FⅨ gene all exons sequencing technology in hemophilia B gene carriers detection and the application of prenatal gene diagnosis / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a feasible protocol to provide genetic diagnosis and prenatal diagnosis in Chinese hemophilia patients and their relatives by direct exon sequencing.</p><p><b>METHODS</b>In our study, genetic diagnosis was performed on 5 unrelated families with informed consent, which included 3 pregnant women who asked for prenatal diagnosis. Umbilical cord blood was obtained from 2 fetuses and amniotic fluid from another fetus. After extraction of the genomic DNA, all of the exons, exon-intron boundaries and their flanks of FⅨ gene were amplified by polymerase chain reaction (PCR). PCR products were detected through direct-sequencing.</p><p><b>RESULTS</b>Sequence analysis indicated that the patients and carriers from 5 families have the pathogenic mutations,c.1022G>A (p.R341Q), c.484 C>T (p. R162X), c.1135C>T (p.R379X), c.799C>T (p.H267Y), c.1232G＞T (p.S411I), respectively. Except c.484 C>T (p. R162X), 4 of the 5 mutations were reported firstly in Chinese population. During prenatal diagnosis, one of the fetuses was found to be affected with c.484C>T; p.R162X. The remaining two fetuses were diagnosed as normal, the results of which were later verified by post-birth diagnosis, and factor FⅨ activities in plasma was 52.7% and 76.2%, respectively.</p><p><b>CONCLUSION</b>In the quest of strict quality control, exon sequence on FⅨ gene was a rapid and accurate method for genetic diagnosis and prenatal diagnosis of hemophilia B.</p>

Genotype of thalassemia genes and the polymorphism of β- globin gene in Cantonese / 中华血液学杂志

<p><b>OBJECTIVE</b>To understand the genotype of α and β-globin, as well as the polymorphism of β-globin gene in Cantonese in recent years, and to provide an effective genetic diagnosis for thalassemia (thal).</p><p><b>METHODS</b>The single-tube complex PCR was used to detect 3 types of deletional α-thal, reverse dot blotting (RDB)/PCR to detect 3 kinds of undeletional α-thal-αCS, αQS, αWSand 18 kinds of β-thal mutations which were common in Chinese population. A total of 454 cases from Guangdong were undergone thal genotype genetic diagnosis. Among the 454 cases, 142 cases were selected to perform the single nucleotide polymorphisms (SNPs) analysis of β- globin gene by denaturing high-performance liquid chromatography (DHPLC)combining the whole gene sequencing.</p><p><b>RESULTS</b>Of the 454 cases, 438 were diagnosed as thalassemia, including 246 of α-thal, 164 of β-thal and 28 of αβ-thal. In 246 α-thal cases, deletions were the dominant mutations, including 197 cases of αα/--(SEA), 20 of αα/-α(3.7) and 9 of αα/-α(4.2). In 164 β- thal cases, heterozygotes accounted for 92.7% (152/164), the main genotypes were CD41- 42, IVS-II-654, ﹣28 and CD17, and the dual heterozygotes and homozygotes accounted for 4.9% (8/164) and 2.4% (4/164), respectively. The result of β-globin gene screening by DHPLC combining with sequencing was consistent with that of RDB. Moreover, we also found 9 kinds of SNP, in which 2 were unreported, the IVS-I-13 G> A and IVS-II- 310 T>C. In the tested samples, the frequency of 4 kinds SNP was high, among which 3 kinds SNPs-rs713040, rs10768683 and rs1609812 were carried together.</p><p><b>CONCLUSION</b>The dominant genotypes were αα/--(SEA) in α-thal cases, CD41-42, IVS-II-654, -28 and CD17 in β-thal. The frequency of β-thal heterozygotes, homozygotes and αβ-thal is high. DHPLC combining the whole β-globin gene sequencing can effectively detect the common β-thal mutation and even new mutations or SNPs. In Cantonese, the frequency of SNP rs713040, rs10768683, rs7480526 and rs1609812 of β-globin gene was high, and there may exist genetic linkage between rs713040, rs10768683 and rs1609812.</p>

