OBJECTIVETo investigate the type and frequency of mutations in exon 7 of phenylalanine hydroxylase (PAH) gene among children with phenylketonuria (PKU) in Ningxia, China and to provide a basis for the genetic diagnosis and prenatal diagnosis of PKU in this region.

METHODSDirect sequencing of PCR product was performed to analyze the sequences of exon 7 and its flanking introns of 146 PAH alleles in 73 children with typical PKU (39 cases of Hui nationality and 34 cases of Han nationality) in Ningxia.

RESULTSSix mutations were detected, including R243Q (14.4%), R241C (6.8%), IVS7+2T→A (2.7%), L255S (0.7%), G247V (0.7%), and G247R (0.7%). The overall frequency of mutations (missense mutation and splice site mutation) in exon 7 was 26.0% (38/146). The detection rate of R241C mutation was significantly higher in children of Hui nationality than in children of Han nationality(10% vs 3%; P<0.05).

CONCLUSIONSIn Ningxia, R243Q mutation in exon 7 of PAH gene is most common in children with PKU, followed by R241C. The frequency of R241C mutation in exon 7 of PAH gene varies between children with PKU of Hui and Han nationality.

China ; ethnology ; Exons ; Humans ; Mutation ; Phenylalanine Hydroxylase ; genetics ; Phenylketonurias ; genetics

AIMTo develop a simple, fast and inexpensive approach as well as an instrument for detection of gene mutation.

METHODSPyrosequencing based on bioluminometry assay was employed to detect gene mutation. Pyrosequencing is a method of sequencing by synthesis step-by-step using four enzymes, DNA-polymerase, ATP sulfurylase, luciferase and apyrase. The signal was produced by detecting pyrophosphate released during a dNTP incorporation. For mutation detection, a DNA fragment was amplified by PCR at first, followed by a single-stranded DNA preparation. In the second step, a short primer was annealed to the position just before the mutation point. Finally, specific dNTPs were added in terms of the template sequence. The mutation species can be readily determined by the sequence.

RESULTSA new instrument was developed for gene mutation detection by pyrosequencing. To iteratively inject small amount of each dNTP for the sequencing reaction, capillaries were used to connect dNTP reservoirs and the reaction chamber. Each dNTP was delivered by adding a gas pressure on the top of a corresponding dNTP reservoir, by which 0.2 microL of dNTP can be exactly added each time. It was theoretically proved that undesired liquid seep through the capillary did not affect the sequencing reactions in pyrosequencing. In addition, the three possible variants (wildtype, mutant and heterozygote) of a mutant point Cys275Ser in P53 gene exon 8 were determined by pyrosequencing using the instrument. A simple method was also described for rapidly distinguishing the type of a variant.

CONCLUSIONThe developed method is very simple, and the corresponding instrument is inexpensive and easy to operate, which can be used to detect many types of mutation.

Exons ; genetics ; Genes, p53 ; genetics ; Humans ; Luminescent Measurements ; Point Mutation

OBJECTIVETo investigate NPM1 gene mutations in patients with primary myelodysplastic syndromes (MDS) and the clinical characteristics of patients with NPM1 mutants.

METHODSGenomic DNA corresponding to exon 12 of NPM1 gene was amplified by polymerase chain reaction (PCR) in 232 patients with primary MDS. Identification of mutants was by direct sequencing and classification of mutation types by sequencing followed by plasmid cloning.

RESULTSNPM1 mutants were found in 9 patients (3.9%). All the mutants were type A. As compared with those with NPM1 wild type, patients with the mutant were of lower ANC \[0.60 (0.12 - 2.91) × 10(9)/L vs 1.02 (0 - 10.23) × 10(9)/L, P = 0.046\], higher blast percent in bone marrow \[0.050 (0 - 0.090) vs 0.025 (0 - 0.190), P = 0.035\], decreased BFU-E \[0 (0 - 0)/10(5) BMMNC vs 6 (0 - 40)/10(5) BMMNC, P = 0.038\] and increased serum vitamin B(12) \[936.40 (373.80 - 2400.00) pmol/L vs 557.85 (17.00 - 3032.10) pmol/L, P = 0.045\] The chromosomal karyotypes of patients with NPM1 mutant were predominantly normal.

CONCLUSIONMDS patients with NPM1 gene mutations have some unique clinical and laboratory features. The results give new hint for the pathogenesis of MDS development and progression.

Exons ; Humans ; Karyotyping ; Mutation ; Myelodysplastic Syndromes ; genetics ; Nuclear Proteins ; genetics

Child ; Exons ; Hepatolenticular Degeneration ; genetics ; pathology ; Humans ; Male ; Sequence Analysis

OBJECTIVE: To analyze the molecular characteristics of a ABO subgroup. METHODS: The ABO phenotype was determined with the tube method. Exons of the ABO gene were analyzed by Sanger sequencing, and haplotypes of exons 6 and 7 were analyzed by cloning sequencing. RESULTS: By forward typing, the red blood cells showed 3+ agglutination reaction with anti-A and 4+ agglutination with anti-B. A weak reaction with A1 cells and no agglutination reaction with B, O cells by the reverse typing. Sequencing results showed heterozygosity including c.297A>G, c.467C>T, c.526C>G, c.608A>G, c.657C>T, c.703G>A, c.796C>A, c.803G>C, c.930G>A. Cloning sequencing revealed a c.608A>G variant in the A allele compared with the ABO*A1.02. CONCLUSION A new variant site of subtype A of c.608G variation has been identified.
ABO Blood-Group System/genetics* ; Alleles ; Exons ; Genotype ; Heterozygote ; Phenotype

