A case of non-b-globin gene linked b thalassaemiain a Dutch family with two additional a-gene defects:the common ¹a3?7 deletion and the rare IVS1–116(A → G) acceptor splice site mutation

P. C. GIORDANO, C. L. HARTE VE LD, H. L. HAAK,* D. BATE LAAN, P. VAN DELF T, R. J. PLUG, M. EMONT S,R. ZANARDINI AND L. F. BERNINI MGC-Department of Human Genetics, Leiden University, and*Leyenburg Hospital, The Hague, The Netherlands

Received 31 May 1998; accepted for publication 23 July 1998

Summary. We describe a family with b thalassaemia, appa-rently not linked to the b-globin gene cluster, in combinationwith a thalassaemia. The propositus, an adult Dutch Cau-casian male, and his son presented with microcytic hypo-chromic parameters. Their lysates displayed the normaladult pattern on electrophoresis. The HbA2 concentration,which is usually increased in b thalassaemia, was normal.The in vitro biosynthetic rate of the globin chains wasstrongly unbalanced even in the presence of a coexisting a-thalassaemia defect. Routine analysis of the b genes, includ-ing the promoter region, was performed repeatedly bypolymerase chain reaction (PCR), denaturing gradient gelelectrophoresis (DGGE) and direct sequencing. No molecularabnormalities were detected. Large b deletions were excludedby haplotype determination, using seven polymorphic markersdistributed over an area of 50 kb, from 1 kb 5 0 of the e gene to4 kb 3 0 of the b gene. The haplotype analysis of the b-gene

cluster revealed that the unaffected daughter had receivedthe same b haplotype as her b-thalassaemic brother fromtheir b-thalassaemic father. These data suggest that the b-gene cluster shared by father and son was not directlyassociated with a reduced b-globin chain expression. Inorder to exclude the remote possibility of a b-locus-controlregion (LCR) rearrangement in the paternal haplotype of thedaughter, the sequence of the HS2 element was examined inthe nuclear family. We compared the haematological andclinical data of this family with the data reported in thelimited number of similar cases. We discuss the possibilitythat the mutation of a trans-acting erythroid factor(s), notlinked to the b-genes cluster, may impair the b-geneexpression of both alleles.

Keywords: b-thalassaemia, a-thalassaemia, globin chainbiosynthesis, EKLF, HS2.

During routine analysis of thalassaemia carriers, the patientmay present with evident hypochromic microcytic para-meters, but with normal or decreased HbA2 levels. In suchcases, having excluded iron deficiency, a biosyntheticanalysis must be done to differentiate between a thalassae-mia and ‘normal A2 b thalassaemia’. In the current case thepropositus (II-2) displayed a normal HbA2 level and a 70%reduction in the biosynthetic output of the b genes, but didnot display the anaemia that is expected in intermediate b

thalassaemia.

MATERIAL AND METHODS

Blood samples were vacuum collected in Li-Heparin and Na-EDTA. The haematological parameters were obtained from asemiautomatic counter Sysmex F300 (Sysmex-Toa MedicalElectronics Co. Ltd, Kobe, Japan). Red cell lysates wereexamined on starch gel electrophoresis at pH 8.6 (Smithies,1965) The HbA2 fraction was estimated by ion exchangecolumn chromatography (Bernini, 1969). The HbF concen-tration was established by alkaline denaturation (Betke et al,1959). The synthetic ratio of the a and non-a globin chainswas determined using a modified method based on standardprocedures (Weatherall & Clegg, 1981). Genomic DNA wasisolated by selective lysis (Weening et al, 1974) followed byhigh salt extraction (Miller et al, 1988). The a-gene deletion

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Correspondence: Dr P. C. Giordano, Institute of Human Genetics,Leiden University, Department of Biochemical Genetics/Haemo-globinopathies, Sylvius Laboratory, Wassenaarseweg 72, 2333ALLeiden, The Netherlands.

