Genetic Diagnosis And Phenotypic Correlation Of Two Hereditary Diseases:Tuberous Sclerosis Complex And Axonal Charcot-Marie-Tooth Disease

Purpose: Tuberous sclerosis complex(TSC)is a group of neurocutaneous syndrome characterized by the development of hamartomas in multiple organ systems,mainly involving the brain,skin,heart,lung and kidney.Inactivating mutations in either of two genes,TSC1 and TSC2 were identified as responsible for TSC.Both proteins form a complex that negatively regulates the activation of the mechanistic target of Rapamycin complex 1(mTORC1),affecting the downstream metabolism such as cell proliferation,survival and other functions.The mutation types varied,including small variants,copy number and mosaic variations and so on.The genetic heterogeneity and phenotypic variability are significant.Patients can be clinically diagnosed with TSC according to the criteria revised by the 2012 international Tuberous Sclerosis Complex Consensus Conference.To date,only a few studies regarding systematic analysis of the genotypic characteristics and clinical manifestations in TSC patients based on Chinese population have been reported.In this study,we explored the genotype and phenotypic features of patients with clinical or suspected diagnosis of TSC.Method: We performed the genotypic analysis of TSC1 and TSC2 genes in 94 clinically diagnosed or suspected diagnosed Chinese TSC patients using direct sanger sequencing to screen small variants.Patients without small mutations identified were then screened copy number variations using Multiplex ligation-dependent probe amplification(MLPA).Lastly,the mosaic variants were suggested to be detected.And patients with suspected diagnosis of TSC may be excluded or using whole exome sequencing and whole-genome sequencing if all the results were negative.Results: Neurological disorders such as subependymal nodules(67%),epileptic seizures(65%)and cutaneous lesions including facial angiofibroma(62%)and hypomelanotic macules(60%)were observed in most patients,all accounting for proportions over 50%.And nervous system was affected in about 88.2% patients.Mutations were identified in a total of 76(80.8%)cases,80.9% and 80% in patients with clinical diagnosis and suspected diagnosis of TSC respectively,including 24 TSC1 mutations and 52 TSC2 mutations.Among the variations detected in TSC1 gene,point mutations were identified in 24 patients.Most were identified with nonsense mutations(9/24,37.5%)and frame shift mutations(7/24,29.1%).Exons 8 and 15 of the TSC1 gene revealed relatively higher frequency of mutations in this study.With regard to the TSC2 locus,52 small mutations were detected,most of which were missense mutations(16/46,32%)and most variants distributed in the GTPase activating-protein(GAP)and Rabaptin domains(spans exon 34 to 40).Large deletions of the TSC2 gene were identified by MLPA in 6 out of 52 patients,accounting for 7.9% of all mutations.The molecular diagnosis of 19.15% patients still remained unknown.Hypomelanotic macules and renal angiomyolipoma occurred significantly more often in patients with TSC2 mutations.Conclusions: Using sanger sequencing and MLPA could make molecular diagnosis for most clinically diagnosed patients(80.8%).Our data expands the spectrum of mutations associated with the TSC loci and will be of value to the genetic counseling in patients with the disease and provide direction for the future disease surveillance and management.Background: Charcot-Marie-Tooth(CMT)is the most frequent form of inherited neuropathy characterized by the muscle weakness and atrophy with or without hypoesthesia,weakening or disappearance of tendon reflexes,hand and foot deformity(such as pes cavus,foot dropping,talipes equinovarus,joint contracture deformity,etc.)and abnormal gait and posture.Median motor nerve conduction velocities(MMNCVs)are used to classify CMT into either the CMT1(MNCV<25 m/s),CMT2(MNCV >45 m/s)or ICMT(25 m/s < MNCV < 45 m/s).Approximately 100 causative genes of CMT have been reported to date.However,it has been estimated that genetic diagnosis of CMT2 still remained unclear in about 75% of clinically diagnosed CMT2 individuals.Many CMT2-casusative genes affect the the peripheral nerve system,including the axonal architecture regulating genes and axonal-transport-related cytoplasmic dynein genes such as HSPB1,NEFL.Some genes encode outer mitochondrial membrane proteins such as MFN2,GDAP1.Charcot-Marie-Tooth disease type 2(CMT2)is a clinically and genetically heterogeneous inherited neuropathy.In this study,we explored the genotype and phenotypic features of patients and the association between disease severity and levels of mitochondrial DNA.Method:Here,we studied a cohort of 35 CMT2 patients of Chinese descent,using whole exome sequencing to investigate gene mutations and then explored relationships amongst genotypes,clinical features,and mitochondrial DNA levels in blood as assessed by droplet digital PCR.Results: Most patients(71.4%;25/35)developed initial symptoms in their childhood or adolescent period.Among the 35 patients,71.4% cases developed muscle weakness in lower limbs initially.Other signs also included muscle weakness in the upper limbs(31.4%,11/35),distal muscle atrophy(54.3%,19/35),hyporeflexia(65.7%,23/35),foot deformity(37.1%,13/35)and sensory disturbance(20%,7/35).Except for these common symptoms,atypical manifestations also appeared in several cases: two patients revealed clinical presentations of neuro-myotonia.And three patients experienced different degree of hearing loss.We identified pathogenic or likely pathogenic variants in 60% of CMT2 patients,with two novel mutations(MFN2: c.614T> C(p.Val205Ala)and YARS: c.1079C>A(p.Pro360Gln)).The most common genetic causes in the cohort were due to MFN2 mutations(42.9,15/35).86.6%(13/15)of the MFN2 variants were located in the GTPase domain.Other identified genes also included HINT1,HSPB1,YARS,GARS,GDAP1.Two patients with typical CMT phenotype and neuro-myotonia harbored compound heterozygous variations in the HINT1 gene.Additionally,we found that the increasing disease severity assessed via the CMT Neuropathy Score(CMTNS)might be associated with reduced mitochondrial DNA(mt-DNA)levels in blood.Conclusion: In conclusion,the mutation detection rate increased in our study by optimized diagnostic process.It can be suggested that assessment of possible HINT1 mutations should be undertaken for CMT2 patients with neuro-myotonia.Moreover,we found that mitochondrial DNA levels in blood may be associated with CMT2 severity,but any such association will require further exploration.