T. A. Stoming

Nuclear aryl hydrocarbon hydroxylase and interaction of polycyclic hydrocarbons with nuclear components.

Aryl hydrocarbon hydroxylase (AHH) activity has been demonstrated in the nucleus by a specific histofluorescent technique. The nuclear AHH is present at 15–20% of the activity of the microsomal enzyme. The properties of the nuclear and microsomal AHH have been compared. Cytochrome P-450 is also present within the nucleus as demonstrated by the CO-binding spectrum. Both the AHH and hemopigment are induced by prior treatment of the rats with 3-methylcholanthrene (3MC). The ratio of induced/ control in nuclei and microsomes was identical. Other inducing agents of the nuclear AHH included pregnenolone 16α-carbonitrile, phenobarbital, arochlor 1254, arochlor 1260, β-naphthoflavone, and benzo(α)pyrene (BP). The control nuclear and microsomal AHH were inhibited to a similar degree by 1-benzylimidazole, diethylmaleate, SKF 525A, and metyrapone; α-naphthoflavone inhibited the induced nuclear and microsomal enzymes to a similar degree. Lung nuclear AHH was induced by 3MC in AKR/J, DBA/J, A/J, C3H/J, and C57B1/6J mice; the most inducible strain was the C57B1/6J, while the least inducible was the DBA/J. High-pressure liquid Chromatographic analysis of the microsomal and nuclear metabolites did not indicate any substantial qualitative differences. [3H]BP was incubated with liver nuclei and a NADPH-generating system. Aralkylation of DNA, histones, and nonhistone components was demonstrated in these in vitro systems. The aralkylation of each of these macromolecules was stimulated by prior treatment of the rats with 3MC. The HIV histones were aralkylated to the least extent. Gel electrophoretic and radioisotopic analysis indicated that the nonhistone components were generally aralkylated. The implications of the nuclear enzyme are discussed.

β-Thalassemia in Turkey

A review is presented of the various β-thalassemia alleles observed in nearly 191 patients with β-thalassemia major and their 182 heterozygous relatives. Determination was by gene amplification and dot-blot hybridization with synthetic probes, specific for 27 different mutations. Eighteen mutations have been observed; six of these acount for nearly 83% of all thalassemia abnormalities (Table I). A new mutation, i.e. a G ↣ C mutation at the acceptor splice site of IVS-I, was found in one teenager who was homozygous for this disease. the high consanguinity among the families was considered the main reason for the high number of patients with a homozygosity for the IVS-I-110 (G ↣ A) mutation. Combinations of different mutations were present in many patients; some were mildly affected because of the specific mutation present on one chromosome. Combinations of classical β-thalassemia and an abnormal hemoglobin mainly concerned Hb S. Hbs Knossos and Lepore were rare occurrences. A comparison of hematological da...

Molecular characterization of β-globin gene mutations in Malay patients with Hb E-β-thalassaemia and thalassaemia major

Summary. This study concerned the identification of the β‐thalassaemia mutations that were present in 27 Malay patients with Hb E‐β‐thalassaemia and seven Malay patients with thalassaemia major who were from West Malaysia. Nearly 50% of all β‐thalassaemia chromosomes carried the G → C substitution at nucleotide 5 of IVS‐I; the commonly occurring Chinese anomalies such as the frameshift at codons 41 and 42, the nonsense mutation A → T at codon 17, the A → G substitution at position −28 of the promoter region, and the C → T substitution at position 654 of the second intron, were rare or absent. Two new thalassaemia mutations were discovered. The first involves a frameshift at codon 35 (‐C) that was found in two patients with Hb E‐β°‐thalassaemia and causes a β°‐thalassaemia because a stop codon is present at codon 60. The second is an AAC → AGC mutation in codon 19 that was present on six chromosomes. This substitution results in the production of an abnormal β chain (β‐Malay) that has an Asn → Ser substitution at position β19. Hb Malay is a‘Hb Knossos‐like’β+‐thalassaemia abnormality; the A → G mutation at codon 19 likely creates an alternate splicing site between codons 17 and 18, reducing the efficiency of the normal donor splice site at IVS‐I to about 60%.

Effect of α‐thalassemia and β‐globin gene cluster haplotypes on the hematological and clinical features of sickle‐cell anemia in Brazil

To compare the features of sickle‐cell anemia in Brazil with those in other locales, we studied the effects of the β‐globin‐like gene cluster haplotype and α‐thalassemia upon the clinical and hematological features in 85 patients. The distribution of haplotypes differed from that in the United States and Jamaica. The Central African Republic (CAR) haplotype predominated; 34% of patients were CAR haplotype homozygotes, 45% CAR/Benin homozygotes, and 11% Benin homozygotes. No Senegal haplotype chromosomes were observed. α‐thalassemia was present in 17.5% of patients. HbF levels were higher in Benin homozygotes, compared with the other two groups (P < 0.05). Nearly half the patients with a CAR haplotype had leg ulcers, compared to 12.5% of the Benin homozygote group; stroke did not occur in α‐thalassemia carriers, but neither result was statistically significant. As in other studies, our results indicate that the CAR haplotype may be associated with more severe disease.