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was identified by standard Southern blot procedure, usingEco-RI and Bgl-II endonucleases and hybridization with a32P-labelled a and z gene probe (Harteveld et al, 1994). Thea-gene point mutation was characterized by DGGE andsequence analysis as previously described (Harteveld et al,1996). The molecular analysis of the b gene was done byselective amplification from genomic DNA of overlappingb-gene fragments followed by denaturing gradient gel elec-trophoresis (DGGE) as previously described (Losekoot et al,1990). Direct solid-phase sequencing of the PCR product(Sanger et al, 1977) was achieved using magnetic beads assolid support (Hultman et al, 1989) and FITC-labelleduniversal M13 sequencing primer on an Automated LaserFluorescent DNA Sequencing apparatus (A.L.F. Pharmacia,Sweden). The sequence gels (6% acrylamide in 7 M urea)were run for 8 h at 1500 V and 44 mA at 458C and scannedby a 5 mV laser beam. The PCR reactions were carried out ina forced water circulation thermocycler (Giordano et al,1989). The haplotyping spanned a DNA fragment of 50 kb,extending from 1 kb 5 0 of the e gene to 4 kb 3 0 of the b gene.The PCR products of seven DNA fragments, each containing apolymorphic site which was recognizable by specific restrictionenzyme digestion, were directly examined on agarose gelelectrophoresis (Varawalla et al, 1992; Sutton et al, 1989). Theinternal b-gene framework was analysed by examining fivepolymorphisms at position 6 of the first exon, and positions 16,74, 81 and 666 of the second intron (Losekoot et al, 1992).The sequence of the 5 0 hypersensitive site 2 (HS2) of the LocusControl Region (LCR) was examined according to Oner et al(1992). Total RNA was isolated from reticulocytes startingfrom 10 ml buffy-coat-free Li-heparin blood, thoroughlypurified from nucleated cells passing through a short 4 mlcolumn of a 2:1 mixture of a-cellulose:Sigmacell type 50(Sigma, St Louis, U.S.A.) (Beutler & Gelbart, 1986). RNA wasextracted using RNAzoltm B, following the supplier’sinstructions (Campro Scientific, Veenendaal, The Nether-lands). Approximately 5 mg of total RNA was transcribed for1 h with 200 Units Moloney Murine Leukaemia virus RNaseH¹ reverse transcriptase (RT) (Gibco BRL, Gaithersburg,U.S.A.), according to the manufacturer’s instructions. Theb-globin-specific mRNA was extended with the b-globingene specific primer M13B (5 0-CGACGTTGTAAAACGACG-GCCAG*TTGCAGCTTGTCA CAGTGCAGCTGACT-3 0).

The cDNA was subsequently amplified with the b-RTprimer (5 0-GACACAACTGTG TTCACTAGC-3 0) specific for theb-globin 5 0 untranslated region and the master primer (5 0-GACGTTGTAAAACGACGGCCAGT-3 0) matching the M13 tagof the M13B primer. The amplified product was analysed bysequencing using automated laser fluorescent DNA sequencer(A.L.F. DNA sequencer, Pharmacia Biotech, Upsala, Sweden).

Evident chromosomal abnormalities were excluded and noevidence of false paternity was found using standard CArepeat analysis on three independent markers on chromo-some 4 (DuS392, DuS1572 and DuS394).

RESULTS

Clinical reportThe propositus, a 51-year-old Dutch Caucasian male (II-2),

presented with chronic microcytic hypochromic parameterswithout anaemia. His 18-year-old son (III-2) was moderatelyanaemic with similar parameters, suggesting the presence ofa hereditary disorder, possibly a mild a-thalassaemia. Ironand enzymes deficiency were excluded and further haemato-logical and molecular analysis of the family was implemented.

Haematological dataSeven members of a two-generation family were examined(Fig 1). Five of them presented with variable microcytichypochromic parameters. On starch gel electrophoresis atpH 8.6 no abnormal bands were detected and no elevatedHbF fractions were measured. The HbA2 levels rangedbetween low and normal values. The results of in vitroHb-chain synthesis determination repeatedly revealed in thepropositus and his son, b/a ratios of 0.3 (0.36, 0.32, 0.27)and 0.2 (0.21, 0.19), respectively. The propositus’ wife, sisterand daughter displayed b/a ratios compatible with milda-thalassaemia defects. The haematological data of thefamily are shown in Table I.

