59

Acknowledgement This work was financially supported by the Council of Scientific and Industrial Research, New Delhi, India

References ‘Dixon, M and Webb, E C (1979) Enzymes, third edition, pp 18-19, Longman, London

zSrere, P A (1987) Ann Rev Biochem 56, 39-124

3Srivastava, D K and Bernhard, S A (1987) Ann Rev Biophys Chew 16, 175-205

40vBdi, J (1991) / Theor Biol152, l-22

5Malhotra, 0 P, Prabhakar, P, Sen Gupta, T and Kayastha, A M (1995) Eur / Biochem 227, 556-562

6Gutfreund, H and Chock, P B (1991) J Theor Biol 152, 117-122

‘Ambasht, P K (1993) PhD Thesis, Banaras Hindu University, Varanasi, India

‘Malhotra, 0 P, Srivastava, D K and Srinivasan, Biochem Biophys 179, 302-309

(1979) Arch

‘Kayne, F J (1973) The Enzymes (Boyer, P D etal, eds), Vol2, pp 353- 382, Academic Press, New York

“‘Winstead, J A and Weld, F (1965) ,2145-2150

“Malhotra, 0 P, Prabhakar, P, Kayastha, A M and Sen Gupta, T (1993) Protein Structure Function Relationshir, (Zaidi. Z H. Abbasi. A. Smith, D L, eds) pp 179-192, TWEL, Gaithersburg, MD

“Neuzil, J, Danielson, H, Welch, R G and Ovadi, J (1990) Biochim Biophys Acta 1037, 307-312

0307-4412(95)00128-X

The Role of Molecular Biology in Characterisation of (Y Thalassaemia

JOHN HOWARTH,a HARRY WATERS,” KEITH

HYDE,” STEVE HEATH,b CHRISTOPHER BOTTRILLb

and JOHN RICHARDSb

a University Department of Clinical Haematology Cobbett House, Manchester Royal Infirmary Oxford Road Manchester Ml3 9WL, UK b Department of Biological Sciences The University of Salford Salford M5 4WT, UK

Introduction Inherited disorders of haemoglobin structure or synthesis constitute a public health problem in many parts of the world, inflicting a significant impact on the individuals concerned, their families and the resources available in the community for the provision of healthcare. The thalassaemias are among the most common genetic diseases of man, the high gene frequency resulting from a probable selective advantage of the thalass- aemia phenotype in heterozygotes where it may afford pro- tection from the most severe effects of malaria. The condition is classified as an hereditary haemolytic anaemia characterised by decreased or absent synthesis of one of the globin subunits of the haemoglobin molecule.’ In the (Y thalassaemias, the decreased synthesis of c1 globin produces accelerated red blood cell destruction resulting from the formation of insoluble inclusions of p globin in mature cells. The imbalance in globin production may produce profound clinical effects, resulting in either death in utero or life-long transfusion therapy in the most severe cases.

BIOCHEMICAL EDUCATION 24(l) 1996

The (Y globin gene cluster spans a distance of approximately 30 kb and has been refined to the distal segment (p 13.3-pter) of the short arm of chromosome 16.* Physical mapping has shown that the cluster lies a short but variable distance (170-430 kb) from the telomere. Norma1 individuals have two a globin genes per haploid and are designated (Y(Y/o((Y. The most common cause of CL thalassaemia is gene deletion within the (Y globin gene cluster.” These deletions result in conditions which may be classified as either a+ or a”, indicating either a reduced (a’) or absent (~1”) output from the affected complex.

In the case of (Y+ thalassaemia, two different size deletions have been described.3,4 One produces a 4.2 kb deletion of DNA and is prevalent in Oriental individuals. The other removes 3.7 kb of DNA and has a worldwide distribution.

Genotypes a-/aa Heterozygous CI+ thalassaemia (Silent carrier) a-/a- Homozygous ~1~ thalassaemia (Thalassaemia trait) - -/acu Heterozygous (Y” thalassaemia (Thalassaemia trait) c-u-/-- Haemoglobin H disease - -/- - Haemoglobin Bart’s hydrops fetalis

Deletion of one o( gene does not significantly alter the quantity of a globin produced. Individuals with this phenotype are termed silent carriers, and although they are carriers of the mutant gene, they are asymptomatic.

The (Y thalassaemia trait producecs a haemotological blood picture resembling iron deficiency. Differentiation of the con- ditions is very important to obviate inappropriate therapy which may result in complications resulting from iron overload.

