Zhao-rui Ren

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.

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.

A NOVEL β‐THALASSAEMIA MUTATION: DELETION OF 4 bp (–AAAC) IN THE 5’TRANSCRIPTIONAL SEQUENCE

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Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats.

The production of valuable pharmaceutical proteins using transgenic animals as bioreactors has become one of the goals of biotechnology. However, the efficiency of producing transgenic animals by means of pronuclear microinjection is low. This may be attributed in part to the low integration rate of foreign DNA. Therefore, a large number of recipients are required to produce transgenic animals. We recently developed a transgenic procedure that combined the techniques of goat oocyte in vitro maturation (IVM), in vitro fertilization (IVF), microinjection, preimplantation selection of the transgenic embryos with nested PCR and transferring the transgenic embryos into the recipient goat uterus to produce transgenic goats. Thirty-seven transgenic embryos determined by nested PCR were transferred to thirty-two recipient goats. In the end, four live-born kids were produced. As predicted, all the live kids were transgenic as identified by PCR as well as Southern blot hybridization, The integration rate was 100% (4/4) which was completely in accordance with the results of embryo preimplantation detection. The results showed a significant decrease in the number of recipients required as only 8 recipients (32/4) were needed to obtain one live transgenic goat. We suggest that the transgenic system described herein may provide an improved way to efficiently produce transgenic goats on a large scale.

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.

A new unstable haemoglobin variant: Hb Shanghai [β131 (H9)Gl→Pro] found in China

A 34–year‐old woman patient was found to have a chronic hereditary haemolytic anaemia. No abnormal haemoglobin band was detected by conventional electrophoresis, but a slow β chain could be separated on urea‐carboxymethyl cellulose chromatography. Investigations of the patients haemoglobin revealed an unstable component. Analyses of chemical structure, including isolation and TPCK trypsin digestion of the abnormal globin chain, HPL chromatography, amino acid composition as well as sequence determination of the abnormal peptide, indicated that a glutamine was replaced by a proline at position β 131 (H9). Biosynthesis studies demonstrated a normal rate of synthesis but relatively fast degradation of the mutant β chain. The new variant is named as Hb Shanghai according to the place where it was discovered.