Research Of Relationship Between Genotype And Phenotype And Changes Of IGF-1 In Tuberous Sclerosis Complex

Tuberous sclerosis complex （TSC） is an autosomal-dominant, neurocutaneous, multisystem disorder characterized by cellular hyperplasia and tissue dysplasia.TSC can cause circumscribed,benign, noninvasive lesions in any organ.The random distribution, number,size, and location of lesions cause varied clinical manifestations, involving the brain, skin,,kidney heart,eyes, and lung.Seizures are the most common neurological symptom of TSC, occurring in about 85% of patients. TSC is associated with a wide range of cognitive, behavioral,and psychiatric manifestations. Subependy-mal giant cell tumors occur in about 5-15% of cases.The most well-known cutaneous manifestations of TSC are facial angiofibromas, which occur in about 75% of TSC patients with onset typically between ages 2 and 5 years. Other skin lesions consist of hypomelanotic macules, ungula or gingival fibromas, and thickened, firm areas of subcutaneous tissue often at the lower back or on the buttocks or torso （shagreen patch） or forehead and face （fibrous plaques）. Renal manifestations of TSC are the third most common clinical feature. Four types of lesions can occur:angiomyolipo-mas（AMLs）, isolated renal cyst（s）, autosomal dominant polycystic kidney disease （PKD）, and renal cell Carcinoma. Cardiac rhabdomyomas are benign tumors of the heart that are rarely observed in non-TSC eaffected individuals. These lesions usually do not cause serious medical problems, but they are highly specific to TSC and often the first noted manifestation of disease. Retinal hamartoma are observed in 30-50% of TSC patients. These lesions have similar histologic features to the tubers located in the brains of TSC patients. Three forms of symptomatic pulmonary involvement in TSC have been described: multifocal micronodular pneumocytes hyperplasia, pulmon-ary cysts, and lymphangioleio-myomatosis（LAM）.TSC is due to an inactivating mutation in one of the two genes, TSC1 （on chro-mosome 9q34） encoding hamartin, or TSC2 （on chromosome 16p13.3） encoding tuberin. The tumour suppressor hamartin is a ubiquitously expressed 130 kDa protein. The human tumour suppressor tuberin is a 198-kDa ubiquitously expressed protein. They are highly evolutionarily conserved proteins. The human TSC1 gene consists of 23 exons with coding sequence from exon 3 to exon 23, and exon 2 is alternatively spliced. Hamartin has a potential transmembrane domain at amino acids 127-144 and a coiled coil domain at amino acids 730-996. The amino acid residues 145-510 are both necessary and sufficient for the activation of Rho GTPase, and amino acid residues 881-1084 interact with the Nterminal of the ezrin-radixin-moezin （ERM） family of actinbinding proteins. TSC2 gene lies approximately 2.25 Mb from the telomere and is immediately adjacent to the polycystic kidney disease （PKD1） gene. The human TSC2 gene has a complex genomic structure comprising 41 exons with small size introns. The 5.5 kb TSC2 transcript encodes a 1784 amino acid tuberin with predicted leucine zipper motive at amino acids 81-98 （exon 3）, two small coiled coil domains at amino acids 346-371 （exon 10） and 1008-1021 （exon 26）, and a small region of similarity with GTPase-activating protein （GAP） GAP3 or Rap1 GAP at amino acids 1593-1631 （exons 34-38）.840 TSCI and 12354 TSC2 unique allelic variants have been reported （www.lovd.nl/TSC1, www.lovd.nl/TSC2）. These mutations comprise the usual mix of nonsense, missense,insertion and deletion mutations, involving nearly all of the exons of TSC1 and TSC2.The pathologic condition of TSC is characterized by cellular hyperplasia and tissue dysplasia affecting multiple organs. The pathogenesis of TSC is closely related to mTOR（mammalian target of rapamycin）. Now upstream stimulatory factor of mTOR signaling pathway is considered including amino acids, glucose, WNT ligand, oxygen, cAMP （cyclic AMP） and insulin/IGF-1. Following the discovery of the TSC1 and TSC2 genes and their respective protein product （hamartin and tuberin）, subsequent genetic and functional studies have identified several downstream targets and signaling cascades. Tsc1 and Tsc2, together with a third protein, TBC1D7, form the TSC protein complex, which regulates multiple cellular processes and importantly acts as a critical negative regulator of the mechanistic target of rapamycin （mTOR） complex 1 （mTORC1）, a serine/threonine kinase that is central to many cell func-tions including cell growth and proliferation. Rheb （Ras homolog enriched in brain） is a specific GTPase downstream of the TSC protein complex that functionally links TSC1/TSC2 to mTORC1. The TSC1/TSC2 complex functions as a GTPase-activating protein （GAP） for