Mutational Analysis And Functional Characterization Of The Pathogenic Genes For Mucolipidosis And Congenial Nystagmus

In this study, one Chinese family with mucolipidosis and two Chinese families with CN have been ascertained. The underlying pathogenic causes for the disease in these three families were analyzed in detail using molecular genetic techniques including linkage analysis and positional cloning. In addition to genetic studies, I also pursued molecular functional studies to broaden my training in molecular biology and cell biology. The FRMD7 gene is the first gene identified for CN, but its function and mechanism for causing CN are not clear. I carried out functional analysis of the FRMD7 gene. The results for these three areas are presented below:(1) Molecular genetics analysis of a family with mucolipidosisA Chinese family with three patients with mucolipidosisâ…¢from non-blood related normal parents has been identified and characterized. Linkage analysis mapped the disease-causing gene in the family between markers D16S3024 and D16S423. The GNPTG gene, one of the mucolipidosis genes, is located at only 0.2 Mb from marker D16S3024 and became the strong candidate gene for the disease in the family. Mutational analysis of all exons and exon-intron boundaries of GNPTG was carried out using direct DNA sequence analysis. Two novel heterozygous mutations (a one bp deletion c.471de1C and a splicing mutation IVS4-1G>C) of GNPTG were found in the proband. The sequencing analysis of the two parents showed that the deletion mutation (c.471de1C) was paternally inherited and the splice-site mutation (IVS4-1G>C) was maternally inherited. The other two affected siblings also inherited the c.471de1C mutation from the father and the IVS4-1G>C mutation from the mother. The deletion mutation (c.471de1C) causes a frameshift at codon 157, resulting in four additional novel amino acids and premature termination at codon 5 in shifted reading frame (p.C157Wfs*5). By the RT-PCR analysis and direct sequencing analysis, we found that the splice-site mutation (IVS4-1G>C) could lead to an insertion of 74bp of intron 4 into the mature RNA. The abnormal transcript contains a premature translation termination codon at the initial intron 4 and results in a non-functional truncated protein. The compound heterozygous mutations c.471de1C and IVS4-1G>C in the GNPTG gene resulted in severely truncated GNPTG proteins. The nonfunctional truncated proteins caused MLIIIC in this family. The two novel heterozygous mutations were not detected in the 50 controls (100 chromosomes). These results indicate that the two novel heterozygous mutations in the GNPTG are the cause of the MLIIIC in this family.(2) Molecular genetics analysis of two Chinese families with congenial nystagmusTwo Chinese families with X-linked inherited CN have been collected and characterized. The linkage analysis using microsatellite markers DXS7108 close to GPR143 in family 1 with X-linked recessive congenial nystagmus showed positive linkage to this disease. Mutational analysis of all exons and exon-intron boundaries of GPR143 was carried out using direct DNA sequence analysis. A novel 4-bp deletion mutation (c.819₈22del) was found in the proband. The proband's mother carried heterozygous mutation. The deletion mutation (c.819₈22del) causes a frameshift at codon 274, resulting in seven additional novel amino acids and premature termination at codon 8 in shifted reading frame (p.T274Ifs*8). The truncated protein was predicted to delete the 7th transmembrane domain and the C-terminal fragment of GPR143. To confirm that c.819₈22del mutation is associated with the disease in the family, single strand conformation polymorphism (SSCP) analysis was carried out. The c.819₈22del mutation was not identified in 50 normal females and 50 normal males. Family 2 show an X-linked dominant CN and is characterized by head nodding and strabismus in the proband and his mother. These results showed that mutation c.819₈22del (p.T274IfsX7) in GPR143 were the cause of CN in Chinese family 1.For the second family with CN, I could not exclude FRMD7 as the causing gene in the family. Direct DNA sequence analysis of all exons and exon-intron boundaries of FRMD7 was carried out in the proband. A novel missense mutation of c.580G>A was identified. The mutation results in the change from a hydrophobic alanine to hydrophilic threonine at codon 194 (p.A194T). Direct sequencing analysis of all individuals in this family showed the presence of the hemizygous mutant allele in all male patients, both wild type and mutant alleles (heterozygous mutation) in females and wild type alleles in normal individuals. This mutation was not detected in the 100 controls (50 males and 50 females). The amino acid at codon 194 is located in the FERM domain of FRMD7 protein and shows highly conservation from caenorhabditis to Homo sapiens. These results showed that mutation c.580G>A (p.A194T) in FRMD7 was the cause of CN in Chinese family 2. (3) Function analysis of the FRMD7 geneTo understand the function of FRMD7 in the processing of neuronal differentiation, EGFP-tagged wild type FRMD7 and mutant FRMD7 with mutation p.A194T, p.H333fs (found before in our lab) and p.G24R (found by Tarpey et al) were overexpressed in HeLa cells, respectively. The transfected cells were visualized under a laser scanning confocal microscope. The results showed that the mutatant protein with p.A194T was localized in the cytoplasm as wild type FRMD7, but the mutatant proteins with p.G24R and p.H333fs were in the nucleus. Those results suggest that FRMD7 protein may have NES (Nuclear export signals) and NLS (Nuclear location signals). How does the FRMD7 protein shuttle back and forth between the nucleus and cytoplasm? Leptomycin B inhibition of FRMD7 nuclear export suggests that the NES-mediated crml/exportinl pathway is involved in the process of cytoplasmic localization. Bioinformatics analysis of the primary sequence shows that theFRMD7 contains a stretch of basic amino acids (aa 234-237, KRKH) similar to a monopartite NLS (PSORTII) and a cluster of hydrophobic amino acids (aa 93-102, LTRYLFTLQI) homologous to classic leucine-rich NES (NETNES). Thus, amino acids 234-237 constitute a monopartite signal for FRMD7 nuclear localization and amino acids 93-102 constitute a classic leucine-rich NES for FRMD7 nuclear export. To understand the possible function of the FRMD7 protein localized in the nuclear region, we performed cotransfection experiments in HeLa cells with expression constructs encoding a wild-type EGFP-FRMD7 and the DsRed protein fused to p53 (DsRed-p53) which localizes into the nucleus and functions in the neuronal differentiation. The results showed that the two proteins did not colocalize in the nucleus in the presence of Leptomycin B. Immunofluorescence microscopy on HeLa cells transiently transfected with expression constructs EGFP-FRMD7 and stained with an anti-coilin antibody showed that the FRMD7 protein did not localize in the Cajal body. Thus, these results showed that the FRMD7 protein could not directly interact with p53 and Cajal body in neuronal differentiation.In conclusion, the disease-causing genes in one family with MLIIIC and two families with CN were characterized by the techniques of molecular genetics and molecular biology. In the study of the family with MLIIIC, we identified the first compound mutation in the disease-causing gene GNPTG in the Chinese population. The results could help the patients in this family to distinguish MLIIIC from the other diseases with similar symptoms (such as MLIIIA and MPSII) and guide individualized therapy. The results from data of family 1 with CN support that the symptoms caused by the mutations of GPR143 in the Chinese population are different from those in European populations. The data from family 2 with CN extend the mutation spectrum of the FRMD7 gene. All results in this project may help accurate diagnosis and individualized therapy (personalized medicine) and improve the health status of families with these hereditary diseases. Meanwhile, I analyzed the mechanism for subcellular localization of the FRMD7 protein. My results will further the understanding of the causes and pathogenic mechanisms of MLIIIC and CN.