Genetic Diagnosis And Prenatal Diagnosis Of Alport Syndrome And SIOD Patients

This thesis will comprise three parts: Part I: The molecular genetics research on Alport syndrome; Part II: The molecular genetics research on Schimke immuno-osseous dysplasia; Part III: Prenatal genetic diagnosis of three Alport syndrome’s families.PART Ⅰ The molecular genetics research on Alport syndromeBackground: Alport syndrome (AS) is an inherited nephropathy characterized by the presence of repeated microscopic or gross hematuria, chronic renal insufficiency, and often associated with ocular lesions or sensorineural deafness. The mutations in the collagen alpha-3, alpha 4, alpha 5(Ⅳ) chain (COL4A3, COL4A4,COL4A5) are responsible for the disorder. There are three main models:autosomal-dominant, autosomal-recessive and X-linked dominant inheritance patterns.While X-linked dominant inheritance is the main patterns of AS, and COL4A5 is the pathogenic gene, account for 85%. The pathologic gene COL4A3, COL4A4,COL4A5 of AS, consisting of 48, 52 and 53 exons, respectively. The mutations across these genes and no hotspot mutation sites existed. AS is a clinical heterogeneity of the basement membrane of kidney disease, sometimes it is easy to be confused with thin basement membrane nephropathy, focal segmental glomerulosclerosis and other kidney diseases, resulted in missed diagnosis or misdiagnosis. At present, the development of next generation sequencing will lay the foundation for AS, make fast and efficient screening of the location and mutation type of pathologic gene is possible.Methods: Six confirmed or suspected AS cases were collected, and using whole exome sequencing to screening for pathogenic gene,confirmed by PCR amplification and sequencing technology. Multiple sequences alignment was used to observe whether the mutations have high conservatism of evolution. Meanwhile, the pathogenic of mutations were analysed by bioinformatics methods. For missense mutations, SIFT and Polyphen-2 were used for hazardous analysis. MaxEntScan was designed for predicting the influence of the splicing mutation. In order to rule out the possibility of polymorphism, sequencing for 200 normal control simultaneously.Results: Five new mutations and two known mutations were detected by whole exome sequencing, included two new mutations c.4348C>T (p.R1450X) or c.352G>A (p.G118R) of COL4A3 gene, two new mutations c.3455-2A>G, c.3817G>A(p.G1273S) or two known mutations c.796C>T (p.R266X), c.2858G>T (p.G953V) of COL4A5 gene, and a new mutation c.1099G>A (p.G367S) of COL4A4 gene. These mutations were identified in the affected members, but absent in the unaffected members from AS families and completely cosegregated with the disease phenotype.Multiple sequences alignment shows those mutations are highly conservatism of evolution. No matter what types of mutations found in our study are harmful for AS families and once mutated will affect protein function.Conclusion: In current study, we found five new mutations and further more to enrich the mutation database. Some mutations will be valuable for pinpointing the genetic basic of a definitive diagnosis and avoid missed diagnosis or misdiagnosis. It suggested that gene diagnosis is of significance for clinical studies. The detection of pathogenic genes undoubtedly have provide reasonable treatment prognosis and allow for genetics counseling or or prenatal diagnosis of the family members.PART Ⅱ The molecular genetics research on Schimke immuno-osseous dysplasiaBackground: Schimke immune-osseous dysplasia (SIOD) is a rare autosomal recessive characterized by spondyloepiphyseal dysplasia, T-cell deficiency, renal dysfunction and special facial features. SIOD is multi-system disease with an incidence of 1:1×106～3×106. And SIOD manifests in approximately 50% of patients due to mutations in the SMARCAL1 gene. SIOD shows phenotypic heterogeneity, and disease severity varies from mild to severe. Nonsense and missene mutations account for a high percentage of SMARCAL1 gene mutations. Most missense mutation always resulted in mild phenotype of SIOD, whereas severe phenotype often resulted from nonsense or nonsense mutations in combination with other types of mutations.However, compared to the literature available abroad, there are few reports about this issue in China.Methods: One suspected SIOD case were collected, PCR amplification and sequencing technology were used to screen for pathogenic SMARCAL1 gene.Multiple sequences alignment was used to observe whether the mutations have high conservatism of evolution. Meanwhile, the pathogenic of mutations were analysed by bioinformatics methods. For missense mutations, SIFT and Polyphen-2 were used for hazardous analysis. MaxEntScan was designed for predicting the influence of the splicing mutation. In order to rule out the possibility of polymorphism,sequencing for 200 normal control, simultaneously.Results: We detected two mutations c.1933C>T (p. R645C) and c.2450G>A(p.R817H) of SMARCAL1 gene by direct sequencing. The patient presented with a well-known maternally inherited missense mutation (p.R645C), and harbored a paternally-derived missense mutation (p.R817H) of SMARCAL1 leading to an arginine-to-histidine substitution. The two mutated sites are highly conserved. It is forecasted to be detrimental to the patient with a score of 0.0 by employing the Sorting Intolerant from Tolerant (SIFT) technique. Similarly, the potential effect of substitution has a detrimental score of 1.000 as calculated by PolyPhen-2.Conclusion: In present study, a novel compound heterozygous mutation of the SMARCAL1 gene leading to mild SIOD. This novel mutation enriches the mutation database and it is also valuable for pinpointing the genetic basic of a definitive diagnosis and avoiding missed diagnosis or misdiagnosis. Furthermore,it suggested that gene diagnosis is of significance for clinical studies. The detection of pathogenic genes undoubtedly have provide reasonable treatment prognosis and allow for genetics counseling or or prenatal diagnosis of the family members.PART Ⅲ Prenatal genetic diagnosis of three Alport syndrome’s familiesBackground: The most prominent clinical manifestations of Alport syndrome is hematuria, but with the growth of the age,it can appear a few or even a large number of proteinuria, and gradually progress to kidney failure and need dialysis or a kidney transplant. There is no effective way to cure and prognosis is poor yet, prenatal diagnosis of AS not only can effectively avoid children born again, but also can reduce the family and the spirit of the national economic pressure. So for the early diagnosis of pregnancy, especially prenatal diagnosis is really essential.Methods: We collected three AS families of the proband and parents of peripheral blood, and gathered for 10 ml of the amniotic fluid of the mothers at 18th,20th and 20th week of gestation, respectively. STR technology is used to eliminate the pollution of the parent cell line and perform PCR amplification, and then to DNA sequencing.Results: Three maternal cells are free from contamination. Sanger sequencing found that the amniotic fluid testing of the family Ⅰ has not yet found the fetus carried the same COL4A5 gene c.1909C>T (p.637Q>X) heterozygous nonsense mutation with the proband, and can normal childbirth. However, the family Ⅱ shows that the fetus carried the same COL4A5 gene c.1185-1186insC heterozygous insertion frameshift mutation with the proband, and chooses to terminate pregnancy. The familyⅢ shows that the fetus without the two compound heterozygous mutations of COL4A3 c.2810G>A and c.4793T>G, and can normal childbirth.Conclusion: Molecular analysis allows early and accurate prenatal genetic diagnosis for AS once mutation is known in a family.