Rheb and stimulates the conversion of Rheb-GTP to a GDP-bound state, thereby inactivating Rheb signaling and thus removing its stimulatory effect on mTORC1. Conversely, loss-of-function mutations in either TSC1 or TSC2 lead to enhanced Rheb-GTP signaling and mTORC1 activation. Constitutively active mTORC1 signaling thus constitutes the molecular basis of TSC.Domestic research on TSC is still relatively weak, which mainly consisting of a small number of cases reported. The relationship between gene mutation and clinical phenotype is rarely reported at present, while research on the pathogenesis of TSC is even less. Based on the current research situation at home and abroad, we carried out two aspects of research:1. In order to clarify the gene mutation results of some TSC patients,TSC1 and TSC2 genes were detected. At the same time, we collected 160 cases of TSC patients with clear pathogenic gene mutation in China. Then statistical analysis was carried out after the summary, we summarize the characteristics of gene mutation and explore the relationship between gene mutation type and clinical pheno-type. In this study,we deeply study TSC in gene level, which will enrich the database of the genetic mutation of TSC in our country and figure out the relationship between genotype and phenotype. It will provides theoretical basis for judging the prognosis of patients and providing individualized treatment.2.IGF-1 is one of upstream stimula-tory factors of mTOR signaling pathway. Recent studies have found that IGF-1 plays an important role in the occurrence and development of tumor. We research the changes of serum IGF-1 level in TSC to explore whether IGF1 can be used to evaluate condition of TSC and as a therapeutic target,and to understand whether the hamartomatosis and IGF-1 are associated in TSC.Part 1 of abstract:the relationship between the gene mutation and clinical phenotype in tuberous sclerosis complex[Objective] To detect the genetic mutations in 8 patients with TSC, and to summarize the characteristics of the genetic mutations of TSC in China, and to explore the relationship between the gene mutation types and clinical phenotypes.[Methods]1. Collect blood samples.:According to Diagnostic criteria for TSC established by International Tuberous Sclerosis Complex Consensus Conference in 1998, we collected 5 families with TSC, including 8 patients and 15 family members. There is no blood relationship between family. Gene detection is carried out after the consent of the patient’s family members. Gene detection methods:the TSC1 and TSC2 genes were detected by target sequence capture and high throughput sequencing technology, and some of the testing procedures were completed by the genetic testing company.2.Collection of literature:using literature check retrieval method, from the domestic Chinese medicine database （China Journal Net, Wanfang Data, VIP medical network,China national knowledge internet） retrieval from 1995 to 2014 about the TSC with gene mutation results, we collectted a total of 30 related articles,including 160 TSC patients. We have eliminated patients in the case of single nucleotide polymorphism in gene mutations,as well as duplicate reports. In addition we get 6 patients with gene mutations in our hospital.Statistical method:use SPSS 16.0 software to analysis. By chi square test or Fisher’s exact test of the following groups of clinical manifestations were compared and analyzed:（1） male group and female group; （2） a family history group and sporadic cases; （3） the TSC1 and TSC2 group; （4） the missense mutation group and other types of mutation group. P<0.05 difference has statistical significance.[Results]1.Mutations were positive in 6 patients and 2 were negative in 5 TSC families, with TSC1 mutation as the main（4 of TSC 1 mutation and 2of TSC2 mutation）.1).TSC2 gene mutation was detected in proband of family 1:TSC2 c.826₈27delAT, p.Met276Valfs*61. cDNA 826-827 del base A and T,then codon 276th encode valine instead methionine. This caused the amino acid sequence premature termination of the password in 337th. No mutations were detected in their parents.2).TSC1 gene mutation was detected in proband of family 2:TSC1 c.1888₁891delAAAQ p.Lys630GlnfsX22. Bases 1888-1891 of code area were deleted, which lead to the 630th codon from the original code lysine into glutamine. No mutations were detected in their parents.3).TSC1 gene mutation was detected in proband of family 3:TSC1 c.836₈37 insT（p.Leu279fs）. Base between 836 and 837 was inserted base T. It causes a shift in the gene to occur. The patient’s father is heterozygous at the same site and mother is normal.4). TSC2 gene mutation was detected in proband of family 4:TSC2 C.2713C＞T p.Arg905Trp. It was a missense mutation.5).There