Rapid prenatal genetic diagnosis of a fetus with a high risk for Morquio A syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To provide rapid and accurate prenatal genetic diagnosis for a fetus with high risk of Morquio A syndrome.</p><p><b>METHODS</b>Based on ascertained etiology of the proband and genotypes of the parents, particular mutations of the GALNS gene were screened at 10th gestational week with amplification refractory mutation system (ARMS), denaturing high performance liquid chromatography (DHPLC), and direct DNA sequencing.</p><p><b>RESULTS</b>DHPLC screening has identified abnormal double peaks in the PCR products of exons 1 and 10, whilst only a single peak was detected in normal controls. Amplification of ARMS specific primers derived a specific product for the fetus's gene, whilst no similar product was detected in normal controls. Sequencing of PCR products confirmed that exons 1 and 10 of the GALNS gene from the fetus contained a heterozygous paternal c.106-111 del (p.L36-L37 del) deletion and a heterozygous maternal c.1097 T>C (p.L366P) missense mutation, which resulted in a compound heterozygote status.</p><p><b>CONCLUSION</b>The fetus was diagnosed with Morquio A syndrome and a genotype similar to the proband. Termination of the pregnancy was recommended. Combined ARMS, DHPLC and DNA sequencing are effective for rapid and accurate prenatal diagnosis for fetus with a high risk for Morquio A syndrome. Such methods are particularly suitable for early diagnosis when pathogenesis is clear. Furthermore, combined ARMS and DHPLC are suitable for rapid processing of large numbers of samples for the identification of new mutations.</p>

Prenatal diagnosis of oculocutaneous albinism type IV and discovery of a novel mutation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To provide guidance for clinical genetic counseling and prenatal diagnosis of oculocutaneous albinism (OCA) in China.</p><p><b>METHODS</b>PCR and automatic DNA sequencing were applied to obtain the genotypes of the patients and their parents in three Chinese albinism families. Prenatal gene diagnoses were performed at early pregnancy by chorionic villus sampling (CVS) or by amniocentesis at mid-pregnancy.</p><p><b>RESULTS</b>The three patients were all OCA4, whose genotypes were G349R/c.870delC, G349R/P419L and G349R/D160H, respectively. The three couples had been diagnosed as carriers. In family 1, the first fetus was diagnosed as affected. Termination of pregnancy was opted following genetic counseling. The second fetus (monozygotic twin) was heterozygous only with the paternal G349R mutation. The fetus in family 2 did not get either one of the two mutations. The fetus in family 3 was heterozygous only with the paternal G349R mutation.</p><p><b>CONCLUSION</b>This study detected three reported pathogenic mutations of the membrane associated transporter protein gene (MATP), including G349R, D160H and P419L, and identified a novel pathogenic mutation c.870delC. The prenatal gene diagnosis of OCA4 will be important to prevent the birth of affected child.</p>

Improvement of I-PCR and its application in gene diagnosis of hemophilia A / 中华血液学杂志

<p><b>OBJECTIVE</b>To improve inversion-polymerase chain reaction (I-PCR) in detection of factor VIII (FVIII) intron 22 inversion for gene diagnosis and prenatal diagnosis of hemophilia A (HA).</p><p><b>METHODS</b>The modified I-PCR was applied to detect FVIII intron 22 inversion in 8 families with HA. The prenatal diagnosis was performed for 2 pregnant women in the families. The fetal blood samples were obtained by cordocentesis at 22 and 26 weeks gestation respectively.</p><p><b>RESULTS</b>Four of 8 HA families were diagnosed to be FVIII intron 22 inversion. Two fetuses were identified to be normal and one of them was born normal.</p><p><b>CONCLUSION</b>The modified I-PCR enables the gene diagnosis and prenatal diagnosis of FVIII intron 22 inversion more accurately and rapidly.</p>