Molecular analysis of the a genesRestriction fragments analysis of the a-gene cluster showedthe presence of the (a,a/¹a3?7) genotype in the propositus(II-2), his sister (II-1) and his daughter. No deletion defectswere found in his wife (II-3) but the analysis of her a genesby PCR, DGGE and direct sequencing revealed the thirdreported case of point mutation a-thalassaemia inducedby the IVS1-116 (A → G) acceptor splice site mutation(Harteveld et al, 1966) (data not shown).

Molecular analysis of the b genesBecause no abnormal patterns were found by DGGE analysis,the entire b gene was amplified as indicated in Fig 2 andextensively sequenced. No molecular abnormalities weredetected in spite of repeated analyses. The framework ofthe b-gene clusters, established using the polymorphic sites

Fig 1. Pedigree of the family.

indicated in Fig 3, showed heterozygous haplotypes for boththe father and his affected son excluding the presence oflarge deletions. The propositus’ daughter carried the samepaternal b haplotype inherited by her brother without show-ing any indication of b thalassaemia (Table II).

Molecular analysis of the b-LCRIn an attempt to extend the haplotype analysis to the LocusControl Region, the sequence of the HS-2 motifs wasexamined in the nuclear family. All four family membersdisplayed normal sequences and the sequence variations

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372 P. C. Giordano et alTable I. Haematological data of the family members.

Individual: II-1 II-2 II-3 II-4 II-5 III-1 III-2Sex/age: F60 M58* F52 M52 F51 F28 M25

Hb (g/dl) 13.0 14.2 14 11.4 15.3 12.7 11.9PCV (l/l) 0.37 0.40 0.48 0.32 0.42 0.37 0.36RBC (1012/l) 4.44 5.77 6.0 3.48 4.61 4.88 5.8MCV (fl) 84.0 69.0 80.0 91.0 92.0 77.1 62MCH (pg) 29.3 24.3 23 32.8 33.2 26.1 20.6MCHC (g/dl) 34.9 35.4 29.2 35.7 35.4 33.8 33.0HbA2 (%) 2.78 2.7 2.13 2.33 3.3 2.7 2.7HbF (%) 0.30 0.55 0.45 3.65† 0.70 0.42 0.31Haptoglobin (g/l)‡ 2.23 1.54 1.36 2.56 1.82 1.37 0.63Osmotic fragility ↑ N ↓ N ↑ N ↑Ferritin (mg/l) 280 154 n.d. n.d. 182 n.d. 156Erythrocyte morphology þ þþ þ þ N þ þþþ

Inclusion bodies neg. neg. neg. neg. n.d. neg. neg.b/a ratio 1.48 0.30 1.43 0.99 n.d. 1.32 0.20a-genotype ¹a/aa ¹a/aa aTa/aa aa/aa aa/aa ¹a/aa aa/aa

a-defect RW RW IVS1-116 n.d. n.d. RW n.d.b-phenotype b/b ¹/b b/b b/b b/b b/b ¹/b

N ¼ normal; ↓ or ↑ ¼ slightly decreased or increased; neg. ¼ negative; n.d. ¼ not determined; þ, þþ, þþþ¼ increase inabnormality.

* Propositus. † Increased HbF % due to NHL. ‡Values <0.50 indicate haemolysis.

Fig 2. Scheme indicating the position of the PCR primers used for the amplification and sequencing of the b-globin gene. The FITC labelledsequencing primer, matching the M13 tag, is included in the Autoread kit (Pharamacia).

