Exploration Of Non-invasive Prenatal Diagnosis Of TSC And The Effect Of Cytomegalovirus On TSC-Related Autism

Backgrounds and objective: Tuberous sclerosis complex(TSC)is an autosomal dominant monogenic disease which leads to birth defect and is caused by heterozygous mutations in the TSC1 or TSC2 genes.Loss of function of TSC1 or TSC2 genes results in overactivation of m TOR pathway and then leads to the development of hamartomas in multiple organs including brain,skin,heart,kidneys,eyes,lungs and so on.Neurological symptoms such as epilepsy,autism spectrum disorders(ASD)and psychomotor development retardation are the main factors affecting the quality of life.Although the etiology and pathogenesis of TSC are clear,its genetic and clinical manifestations are highly heterogeneous.There were no mutation hotspots in TSC1 or TSC2 genes and no specific gene-phenotypic relationship,so it is impossible to predict the severity of disease and future quality of life based on gene mutations.Recently,more and more attention has been paid to the influence of environmental factors on disease,especially neural development disease,which is precisely regulated by both genetic and environmental factors.For heterogeneous birth defect caused by monogenic mutation such as TSC,prevention of birth by non-invasive prenatal diagnosis(NIPD)is of great significance in improving birth quality and alleviating social burden.On the other hand,explore the influence of environmental risk factors on TSC can help explore the role of environmental factors on the heterogeneity of diseases and help to prevent the severity of diseases from the perspective of environment factors.Human cytomegalovirus(HCMV)is a common virus that affects neurological development and function and is considered as an environmental risk factor for ASD.Besides,the infection activities of HCMV are closely related to m TOR pathway.At present,there is no systematic report on noninvasive prenatal diagnosis of TSC and no report on the effect of HCMV on TSC.The aim of this study was to explore the feasibility of noninvasive prenatal diagnosis based on cell-free DNA in TSC and to explore the impact of HCMV on TSC-related autism,thus to explore the prevention of monogenetic disease from the aspects of genetic and environmental factors.Methods: From June 2016 to December 2021,14 families with TSC probands and single pregnancy were recruited,the pathogenic gene mutation of proband and parental genotypes were identified by gene sequencing.Then,cell-free DNA(cf DNA)extracted from maternal peripheral blood and white blood cell genomic DNA(wbc DNA)extracted from maternal peripheral blood were sequenced in parallel with next generation sequencing.Fetal fraction(FF)and mutation ratio(MR)were calculated and the fetal genotype is deduced from the deviation between the allele of cf DNA and that of maternal background DNA(wbc DNA).The accuracy of NIPD results was validated by both gene sequencing and clinical manifestations of the born children.From September 2011 to March 2021,peripheral blood samples of children aged over 6months of 315 TSC children and 93 healthy children were collected in the Department of Pediatrics of the First Medical Center of the General Hospital of the People’s Liberation Army.The serum positive rate of HCMV Ig G was detected by indirect chemiluminescence immunoassay using magnetic particles.The serum positive rate of children with TSC was compared with that of healthy children,and the basic demographic information of children with TSC was collected to analyze the risk factors of HCMV infection in children with TSC.From August 2020 to March 2021,brain samples from 17 TSC patients and 19 non-TSC patients who underwent surgical resection of intracerebral lesions were collected in Yuquan Hospital of Tsinghua University.Nested PCR and Western Blot were used to detect IE1,UL55 and UL83 DNA and corresponding proteins of HCMV.The differences of HCMV infection in TSC patients and non-TSC patients were analyzed.206 TSC patients with ASD records were divided into ASD group and non-ASD group.Clinical data including epilepsy,psychomotor development and gene mutation were collected.A case-control study was conducted to analyze whether HCMV infection was a risk factor for TSC related ASD by univariate and multivariate analysis.Among patients detected HCMV in brain,7 TSC patients has ASD records,the differences of HCMV in brain of patients with and without ASD were compared.Results: 1.Among 14 NIPD families,12(85.71%)were TSC2 gene mutations and two(14.29%)were TSC1 gene mutations.The specific mutation types included five(35.71%)missense mutations,four(28.57%)frame-shifting mutations,three(21.43%)nonsense mutations,and two(14.29%)splicing-site mutations.The mutation included 11 de-novo mutations,two maternal mutations,and one paternal mutation.Among 13 probands with complete clinical data,12 had epilepsy,including 4 refractory epilepsies;five had psychomotor retardation,and four had ASD,cutaneous lesions and intracerebral lesions occurred in 13 patients,cardiac hamartoma occurred in 5 patients,and renal disease(hamartoma or polycystic kidney disease)in 5 patients.The mean gestational age of NIPD was 17.18±5.83 