Yi-Tao Zeng

Hydroxyurea therapy in β‐thalassaemia intermedia: improvement in haematological parameters due to enhanced β‐globin synthesis

Summary. The β‐thalassaemias represent a heterogenous group of diseases resulting from decreased erythroid β‐globin mRNA expression and imbalanced a/β‐globin chain synthesis which are manifest clinically by ineffective erythropoiesis and excessive haemolysis. Increasing levels of haemoglobin F (HbF) by pharmacological agents has been proposed to ameliorate the severity of the disease by improving the balance in globin chain synthesis. Hydroxyurea (HU), as an effective agent with low toxicity for activating 7‐globin gene, has been shown to enhance HbF synthesis in experimental animals and in patients with sickle cell anaemia. However, previous trials of HU in β‐thalassaemia patients are ambiguous, with a small number having increased HbF synthesis. In a recent study of HU effects in Chinese j3‐thalassaemia patients we unexpectedly found that two unrelated patients with β‐thalassaemia intermedia demonstrated an improvement in the effectiveness of erythropoiesis reflected by an increase in haemoglobin concentration (from 4‐1 to 6‐3 g/dl, patient 1; from 6‐5 to 97 g/dl, patient 2) and in red cell volume (from 68 to 104 fl, patient 1; from 68 to 85 fl, patient 2) after a period of excess of 300 d of low‐dosage HU treatment. These effects, however, appear to be due to increased,3‐globin biosynthesis, because the percentage of HbF decreased in each patient as total Hb increased. This was reflected by changes in the β/a ratio (from 0′301 to 0‐581, patient 1; from 0′348 to 0‐487, patient 2) with minimal changes in 7‐globin biosynthesis. We conclude that in addition to its known effects in stimulating 7‐globin production, hydroxyurea may have a more general role in augmenting globin synthesis, including β‐globin in some thalassaemia intermedia patients who maintain the capacity to express normal β‐globin chains.

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.

RNA transcripts of the β-thalassaemia allele IVS-2–654 C T: a small amount of normally processed β-globin mRNA is still produced from the mutant gene

Summary. IVS‐2–654 C → T is a common Chines β‐thalassaemia mutation. Previous studies report that this mutation resulted in the formation of an abnormally spliced mRNA and the absence of detectable normal β‐globin mRNA, hence the mutation was considered to cause βd́; ‐ thalassaemia. We recently used the method of PCR amplified cDNA copies of circulating erythroid cell mRNA to analyse the mutant gene transcripts and found that this IVS‐2–654 mutation does not abolish normal RNA processing entirely, but that a significant amount (over 15%) of normally processed β‐globin mRNA is produced. Microglobin chain biosynthetic analysis using the HPLC method showed that β‐globin chain was also present in the blood of patients with IVS‐2–654 C → T mutation. Accordingly, this mutant allele leads to a β+‐thalassaemia. Further, the methodology described in this paper provides a new approach towards the detection of RNA transcripts in β‐thalassaemia and other genetic diseases.

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.

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.

Construction of antisense RNA expression vectors and correction of splicing defect in human β-globin gene (IVS-2-654 C→T mutant) in HeLa cells

The antisense fragments, which were available inin vitro system, were cloned into the mammalian expression vector pcDNA3, and were transfected into H654 cells, a mammalian cell line stably expressing the thalassaemic (IVS-2-654 C→T) human β-globin gene. In these transfected cells, the level of correctly spliced β-globin mRNA in total β-globin mRNA (β/(β + β*)) was improved from 0.07 (0 d) to 0.22 (3 d), and this effect persisted for up to 15 d post transfection. All the results demonstrated that antisense RNAs were able to be transcribed from the antisense fragment expression vectors stably and effectively suppressed aberrant splicing pattern of the mutated β-globin gene (IVS-2-654 C→T) and restored correct splicing pathway. This work provided a novel approach with potential clinical significance to gene therapy of this kind of splicing mutants including β-thalassaemia (IVS-2-654 C→T) by antisense RNAs.