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described by Oner et al (1992) at position ¹10924, ¹10905and ¹10390 were not present. However, the repetitive regionbetween position ¹10623 and ¹10570, which contains vari-able TA and CA motifs, was found informative:

Homozygous reference (TA)10 (CA)2 (TA)2Father (II-2) (TA) 8/9 (CA)3/2 (TA)1/1Mother (II-3) (TA)10/9 (CA)2/2 (TA)2/2Daughter (III-1) (TA)10/9 (CA)2/2 (TA)2/1Son (III-2) (TA)10/9 (CA)2/2 (TA)2/1

This result indicated that the same b haplotype of theb-thalassaemic father, (II-2) which was inherited by bothchildren (III-1 and III-2), included the same paternal HS-2allele.

Expression analysis of the b genesThe heterozygous presence of the (cd2: CAC/CAT) poly-morphism in the coding region of the b-globin genes ofthe b-thalassaemia phenotypic father (II-2), demonstrated,by RT-PCR, that the messenger RNA expression of bothalleles was present.

The cDNA obtained from total RNA, isolated from reticulo-cytes, transcribed with murine leukaemia virus reversetranscriptase and extended with the b-globin gene specificprimers, was amplified and sequenced. The amplified pro-ducts of both the cd2 CAC and CAT sequences were presentin about the same proportion, demonstrating a balancedmRNA production by both alleles (Fig 4). This finding is at

variance with the expected deficient production or stability ofmessenger, secondary to b-thalassaemia mutations whichaffects the transcription, processing or even translation (non-sense mutations) of mRNA (Kugler et al, 1995).

DISCUSSION

Few cases of b thalassaemia not linked to the b cluster areknown. Gibbons et al (1992) described an X-linked erythroidspecific factor that selectively decreased a-gene expression,associated with an HbH disease phenotype. Because in ourcase the b thalassaemia was inherited from father to son, asimilar X-linked erythroid specific factor acting on the b

cluster is excluded. Furthermore, only four cases of docu-mented, autosomally inherited, non-b-cluster linked b-thalassaemia defects have been reported. (1) A family fromcentral Italy in which an apparently normal b gene inducedthalassaemia major in association with a common b39defect (Murru et al, 1992). (2) An English family (Thein et al,1993) with a b-thalassaemic trait inherited through threegenerations, which segregated independently from the b-gene haplotype, with both normal and elevated HbA2 levels.(3) Five members of a Portuguese family with non-b-gene-linked high HbA2 b thalassaemia, and HbS heterozygosity(Pacheco et al, 1995). (4) A case of thalassaemia intermediain a patient heterozygous for the 39 nonsense mutation asthe sole abnormality (Gasperini et al, 1998).

The present case shows some similarity with the Englishfamily (Thein et al, 1993) in which mild haematologicalabnormalities and normal HbA2 levels were also present. Inour case, however, the b/a ratios were more unbalanced,even in the presence of a coexisting ¹a3?7 a-thalassaemiadeletion, indicating a determinant with a strong suppressingeffect.

The haematological data of our family may seem quitemild in relation to the biosynthetic rate. A 70% reduction ofthe b-globin gene expression in the propositus, in thepresence of aþ thalassaemia, and 80% reduction in his sonwith b thalassaemia only, were nevertheless repeatedlyconfirmed.

Table II. Results of the b-cluster haplotype of five family members.

Fragment Position Enzyme II-1 II-2* II-3 II-4 III-1 III-2

1 5 0-e Hinc II ¹/¹ ¹/¹ þ/þ ¹/¹ ¹/þ ¹/þ2 Gg Hind III þ/þ þ/þ ¹/¹ þ/þ þ/¹ þ/¹3 Ag Hind III ¹Oþ ¹/þ ¹/¹ ¹Oþ ¹/¹ ¹/¹4 wb Hinc II þO¹ þ/¹ ¹/¹ þO¹ þ/¹ þ/¹5 3 0-wb Hinc II þ/þ þ/þ ¹/¹ þ/þ þ/¹ þ/¹6 5 0-b Ava II þ/þ þ/þ þ/þ þ/þ þ/þ þ/þ7 3 0-b Hinf I þ/þ þ/þ þ/þ þ/þ þ/þ þ/þ

Haplotype 3/2 3/2 1/1 0 3/2 3/1 3/1 0

Internal b-gene framework A/C A/D A/C A/D A/A 0

b-Thalassaemia phenotype Present Absent Present

* Propositus. O, Phase uncertain.

