Whole Exome Sequencing Identifies Pathogenic Genes Of Two Mendelian Genetic Disease Pedigrees And Function Characterization

At the present time,precision medicine is rapidly developing in China.Finding out the disease-causing genes,exploring the functions of disease genes,and understanding the pathogenic mechanisms are required to move precision medicine forward rapidly.Genetic pedigree resources provide a valuable tool to explore the relationship between disease-causing genes and diseases.In the past,the analysis of Mendelian inherited diseases and identification of disease genes were accomplished using genome-wide linkage analysis,fine mapping and candidate gene sequencing.However,due to the incompleteness of the pedigree data,reduced disease penetrance,late onset,and limitations of molecular marker polymorphisms,it is often impossible to find the target region of the disease genes or identify the underlying disease genes.With the development of high-throughput next generation sequencing technologies,the discovery of pathogenic genes has been greatly accelerated.This study was based on the whole-exome sequencing technology and identified two pathogenic genes in two Mendelian genetic pedigrees.We then studied the function of the genes and mutations.Our findings highlight that the next generation sequencing technology and clinical examinations complement each other and together they facilitate the characterization of the genotype-phenotype correlation.Our studies also identified novel molecular pathogenic mechanisms for diseases.Thus,our data provide important new insights into the pathogenesis of diseases and may help with the accurate genetic testing and the development of new medical treatment for the diseases under the study.In the first section of the thesis,we collected and characterized a three-generation family with early-onset sick sinus syndrome（SSS）,cardiac conduction disorder（CCD）,dilated cardiomyopathy（DCM）,and sudden cardiac death（SCD）.The mean age of SCD in the family is 39 years.We employed whole exome sequencing（WES）to genetically analyze and identify the disease-causing gene and mutation in the family.We identified a novel frameshift mutation with a 1-bp duplication（c.1433dupT,or p.（Leu478Phefs*74））in the lamin A/C（LMNA）gene（NM₁70707.3/NM₀05572.3）.This mutation was predicted to generate a truncation of the lamin A/C protein with disruption of the important Ig-like domain.We hypothesized that the LMNA mutation affects the function of the cardiac sodium channel Na_v1.5 encoded by the SCN5A gene.Whole-cell patch-clamping was performed in HEK/Na_v1.5 cells over-expressing the mutatant LMNA（p.Leu478Phefs*74）as compared with the wild type LMNA revealed a significant decrease in the sodium current density by the mutation.The p.Leu478Phefs*74 mutation did not affect the recovery from inactivation,and kinetics of activation and inactivation.For the first time,we showed that the mutant LMNA significantly decreased the cell surface expression level of Na_v1.5,however,it did not affect the expression level of total Na_v1.5 protein in cells.Fluorescence microscopy showed that wild type LMNA was homogeneously dispersed adjacent to the nuclear envelope in normally-shapped nuclei,however,the mutant LMNA aggregated and caused misshaped nuclei.Together,our data indicate that the novel heterozygous mutation p.Leu478Phefs*74 in LMNA causes an interesting autosomal dominant syndrome of SSS,CCD,DCM and SCD.Our data suggest that the p.Leu478Phefs*74 mutation in LMNA is associated with high clinical heterogeneity of a variety of arrhythmias,DCM and sudden death,which expands the genetic spectrum of laminopathies.Our functional study suggests that the truncated protein LMNA（p.Leu478Phefs*74）produced by the frame-shifted LMNA mutation has caused a significant decrease of the sodium current density and the number of Na_v1.5 ion channels on the cell membrane,impaired the assembly and distribution of lamin A/C,and disrupted the structure of the nuclei.These negative dominance effects and haploinsufficiency may together lead to the occurrence and development of the disease.These results provide important insights into the molecular mechanism for the pathogenesis of laminopathies.In the second section of the thesis,we investigated an Amish family in which three siblings presented with an early-onset childhood retinal dystrophy inherited in an autosomal recessive fashion.Genome-wide linkage analysis identified significant linkage to marker D2S2216 on 2q11 with a two-point LOD score of 1.95 and a multi-point LOD score of 3.76.Whole exome sequencing was then performed for three affected individuals and identified a homozygous nonsense mutation（c.1813C>T,p.R605X）in the cyclin and CBS domain divalent metal cation transport mediator 4（CNNM4）gene located within the 2p14-2q14 Jalili syndrome locus.The initial assessment and collection of the family were performed before the clinical delineation of Jalili syndrome.Another assessment was made after the discovery of the responsible gene and the dental abnormalities characteristic of Jalili syndrome were retrospectively identified.The p.R605X mutation represents the first and probably founder Jalili mutation identified in the Amish community.The molecular mechanism underlying Jalili syndrome is unknown.Here we show that CNNM4 interacts with IQCB1,which causes Leber Congenital Amaurosis（LCA）when mutated.A truncated CNNM4 protein starting at R605significantly increased the rate of apoptosis,and significantly increased the interaction between CNNM4 and IQCB1.Mutation p.R605X may cause Jalili syndrome by a nonsense-mediated decay mechanism,affecting the function of IQCB1 and apoptosis,or both.Our data,for the first time,functionally link Jalili syndrome gene CNNM4 to LCA gene IQCB1,providing important insights into the molecular pathogenic mechanism of retinal dystrophy in Jalili syndrome.