weeks(range 8-29 weeks),and all 14 NIPD results were confirmed to be accurate.Two fetuses were confirmed to carry genetic mutations by amniocentesis and Sanger sequencing,which were paternal mutations and maternal mutations respectively.All the two families terminated pregnancy voluntarily.The other 12 fetal genotypes were normal,nine of which were confirmed by amniocentesis and Sanger sequencing.two were confirmed by postanal Sanger sequencing,and one did not undergo any gene sequencing verification.All 12 children were free of TSC related manifestation at follow-up at 2.74±0.96 years of age.2.There were 181 males(57.46%)and 134 females(42.54%)in 315 TSC children with serum HCMV Ig G detection.The median age of detection was 3.50(5.00)years.Children with TSC came from 27 provinces,autonomous regions and municipalities directly under the Central Government in China,almost evenly distributed in rural and urban areas.Among TSC children,89.15%(263/295)were full-term delivery,8.14%(24/295)were preterm delivery,and 2.71%(8/295)were overdue delivery.The average birth weight was 3.38±0.55 Kg.46.39%(135/291)had vaginal delivery,52.92%(154/291)had cesarean delivery,and 0.69%(2/291)had aspirator or forceps delivery.76.03%(111/146)were breast-fed,and 23.97%(35/146)were not breastfed.Most mothers(88.36%,243/275)had education from junior middle school to university/college level,and most of the mothers(199/270)had their delivery under 30 years of age.The median age of 93 healthy children tested for plasma HCMV Ig G was 3.91(0.42)years,with 45 males and 48 females.There was no difference in HCMV seroprevalence between TSC population and healthy children(74.28% vs 72.04%,P=0.666).The HCMV infection rate of TSC children increased with age.The duration of breast-feeding after birth was different between the two groups with seropositive or seronegative HCMV.The average duration of breast-feeding in the seropositive group(10.0(14.0)months)was longer than that in the seronegative group(4(12.0)months),P=0.009.There was no difference between the two groups in the level of maternal education and reproductive age,rural or urban population,mode of production,birth weight and other socioeconomic factors.Logistic regression analysis showed that breast-feeding increased the risk of HCMV seroprevalence in TSC children,but the risk was small(OR 1.06,95%CI 1.007-1.117).There was no difference in HCMV detection rate between TSC patients and non-TSC patients.3.The median age of diagnosis of TSC-related autism patients was 4.75(4.58)years old,which was mostly male.Children with TSC autism were mostly accompanied by developmental retardation and refractory epilepsy,and the autism of TSC patients was mainly manifested as the disorder of self-care ability in daily life.Univariate analysis showed that the serum positive rate of TSC patients with ASD was higher than that of TSC patients without ASD(89.2% vs75.1%,P=0.063).Multivariate analysis showed that HCMV serum positive rate was a risk factor for ASD in TSC patients(OR=3.976,95%CI 1.093-14.454).In addition,HCMV is more easily detected in cortical malformations in TSC patients with ASD.Conclusions: 1.Noninvasive prenatal diagnosis of TSC based on cf DNA is applicable to pregnant women with single pregnancy and a proband with a clear pathogenic mutation.NIPD for TSC can be carried out in the early stage of pregnancy(the earliest gestational week in this study is 8 weeks)and are feasible for missense mutations,nonsense mutations,splicing site mutations and frames shifting mutations which arise from de-novo,paternal and maternal,but the accuracy of our methods needed to be verified by more cases.2.The susceptibility of TSC children to HCMV is not differ from that of healthy children and children with cortical dysplasia caused by other genetic reasons,which indicates that activation of m TOR pathway caused by TSC1 or TSC2 gene mutation does not lead to increased susceptibility to HCMV.HCMV seroprevalence of TSC children increases with age,and breast-feeding is one of the ways of infecting infants with HCMV.However,prolonged breastfeeding time increases the risk of HCMV infection in TSC children,so it is not recommended to reduce breast-feeding time for fear of HCMV infection.Mode of production and social,economic and cultural level don’t affect the HCMV seroprevalence of TSC patients.3.Autism is a highly genetic and heterogeneous disorder that is influenced by the interaction of gene-environmental factors(G×E).Our study suggests that even autism with a clear genetic etiology can be influenced by environmental factors,the genetic background of TSC patients may increase autism susceptibility to HCMV,and that HCMV seropositivity is a risk factor for autism in TSC patients.In conclusion,we have explored the prevention and management of TSC from both genetic and environmental factors.NIPD can effectively prevent high-risk families from having another TSC children.Reinforcing the management of HCMV infection can help prevent or develop symptoms of autism related TSC.Both genetic prevention and environmental management are of great significance in reducing the family and social burden caused by TSC and improving the quality of life of patients.