Carrier diagnosis of F9 gross deletion by multiple ligation-dependent probe amplification in hemophilia B / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the application of multiplex ligation-dependent probe amplification (MLPA) in the gene diagnosis of hemophilia B (HB).</p><p><b>METHODS</b>MLPA and linkage analysis of short tandem repeat (STR) were used for gene diagnoses of two HB families with gross deletions of F9 gene, which were negative by sequencing.</p><p><b>RESULTS</b>The MLPA results indicated the loss of one or two exons in the two patients with the ratio lower than 0.10. Their mothers showed a ratio average of 0.50 ± 0.05 for the corresponding probes, which revealed she was carrier of large deletions of the F9 gene. The ratios of three sisters of the HB patients were normal, which indicated they were non-carriers. Linkage analysis was consistent with MLPA, but sequencing was not conclusive.</p><p><b>CONCLUSION</b>This report illustrated that MLPA technique represented an efficient method to screen F9 gene gross deletions in sequencing undiagnosed carriers of hemophilia B.</p>

Application of multiplex ligation-dependent probe amplification to diagnosis and prenatal diagnosis of common aneuploidies / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the application value of the multiplex ligation-dependent probe amplification (MLPA) technique in diagnosis and prenatal diagnosis of chromosomes 13, 18, 21, X and Y aneuploidy.</p><p><b>METHODS</b>Forty-four cases including 30 peripheral blood samples, 10 fetal cord blood samples, and 4 amniotic fluid samples were collected in this study. DNA was isolated from the samples and detected by MLPA, followed by analyzing in ABI310 Genetic Analyzer. Analysis of copy number changes for chromosomes 13, 18, 21, X and Y was carried out with RH-MLPA-analysis software. The routine karyotype analyses were also done for all the samples.</p><p><b>RESULTS</b>Of 44 samples, the results of 42 by MLPA method was consistent with that by chromosome karyotyping. Only one case with trisomy 21 chimerism was failed to reach conclusion. In addition, one case of mark chromosome segment was identified as Y-chromosome segment by MLPA, while karyotyping failed to make judgment. The accurate rate of MLPA was 97.7% (43/44).</p><p><b>CONCLUSION</b>The MLPA technique can simultaneously detect dozens of different target sequences and their copy number changes in a single reaction. It showed high specificity, good reproducibility, was fast and high-throughput. The MLPA technique can be applied to diagnosis and prenatal diagnosis of the common chromosomal aneuploidy.</p>

Detection of common deletions and mutations causing α-thalassemia in Southeast Asians and Southern Chinese with denaturing high performance liquid chromatography / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a comprehensive and simple assay using denaturing high performance liquid chromatography (DHPLC) for the diagnosis of most common mutations and deletions of α-thalassemia gene in Southeast Asians and Southern Chinese.</p><p><b>METHODS</b>This assay has included a duplex polymerase chain reaction (PCR) followed by DHPLC analysis. An improved PCR was also performed followed by DHPLC analysis. With this assay, a blinded study of 160 samples was screened for three common mutations and three common deletions.</p><p><b>RESULTS</b>The duplex PCR-DHPLC combined with the improved PCR-DHPLC analysis has detected all mutations and the wild-type allele. The results were consistent with those by the original methods.</p><p><b>CONCLUSION</b>This molecular assay may be used for the diagnosis of α-thalassemia patients from this geographical region. The method is accurate, rapid, semi-automatic and cost-effective, which makes it suitable for large-scale screening.</p>

Improvement and application of DXS52(St14) in gene diagnosis of hemophilia A / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To improve the experimental method of DXS52 (St14) and apply it to genetic testing for hemophilia A (HA).</p><p><b>METHODS</b>PCR of DXS52 and agarose gel electrophoresis were performed for genetic testing in 61 non-inversion HA families. Linkage analysis of 7 loci within the FVIII gene including Bcl I, Hind III, Xba I, STR1, STR13, STR22 and STR24 were also carried out for the 61 families.</p><p><b>RESULTS</b>DXS52 can provide information in 43 out of 61 families and the diagnostic rate was 70.5%. Eight families can be diagnosed only by DXS52 locus, accounting for 13.1%. Two families were found to have recombination between DXS52 and FVIII.</p><p><b>CONCLUSION</b>The new experimental conditions can reach accurate and clear results in DXS52 genetic testing. This gene maker has high diagnostic rate, so it is an indispensable linkage analysis method in HA gene diagnosis. More caution should be paid when using the extragenic locus DXS52 to perform gene diagnosis because of its high recombinant rate with FVIII.</p>