An individual with only one functioning a gene suffers from haemoglobin H disease which results in a moderately severe haemolytic anaemia.

Deletion of all four (Y genes is invariably fatal. This condition usually results in fetal death close to full-term, posing a potential threat to the health of the mother. It is very important to distinguish homozygous uf from heterozygous u0 thalassaemia since Hb Bart’s hydrops fetalis only occurs when both parents have the latter genotype. Such individuals require specialised genetic counselling.

Laboratory Techniques There are no ‘simple’ laboratory tests for the definitive recognition of LY thalassaemia. Diagnosis may often be achieved by the process of eliminating other possible causes of the haematological picture. This involves electrophoretic and chromatographic techniques prior to the application of more specialised procedures for a final delineation of the disorder. Accurate diagnosis may be carried out by restriction endo- nuclease digestion with Barn Hl and Bgl II, followed by hybridisation with DNA probes directed against the (Y and 5 genes of chromosome 16:-’ We have used probes which were kindly supplied by Dr D Higgs and Dr J Old of the MRC Molecular Haematology Unit, John Radcliffe Hospital, Oxford.

The (Y probe is a 1.3 kb Pst I a-globin specific fragment and the 5 probe is a 1.8 kb Sac 1 <-globin specific fragment, both derived from the plasmid pBR322. Although robust, the methodology is inappropriate for large-scale screening as it is both expensive and laborious. Recent developments in appli- cation of the polymerase chain reaction (PCR) have advanced the possibility of large-scale characterisation of (Y globin gene deletions.

As a consequence of the ethnic mix within the Manchester conurbation, and the potential for Hb H disease and Hb Bart’s hydrops fetalis, our work has concentrated on the screenin

5 of

antenatal patients and their partners at risk for the -- EA determinant. This is a large deletion encompassing approxi- mately 20 kbp which removes the $a2 - +a1 - cu2 - al - 01 globin genes, whilst sparing the 52 and +<l genes.

60

Materials and Methods A number of papers have recently been published citing the application of PCR for the characterisation of o! thalass- aemia.x-‘2 Our experimental work comprised a modification of original work previously described by Bowden et al” which also describes primer sequences for the detection of the Mediter- ranean (--MED) and --c1 20.5 determinants. Reference to the original paper is recommended for fuller details of the relevant methodology.

Oligonucleotides These were obtained from Genosys Bio- technologies Europe. They were identical to those described by previous investigators. ”

Primer name S-3’ sequence S’SEA 5’-CTCTGTGTTCTCAGTATTGGAG-3’ S’SEA 5’-ATATATGGGTCTGGAAGTGTATC-3’ 3’SEA N .5’-TGAAGAGCCTGCAGGACCAGTCA-3’

S’SEA and 3’SEA N were used to amplify DNA from the normal chromosome. S’SEA and 3’SEA were used to amplify DNA from the abnormal chromosome.

The polymerase chain reaction All the reactions were carried out using the materials supplied in a GeneAmp PCR Reagent kit from Perkin Elmer Cetus. The water used was supplied by a Mini-Q plus system. A ‘touch-down’ program designed to limit the number of non-specific products was used in a Hybaid Thermal Cycler (Table 1).

The amplification reactions were carried out using the following conditions:

Template DNA 100.0 ng (5 t.~l of 20 ng/pI) Primers 100.0 pM (each) t{lOjreaction buffer 10.0 tL1

AmpliTaq DNA polymerase ‘2:: I-$ (each)

Water 71.5 /.‘.I

The polymerase was added after the other reagents had been heated to 95°C. The polymerase enzyme was diluted first with

Table I Conditions for PCR ‘touch-down’ program

Cycles Temperature eC) Time (min)

_ 95 3

1 95 1 55 1 72 2

1 95 1 50 1 72 2

1 95 1 45 1 72 2

26 95 1 40 1 72 2

1 95 1 40 1 72 5

sufficient water to allow 10 t.~l of solution to be added. This permitted accurate addition of the reagent. The 10X buffer, water and dNTPs were made up as a Master Mix for the same reason. Each tube was overlaid with 100 ~1 reagent grade liquid paraffin.

The products of the reactions were visualised by electrophor- esis in a 2% NuSieve 3:l agarose gel, containing ethidium bromide at a concentration of 0.2 mgll which was run at 6Ov for 2 hours. The running buffer contained ethidium bromide at the same concentration as in the gel. 10 ~1 PCR solution was added to 2 ~1 bromophenol blue loading buffer (15% Ficol, 10 mM Tris pH 8.0, 10 mM EDTA, 0.1% SDS, 0.1% bromophenol blue) prior to being loaded on to the gel. The marker was h DNA cut with Hind III and EcoRl. One oligonucleotide was also run alongside the PCR product to ensure that the bands shown were due to true PCR products and not excess primer unused after the reaction period.

