OBJECTIVE: To detect potential variants of MECP2 gene in three pedigrees affected with Rett syndrome (RTT). METHODS: All exons and their flanking regions of the MECP2 gene were subjected to Sanger sequencing and multiplex ligation-dependent probe amplification assay. RESULTS: The probands of pedigrees 1 and 2 have respectively carried a c.965C>G and a c.1157_1197del41 variant of the MECP2 gene, while the proband of pedigree 3 carried a heterozygous deletional variant in exon 4 of the MECP2 gene. CONCLUSION Variants of the MECP2 gene probably underlay the RTT in the three pedigrees. Above finding has enriched the spectrum of MECP2 gene variants, and provided a guidance for the patients upon preimplantation genetic testing and prenatal diagnosis.

OBJECTIVETo identify potential mutations of PKD1 gene in a family affected with autosomal dominant polycystic kidney disease (ADPKD).

METHODSThe coding regions of the PKD1 gene were subjected to PCR and Sanger sequencing. Reverse transcription-PCR (RT-PCR) was used to determine the relative mRNA expression in the patient.

RESULTSA splicing site mutation, c.8791+1_8791+5delGTGCG (IVS23+1_+5delGTGCG), was detected in the PKD1 gene in all 5 patients from the pedigree but not in 6 phenotypically normal relatives and 40 healthy controls. Sequencing of RNA has confirmed that there were 8 bases inserted in the 3' end of exon 23 of the PKD1 gene.

CONCLUSIONThe novel c.8791+1_8791+5delGTGCG mutation has created a new splice site and led to a frameshift, which probably underlies the ADPKD in the family. Above finding has enriched the mutation spectrum of the PKD1 gene.

Adult ; Female ; Humans ; Male ; Mutation ; genetics ; Pedigree ; Polycystic Kidney, Autosomal Dominant ; genetics ; RNA Splicing ; genetics ; TRPP Cation Channels ; genetics ; Young Adult

OBJECTIVETo detect the disease-causing mutation in a pedigree affected with autosomal dominant congenital cataract.

METHODSGenomic DNA was extracted and purified from peripheral blood samples from members of the pedigree and 100 healthy controls. Coding regions of 18 candidate genes were screened with PCR and Sanger sequencing. Identified mutations were verified among 100 healthy individuals to exclude single nucleotide polymorphisms.

RESULTSA heterozygous nonsense mutation c.471G>A of the CRYGD gene, which resulted in p.Trp157Term, was identified in all three patients. The same mutation was not found in the two normal individuals from the family and 100 healthy controls. The nonsense mutation was predicted to be "disease causing" by Mutation t@sting program.

CONCLUSIONThe nonsense mutation c.471G>A of the CRYGD gene probably underlies the congenital cataract in the pedigree.

Cataract ; etiology ; genetics ; Child ; Codon, Nonsense ; Humans ; Male ; Sequence Analysis, DNA ; gamma-Crystallins ; genetics

OBJECTIVETo identify potential mutation of PHEX gene in two patients from a family affected with X-linked hypophosphatemia (XLH).

METHODSPCR and Sanger sequencing were performed on blood samples from the patients and 100 healthy controls. Reverse transcription-PCR (RT-PCR) was used to determine the mRNA expression in patient samples.

RESULTSA splicing site mutation, IVS21+2T>G, was found in the PHEX gene in both patients but not among the 100 healthy controls. RT-PCR confirmed that exon 21 of the PHEX gene was deleted.

CONCLUSIONThe novel splicing mutation IVS21+2T>G of the PHEX gene probably underlies the XLH in this pedigree. At the mRNA level, the mutation has led to removal of exon 21 and shift of the open reading frame (p.Val691fsx), resulting in premature termination of protein translation.

Adult ; Base Sequence ; DNA Mutational Analysis ; Exons ; Familial Hypophosphatemic Rickets ; genetics ; Female ; Genetic Diseases, X-Linked ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; PHEX Phosphate Regulating Neutral Endopeptidase ; genetics ; Pedigree ; RNA Splicing ; Young Adult

OBJECTIVE: To explore the genetic basis for a patient with tuberous sclerosis complex. METHODS: Genomic DNA was extracted from peripheral blood samples from members of his family and 100 unrelated healthy controls. The proband was subjected to next-generation sequencing, and candidate variant was confirmed by multiple ligation-dependent probe amplification (MLPA) and Sanger sequencing. Reverse transcription-PCR (RT-PCR) was carried out to determine the relative mRNA expression in the proband. RESULTS: The patient was found to harbor a c.2355+1G>C splicing variant of the TSC2 gene. Sequencing of cDNA confirmed that 62 bases have been inserted into the 3' end of exon 21, which has caused a frameshift producing a truncated protein. CONCLUSION The novel splicing variant c.2355+1G>C of the TSC2 gene probably underlay the TSC in the proband. Above finding has expanded the variant spectrum of TSC2 and provided a basis for preimplantation genetic testing and/or prenatal diagnosis.
Female ; Humans ; Mutation ; Pregnancy ; RNA Splicing/genetics* ; Tuberous Sclerosis/genetics* ; Tuberous Sclerosis Complex 1 Protein/genetics* ; Tuberous Sclerosis Complex 2 Protein/genetics*