Identification of seven novel mutations in the factor VIII gene in 18 unrelated Chinese patients with hemophilia A / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hemophilia A (HA) is an X-linked inherited bleeding disorder caused by decreased activity of factor VIII (FVIII) due to heterogenous mutations in the FVIII coding gene (F8). The type of mutation plays an important role in the FVIII inhibitor formation. To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we reported the distribution of the F8 gene mutations in 18 unrelated Chinese patients with HA.</p><p><b>METHODS</b>Intron 22 and intron 1 inversions in the F8 gene were screened in 158 unrelated patients with HA using a long-distance PCR and multiplex PCR method. Direct sequencing of the coding region of the F8 gene was used to identify the mutations responsible for HA in 18 unrelated Chinese HA patients who were negative for intron 22 and intron 1 inversions; sequences were compared with the HAMSTeRS database. A clotting method was used to assay the FVIII activity level and the Bethesda assay was used to detect the FVIII inhibitor.</p><p><b>RESULTS</b>A total of 18 different HA F8 mutations were identified, seven of which were described for the first time. These novel mutations included five small deletions, one point mutation and one small insertion. One novel mutation (4382-3 AC deletion) was associated with inhibitor development.</p><p><b>CONCLUSION</b>These data extend our insight into the mechanisms by which novel amino acid mutations may lead to HA and how the HA patient genotypes influence the risk of FVIII inhibitor.</p>

Detection of factor VIII intron 1 inversion in severe haemophilia A / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Screening the intron 1 inversion of factor VIII (FVIII) in the population of severe haemophilia A(HA) in China and performing carrier detection and prenatal diagnosis.</p><p><b>METHODS</b>Using LD-PCR to detect intron 22 inversions and multiple-PCR within two tubes to intron 1 inversions in severe HA patients. Carrier detection and prenatal diagnosis were performed in affected families. Linkage analysis and DNA sequencing were used to verify these tests.</p><p><b>RESULTS</b>One hundred and eighteen patients were seven diagnosed as intron 22 inversions and 7 were intron 1 inversions out of 247 severe HA patients. The prevalence of the intron 1 inversion in Chinese severe haemophilia A patients was 2.8% (7/247). Six women from family A and 2 from family B were diagnosed as carriers. One fetus from family A was affected fetus.</p><p><b>CONCLUSION</b>Intron 1 inversion could be detected directly by multiple-PCR within two tubes. This method made the strategy more perfective in carrier and prenatal diagnosis of haemophilia A.</p>

A study on the (CA)n in FVIII gene in Han ethnic group in Guangxi Zhuang Autonomous Region by amplification polymorphisms combined with silver staining / 中华儿科杂志

<p><b>OBJECTIVE</b>Hemophilia A is an inherited bleeding disorder caused by defects in factor VIII (FVIII) gene. In the present study, the frequencies of the microsatellite alleles at introns 13 and 22 in the factor VIII gene were analyzed in the group of Han nationality in Guangxi Zhuang Autonomous Region to explore their diagnostic value for hemophilia A. These two sites were also used to detect the carriers in 13 hemophilia A families.</p><p><b>METHODS</b>Ninty-one individuals of Han ethnic group in Guangxi Zhuang Autonomous Region (135 X chromosomes) and 13 HA families were subjected to molecular studies. First, these two fragments were PCR amplified simultaneously. Then, silver staining was used later to show their polymorphisms. The investigators selected one sample at random to obtain its lengths of the PCR products at these two sites by ABI310 PCR amplifier. After counting its repeated numbers of (CA) according to the documents concerned, the repeated numbers of the other samples could be counted easily.</p><p><b>RESULTS</b>In the 91 individuals, 6 and 4 alleles were detected at these two sites, respectively. At intron 13 the allele frequencies ranged from 0.0002 to 0.5408 and polymorphism information content (PIC) was 0.5899. At intron 22 the allele frequencies ranged from 0.0444 to 0.4963 and its PIC was 0.5359. The actual heterozygosity for intron 13 and intron 22 were 0.6364 (28/44) and 0.5227 (23/44), respectively. In 13 hemophilia A families with positive history, 9 of them were diagnosed by this method and the diagnosis rate was 69%.</p><p><b>CONCLUSION</b>With high PICs, (CA)n at intron 13 and intron 22 were two valuable sites in the diagnosis of hemophilia A in the population of Han ethnic group in Guangxi Zhuang Autonomous Region. Compared with some other HA restrictive fragment length polymorphisms (RFLP), intron 22 (GT)n (AG)n was more informative.</p>