Results The amplified products were identical to those previously reported by Bowden et al ” thereby permitting the discrimin- ation of the normal from the abnormal genotype. The relevant fragment sizes are tabulated below.

Primer Combination Haplotype S’SEA and 3’SEA N o.(Y S’SEA and 3’SEA __=A

Fragment size 0.98 kb 0.66 kb

Further developments Detection of a+ thalassaemia Recent work published by Baysal and Huisman has resulted in the development of the primer sequences described below for selective amplification of the az and ol genes and subsequent identification of the -03.’ and -IX’.’ deletions.” A full description of the methodology may be obtained by reference to the original paper.

Primer A 5’-CTTTCCCTACCCAGAGCCAGGTT-3’ Primer B 5’-CCCATGCTGGCACGTTKTGAGG-3’ Primer C 5’-CCATTGTTGGCACATTCCGGGACA-3’ Primer D 5’-CCTTCCTCTCACTTGGCCCTGAG-3’ Primer E 5’-CCCTGGGTGTCCAGGAGCAAGCC-3’ Primer F 5’-GGCACAGGCCGGGACAGAGAGAA-3’ Primer G 5’-CCGGTTTACCCATGTGGTGCCTC-3’

For the -a3.’ kb deletion, primers A and B amplify the abnormal segment; primers A and C amplify the normal sequence. For the -(Y~.~ kb deletion, primers D and E or G and E amplify the abnormal segment; primers D and F or G and F amplify the normal sequence.

Amplified DNA assays Following the optimisation of PCR, the potential of an amplified DNA assay (ADA) carried out on microtitre plates is to be evaluated for the detection of both the milder and more severe forms of (Y thalassaemia.13 The features of the system are its high specificity and sensitivity, the rapidity of the reaction, its applicability to mass screening and the fact that it does not require hazardous reagents. The test requires DNA amplification using oligonucleotide primers, one of which is biotinylated and the second of which incorporates a specific 12 bp recognition sequence (S’GGATGACTCATT) for a double-stranded DNA binding probe, GCN4. In brief, the methodology consists of performing PCR for twenty cycles with two oligonucleotides as primers of the target sequence of interest. Two further oligonucleotides are then nested between the primer oligonucleotides - one is linked to a biotin moiety, the other bears the 12 bp recognition sequence that can bind to GCN4 when double stranded. The PCR product is then transferred to GCN4 fusion protein-coated plates. The amplified

BIOCHEMICAL EDUCATION 24(l) 1996

61

target sequence is then detected with a Streptavidin Horse Radish Peroxidase (HRPO) calorimetric system. The methodol- ogy is marketed by Pharmacia Biotech as the CAPTAGENE- GCN4 kit for the detection of amplified DNA.

Discussion The development and refinement of PCR should facilitate its application as a cost-effective method for the characterisation of cx thalassaemia in countries where resources are severely limited but the incidence of the disorder has been reported to be high. The importance of developing a specific screening test for the identification of carriers of IY thalassaemia may be eloquently summarised by the following quotation:

From a practical point of view it is important to remember that millions of individuals throughout the world are carriers of CL thalassaemia and every year many thousands of pregnancies are at risk of producing children with severe (Y thalassaemia syndromes

. However, because this is a genetic disease that predominantly affects people from countries with limited health resources, simpler and cheaper methods of screening and diagnosis will have to be developed before this information has a significant impact on the attendant morbidity and mortality.‘4

References ’ Weatherall, D J and Clegg, J B (1981) In The Thalassaemia Syndromes, third edition, Blackwell Scientific Publications, Oxford, p 85

‘Nicholls, R D, Jonasson, J A, McGee, J 0 D, Patil, S, Ionasescu, V V, Weatherall, D J and Higgs, D R (1987) J Med Gener 24, 39-46

‘Higgs, D R, Vickers, M A and Wilkie, A 0 M (1989) Blood 73, 1081- 1104

‘Embury. S H, Miller, J A, Dozy, A M. Kan, Y W, Chan, V and Todd, D (1980) J Ckn Inuesf 66, 1319

‘Pressley, L, Higgs, D R, Clegg, J B and Weatherall, D J (1980) Proc Nat Acad Sci USA 77, 3586-3589