OBJECTIVE: To explore the genetic basis for a pedigree affected with Alport syndrome. METHODS: Next generation sequencing and Sanger sequencing was carried out to detect potential variant of the COL4A5 gene among members from the pedigree and 100 unrelated healthy controls. RESULTS: A novel missense c.3293G>T (p.Gly1098Val) variant was found in the COL4A5 gene among 6 affected members but not the unaffected members of the pedigree or the 100 healthy controls. According to the American College of Medical Genetics and Genomics standards and guidelines, the c.3293G>T variant was classified as pathogenic (PP1-strong+PM1+PM2+PP3+PP4). CONCLUSION By destructing the Gly-X-Y structure of its protein product, the c.3293G>T variant of the COL4A5 gene probably underlies the Alport syndrome in this pedigree. Above finding has enriched the spectrum of COL4A5 variants.

OBJECTIVE: To analyze variant of IDS gene in a pedigree affected with mucopolysaccharidosis type II (MPS II). METHODS: The proband was subjected to next generation sequencing and Sanger sequencing to identify potential variants. Suspected variant was analyzed by its co-segregation with the disease in the pedigree. Its impact on mRNA splicing was analyzed by using reverse transcription PCR (RT-PCR). RESULTS: A hemizygous IVS1-3T>G variant was found in the IDS gene in the proband. RT-PCR results revealed two abnormal cDNA fragments of 600 bp and 300 bp. The 600 bp fragment had inserted 216 nucleotides at the 3' end of intron 1, while the 300 bp fragment had lost 109 nucleotides at the 5' end of exon 2, which resulted in two truncated proteins comprising 38 and 92 amino acids, respectively, instead of the normal product (550 amino acids). The proband and his mother were respectively hemizygous and heterozygous for the variant. The same variant was not found among 100 normal controls. CONCLUSION The IVS1-3T>G variant of the IDS gene probably underlies the MPS II in this pedigree by causing reduction or elimination of the IDS protein.

OBJECTIVETo explore the clinical application of droplet digital PCR (ddPCR) for genetic testing and prenatal diagnosis of spinal muscular atrophy (SMA) with deletion of SMN1 gene exon 7.

METHODSA total of 138 clinical samples, including 121 peripheral blood, 13 amniotic fluid, 2 umbilical cord blood and 2 chorionic villi from 56 SMA families, were tested by both ddPCR and multiplex ligation-dependent probe amplification (MLPA). Results of the two approaches were analyzed with commercial software QuantaSoft (ddPCR) and Coffalyser (MLPA), respectively.

RESULTSAmong the 138 cases, 25 had two copies, 84 had one copy, and 29 had null copy of exon 7 of the SMN1 gene. The results of ddPCR and MLPA were completely consistent.

CONCLUSIONAs a rapid, precise and economically efficient method, ddPCR will provide a new choice for genetic testing of SMA.

Adult ; DNA Copy Number Variations ; Family Health ; Female ; Gene Dosage ; Genetic Predisposition to Disease ; genetics ; Genetic Testing ; methods ; Humans ; Male ; Multiplex Polymerase Chain Reaction ; methods ; Muscular Atrophy, Spinal ; diagnosis ; embryology ; genetics ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; methods ; Reproducibility of Results ; Sensitivity and Specificity ; Sequence Deletion ; Survival of Motor Neuron 1 Protein ; genetics

OBJECTIVETo detect mutation of GLI3 gene in a family affected with autosomal dominant synpolydactyly.

METHODSGenomic DNA was extracted from peripheral blood samples from members of the family and 100 unrelated healthy controls. Potential mutation was screened by next-generation sequencing and confirmed by Sanger sequencing.

RESULTSA heterozygous frameshift mutation c.480dupC was identified in the GLI3 gene among all patients from the family. The same mutation was not found in unaffected family members and the 100 healthy controls.

CONCLUSIONThe c.480dupC of the GLI3 gene probably underlies the synpolydactyly in this family.

Adolescent ; Adult ; Amino Acid Sequence ; Female ; Humans ; Male ; Middle Aged ; Mutation ; genetics ; Nerve Tissue Proteins ; genetics ; Pedigree ; Syndactyly ; genetics ; Zinc Finger Protein Gli3 ; genetics

OBJECTIVETo assess the value of droplet digital PCR (ddPCR) for non-invasive prenatal diagnosis of single gene disease in two families.

METHODSPaternal mutation in cell-free DNA derived from the maternal blood and amniotic fluid DNA was detected by ddPCR. Suspected mutation in the amniotic fluid DNA was verified with Sanger sequencing.

RESULTSThe result of ddPCR and Sanger sequencing indicated that the fetuses have carried pathogenic mutations from the paternal side in both families.

CONCLUSIONDroplet digital PCR can accurately detect paternal mutation carried by the fetus, and it is sensitive and reliable for analyzing trace samples. This method may be applied for the diagnosis of single gene diseases caused by paternal mutation using peripheral blood sample derived from the mother.

Fathers ; Female ; Genetic Diseases, Inborn ; diagnosis ; Humans ; Male ; Maternal Serum Screening Tests ; Mutation ; Polymerase Chain Reaction ; methods ; Prenatal Diagnosis ; methods ; Sequence Analysis, DNA