Detection of alpha thalassemia using real-time PCR and dissociation curve analysis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish an automatic, high throughput, quick detection method of alpha thalassemia.</p><p><b>METHODS</b>The genotypes of -alpha(4.2) and -alpha(3.7) were detected by two real-time fluorescence PCRs using SYBR-Green1 (SYBR-PCR) with the analysis of dissociation curve (DC) and melting temperature (Tm). The PCR products were recombined into T-vector and the correct cloning was selected as positive control. Sensitivities were gained from a serious dilution of the recombinant as SYBR-PCR template, from which detection deadlines for two kinds of alleles could be determined. Totally 110 samples were detected by this technique.</p><p><b>RESULTS</b>The length of product of -alpha(4.2) and -alpha(3.7) were 1.65 kb and 1.9 kb respectively, and the Tm were (81.5+/-0.5) degrees C and (82.5+/-0.5) degrees C respectively. The detection deadline were 9 x 10(2 ) copies and 4.3 x 10(2) copies respectively. The sensitivity of the technique was much higher than that of regular PCR plus gel electrophoresis method, and the detection results of the technique were the same as that of multiplex PCR.</p><p><b>CONCLUSION</b>The genotypes of -alpha(4.2), -alpha(3.7), non-deletion alpha alpha/and --(SEA) can be sensitively, exactly diagnosed by the real-time PCR with SYBR-Green1 combined with the dissociation curve analysis. The assay is automatic, low-cost, high throughput, and easy for quality control without fluorescence probe.</p>

Improvement of gene analysis method in hemophilia A and its application of prenatal diagnosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a simple and rapid system for carrier detection and prenatal diagnosis in hemophilia A (CHA) family.</p><p><b>METHODS</b>Long distance-polymerase chain reaction (LD-PCR) was selected for detection factor VIII intron 22 inversion. Polymorphism of factor VIII intragenic restriction fragment length polymorphism (RFLP) of Xba I and Hin d III, short tandem repeat (STR) within intron 13 and 22, as well as extragenic DXS52 (ST 14) variable number of tandem repeat (VNTR) were assayed by PCR and linkage analysis.</p><p><b>RESULTS</b>Seventy-one females were diagnosed as carriers within 52 HA families. Twenty-one families were diagnosed to be factor VIII intron 22 inversion and 28 families were diagnosed by linkage analysis, whereas 3 families could not been diagnosed. Seventeen of 18 fetuses at risk were male. Ten of 17 male fetuses were shown to be affected and were subsequently aborted. Seven male fetuses were diagnosed to be not affected. One female fetus was identified to be HA carrier. One-year follow-up study demonstrated that these babies were normal and living well.</p><p><b>CONCLUSION</b>LD-PCR combined with multiple locus linkage analysis enables the direct and indirect detection of HA for carrier testing and prenatal diagnosis.</p>

