OBJECTIVETo study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron structure might be related to intron instability.

METHODSJunction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed.

RESULTSAn analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5' and 3' breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34.8% of the overall dystrophin intron sequences is composed of repeat sequences.

CONCLUSIONRepeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.

Dystrophin ; genetics ; Humans ; Introns ; genetics ; Sequence Deletion

Choriocarcinoma/pathology* ; Female ; Gene Deletion ; Humans ; PTEN Phosphohydrolase/genetics* ; Pregnancy ; Sequence Deletion ; Stomach Neoplasms/surgery*

OBJECTIVETo investigate the polymorphism of mitochondrial DNA region V in Bouyei people and Miao people living in Guizhou province of China.

METHODSPolymerase chain reaction and direct sequencing were used in the study.

RESULTSOnly two kinds of polymorphism were found in Bouyei and Miao people. One was standard pattern, the other short pattern. And the frequencies of short pattern(9 bp deletion) in Bouyei and Miao people were 31.1% and 33.3% respectively.

CONCLUSIONThe polymorphisms of mitochondrial DNA region V in Bouyei people and Miao people living in Guizhou province of China are similar, but they are different from those in other people.

Base Sequence ; China ; DNA, Mitochondrial ; chemistry ; genetics ; Female ; Gene Frequency ; Humans ; Male ; Polymorphism, Genetic ; genetics ; Sequence Analysis, DNA ; Sequence Deletion

OBJECTIVETo explore the molecular basis of an individual with Bel variant of the ABO blood group.

METHODSThe ABO antigen and serum antibody of the individual were detected by serological method. All coding regions and flanking introns of the ABO gene were amplified with PCR and sequenced bidirectionally. The haplotypes of the individual were analyzed by cloning and sequencing. A three dimensional model of the mutant protein was constructed and analyzed.

RESULTSThe individual has expressed a very weak B antigen on its red blood cells by absorption and elution testing, which was identified as a Bel variant phenotype. The heterozygous sites in exon 6 (261del/G) and exon 7 (297A/G, 484del/G, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 930G/A) of the coding region of the ABO gene were identified by direct sequencing. Haplotype analysis showed that the individual has carried an O01 allele and a novel B allele. The sequence of the novel B allele was identical to B101 except for a del G at nucleotide position 484 (484delG), which was nominated as B120 by the Blood Group Antigen Gene Mutation Database (dbRBC NCBI). The 484delG mutation of the B allele has led to a reading frame shift and created a premature terminal codon for the glycosyltransferase (GT) enzyme. Prediction of the 3D structure suggested that the GT enzyme has become an incomplete protein only with its N-terminal region.

CONCLUSIONThe 484delG mutation of the glycosyltransferase B gene has probably abolished or reduced the enzymatic activity and resulted in the Bel variant phenotype.

ABO Blood-Group System ; genetics ; Alleles ; Base Sequence ; Exons ; Female ; Genotype ; Glycosyltransferases ; genetics ; Humans ; Molecular Sequence Data ; Mutation ; Sequence Deletion

OBJECTIVE: To explore the potential pathogenetic mutations of primary hypereosinophilia（HEN）by sequencing FGFR1 FLT3, MPL and JAK2 genes, and to clarify their effect on clinical manifestation and prognosis of HEN patients. METHODS: The direct DNA sequencing was employed to detect the gene mutations of FGFR1, FLT3, MPL and JAK2 in HEN patients. RESULTS: One deletion mutation (2654_2753del) within tyrosine kinase domain of FLT3 gene was found in a patient suffered from severe symptoms and ended with dismal outcome, which induced a premature stop codon (G885fsX888). For FGFR1, a new variation described as 1014_1019del AACAGT for nucleotide change was found in 19 cases, resulting in T339_V340del at the protein level. CONCLUSION The deletion of 6 bases in the FGFR1 gene (1014_1019del AACAGT) is first reported as non-synonymous SNP (nsSNP) site in the patients with primary hypereosinophilia. Deletion mutations in the FLT3 gene may be related with malignant clinical features and poor prognosis.
Base Sequence ; Humans ; Hypereosinophilic Syndrome ; genetics ; Mutation ; Receptors, Thrombopoietin ; Sequence Deletion ; fms-Like Tyrosine Kinase 3

OBJECTIVETo detect potential mutation of F8 gene in a family affected with hemophilia type A.

METHODSInverse-shifting PCR (IS-PCR), next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), and short tandem repeat (STR) assays were used.