The detection of β-globin gene mutations in β-thalassemia using oligonucleotide probes and amplified DNA

Abstract DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in β-thalassemia. A series of oligonucleotide primers are synthesized which span the β-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the δ gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the β-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25–30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous β-thalassemia mutations.

Two novel polyadenylation mutations leading to β+‐thalassaemia

In an ongoing effort to identify point mutations causing β‐thalassaemia, we have found two previously unreported mutations which are located in the Poly A site of the β‐globin gene. The screening programme used amplified DNA and dot‐blot hybridization with several 32P‐labelled oligonucleotide probes. DNA samples which remained unidentified by this methodology were subjected to sequencing with 32P‐labelled primers and modified T7 DNA polymerase. The newly discovered mutations were confirmed by the dot‐blot hybridization technique. One type concerned an AATAAA→ÁTGAA mutation in the polyadenylation site and was found in one family from Yugoslavia (including one patient with the C→T mutation at codon 29 in trans), one from Bulgaria (the patient had the G→A mutation at IVS‐I‐110 in trans), and one from Greece (this patient had the C→G mutation at IVS‐II‐745 in trans). Haematological data for three simple heterozygotes suggested a rather mild β+‐thalassaemia. The second type involved an AATAAA→ÁTAGA mutation and was found in one family from Malaysia. The propositus had the βE mutation on the other chromosome, was originally diagnosed as mild Hb E‐β+‐thalassaemia, and had Hb A and Hb E percentages which were nearly the same.

Gas Chromatographic Assay of Epoxide Hydrase Activity with 3-Methylcholanthrene-11,12-Oxide

Epoxide hydrase has been measured in rat tissue with 3-methylcholanthrene-11,12-oxide as substrate; diol formation was assayed by gas chromatographic separation of the trimethylsilylated derivative of trans-11,12-dlhydro-11,- 12-dihydroxy-3-methylcholanthrene from the corresponding derivative of the 11 (or 12)-hydroxy-3-methylcholanthrene on 3 percent OV-17, which is formed from the 11,12-oxide during the derivatization. The polycyclic hydrocarbons were extracted initially from the incubation mixture with ethyl acetate. The assay is simple, inexpensive, and sensitive.

Severe Hb S‐β°‐thalassaemia with a T → C substitution in the donor splice site of the first intron of the β‐globin gene

Through direct sequencing and dot‐blot analyses with synthetic oligonucleotide probes of amplified DNA, a new nucleotide substitution was discovered in a Black teenager with severe Hb‐S‐β°‐thalassaemia. The substitution involved a T → C replacement at the second position of the donor splice site of the first intervening sequence of the β‐globin gene. The clinical and haematological observations made in Black subjects with Hb S‐β°‐thalassaemia, with different types of thalassaemia, suggest severe disease resembling sickle cell anaemia. Only an occasional patient had a milder clinical course, perhaps because of a greatly increased production of fetal haemoglobin.

Types of Thalassemia Among Patients Attending a Large University Clinic in Kuala Lumpur, Malaysia

We have identified the beta-thalassemia mutations in 59 patients with thalassemia major and 47 patients with Hb E-beta-thalassemia, and the deletional and nondeletional alpha-thalassemia determinants in 23 out of 24 patients with Hb H disease. All persons were attending the Haematology Clinic at the National University of Malaysia in Kuala Lumpur (Malaysia). Most patients (76) were of Malay descent, while 52 patients were Chinese, and two came from elsewhere. The most frequently occurring beta-thalassemia alleles among the Malay patients were IVS-I-5 (G----C) and G----A at codon 26 (Hb E), while a few others were present at lower frequencies. The Chinese patients carried the mutation characteristic for Chinese [mainly codons 41/42 (-TTCT) and IVS-II-654 (C----T)]; Malay mutations were not observed among Chinese and Chinese mutations were virtually absent in the Malay patients. The large group of patients with Hb E-beta-thalassemia and different beta-thalassemia alleles offered the opportunity of comparing hematological data; information obtained for patients with Hb E-beta-thalassemia living in other countries was included in this comparison. Twenty-three patients with Hb H disease carried the Southeast Asian (SEA) alpha-thalassemia-1 deletion; 13 had the alpha CS alpha (Constant Spring) nondeletional alpha-thalassemia-2 determinant, while the deletional alpha-thalassemia-2 (-3.7 or -4.2 kb) was present in 10 subjects. The --/alpha CS alpha condition appeared to be the most severe with higher Hb H values. Both deletional and nondeletional types of alpha-thalassemia-2 were seen among Malay and Chinese patients.

β-Thalassemia in Yugoslavia

This study concerned the evaluation of β-thalassemia alleles in nearly 50 patients with β-thalassemia major and in 130 -thalassemia heterozygotes using gene amplification and dot-blot hybridization with synthetic probes. Fourteen different mutations were observed (Table I); of these, three (IVS-I-110; IVS-I-6; IVS-I-1) account for some 75% of all β-thalassemia alleles. Newly discovered variants, i.e. T ↣ C in the initiation codon and AATAAA ↣ AATGAA in the poly A site were observed in a few patients. the poly A mutation with classicall β-thalassemia alleles result in thalassemia intermedia. Hb Lepore is a rather common abnormality and combinations of this variant with β-thalassemia often result in severe disease; a search for β-thalassemia mutations among patients affected with this disease should include an analysis to detect this hemoglobin abnormality.