Shu-Zhen Huang

Missense mutations prevalent in orientals with phenylketonuria: Molecular characterization and clinical implications

Two missense mutations in the phenylalanine hydroxylase (PAH) genes of Orientals with phenylketonuria (PKU) have been identified. A G-to-A transition in exon 7 of the gene results in the substitution of Gln243 for Arg243 (R243Q) and accounts for 18% of all PKU chromosomes among Chinese. An A-to-G transition in exon 6 of the gene results in the substitution of Cys204 for Tyr204 (Y204C) and identifies about 13 and 5% of all PKU chromosomes in the Chinese and Japanese populations, respectively. The R243Q construct produced less than 10% of normal PAH activity in in vitro expression analysis in a eukaryotic cell system, and patients homozygous for this substitution exhibit a severe clinical phenotype. These results are consistent with previous findings in this expression system. The Y204C construct, however, produced near normal levels of PAH enzyme activity and immunoreactivity in this in vitro expression system. Because this substitution is present only on PKU chromosomes, it is a valuable marker for identifying the corresponding mutant allele for carrier screening of PKU. With the characterization of these two substitutions, about 60% of PKU alleles in China can now be identified. The continuing search for additional PKU mutations will permit effective carrier screening and prenatal gene diagnosis of PKU in East Asia.

Detection of β-thalassemia mutations in the Chinese using amplified DNA from dried blood specimens

SummaryThis paper describes DNA polymerase chain reaction (PCR) amplification directly from dried blood specimens for the detection of the β-thalassemia mutation in China. Target DNA was amplified to span the β-globin gene regions, which included ten types of mutation sites specific for Chinese β-thalassemias. Ten kinds of oligonucleotide probes were constructed and used to hybridize with the amplified DNA. A total of 170 β-thalassemia alleles originating from eastern, southwestern and southern China were analyzed. The results revealed that the distributions of different types of mutations were different in the three regions. The most common types in southern China were a frameshift at codons 41/42 and a C→T substitution at IVS II n.654, the most frequent types in south-western China were codon 17 and IVS II n.654 mutations, and the predominant mutations in eastern China were frameshifts at codons 41/42 and 71/72.

Identification of three novel missense PKU mutations among Chinese

Three novel missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese individuals afflicted with various degrees of phenylketonuria (PKU). A T-to-C transition was observed in exon 5 of the gene, resulting in the substitution of Phe161 by Ser161. Two substitutions, G-to-T and T-to-G, were observed in exon 7, resulting in the substitution of Gly247 by Val247 and Leu255 by Val255, respectively. Expression analysis demonstrated that these mutant proteins produced between 0 and 15% of normal PAH enzyme activity. Population screening of a Chinese sample population indicates that these mutations are quite rare, together accounting for only about 4% of all PKU alleles among the Chinese. The P161S and G247V mutations were each present on a single PAH RFLP haplotype 4 chromosome in patients form Northern China, while the L255V mutation was present on chromosomes of both haplotypes 18 and 21 in patients from Southern China. These results suggest that the remaining 30% of uncharacterized PKU alleles in the Chinese population may bear a large number of relatively rare PAH mutations.

Identification of three novel PKU mutations among Chinese : evidence for recombination or recurrent mutation at the PAH locus

Three novel mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese classical phenylketonuria (PKU) patients. Two of these substitutions (W326X and Y356X) result in the generation of a premature stop codon, while the third (IVS-7nt2) alters an invariant dinucleotide splicing signal. These mutations together account for about 10% of all PKU alleles in the Chinese population. The W326X mutation is associated with PAH RFLP haplotype 4, the most common haplotype in Orientals, while the IVS-7nt2 mutation occurs once on a haplotype 7 chromosome. The Y356X mutation is associated with multiple haplotypes, possibly due to crossover, gene conversion, or recurrent mutation.

Reversal of aberrant splicing of β-thalassaemia allele (IVS-2-654 C→T) by antisense RNA expression vector in cultured human erythroid cells

