TSC1,TSC2 Gene Mutation Analysis And Prenatal Diagnosis Of Tuberous Sclerosis Complex

Research Background:Birth defect refers to the baby born with body structure,function or metabolic abnormalities,including congenital malformation,genetic metabolic defects.congenital functional disability,autoimmune disease.mental retardation,etc.It can be caused by genetic factors,environmental factors or interaction of genetic and environmental factors.With the progress of modern medicine,maternal mortality and child mortality rates gradually reduce and birth defects become prominent public health problems or even social problems.China is a country with large population and high incidence of birth defects.Birth defects has become a monumental problems that influences our country's population health quality.It not only leads to spontaneous abortion,perinatal death,infant death and congenital deformity,but is also serious harm to child survival and health.It influences the happiness of a family and causes society and family to bear a heavy financial burden.According to statistics,the total incidence of birth defects in our country is about 5.6%(China's birth defects prevention report,2012).According to the report of WHO incidence of birth defects is 6.42%,5.57%and 4.72%respectively in low-income countries,middle-income countries and high-income countries.In contrast,the incidence of birth defects in our country is close to one of middle-income countries.Howerver,the abslute number of birth defects is enormous in our country because of the large population.It is a effective method to carry out prenatal diagnosis so as to reduce birth defects.Tuberous sclerosis complex(TSC)is a monogenetic disease and one of the most common birth defects.The incidence of TSC is 1/6000-10000.The tumors of central nervous system is the major cause of death.The next death cause is kidney disease.The severity of clinical manifestations of TSC is variable,manifesting hypomelanotic macules,epilepsy,intellectual disability,autism,and multiple hamartomas in different organs.Although there are well-defined clinical diagnostic criteria for TSC,it can still be difficult to establish a clinical diagnosis of TSC,particularly in young patients who do not yet exhibit typical TSC lesions but may have severe but nonspecific symptoms such as epilepsy,intellectual disability,and/or autism.In these cases,a molecular diagnosis can be helpful.Although the cardiac rhabdomyoma can detect through echocardiography and the subependymal nodules can detect though magnetic resonance imaging(MRI)prenatally,the symptomatic appearing time is uncertain and the images are nonspecific.Therefore,genetic detecting should be the gold standard of prenatal diagnosis for fetuses with TSC.The genetic testing for TSC is very complicated in view of bigger structure of TSC1 and TSC2,mosaic and nonspecific pathogenic mutations.After 40 years of development,DNA sequencing technologies have experienced from the first generation to the third generation.The speed and cost of DNA sequencing meets a condition of clinical requirement.DNA sequencing technologies can be divided into three generations.The second and third generation sequencing technologies are also called next generation of high-throughput sequencing technology.Sanger sequencing is first generation sequencing technology.It is high labor cost and time-consuming.Sanger sequencing depands on gel electrophoresis and is unable to further expand the sequencing's flux and speed.The next generation's sequencing technologies are highly parallel.It means that many sequencing reactions can be done on a platform at the same time,the reaction system becomes smally,the time to get a lot of base information becomes shortly and cost is greatly reduced.Objective:To identify the pathogenic mutations for the probands with TSC and carry out prenatal disgnosis for fetuses with high risk of TSC.Methods:All exons and their flanking 10 bp regions of gene TSC1 and TSC2 were sequenced by next-generation sequencing(NGS)method.The muatations identified by NGS were then to be further varified by Sanger's DNA sequencing.After pathogenic mutatations found in probands were confirmed,pedigree analysis were carried out.The fetuses with high risk of TSC were genetic diagnosed prenatally by Sanger DNA sequenccing using amniotic fluid cells or chorioric villi.After dilivery,the cord blood's or umbilical samples were detcted again to confirm the results of prenatal testings.Results:13 mutations were identified,including 12 muations of gene TSC2,1 mutations of TSC1 from 13 families.Among these 13 mutations,there are 10 known pathogenic mutations and 3 likely pathogenic mutations.12 fetuses with high risk of TSC were carried out prenatal genetic diagnosis.Among them,there were 2 fetuses to be diagnosed as TSC and 10 fetuses to be diangosed as normal.The 2 fetuses with TSC were terminated according to dicision of their parents.The another 12 fetuses were followed up to be normal and be in accordance with prenatal testings after birth.Conclusion:3 mutations of TSC2(c.2415-2416insGT,c.3981-3982 insA,c.4013-4014 delCA)are likely pathogenic mutations.They belong to frame shift mutations.These three mutations are not be reported so far and enrich the TSC gene mutation's spectrum.We should make a further efford to identify their pathogenic in later research.12 mutations of TSC2 were successfully identified,including TSC1 c.2074C>T,TSC2 c.1832G>C,c.5227C>T,c.2713C>T,c.2415-2416insGT,c.3352C>T,c.4013-4014 delCA,c.5024C>T,c.1432C>T,c.3981-3982insA,c.880G>A and c.4541-4544 delCAAA.