Molecular Genetic And Fuctional Studies Of Human Diseases, Including Type Ⅱ Dentinogenesis Imperfecta (DGI),Congenital Fibrosis Of The Extraocular Muscles Type 1 (CFEOM1), Long QT Syndrome And Atrial Fibrillation

Identification of a disease-causing gene for a human disease can provide remarkable insights into the molecular mechanism for the pathogenesis underlying the disease.The most common method for disease gene identification is positional cloning,which is one of the powerful technologies developed in genetics.Positional cloning has been successful in discovering many human disease genes,including over 30 cardiac disease genes. Positional cloning revolutionized the field of medicine as the identification of the disease gene not only provides the molecular understanding of the disease as well as the function of the gene in biology,but also enchance the development of diagnostic tests and guides gene-specific treatments for patients.Functional studies of disease-causing genes will provide the mechanism to understand the pathogenesis and treatment of disease,and will also strengthen the understanding of structure and function of the gene.In this thesis project,I identified some mutations in disease genes for TypeⅡdentinogenesis imperfecta (DGI).congenital fibrosis of the extraocular muscles type 1(CFEOM1),long QT syndrome (LQTS) and used biochemical,molecular biological,cell biological methods and a knockout mice model to understand the function of NUP155,a newly identified gene for atrial fibrillation.This thesis is composed of four main parts:PartⅠ:A novel DSPP mutation is associated with typeⅡdentinogenesis Imperfecta in a chinese familyHereditary defects of tooth dentin are classified into two main groups:dentin dysplasia (DD)(typesⅠandⅡ) and dentinogenesis imperfecta(DGI)(typesⅠ,Ⅱ,andⅢ).TypeⅡDGI is one of the most common tooth defects with an autosomal dominant mode of inheritance.One disease-causing gene,the dentin sialophosphoprotein(DSPP) gene,has been reported for typeⅡDGI.In this study,we characterized a four-generation Chinese family with typeⅡDGI that consists of 18 living family members,including 8 affected individuals.Linkage analysis with polymorphic markers D4S1534 and D4S414 that span the DSPP gene showed that the family was linked to DSPP.All five exons and exon-intron boundaries of DSPP were sequenced in members of typeⅡDGI family. Direct DNA sequence analysis identified a novel mutation(c.49C→T,p.Prol7Ser) in exon 1 of the DSPP gene.The mutation spot,the Prol7 residue,is the second amino acid of the mature DSP protein,and highly conserved during evolution.The mutation was identified in all affected individuals,but not in normal family members and 100 controls.These results suggest that mutation p.Prol7Ser in DSPP gene causes typeⅡDGI in the Chinese family.This study identifies a novel mutation in the DSPP gene,and expands the spectrum of mutations that cause DGI.PartⅡ:Mutation p:Arg954Trp of KIF21A Causes Congenital Fibrosis of the Extraocular Muscles in a Chinese FamilyCongenital fibrosis of the extraocular muscles type 1(CFEOM1) is an autosomal dominant strabismus disorder associated with defects of the oculomotor nerve.In this study,we identified a four-generational Chinese family with CFEOM1.Linkage analysis mapped the causative gene of the family to 12q with a LOD score 2.1 for polymorphic marker D12S85,where KIF21A is located.Direct DNA sequence analysis identified a 2860C→T change in exon 21,resulting in a tryptophan substitution for arginine in codon 954 of KIF21A.SSCP analysis showed that mutation p.Arg954Trp of KIF21A cosegregated with affected members,but was absent in unaffected individuals in the family and 150 normal controls.Our results indicate that mutation p.Arg954Trp of the KIF21A is the genetic basis of the Chinese family with CFEOM1.PartⅢ:Identification of novel KCNQ1 and KCNH2 mutations and the protective effect of KCNH2 SNP K897T in LQTS familiesKCNH2 is one of the most common potassium channel gene causing long QT syndrome (LQTS),a cardiac arrhythmia associated with prolongation of QTc on electrocardiograms and sudden death.In a cohort of LQTS families and patients,one novel mutations, 2020insAG in KCNH2 was identified.In another family,KCNH2 mutation A490T co-segregated with a common SNP K897T in KCNH2.Bradycardia was prevalent in carriers with A490T and K897T as well as the patient with another KCNH2 mutation R366X.SNP K897T in KCNH2 was reported to exert a modifying effect on QTc,but it remains controversial whether it confers a risk or protective effect.We found that seven carders for mutation A490T and the minor allele T of SNP K897T showed shorter QTc (0.45±0.2s) and fewer symptoms than carriers with A490T alone or A490P(P＜10^-4), suggesting that KCNH2 SNP K897T confers a protective effect on QTc prolongation.PartⅣ:Link of the Nuclear Pore Complex to Cardiac Arrhythmias:NUP155 Mutation Causes Neonatal Atrial Fibrillation and Sudden DeathAtrial fibrillation(AF) is the most common clinical arrhythmia.We previously mapped an AF locus to chromosome 5p13 by genome-wide linkage scan.Here we show that the specific AF gene underlying this linkage is NUP155.It encodes a member of the nucleoporins,with a molecular weight of 155k Da,the components of the nuclear pore complex(NPC).We identified a homozygous mutation,R391H,in NUP155 that co-segregated with AF in the family,and R391H mutation in NUP155 gene affected nuclear localization of NUP155. NUP155 was strongly expressed in atrial myocytes.Homozygous NUP155 knockout mice died before E8.5,but heterozygous NUP155^+/- mice showed AF phenotype.Studies of the human and mouse indicate that loss of NUP155 function causes AF,and for the first time link the NPC to cardiovascular disease.These results also provide a non-ion channel genetic basis for AF,and suggest a new mechanism for the pathogenesis of AF,which may provide a potential diagnostic and therapeutic target for AF.