Fifteen short tandem repeat polymorphisms in Han race of North China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To research on the genetic polymorphism distributions of 15 short tandem repeat (STR) loci in Han race of North China and the genetic data of population genetics.</p><p><b>METHODS</b>The capillary electrophoresis and five-color fluorescent multi-amplifying were applied to detect the genotypes of 15 STR loci in 597 unrelated Chinese Han individuals.</p><p><b>RESULTS</b>No significant deviation from the Hardy-Weinberg Equilibrium was observed. High polymorphism was detected in the loci. Statistical analysis was carried out to obtain some parameters of forensic medicine. The heterozygosity of 15 loci was above 0.62. The values of discrimination power (DP) at 15 STRs ranged from 0.802 to 0.967. The values of excluding probability of paternity (EP) ranged from 0.320 to 0.697. The values of probability matching (Pm) ranged from 0.033 to 0.198. The fifteen loci showed an accumulated total discrimination power (TDP) more than 0.999999, a cumulative excluding probability of paternity (CEP) as 0.99999571, and total probability matching to be 8.93 x 10(-18).</p><p><b>CONCLUSION</b>The data indicated that detecting combined 15 STRs is sensitive and reliable, and can be used to forensic and individual identification cases in Chinese group.</p>

Analysis of X ba I polymorphism of FVIII gene and its application on prenatal diagnosis for hemophilia A / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish the linkage methods of X ba I polymorphisms specific for FVIII gene intron 22, and to find a rapid and simple system for haemophilia A (HA) carrier detection and prenatal diagnosis.</p><p><b>METHODS</b>A long PCR to amplify FVIII gene intron 22 followed by X ba I digestion was used to assay the gene rate and heterozygosity rate of 206 unrelated people. Detection of intron 22 inversion by long distance PCR (LD-PCR) and XbaI, BclI, Hind III, DXS52, STR polymorphism within intron 13 and 22 by hereditary linkage analysis were assays in 20 HA pedigrees.</p><p><b>RESULTS</b>The gene rate and polymorphism information contents of 206 people were 0.5475 and 0.4955 respectively, 7 of 20 HA families were diagnosed as intron 22 inversion, 6 of 13 non-inversion HA families were diagnosed by X ba I linkage analysis, 8 of 13 non-inversion HA families were diagnosed by two or more linkage analysis.</p><p><b>CONCLUSIONS</b>The improved X ba I linkage analysis is a specific and useful molecular diagnosis marker. LD-PCR and five-linkage analysis can be used in prenatal HA gene diagnosis.</p>

High-level expression of phenylalanine ammonia-lyase in Lactococcus Lactis via synthesized sequence based on bias codons / 生物工程学报

To construct a safer and more efficient gene engineering Lactococcus Lactis for expressing phenylalaine ammonia lyase (PAL) which will be benefit for PKU therapy, pal cDNA of Parsly and synthesized sequence based on Lactococcus Lactis bias codons were recombined into two Lactococcus Lactis NICE systems. The activities of the expressed PAL were detected, and the effect of Lactococcus Lactis bias codons on the expression of exterior protein was analyzed. The results showed that the expression level of PAL was increased by using Lactococcus Lactis bias codons in both Lactococcus Lactis NICE systems. Through which several safer andmore efficient strains of the gene engineering Lactococcus Lactis were obtained.

Study on genetic diagnosis and prenatal diagnosis of alpha-thalassemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To develop a single-tube multiplex polymerase chain reaction (mPCR) technique to detect three common deletional alpha-thalassemias (alpha-Thal) in Chinese, and to perform genetic diagnosis and prenatal diagnosis for an alpha-Thal family from Hebei province, China.</p><p><b>METHODS</b>Fourty-two blood samples including samples from one alpha-Thal family from Hebei province were assayed. The mPCR containing 7 primers, gel electrophoresis and DNA sequencing were used for the genetic diagnosis and prenatal diagnosis.</p><p><b>RESULTS</b>The gene types of the fourty-two DNA samples analyzed by the mPCR-gel electrophoresis technique were in accordance with the results by Southern blot and three separate PCR techniques. A HbH child and a fetus of the alpha-Thal family were diagnosed as--(SEA)/alpha(cs)alpha and alpha alpha/alpha alpha respectively by using the mPCR and DNA sequencing. The result of postnatal analysis of the cord blood was consistent with the prenatal result (alpha alpha/alpha alpha).</p><p><b>CONCLUSION</b>The developed mPCR technique can be used for genetic diagnosis and prenatal diagnosis of the 3 deletional alpha-Thal in Chinese.</p>