OBJECTIVE: To analyze the different subtypes caused by c.721C>T substitution in the exon 7 of the ABO gene, and to investigate the related molecular mechanism of different antigens expression. METHODS: ABO subtypes in 7 samples were identified by standard serological methods. The exons 6, 7, and adjacent intron of ABO gene were amplified by Polymerase Chain Reaction (PCR), and the PCR products were analyzed by direct DNA sequencing and cloning sequencing. RESULTS: ABO subtypes phenotypes were A CONCLUSION c.721C>T substitution in the ABO gene causes p.Arg241Trp exchange resulting in the decreasing of GTA or GTB activities and weaker antigen expression. O.01.07 is a null allele which cannot form a functional catalytic enzyme has no effect on A
ABO Blood-Group System/genetics* ; Alleles ; Exons ; Genotype ; Mutation, Missense

OBJECTIVE: To determine the genotype of an individual suspected for Aw through DNA sequencing. METHODS: Serologic testing was carried out with standard methods. Exons 6 and 7 of the ABO genes were amplified by PCR and subjected to direct sequencing or sequenced after gene cloning. RESULTS: Serological testing showed that the forward typing and reverse typing were Aw and A, respectively. DNA sequencing revealed that the individual has carried an Aw allele and an O allele. Haplotype sequencing of each allele has revealed a nt543 variant (543G>C) in the Aw allele. CONCLUSION The individual was verified as a rare A subtype, which was previously unreported in mainland China.
ABO Blood-Group System/genetics* ; Alleles ; Exons ; Genotype ; Humans ; Phenotype

OBJECTIVE: To detect pathogenic variants in a pedigree affected with propionic acidemia (PA). METHODS: The proband was subjected to high-throughput next-generation sequencing. Suspected variants were validated by Sanger sequencing of his family members. mRNA was extracted from peripheral blood lymphocytes from the proband's father in order to verify the impact of the splicing variant by RT-PCR combined with Sanger sequencing. The pathogenicity of the missense variant was predicted by using PolyPhen-2, Mutation Taster, SIFT, COBALT and HOPE software. RESULTS: The proband was found to harbor compound heterozygous variants of the PCCB gene, namely c.184-2A>G and c.733G>A (p.G245S), which were respectively inherited from his father and mother. RT-PCR combined with Sanger sequencing confirmed skipping of exon 2 during transcription. Bioinformatic analysis indicated the c.733G>A (p.G245S) variant to be damaging. CONCLUSION The two variants of the PCCB gene probably underlay the disease in this patient. Above findings have enriched the spectrum of PCCB gene variants.
Exons ; Humans ; Mutation ; Mutation, Missense ; Pedigree ; Propionic Acidemia/genetics*

OBJECTIVETo investigate the binding mechanisms of FVIII Trp1707Ser mutation-associated inhibitor.

METHODSThe APPT, PT, TT, Fg and FVIII:C were detected to make phenotypic diagnosis of haemophilia A. Inhibitors titer were measured by Bethesda method. Long distance-PCR (LD-PCR) and sequence-specific PCR were adopted for screening the intron 22 and intron 1 inversions respectively. FVIII coding and boundary sequences were analyzed by direct DNA sequencing. Inhibitor was reacted with different segments of FVIII, including heavy chain and its components A1 and A2, light chain and its components A3, C1 and C2. Corrected test was used to measure the remaining F VIII:C (% ) by adding pooled normal plasmas. After labeling purified inhibitors with biotin, western blot was performed to further confirm the binding reactions between inhibitors and segments.

RESULTSThe haemophilia A patient had mild deficiency of FVIII:C (1.1%) and had high FVIII inhibitor titer of 18.4 BU. A mutation c.97223C>G in exon 14 of F8 gene resulted to p.Trp1707Ser was identified by DNA sequencing. Corrected test showed that the remaining F VIII:C was increased when inhibitors reacted with heavy chain and light chain, especially with heavy chain. The remaining FVIII:C was also increased in the A2 and C2 domain reactions. No significant differences were seen in the A1, A3 and C1 domain reactions. Antigen-antibody reaction bands were confirmed by western blots when degenerated B-domain deleted recombinant FVIII, A2 and C2 were used as antigens.

CONCLUSIONThe binding sites of FVIIITrp1707Ser mutation inhibitor were the A2 domain of heavy chain and C2 domain of light chain. The binding reaction with heavy chain was more intense.

Binding Sites ; genetics ; Exons ; Factor VIII ; antagonists & inhibitors ; genetics ; Hemophilia A ; genetics ; Humans ; Male ; Mutation ; Young Adult

Asian Continental Ancestry Group ; Exons ; genetics ; Humans ; Mutation ; Tuberous Sclerosis ; genetics ; Tumor Suppressor Proteins ; genetics