Fig 3. Scheme indicating the position of the seven polymorphic siteswith their specific restriction enzymes (Hc ¼ Hinc-II or Hind-II;Hd ¼ Hind-III; Hf ¼ Hinf-I) (according to Varawalla et al, 1992, andSutton et al, 1989). The position of the internal b-gene framework isindicated by x (Losekoot et al, 1992).

An efficient post-translational and proteolytic mechanism,combined with a compensatory erythropoietic increase (highred cell count) and the coexistence of a thalassaemia, mayaccount for the normal haemoglobin level in the father.

Similarly, the same mechanisms could explain the mod-erate anaemia in the son, in the absence of a thalassaemia.The normal HbA2 levels in both carriers could indicate thatthe excess of free a chains in the mature reticulocyte islimited, supporting the hypothesis of an efficient post-translational and proteolytic mechanism (Wickramasinghe& Lee, 1998; Giordano et al, 1998). Alternatively, the thalas-saemia determinant may be acting not only on the b- butalso on the d-globin gene expression.

The haematological data reported for the English family(Thein et al, 1993) seems to be more in agreement with thefirst hypothesis, because the carriers with increased HbA2

values showed lower Hb levels than the carriers with normalHbA2 without a correlation to the degree of biosyntheticreduction of the b chain.

Alternatively, a gradient of severity, mild in the early redcell precursors and more severe in the mature reticulocytes,could also explain the mild phenotype in spite of the stronglyunbalanced b/a ratios measured in the reticulocytes. Thiswould implicate that a possible anomalous erythroid factorcould have a deleterious effect on the transcription mechan-ism, leaving little messenger to be translated in the maturereticulocytes, which than will reveal a strongly unbalancedsynthetic ratio.

Having excluded spurious paternity, the presence of the samepaternal haplotype in both the affected son and the non-thalassaemic daughter could be explained in two ways: (1) Theb

thalassaemia is determined ‘in cis’ by a structural abnormality ofthe b-LCR region. The daughter (III-1) would have theninherited a chromosome from the father (II-2) in which thedefect involving the regulatory area has been exchanged bycrossing-over with the normal homologue. (2) The b thalassae-mia is caused ‘in trans’ by an abnormal soluble factor(s)expressed by a gene(s) not closely linked to the b-globin cluster.

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374 P. C. Giordano et al

Fig 4. Sequence diagrams of RT-PCR productsshowing: (a) the first few nucleotides of theb-gene sequence in a control with cd2 C/Chomozygosity; (b) the presence of the cd2 C/Tpolymorphism in the father (II-2), indicated byan arrow, demonstrating the presence ofexpression from both alleles.

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We suggest that this family is affected by a form of b

thalassaemia which is not linked to the b-globin gene cluster.Our conclusion is based on the following evidence: (1) Thesequence analysis of the HS-2 motifs intended to exclude across-over event in the b-LCR has shown that the same HS-2allele of the father was inherited by both children. (2) Unlikethe carriers of large b-LCR deletions described in the litera-ture, the b/a ratio is in this case extremely imbalanced, evenin coexistence with a thalassaemia in the propositus. Wethink, therefore, that such an imbalance is more in agree-ment with a regulation failure of both the b-alleles due to amutation of a non-linked transcription factor. (3) Themessenger RNA expression of both the cd2 CAC and CATalleles was present in the father with a b-thalassaemiaphenotype.

No material or medical history was available from thegrandparents and we were therefore not able to verify whetherthe propositus (II-2) had an inherited or a de novo defect. Forthe same reason it was not possible to establish the phase ofthe third and fourth polymorphic marker in the brother(II-4) and sister (II-1) of the propositus.

Because several co-acting factors appear to be involved inthe expression of the globin genes, one could expect a largernumber of thalassaemia patients with non-gene-relateddefects induced by disturbances of ‘in trans’ acting elements.Conversely, only few cases of unmistakable non-gene-relatedthalassaemias are described.