Denaturing high-performance liquid chromatography technique platform applied to screen G6PD deficient variants / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Of denaturing high performance liquid chromatography (DHPLC), a technique platform was developed for screening G6PD deficient variants.</p><p><b>METHODS</b>When applied to screen and identify the G6PD deficient variants from 124 patients who come from 11 nations in China, the DHPLC was compared with amplification refractory mutation system (ARMS) or DNA sequence technique and assessed carefully in its accuracy, sensitivity, efficiency and the cost of experiment.</p><p><b>RESULTS</b>The G6PD-deficient variants, such as 1388 G-->A (36/124 cases), 1376 G-->T(35), 95 A-->G (14), 1024 C-->T (3), 392 G-->T (4), 871 G-->A /1311 C-->T /IVS XI +93 t-->c (9), 871 G-->A (1), 1311 C-->T/IVS XI +93 t-->c (4), 1376 G-->T /1388 G-->A (1) and so on, were characterized as sharp peaks by DHPLC and verified by DNA sequence. Further, the standard chromatograms were put into database for 8 kinds of common G6PD deficient variants in Chinese populations. And also DHPLC found 3 G6PD variants (1388 G-->A) from 103 negative controls. With taking 8.8 minutes and costing 1 dollar for each sample, DHPLC successfully detected and identified 34 heterozygous females from patients with G6PD deficiency. However, ARMS checked 83 positive controls but got 12 false G6PD mutants, of which 5 were false positive, 7 false negative. Above results show that DHPLC sounds like to be more convenience, sensitive and accurate than ARMS and DNA sequence techniques for checking G6PD mutants.</p><p><b>CONCLUSION</b>DHPLC is of great advantage to screen the G6PD deficient variants with accuracy, convenience, automation and less cost, and significantly to identify the female heterozygote and clinical type IV individuals with G6PD deficiency.</p>

Complex mutations of 1311 C-->T in exon 11 and 93 T-->C in intron 11 in G6PD gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between complex 1311 mutation of C-->T in exon 11 and 93 T-->C in intron 11 of G6PD gene and the G6PD deficiency.</p><p><b>METHODS</b>Using NBT paper strip method to screen and quantitative NBT method to confirm G6PD deficiency. PCR-SSCP technique was used to find the abnormal exon 11 and the amplification refractory mutation system (ARMS) to identify 1311 mutation, and DNA sequencing to identify the complex mutation at 1311 in exon 11 and 93 in intron 11.</p><p><b>RESULTS</b>Abnormal band in exon 11 was found in 12 cases. DNA sequencing showed that they were 1311 mutation together with 93 mutation.</p><p><b>CONCLUSION</b>This complex mutation may be the cause of reduced activity of G6PD enzyme.</p>

Identification of G6PD gene variants from Hakka population in Guangdong province / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Studying on G6PD polymorphism from Hakka population in Guangdong province.</p><p><b>METHODS</b>Identifying the variants of G6PD gene and determining the frequencies respectively with the use of amplified refractory mutation system(ARMS), polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) and ABI 3100 DNA sequencing technologies.</p><p><b>RESULTS</b>Mutations of G6PD gene in cDNA 1388 (G-->A), 1376 (G-->T), 95 (A-->G), 392 (G-->T), 1024 (C-->T), 1311 (C-->T) have been found.</p><p><b>CONCLUSION</b>G6PD cDNA 1388 (G-->A), 1376 (G-->T), 95(A--> G), 392 (G-->T), 1024 (C-->T) and 1311 (C-->T) accompanied with intron 11 (93 T-->C) are the common mutations in Chinese population. cDNA 1388 (G-->A), cDNA 1376 (G-->T) are the most popular G6PD gene variants in Hakka population. In this study, no new type of G6PD gene mutation was found in the Hakkas of Guangdong.</p>