polymorphic loci linked together on a chromosome.STR markers,D5S318,D5S299, D5S82,D5S134 and D5S346,are available for the gene diagnosis of familial APC,which are located 1-10cM from the APC gene.Among them,D5S134,and D5S346 are CA repeats located on 1cM upstream and 30-70kb downstream of APC gene respectively. The analyses of those linked markers will enable the clinicians to perform gene diagnosis of pre-symptomatic or prenatal patients in the FAP family without identified mutations of APC gene.For the screening of APC gene mutation and the establishment of gene diagnosis method in FAP,the study of APC gene mutation and linked STR markers in a family with familial adenomatous polyposis is carried out. Section One Detection of APC gene mutation in the family of FAPObjectiveTo screen APC gene mutations in a FAP family with five generations,analyze the association of the alteration of APC gene sequence with tumorigenesis in the FAP patients and find the germline mutation of APC gene in the FAP family for genetic counseling,prenatal and preimplantation genetic diagnosis.Material and Methods(1)Family members:The figure 1 is the pedigree of a FAP family.There are total 71 members of five generations.The man in generationⅠwas known as the first patient in this family.In generationⅡandⅢ,there are 12 patients in 24 kindreds and 7 patients have been dead of CRC.In generationⅣ,onlyⅣ-1 has been diagnosed as FAP patient,no clinic signs of FAP have been found in the rest children.At present,there is only one child at age of 3 in generationⅤ. (2)Genomic DNA was extracted from 10 ml of whole blood of followed 39 family members:Ⅱ-6～Ⅱ-12,Ⅲ-4,Ⅲ-7,Ⅲ-10,Ⅲ-13,Ⅲ-15～Ⅲ-19,Ⅲ-23,Ⅲ-25,Ⅲ-27,Ⅲ-30～Ⅲ-33,Ⅳ-1,Ⅳ-3,Ⅳ-5,Ⅳ-6,Ⅳ-8,Ⅳ-9,Ⅳ-10,Ⅳ-11,Ⅳ-13～Ⅳ-18,Ⅳ-20 andⅣ-23.As the control,genomic DNA was also extracted from 10 ml of whole blood of 37 normal individuals.(3)According to the corresponding references,35 pairs of PCR primers were designed,that are e1 to e14 for the exon 1 to exon 14 of APC gene and e15-1 to e15-21 for exon 15 respectively.The all amplification products cover the whole encoding region of APC gene.(4)First,5 patients with FAP were under detection.The DNA samples were amplified by 35 pairs of PCR primers and the products were sequenced and the mutation was confirmed by restriction enzyme.(5)According to the finding of mutation in the FAP patients,PCR,sequencing and enzymatic analyses were carried out in rest 34 FAP family members.(6)According to the finding of FAP patients,PCR and enzymatic analyses were carried out in 37 control individuals.(7)The ratios of APC mutation between the FAP patients and the healthy kindreds of the family and the normal controls were compared.Results(1)There was no mutation found in exon 1～14,exon 15-1～15-11,exon 15-13～15-21 by sequencing of the PCR products from five patients.The variant at codon 1822(Asp1822Val)of exon15-12 was identified in two patients.The results were confirmed by restriction enzyme BpiI(5'-GAAGAC(N)2↓-3',3'-CTTCTG(N)6↓-5')。The frequency of the Asp1822Val allele at codon 1822 was 2/5(40.0%)in patients.(2)Two cases of Asp1822Val were identified in rest members of the family.One case was the spouse of patientⅢ-15 and another was their daughter.Therefore the frequency of the Asp1822Val in the examined kindreds of the family was 1/25(4.0%) and in the examined spouse of the kindreds was 1/9(11.1%).(3)The frequency of the Asp1822Val in normal controls was 7/37(18.92%).(4)Statistical analyses by Fisher's exact test of SPSS 15.0 showed that there was no significant difference of Asp1822Val frequencies among FAP patients and control group.Conclusion(1)There have been no mutation found at exon 1～14 in this FAP family.(2)Asp1822Val variant of exon 15 is a common polymorphism without clinical implications.(3)It is necessary to process the study of diagnosis of APC gene by STR linkage analysis in this FAP family. Section Two The study of diagnosis of APC gene in the FAP family by STRObjectiveTo detect STR polymorphic markers highly linked to mutated APC gene in the FAP family,establish the technique of gene diagnosis in the family by STR linkage analysis, direct genetic counseling and pre-symptomatic diagnosis among family members at risk and provide the method for prenatal diagnosis and the basis for preimplantation genetic diagnosis in this family.Methods(1)Family data was the same as section one.(2)Genomic DNA was extracted from 10 ml of whole blood of 39 family members, which is the same as section one.(3)Two highly polymorphic markers,D5S134 and D5S346 were amplified by PCR.(4)The PCR products were separated on 8%polyacrylamid gel for 4 hours and silver- stained.(5)PCR products of D5S346 were named as A1,A2,A3,A4 according to electrophoresis velocity.The products of D5S134 were named as B1,B2,B3,B4 by the same way.Results(1)The genotypes of five clinical FAP patients are A1B2/A1B4,A1B1/A1B4, A3B3/A1B4,A1B2/A1B4 and A1B2/A1B4.All of them shared the same haplotype of A1B4. (2)No A1B4 haplotype was found in healthy kindreds of the family exceptⅢ-13.(3)In generationⅣ,Ⅳ-5,Ⅳ-6,Ⅳ-9,Ⅳ-11,Ⅳ-14 andⅣ-23 were found to have the same haplotype of A1B4.All of them are the children of FAP patients butⅣ-11,who is the son ofⅢ-13,a healthy kindred in the family.Conclusion(1)A1B4 haplotype of STR loci,D5S134 and D5S346,is highly linked to the mutated APC gene in this FAP family.(2)It is most probably thatⅣ-5,Ⅳ-6,Ⅳ-9,Ⅳ-14 andⅣ-23 are the pre-symptomatic patients with FAP.Intensive monitoring and effective treatments are essential for them.(3)It is essential forⅢ-17 and other children of FAP patients with A1B4 haplotype to have prenatal diagnosis by linkage analysis of STR markers D5S134 and D5S346 if they or their wives get pregnancies.The STR analyses will avoid the birth of children with FAP and stop the transmission of the disorder through the generations.(4)It is necessary to develop the technique for preimplantation genetic diagnosis using the STR analysis.Then the prenatal diagnosis could be finished before pregnancy Occurs.(5)Considering the possibility of crossing-over and recombination,suitable examinations for the offsprings of FAP patients without A1B4 haplotype are still necessary.