Thalassaemia induced by ‘in trans’ acting factors couldperhaps produce mild changes in the red cell metabolism andtherefore not be selected by malaria. More likely, however,defects in essential DNA binding proteins could induce astrong bi-allelic inhibition of crucial genes leading to fatalproblems early in ontogenesis.

Nevertheless the number of reported cases seems to beslowly increasing and this report and recent congress pre-sentations (unpublished observations) could indicate thatdefects of ‘trans-acting factors’ could be responsible for atleast a part of those expression defects for which no mol-ecular anomalies of the b-globin gene have been found.

‘In cis’ interaction between elements of the globin genespromoter and the LCR region is accomplished through ‘intrans’-acting erythroid specific factors (TF) such as theKruppel-like factor (EKLF), NF-E1, NF-E2, etc. These factorsare proteins able to recognize specific regulatory elementsand promoter sequences involved in the transcription of theglobin genes (Feng et al, 1994; Yamamoto et al, 1992). Weintend to continue the molecular study of this family,examining the sequences of the EKLF, NF-E1 and NF-E2factors.

REFERENCES

Bernini, L.F. (1969) Rapid estimation of haemoglobin A2 by DEAEchromatography. Biochemical Genetics, 2, 305–310.

Betke, K., Marti, H.R. & Schlicht, I. (1959) Estimation of smallpercentages of foetal haemoglobin. Nature, 184, 1877–1878.

Beutler, E. & Gelbart, T. (1986) The mechanism of removal ofleucocytes by cellulose columns. Blood Cells, 12, 57–64.

Feng, W.C., Southwood, C.M. & Bieker, J.J. (1994) Analyses of

b-thalassemia mutant DNA interaction with erythroid Kruppel-like factor (EKLF), and erythroid cell-specific transcription factor.Journal of Biological Chemistry, 269, 1493–1500.

Gasperini, D., Perseu, L., Melis, M.A., Maccioni, L., Sollaino, M.C.,Paglietti, E., Cao, A. & Galanello, R. (1988) Heterozygous b-thalassemia with thalassemia intermedia phenotype. AmericanJournal of Hematology, 57, 43–47.

Gibbons, R.J., Sutthers, G.K., Wilkie, A.O.M., Buckle, V.J. & Higgs,D.R. (1992) X-linked a-thalassemia/mental retardation (ATR-X)syndrome: localisation to Xq12-q21.31 by X inactivation andlinkage analysis. American Journal of Human Genetics, 51, 1136–1149.

Giordano, P.C., Fodde, R., Losekoot, M. & Bernini, L.F. (1989) Designof a programmable automatic apparatus for the DNA polymerasechain reaction. Technique, 1, 16–20.

Giordano, P.C., Harteveld, C.L., Michiels, J.J., Terpstra, W., Schelf-hout, L.J.D.M., Appel, I.M., Batelaan, D., van Delft, P., Plug, R. &Bernini, L.F. (1998) Phenotype variability of the dominant b-thalassemia induced in four Dutch families by the rare cd121(G → T) mutation. Annals of Hematology, (in press).

Harteveld, C.L., Heister, J.G.A.M., Giordano, P.C., Batelaan, D., vanDelft, P., Haak, H.L., Wijermans, P.W., Losekoot, M. & Bernini, L.F.(1996) An IVS1–116(A → G) acceptor splice site mutation in thea2 globin gene causing aþ thalassaemia in two Dutch families.British Journal of Haematology, 95, 461–466.

Harteveld, C.L., Losekoot, M., Haak, H.L., Heister, J.G.A.M., Gior-dano, P.C. & Bernini, L.F. (1994) A novel polyadenylation signalmutation in the a2-globin gene causing a-thalassaemia. BritishJournal of Haematology, 87, 139–143.

Hultman, T., Stahl, S., Hornes, E. & Uhlin, M. (1989) Direct solidphase sequencing of genomic and plasmid DNA using mag-netic beads as solid support. Nucleic Acids Research, 17, 4937–4946.