‘Nicholls, R D, Higgs, D R, Clegg, .I B and Weatherall, D J (1985) Blood 65, 1434-1438

‘Nicholls, R D, Fischel-Ghodsian, N and Higgs, D R (1987) Cell 49, 369-378

‘Dade, C, Rochette, J and Krishnamoorthy, R (1990) Brit J Haematol 76, 275-281

“Jan-Gowth, C, Long-Shyong, L, Che-Pin, L, Pao-Huei, C and Chih- Ping, C (1991) Blood 78, 853-854

“‘Tsang-Ming, K, Li-Hui, T, Fon-Jou, H, Pi-Mei, H and Tzu-Yao, L (1992) Human Genet 88, 245-248

” Bowden, D K, Vickers, M A and Higgs, D R (1992) Brit J Haematol 81, 104-108

“Baysal, E and Huisman, T H J (1994) AmerJ Haematol49(3), 208-213

13Kemp, D J. Smith, D B. Foote, S J, Samaras, N and Gregory Peterson, M (1989) Proc Nat Acad Sci, USA 86, 2423-2427

“Higgs, D R (1993) Bailliere’s Clinical Haematology: The Haemo- globinopathies (edited by Higgs, D R and Weatherall, D J), Bailliere Tindall 6. 144

Letters to the Editor

From A S Rao

Terminology in Microbial Metabolism

Dear Sir (1) In microbial metabolism, the terms “homolactate” fermenta- tion or “homoacetate” fermentation etc are used. They indicate that in the respective fermentation, lactate or acetate is the sole end product. Thus the prefix homo indicates the exclusive for- mation of the product, lactate or acetate etc, as opposed to hetero or mixed fermentations in which there is more than one end product.’ However, metabolites such as homoserine, homo- cysteine, homocitrate, etc, which are related to serine, cysteine and citrate respectively also carry the prefix home.’ So the prefix homo can be misleading and thus inappropriate while referring to fermentations. For example when we say, homoserine fer- mentation it does not mean that serine is the sole end product (the way we mean that lactate or acetate is the sole end product in homolactate and homoacetate fermentation respectively). Rather it refers to the formation of homoserine - a metabolite different from but homologous to serine. Conversely, when we say homoacetate fermentation, we do not refer to any com- pound ‘homoacetate’ different from acetate (the way homo- serine is different from serine or homocysteine is different from cysteine, etc). Hence, the routinely used fermentation termin- ology with prefix ‘homo’ is confusing although admittedly sanc- tioned by long usage. I suggest that the prefix homo be removed while referring to fermentation processes in view of the occur- rence of metabolites like homoserine, homocysteine, etc. Instead, the terms ‘simple’ or ‘straight’ may be used to refer to homofermentations. Thus homolactate fermentation and homo- acetate fermentation may be referred to as simple or straight lactic or acetate fermentation respectively. However the terms hetero and mired may be retained, which refer to the formation of more than one end product. I feel even the term hetero may be dropped from usage and that it is better to use only the expres- sion, mixed fermentation. The above changes in usage would make the terms more clear and less confusing to students and teachers alike.

(2) With respect to DNA transcription I feel the terms sense strandlcoding strandltemplate strandiantisense strand are con- fusing. Unfortunately many standard books do not even men- tion or explain these terms and even standard text books differ in their definitions.

According to Lewin (1994),’ ‘The DNA strand that bears the same sequence as the mRNA (except for possessing T instead of U) is called the coding strand or sense strand. The other strand of DNA which directs synthesis of mRNA via complementary base pairing is called the template strand or antisense strand (my italics).’

Now let us see the statement from Stanier (1986),’ ‘Initiation of transcription occurs at specific points on the bacterial chromosome that are defined by short sequences of bases of DNA called promoters. The sequences also determine which strand (termed the sense strand) will be transcribed and hence, because polymerisation always occurs 5’ to 3’, the direction of transcription’. According to Kuchel and Ralston (1988),” ‘The sequence of nucleotides within the single stranded mRNA is assembled according to complementary base pairing instruc- tions of one of the strands of duplex DNA, which contains the gene. Since this DNA strand provides the template for transcrip- tion it is called the sense strand’.

From the above I feel that the terminology (explanation) related to sense strand etc, must be clarified to make it easier to understand and remember. I feel the strand that is transcribed, ie the one that directs the synthesis of mRNA should be termed the ‘sense strand’. The terms ‘coding strand’ and ‘template

BIOCHEMICAL EDUCATION 24(l) 1996