Detection of gene expression alteration of myeloma cells treated with arsenic trioxide / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of arsenic trioxide on multiple myeloma (MM) cell gene expression and explore the molecular mechanism of arsenic trioxide therapy for MM.</p><p><b>METHODS</b>U266 cells were divided into two groups, group A as control group and group B as test group. Cells were cultured for 48 hours, and total RNA and mRNA were extracted. Suppression subtractive hybridization (SSHs) was performed to distinguish the differentially expressed genes. The products were cloned into pGEM-T Easy Vector, and transfected into the competent host JM109 to construct two subtractive libraries. Positive colonies were selected by blue-white screening, and the plasmids were extracted. Homologous comparison was conducted in GenBank.</p><p><b>RESULTS</b>Five downregulated clones were isolated in the first SSH: (1) Aminopeptidase N, (2) Homosapiens tumor translationally-controlled protein 1, (3) Human ATP synthetase A chain, (4) Signal recognition particle A10, (5) Mitochondrial ATP synthetase/ATPase subunit 6. Four upregulated clones were isolated in the second SSH: (1) Calcium-binding protein A10, (2) Keratin 6A, (3) 45 kD MIP repetitive element containing splicing factor and (4) poly(A)-binding protein.</p><p><b>CONCLUSIONS</b>Arsenic trioxide exerts proliferation inhibition and apoptosis induction on MM cells by regulating genes expression.</p>

Relationship between polymorphisms of genes encoding microsomal epoxide hydrolase and glutathione S-transferase P1 and chronic obstructive pulmonary disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD). However, only 10% - 20% of chronic heavy cigarette smokers develop symptomatic disease. COPD is most likely the result of complex interactions between environmental and genetic factors. Genetic susceptibility to COPD might depend on the variations in enzyme activities that detoxify cigarette smoke products, such as microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST). In this study, we investigated the relationship between polymorphisms in the genes encoding mEH and glutathione S-transferase P1 (GSTP1) and COPD in a Chinese population.</p><p><b>METHODS</b>Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find mEH polymorphism in exon 3 (Tyr113-->His), exon 4 (His139-->Arg) and GSTP1 polymorphism in exon 5 (Ile105-->Val) in 100 COPD patients and 100 age- and sex-matched healthy controls.</p><p><b>RESULTS</b>The proportion of mEH exon 3 heterozygotes was significantly higher in patients with COPD than that in the control subjects (42% vs 32%). The odds ratio (OR) adjusted by age, sex, body mass index (BMI) and cigarette years was 2.96 (95% CI 1.24 - 7.09). There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and the controls. When COPD patients were non-smokers, the OR of very slow activity genotype versus other genotypes was more than 1.00; and when COPD patients were smokers (current smokers and ex-smokers), the OR was less than 1.00. There was no significant difference in GSTP1 polymorphism adjusted by age, sex, BMI and smoking between COPD patients and the controls.</p><p><b>CONCLUSIONS</b>mEH exon 3 heterozygotes might be associated with susceptibility to COPD in China. The interaction might exist between mEH genotype and smoke. The gene polymorphism for GSTP1 might not be associated with susceptibility to COPD in the Chinese population.</p>

The progressive study on gene therapy for hyperphenylalaninemia rats / 生物工程学报

To construct a new high effective genetic engineering strain which can express active PAL enzyme in Lactococcus lactis (L.L), and acquire better effect on curing hyperphenylalaninemia rats, Firstly translational fusion vector and transcriptional fusion vector were constructed in E. coli MC1061, and then PAL cDNA was transformed into L.L. Two kinds of high effect strain were compared with their enzyme activity and animal experiment was carried out. The results showed: (1) Two kinds of engineering L.L. were obtained and translational fusion strain has higher level enzyme activity. (2) The amount of transcinnamic aicd reach peak when induced for 6 hours. (3) The blood phe level of the treated rats was significantly reduced compared with non-treated rats when receiving fresh p(NZ8048-PAL)1/NZ9000. The engineering L.L(translational fusion strain) can significantly reduce the blood phe level of the hyperphenylalaninemia rats, which has more superiority than pMG36e-PAL/L. L.