Kugler, W., Enssle, J., Hentze, M.W. & Kulozik, A.E. (1995) Nucleardegradation of nonsense mutated beta-globin mRNA: a post-transcriptional mechanism to protect heterozygotes from severeclinical manifestations of beta-thalassemia. Nucleic Acids Research,23, 413–418.

Losekoot, M., Fodde, R., Harteveld, C.L., van Heeren, H., Giordano,P.C. & Bernini, L.F. (1990) Denaturing gradient gel electrophoresisand direct sequencing of PCR amplified genomic DNA: a rapid andreliable diagnostic approach to b-thalassaemia. British Journal ofHaematology, 76, 269–274.

Losekoot, M., van Heeren, H., Schipper, J.J., Giordano, P.C., Bernini,L.F. & Fodde, R. (1992) Rapid detection of the highly polymorphicb-globin framework by denaturing gradient gel electrophoresis.Journal of Medical Genetics, 29, 574–577.

Miller, S.A., Dykes, D.D. & Polesky, H.F. (1988) A simple salting outprocedure for extracting DNA from human nucleated cells. NucleicAcids Research, 16, 1215.

Murru, S., Loudianos, G., Porcu, S., Sciarratta, G.V., Agosti, S.,Parodi, M.I., Cao, A. & Pirastu, M. (1992) A b-thalassaemiaphenotype not linked to the b-globin cluster in an Italian family.British Journal of Haematology, 81, 283–287.

Oner, C., Dimowsky, A.J., Altay, C., Gurgey, A., Gu, Y.C., Huisman,T.H.J. & Lanclos, K.D. (1992) Sequence variation in the 5 0

hypersensitive site-2 of the locus control region of bS chromo-somes are associated with different levels of fetal globin inhemoglobin S homozygotes. Blood, 79, 813–819.

Pacheco, P., Peres, M.J., Faustino, P., Pischedda, C., Goncalves, J.,Carvajales-Ramos, M., Seixas, T., Martins, M.C., Moi, P. & Lavinha,J. (1995) b-thalassaemia unlinked to the b-globin gene interactswith sickle-cell trait in a Portuguese family. British Journal ofHaematology, 91, 85–89.

Sanger, F., Nicklen, S. & Coulson, A.R. (1977) DNA sequencing withchain terminating inhibitors. Proceedings of the National Academy ofSciences of the United States of America, 74, 5463.

Smithies, O. (1965) Characterization of genetics variants of bloodproteins. Vox Sanguinis, 10, 359–369.

Sutton, M., Bouhassira, E.E. & Nagel, R.L. (1989) Polymerase chainreaction amplification applied to the determination of b-like globingene cluster haplotypes. American Journal of Hematology, 32, 66–69.

Thein, S.L., Wood, W.G., Wickramasinghe, N.S. & Galvin, M.C. (1993)b-thalassemia unlinked to the b-globin gene in an English family.Blood, 82, 961–967.

Varawalla, N.Y., Fitches, A.S. & Old, G.M. (1992) Analysis of b-globingene haplotypes in Asian Indians: origin and spread of b-thalas-saemia on the Indian subcontinent. Human Genetics, 90, 443–449.

Weatherall, D.J. & Clegg, J.B. (1981) Screening procedures forquantitative abnormalities in haemoglobin synthesis. Methods inEnzymology, 76, 751–763.

Weening, R.S., Roos, D. & Loos, J.A. (1974) Oxygen consumption ofphagocytising cells in human leucocyte and granulocyte prepara-tions: a comparative study. Laboratory and Clinical Medicine, 83,570–574.

Wickramasinghe, S.N. & Lee, M.J. (1998) Evidence that the ubiquitinproteolytic pathway is involved in the degradation of precipitatedglobin chains in thalassaemia. British Journal of Haematology, 101,245–250.

Yamamoto, M., Ko, L.J., Leonard, M.W., Beug, H., Orkin, S.H. &Engel, J.D. (1992) Activity and tissue-specific expression of thetranscription factor NF-E1 multigene family. Genes and Develop-ment, 4, 1650–1662.

q 1998 Blackwell Science Ltd, British Journal of Haematology 103: 370–376

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