The Phenotypic Analysis And Genetic Diagnosis Of Tuberous Sclerosis Complex

BackgroundTuberous sclerosis complex (TSC) is an autosomal dominant genetic neurocutaneous syndrome in the form of hamartoma involving multiple organs and systems, which is first reported by Bourneville in 1880, and so is often called Bourneville disease. The typical clinical features are mental retardation, epilepsy and adenoma sebaceum. The incidence is about 1:10000-1:6000, and the ratio of male to female is about 2:1. There are no significant ethnic differences. Its skin lesions appear earlier than other manifestations, so patients often go to dermatology clinic first. But serious complications caused by the pathological changes of the nervous system, kidneys, lungs, heart and other organs lesions result in poor prognosis and even death. Therefore, early diagnosis and early treatment are important. At present, the diagnosis of the disease mainly relies on clinical manifestations and imaging changes. Because of the genetic heterogeneity, the clinical manifestations among different patients with tuberous sclerosis are distinct, even among the different members in the same family. This disease involves multiple disciplines, and is rare, at the same time the clinical doctors are lack of enough knowledge, so the disease is easy to be miss-diagnosed. Correct identification of tuberous sclerosis phenotype is one of the important steps for clinicians in the diagnosis of the disease, and it is also useful for the early detection of fatal complications in patients with tuberous sclerosis.Tuberous sclerosis is closely related to the two tumor suppressor genes TSC1 and TSC2, which are located in 9q34.2 and 16p13.3, respectively. TSC1 and TSC2, as two tumor suppressor genes, are the cause of the disease. TSC 1 gene contains 23 exons, encoding the transcripts of 1164 amino acids called hamartoma protein (hamartin). TSC2 gene contains 42 exons, encoding the transcripts consisting of 1807 amino acids callled potato globulin (tuberin). Hamartin and tuberin proteins form heterodimer protein in the cell, regulating the cell proliferation and further biological functions involved in cell adhesion and etc through the TSC-Rheb-TORS6K1/4EBP1 pathways. TSC1 gene mutations are mainly point mutations resulting in nonsense or miss-sense mutation, as well as the small insertions or deletions causing frame-shift mutations. While TSC2 gene has a variety of mutation forms, including nonsense mutations, missense mutation, shear mutation, small insertion or deletion, and the lack of large fragments,85% of which are small mutations and 15% are the lack of large or rearrangement. Both home and abroad are focusing on the hot spots of the two gene mutations. TSC1 gene mutations in exon 15,17,18 are reported frequently, such as 2105 delAAAG, R786X occurring in many familial and sporadic patients. Van Slegtenhorst et al suggest/report more than half of TSC1 gene mutations are in exon 15 and 17, but hot spot mutations researches on TSC2 gene are relatively few, so a unanimous conclusion is yet to be obtained. Research in abroad found TSC gene mutation are complex in type and form; Domestic TSC molecular genetic research is in its infancy, and this may be related to more exons of TSC1 and TSC2 gene resulting in more difficulty and high cost in research.In this study, we analyzed the phenotypic characteristics of tuberous sclerosis in children, at the same time established a gene diagnosis of tuberous sclerosis method, to analyze its pathogenic gene mutation, in order to get an early diagnosis of the disease and an early detection of the pathological changes in important internal organs. This is useful to improve the diagnosis and treatment of the disease, especially in prenatal diagnosis level. Improving the prognosis of patients is of great significance.Chapter Ⅰ The phenotypic analysis of tuberous sclerosis complexObjective:To analyze and summarize the phenotypic characteristics of tuberous sclerosis in children in order to improve the understanding of the disease. Thus achieving of early diagnosis, early treatment and early detection of the pathological changes in important internal organs is useful to improve the prognosis of patients and reduce the morbidity and mortality.Methods:We collected 31 cases of tuberous sclerosis coming for medical diagnosis and treatment in Qilu children’s hospital, Shandong University between September 2009 and September 2014. For each case, detailed medical history data was collected, and systematic examination was performed. General data and clinical manifestations were conducted by self-made questionnaire. Most patients received head CT scan and/or magnetic resonance imaging (MRI). Ultrasound of liver, kidneys, bravery, lienal, pancreatic was also performed. The positive and lateral X ray radiography of chest, hand and foot were collected. Eye examinations including fundus examination were also obtained. Some patients experienced echocardiography and peripheral blood karyotype analysis (including conventional karyotype analysis). PRATIC type spiral CT scanner and GE Sytec type 4000 systemic CT scanner in Hitachi were used to make skull CT plain scan and enhanced scan. Magnetic resonance imaging was done by 0.2 T permanent magnet MRI scanner of GE company.Results:The most common clinical manifestations in 31 cases of sporadic and familial children with tuberous sclerosis are subependymal nodules, followed by loss of pigment spots, seizures, and cerebral cortical tubers.Conclusion:Tuberous sclerosis is a systemic disease, involving multiple organs. It is phenotypically diverse. Children often have lesions of neurologist and dermatologist. Therefore clinical doctors should be aware of the clinical manifestations of tuberous sclerosis in all the subjects and understand the organ system often involved. Once a clear diagnosis is obtained, more attention should be paid to those serious complications often caused by the disease in order to take early treatment.Chapter Ⅱ:The genetic diagnosis research of tuberous sclerosis complexObjective:By establishing and applicating the method of the gene diagnosis of TSC, we analyzed the TSC gene mutations in pediatric patients, and improved the level of the diagnosis of tuberous sclerosis, especially at the prenatal diagnosis level.Methods:The peripheral venous blood of children with tuberous sclerosis was collected, the genomic DNA was extracted, and then all exons of TSC1 and TSC2 gene and gene splicing area were obtained by PCR amplification. We got gene diagnosis by screening the TSC1 and TSC2 gene mutations of the patients.Results:(1) We successfully amplified the exon 1- 23 of TSC 1 gene and the exon 1-41 and splicing area of TSC2 gene; (2) we discovered and reported two new mutations 1VS 29+1 G>C and c.5090-5092 delCCA-inAG.Conclusion:(1) We established the gene diagnosis method of tuberous sclerosis, which contributes to the gene diagnosis of tuberous sclerosis, especially in prenatal diagnosis; (2) We found and reported two new TSC2 gene mutations, adding new knowledge to the database of tuberous sclerosis gene mutations.