Using Exon Traipping Technology To Evaluate The Effect On Splicing Of BRCA1 Variants And The Aunlication Of Whole-exome Sequcencing In The Differential Diagnosis Of Genetic Diseases Klippel-Trenaunay Syndrome And Parkes-Weber Syndrome

Part?Using exon trapping technology to evaluate the effect on splicing of BRCA1 variantsBackgorund:Breast cancer(BC)is a common cancer among women worldwide,with an age-adjusted incidence of about 73% in developing countries and 23% of all types of cancer.BRCA1 mutations have been proven as the cause of BC,Women with BRCA1 mutations will have a 87% risk of breast cancer for whole life.Idetification of the BRCA1 varations is important for breast cancer clinical research.However,more than half of the BRCA1 varations in public database are still lacking experimental evidence,resulting in the classification of unknown clinical significance.To fully verify the pathogenicity of BRCA1 mutations,a comprehensive data will be required.Some DNA mutations can cause the aberrant splicing site.Exon trapping technology is a powerful tool to assess the impact of sequence variants on splicing.Method: A genomic segment encompassing the variant sequence of interest along with flanking intronic sequences is PCR-amplified from patient genomic DNA and is cloned into a minigene vector.After transient transfection into COS-7 cells,the splicing patterns of the transcripts generated from the wild-type and from the variant constructs are compared by reverse transcription-PCR analysis and sequencing.Result:A Novel Mutation BRCA1 c.5137G>A(p.Asp1713Asn)Causing Exon 17 Skipping,provide direct evidence of its pathogenicity.Other two mutations BRCA1 c.627T>C(p.Pro209Pro)and BRCA1 c.4347A>G(p.Thr1449Thr)do not cuase the aberrant splicing.Conclusion:In this study,we confirmed a novel mutation BRCA1 c.5137G> A(p.Asp1713Asn)would cause exon 17 skipping by exon trapping technology.Mutation has broken the BRCT domain,may resulting in an abnormal structure and function of the BRCA1 protein.The mutation is pathogenic and responsible for the breast cancer.Part? The application of Whole-exome sequcencing in the differential diagnosis of genetic diseases Klippel-Trenaunay syndrome and ParkesWeber syndromeWith the development of high-throughput sequencing(NGS),new diagnostic approach has also been developed for genetic diseases.Whole-Exome Sequencing technology(WES)is based on target sequence capture technology.It uses probes to capture the DNA sequence of the exon region,and then detects related genetic mutations through next-generation sequencing(NGS).Compared with the Whole-Genome Sequencing(WGS),WES has more advantages in a shorter cycle,more data,lower cost,and deeper coverage.It has been widely used in clinical diagnosis of some genetic diseases,especially for those extremely rare diseases.Klippel-Trenaunay syndrome and Parkes-Weber have similar symptoms,but the treatment and prognosis are different,and are often misdiagnosed.The KlippelTrenaunay syndrome is currently found to be mainly cuased by mutation of AGGF1,while Parkes-Weber syndrome is mainly cuased by the mutation of RASA1.With the help of WES,we can easily differentially diagnose this two diseases.In this study,a new mutation in RASA1 was detected by WES in a patient who was previously misdiagnosed with Klippel-Trenaunay syndrome.Thus eventually the patient was diagnosed as Parkes-Weber syndrome,which will helps for correct treatments.