RESULTSIS-PCR showed that no inversion of F8 gene has occurred in the family. NGS detected no point mutation or small InDel in the proband, but suggested that the exon 2 of the F8 gene may be deleted. MLPA also showed that exon 2 of the F8 gene was absent in the proband, while the carriers were heterozygous for the deletion, though STR analysis yielded a paradoxical result.

CONCLUSIONNGS analysis has identified a large deletion of exon 2 of the F8 gene in a family affected with hemophilia A. Discretion is required when STR analysis was used for carrier screening and antenatal diagnosis. Combination of multiple methods can improve the accuracy for the detection of F8 gene mutations.

Child ; Exons ; genetics ; Factor VIII ; genetics ; Genetic Testing ; methods ; Hemophilia A ; Humans ; Male ; Pedigree ; Sequence Deletion ; genetics

OBJECTIVETo investigate the frequencies of chemokine (C-C motif) receptor 5 gene (CCR5)Δ32 deletional mutation of in Han and Dai populations from Yunnan province. Immortalized cell lines were derived from a family carrying the CCR5Δ32 mutation.

METHODSBlood samples of 346 Han and 355 Dai individuals were collected for genotyping. The coding regions of CCR5 gene were amplified with PCR followed by agarose gel electrophoresis. Suspected mutations were verified with DNA sequencing. Immortalized cell lines were constructed by using Epstain Barr virus and cyclosporine A. The difference between the cell lines and original blood samples was verified with PCR.

RESULTSOne ethnic Han individual was confirmed to be heterozygous for a deletional mutation by sequencing, which has led to discovery of a family with CCR5Δ32. Nine immortalized cell lines were established from this family, and no difference between the cell lines and original blood samples was detected by PCR.

CONCLUSIONTogether with previous reports, this study has indicated a significant difference in CCR5Δ32 among different ethnic groups in China. Established immortalized cell lines can also provide material for future research.

Base Sequence ; China ; Ethnic Groups ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Receptors, CCR5 ; genetics ; Sequence Deletion

OBJECTIVETo explore F (13) A gene mutation in a pedigree with hereditary coagulation factor XIII (FXIII) deficiency.

METHODSThe FXIII deficiency was diagnosed by clot solubility test and other standard laboratory clotting tests. All exons, exon-intron boundary sequences of F(13) A gene were amplified by PCR and the products were sequenced directly. Any mutation identified by direct sequencing was confirmed by reverse sequencing. The mutation identified in the proband was screened in the family members.

RESULTSThe assays of PT, Qiulan, fibrinogen leveling, platelet counts, bleeding time were normal and the clot solubility test was positive in the proband. The homozygous deletion of 33 nucleotides (127067de133) in exon 10 of F(13) A gene which resulted in deletion of 11 amino acids in FXIIII A protein with 720aa residues was identified in the proband. Family studies showed that the mutation was inherited from the parents both of whom carried the heterozygous deletion mutation.

CONCLUSIONThe homozygous 127067de133 mutation of F(13) A gene is responsible for the disorder of the pedigree.

Adolescent ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Heterozygote ; Homozygote ; Humans ; Male ; Pedigree ; Sequence Deletion

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.
Chromosomes, Bacterial ; genetics ; DNA ; Endonucleases ; metabolism ; Escherichia coli ; genetics ; Genetic Engineering ; methods ; Recombination, Genetic ; Sequence Deletion

OBJECTIVE: To explore the characteristics of PAH gene variants among 113 phenylketonuria patients from Henan Province. METHODS: The 13 exons of the PAH gene were subjected to PCR amplification and direct sequencing. Large fragment deletion and duplication of the PAH gene were detected with a multiple ligation-dependent probe amplification (MLPA) assay. RESULTS: In total 195 point variants and 3 large fragment deletions were detected among the 226 alleles, with the detection rates being 86.28% and 1.33%, respectively. Variants of p.Arg243Gln (18.14%), p.Arg111X (6.19%), p.Arg53His (5.31%), EX6-96A>G (5.31%), p.Tyr356X (4.87%) and p.Val399Val (4.42%) were relatively common. Most of the variants were located in exons 7, 11, 3 and 6. Missense variations were most common. Four novel variations were detected, which included c.1016C>A (p.Ser339Tyr), c.1000T>C (p.Cys334Arg), c.1110G>T (p.Glu370Asp), and IVS6+1G>T. CONCLUSION The PAH gene variations in Henan Province have featured extensive allelic heterogeneity and variety.
China ; Exons ; Humans ; Mutation, Missense ; Phenylalanine Hydroxylase ; genetics ; Phenylketonurias ; genetics ; Point Mutation ; Sequence Deletion