were 4 TSC patients in family of 5,2（1113,1115） of them were detected gene mutation:TSC1 exon7 del.There was no significant abnormality in gene detection in the other 2 patients （II3, II5） and 2 healthy individuals（II 4,III4）.Clinical features of 6 TSC cases:All of them had seizure, subependymal nodules or calcification,skin lesion （hypomelanotic macules or shagreen patche）.In 6 patients,4 cases had mental retardation, cardiac rhabdomyoma in 2 cases,1 cases with subependymal giant cell tumor.2.Among 160 patients of TSC,TSC2 mutation accounted for 81.9%（131/160） and TSC1 accounted for 18.1%（29/160）1). The mutation distribution:28 cases got mutation in TSC1 gene, exon 15,21, 18 of TSC1 gene got more mutation. The TSC1 gene consists of 23 exons, the exonl5 is longer and owns the largest proportion of mutation（34.5%）.Exon 37,40 and 33 of TSC2 gene get more mutation, proportion of mutation is 9.9% （13case）,7.6%（lOcase） and 6.9%（9 case）.2). Gene mutation type:deletion （31.0%） and missense （31.0%） mutation is common in TSC1,which missense （32.1%）,deletion （21.4%） and nonsense （20.6%） mutation in TSC2.3). Clinical characteristics of 160 cases of tuberous sclerosis:the most common clinical manifestation of 160 cases of TSC is:hypomelanotic macules （incidence rate 79.8%） and facial angiofibromas （75.6%）, the subependymal nodules or calcification （73.6%）, epilepsy （71.6%）.3.Combined the gene and clinical data of 166 cases （6 cases in this paper, litera-ture search 160 cases） of patients with TSC, and analysis the relationship between genotype and clinical phenotype:1).TSC1 versus TSC2 mutation:TSC1 mutation in 33 cases, TSC2 mutation in 133 cases. The incidence of hypomelanotic macules in TSC1 group was 60.0% （15/25）, while in TSC2 group was 84.0%（84/100）.Incidence of TSC2 group was higher than TSC1 group. The difference between the two groups was statistically significant （x2=6.993, P=0.008）. The incidence of subependymal nodules of TSC 1 group was 30.8%（4/13）,while in TSC2 group was 64.6%（31/48）.Incidence of subependymal nodules of TSC2 group was higher than TSC1 group （x2=4.783. P=0.029）. Wilcoxon signed rank test was used to compare the incidence of total clinical manifestation between TSC1 group and TSC2 group, calculated statistics Z=-2.691,P=0.007<0.05。 Therefore, it can be considered that the incidence of the overall clinical manifestations of the TSC2 group is higher.The TSC2 mutation group was more severe than the TSC 1 mutation group.2). Missense mutation versus other types of mutation:In our study, missense mutations were 52 cases, other types of mutations were 114 cases:nonsense mutations （31 cases）, insert mutation（22 cases）, deletion mutation （39 cases）, splicing mutation （15 cases） and gene large fragment deletion （7 cases）. Among them 1 case deletion mutations lead to encode functional proteins lack of six amino acids and the rest of the deletion mutation and insertion mutation caused a frameshift mutation, nonsense mutation resulted in coding part of the protein truncation, splicing mutation and gene deletion caused large fragment gene insertion or deletion. Missense mutation led to only one amino acid change. Missense mutations and other types of mutations were compared, to study whether there are differences in clinical phenotype after gene mutations in proteins encoded by a single amino acid change and multiple amino acid changes. The result showed that there were no significant difference between missense mutation and other types of mutation.[Conclusion]1. In our country, the majority of TSC gene mutations in patients are mainly in the TSC2 gene.2. The common clinical phenotype in patients with TSC is epilepsy, subepen-dymal nodules or calcification,et.al. Patients with TSC2 gene mutation more likely get hypomelanotic macules and subependymal nodules. The TSC2 mutation group was more severe than the TSC1 mutation group。part 2 of abstract:Changes of insulin like growth factor-1 in tuberous sclerosis complex[Objective】 To research changes and clinical significance of insulin like growth factor-1 （IGF-1） in tuberous sclerosis complex（TSC）.[Methods] The levels of serum IGF-1 were detected by chemiluminescence method in 30 patients with TSC（experimental group） and 30 healthy people（control group）,which were matched with sex and age. Test method:using the Wilcoxon signed rank sum test. P＜0.05 difference was statistically significant.[Results] The level of IGF-1 in serum of experimental group was 78.95（43.53,135.75）ng/mL,while control group was 83.05（55.08,173.50） ng/mL.There was no statistically significant difference between them.[Conclusion] The level of IGF-1 does not change significantly in serum of TSC patients. Serum IGF-1 can be used as evaluations of TSC needs further study.