The antisense fragment targeting the aberrant splice sites of the β‐thalassaemia allele, IVS‐2‐654 C→T (β654), pretranscript was cloned into the mammalian expression vector, pcDNA3. The recombinant construct, pCMVA, was then used to repair the defective splicing of the β654 mutant pretranscript in cultured β654 erythroid cells by the lipofectin‐mediated DNA transfection method. The total RNA was extracted at given time points after transfection and the effect of antisense RNA was studied by reverse transcription polymerase chain reaction (RT‐PCR)‐mediated mRNA quantitative assay, as well as globin chain microbiosynthesis. The antisense fragment transcribed from pCMVA effectively improved the β654 splicing pattern in cultured erythroid cells. The level of correctly spliced transcript increased from 0·19 (day 0 after transfection) to 0·58 (day 8) in β654/β654 homozygous erythroid cells, and from 0·45 (day 0) to 0·83 (day 8) in β654/βA heterozygous erythroid cells, as determined by the ratio of normally spliced β‐globin transcript over total β‐globin transcript. Correspondingly, the ratios of globin chain biosynthesis (β/α) increased from 0·16 (day 0) to 0·52 (day 8) in β654/β654 erythroid cells, and from 0·39 (day 0) to 0·84 (day 8) in β654/βA erythroid cells. Antisense RNA had no significant effect on the splicing pattern in βA/βA erythroid cells. The splicing pattern in transfected cells with pCMVA showed significant changes compared with that in untransfected cells and that in transfected cells with the control antisense fragment (human SRY gene sequence). In addition, we did not observe side‐effects on cytological features after the introduction of pCMVA. All these results indicated that the antisense RNA transcribed from the mammalian expression vector pCMVA could efficiently and specifically suppress the aberrant splicing pattern of β654 mutant pretranscript and restore the correct splicing pathway in vivo, leading to the improvement of globin chain biosynthesis in thalassaemic cells.

The delta-globin RNA transcript level in beta-thalassemia carriers.

Increased levels of hemoglobin A2 (HbA2) are present in most β-thalassemia carriers. The mechanism of this effect is not understood, although the increase may result from transcriptional and posttranscriptional changes. In the present study, we quantitate δ-globin mRNA levels in peripheral-blood-enriched reticulocytes and characterize the variation of δ-mRNA levels in 30 β-thalassemia heterozygotes who individually carry one of the four common Chinese β-thalassemia alleles [codons 41/42 (–TTCT); codon 17 (A→T); IVS-II-654 (C→T); –28 (A→G)]. A sensitive and quantitative competitive reverse-transcriptase polymerase chain reaction method was developed and used to assess the absolute amounts of δ-mRNA transcripts in these peripheral erythroid cells. The results showed a large increase in δ-mRNA amounts in all the carriers examined (72.3 ± 9.0 amol/μg RNA) as compared with those in 12 controls (1.2 ± 0.2 amol/ μg RNA). There was a direct correlation between the δ-mRNA levels and types of β-thalassemia alleles; generally, the δ-mRNA levels are higher in heterozygotes for β⁰-thalassemia mutations than β+-thalassemia mutations. The δ-mRNA levels correlated inversely with hemoglobin and red cell indices but directly with HbA2 levels in heterozygotes of each of the group of β-thalassemia mutations. These results suggest that a greater impairment in β-globin gene expression results in increased transcription of δ-globin gene and in a higher level of HbA2.

High expression of human serum albumin in milk of transgenic mice directed by the goat β-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene promoter, 5′upstream regulatory region, exons 1,2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5♀, 3 ♂) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.

Expression of biologically active human clotting factor IX (hF IX) in the mammary gland of transgenic mice

The DNA of human factor IX (hF IX) gene vector pMC IX m, which had been proven to be able to express inin vitro and living cells, was introduced into 586 zygotes of Kunming Whlte Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F, pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genornes, giving an integration frequency of 3% (6/216). Two F0 female transgenic mice could express hF IX protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hF IV in the milk of two F0 mike were 44.67 % and 79.43 %, respectively.

PAH 399 GTA(Val)→GTT(Val), a new silent mutation found in the Chinese

SummaryA silent mutation or sequence polymorphism, an A to T substitution at codon 399 in exon 11 of the phenylalanine hydroxylase (PAH) gene has been identified by DNA sequence analysis in the Chinese. The frequencies of this new mutation in normal and abnormal (phenylketonuria; PKU) genes are 0.005 and 0.09, respectively, based on the analyses of 100 apparently normal individuals and 39 PKU patients, as demonstrated by DNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridization methods. The results suggest that there is linkage disequilibrium between this polymorphism and PKU mutations in the PAH gene; approximately 10% of defect PAH alleles in the Chinese population may be identified with this sequence polymorphic marker.

Prenatal Diagnosis of β-Thalassemia Using Multiplex Allele-Specific Amplification (MASPCR) in Chinese Families

Combining allele-specific amplification (ASPCR) with multiplex PCR, we developed a new approach-multiplex allele-specific amplification (MASPCR) to detect five common types of β-thalassemia mutations (CD 41-42(-4bp), CD 17 A→T, CD 71-72 (+ A), -28 A→ G and IVS-2-654 C→ T) which account for approximately 80% of all β-thalassemia alleles in Chinese individuals. Using this technique, prenatal diagnoses were performed for ten pregnancies at risk for β-thalassemia. All the results were confirmed by PCR/ASO probe hybridization or DNA sequencing. This study suggests that this method is a simple, accurate approach that may further improve the prevention programs for β-thalassemia that have already dramatically lowered the birth rate of affected children in some parts of the world.