Objective To study exogenous expression and subcellular localization of wild type (WT ) and mu-tant PAX3 proteins in vitro by generating their expression plasmids for further study of pathogenesis of Waarden-burg syndrome (WS) .Methods The plasmids pECE-PAX3 and pcDNA3 .0-HA were ligased after they were cut by double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pcDNA3 .0-PAX3-HA ,which was as a template to generate expression plasmids pcDNA 3 .0 -H80D -HA and pcDNA3 .0-H186fs-HA of novel mutations H80D and H186fs of PAX3 gene .All constructs were verified by di-rect nucleotide sequencing .NIH3T3 cells were transfected transiently with the expression plasmids of PAX3 ,H80D and H186fs respectively .The exogenous expression of WT PAX3 protein and mutant H80D ,H186fs proteins were analysed using Western blot assay ,while their subcellular distribution were observed using immunofluorescence as-say .Results The DNA sequences of expression plasmids of PAX3 and its mutant H80D ,H186fs were correct . Both WT and mutant PAX3 proteins were detected at the expected size .WT PAX3 and H80D proteins were only lo-calized in the nucleus ,whereas H186fs protein showed aberrant localization in both cytoplasm and nucleus .Conclu-sion We successfully generated the recombinant eukaryotic expression plasmids of PAX 3 gene and its mutants and drew preliminary conlusion of gene mutation having effect on subcellular distribution of WT PAX 3 proteins in vitro , which lays experimental basis for further study of the moceluar mechanism of WS caused by PAX3 gene mutations in China .

OBJECTIVETo study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome.

METHODSEukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays.

RESULTSExpression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus.

CONCLUSIONThis study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.

Amino Acid Sequence ; Animals ; Cell Line, Tumor ; Genetic Predisposition to Disease ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Luciferases ; genetics ; metabolism ; Mice ; Microphthalmia-Associated Transcription Factor ; genetics ; metabolism ; Microscopy, Confocal ; Monophenol Monooxygenase ; genetics ; metabolism ; Mutation ; NIH 3T3 Cells ; Nuclear Localization Signals ; genetics ; Transcriptional Activation ; Transfection ; Waardenburg Syndrome ; diagnosis ; genetics ; metabolism

Objective: To explore the genetic basis for a patient with autism. Methods: High-throughput sequencing was carried out to detect copy number variations in the patient. Results: DNA sequencing found that the patient has carried a 0.11 Mb deletion in distal 2p16.3 spanning from genomic position 50 820 001 to 50 922 000, which resulted removal of exon 6 and part of intron 7 of the NRXN1 gene. The same deletion was not found his parents and brother. Conclusion Partial deletion of the NRXN1 gene may underlie the disease in this patient.

OBJECTIVETo study the exogenous expression and subcellular localization of wild type (WT) and mutant SOX10 proteins in vitro through generation of expression plasmids in order to reveal the pathogenesis of Waardenburg syndrome (WS).

METHODSThe plasmids pECE-SOX10 and pCMV-Flag were ligated after they were subjected to double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pCMV-SOX10-Flag, which was as a template to generate expression plasmids for novel mutations G37fs, G38fs and E248fs of the SOX10 gene. The constructs were verified by direct sequencing. NIH3T3 cells were transiently transfected with the expression plasmids of wide type SOX10, G37fs, G38fs and E248fs, respectively. The exogenous expression of WT SOX10 protein and mutant G37fs, G38fs and E248fs proteins were analyzed using Western blot assay, while their subcellular distribution were observed with an immunofluorescence assay.

RESULTSThe DNA sequences of expression plasmids for SOX10 and its mutant G37fs, G38fs and E248f were all correct. Both WT and mutant SOX10 proteins were detected at the expected site. WT SOX10 and E248fs proteins have only localized in the nucleus, whereas G37fs and G38fs proteins showed aberrant localization in both cytoplasm and nucleus.

CONCLUSIONRecombinant eukaryotic expression plasmids for the SOX10 gene and its mutants were successfully constructed. Preliminary analysis showed that the mutations have affected the subcellular distribution of WT SOX10 proteins, which has laid a basis for further study of the molecular mechanism of WS caused by SOX10 gene mutations.

Animals ; Base Sequence ; Humans ; Mice ; Molecular Sequence Data ; Mutation ; NIH 3T3 Cells ; Plasmids ; Recombination, Genetic ; SOXE Transcription Factors ; genetics ; Waardenburg Syndrome ; genetics

OBJECTIVE: To explore the genetic basis for a patient with autism. METHODS: High-throughput sequencing was carried out to detect copy number variations in the patient. RESULTS: DNA sequencing found that the patient has carried a 0.11 Mb deletion in distal 2p16.3 spanning from genomic position 50 820 001 to 50 922 000, which resulted removal of exon 6 and part of intron 7 of the NRXN1 gene. The same deletion was not found his parents and brother. CONCLUSION Partial deletion of the NRXN1 gene may underlie the disease in this patient.
Autistic Disorder ; genetics ; Cell Adhesion Molecules, Neuronal ; genetics ; DNA Copy Number Variations ; Gene Deletion ; Humans ; Male ; Nerve Tissue Proteins ; genetics