Characterisation and confirmation of rare beta-thalassaemia mutations in the Malay, Chinese and Indian ethnic groups in Malaysia

Pathology (October 2006) 38(5), pp. 437–441

HAEMATOLOGY

Characterisation and confirmation of rare beta-thalassaemia mutations in the Malay, Chinese and Indian ethnic groups in Malaysia JIN AI MARY ANNE TAN*, PUI SEE CHIN*, YEAN CHING WONG*, KIM LIAN TAN*, LEE LEE CHAN{ AND ELIZABETH GEORGE{

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Departments of *Molecular Medicine and {Paediatrics, Faculty of Medicine, University Malaya, Kuala Lumpur, and {Department of Clinical Laboratory Sciences, Faculty of Medicine, University Putra Malaysia, Selangor, Malaysia

Summary Aims: In Malaysia, about 4.5% of the Malay and Chinese populations are heterozygous carriers of P-thalassaemia. The initial identification of rare P-globin gene mutations by genomic sequencing will allow the development of simpler and cost-effective PCR-based techniques to complement the existing amplification refractory mutation system (ARMS) and gap-PCR used for the identification of P-thalassaemia mutations. Methods: DNA from 173 P-thalassaemia carriers and five Pthalassaemia major patients from the Malay, Chinese and Indian ethnic groups were first analysed by ARMS and gapPCR. Ninety-five per cent (174/183) of the 183 P-globin genes studied were characterised using these two techiques. The remaining nine uncharacterised P-globin genes (4.9%) were analysed using genomic sequencing of a 904 bp amplified PCR product consisting of the promoter region, exon 1, intervening sequence (IVS) 1, exon 2 and the 59 IVS2 regions of the P-globin gene. Results: The rare P-globin mutations detected in the Chinese patients were CD27/28 (+C) and CD43 (GAG-TAG), and 288 (C-T) in an Indian patient. Beta-globin mutations at CD16 (2C), IVS1-1 (G-A), IVS2-1 (G-A), 286 (C-G) and Haemoglobin South Florida (CD1, GTG-ATG) were confirmed in the Malay patients. Conclusions: The seven rare P-globin mutations and a rare haemoglobin variant confirmed in this study have been described in other populations but have not been previously described in Malaysian P-thalassemia patients. Key words: Rare P-thalassaemia mutations, Chinese, Malay, Indians, Malaysia, genomic sequencing, ARMS, gap-PCR. Received 3 April, revised 17 May, accepted 25 May 2006

INTRODUCTION The haemoglobinopathies and thalassaemias represent the most common autosomal recessive disorders in the world.1 Beta-thalassaemia major results in severe transfusiondependent anaemia and is caused by the inheritance of two P-globin gene mutations either in a compound heterozygous or homozygous state. Beta-thalassaemia trait (minor) is usually asymptomatic and is associated with the

inheritance of a single P-globin gene defect. Beta-thalassaemia intermedia is of moderate severity and the majority of affected individuals do not require regular blood transfusions.2 The mutations responsible for P-thalassaemia are regionally specific with each population or ethnic group having its unique spectrum of P defects. Despite the large numbers and heterogeneity of mutations causing Pthalassaemia, with about 200 being reported,3 it is fortunate that only a few common mutations together with a variable number of rare mutations account for most of the P-thalassaemia in each ethnic group or population.4 Malaysia has a multi-racial population of 21.89 million citizens, consisting of 65.1% Malays, 26.0% Chinese, 7.7% Indians and 1.2% others.5 Beta-thalassaemia is common in Malaysian Malays and Chinese with a heterozygous carrier rate of about 4.5% in these two ethnic groups.6 There are five common P-globin mutations in the Chinese: CD 41/42 (–TTCT), IVS2-654 (C-T), –28 (A-G), CD17 (A-T) and CD71/72 (+A) and these account for about 90% of the Pthalassaemia.7 Four other mutations at CD26 (HbE, G-A), CD15 (G-A), Cap (+1) (A-C) and –29 (A-G) account for a further 4% of P-thalassaemia in the Malaysian Chinese population.8 The P mutations in the Malaysian Chinese are quite homogeneous as the Chinese are mainly from the South-Eastern provinces of China such as Kwangtung, Fukien and Kwangsi.9 The P-globin mutations in the Malaysian Malays were found to be more heterogeneous. In a study carried out by Tan et al. in 1998, three mutations at CD26 (HbE), IVS1-5 (G-C) and IVS1-1 (G-T) were responsible for about 73.1% of P-thalassaemia; six other P-globin mutations – CD41/42, IVS2-654, CD8/9 (+G), CD17, CD19 (A-G) and Cap (+1) – made up the remaining 11.52% of the identified mutations in this ethnic group, while 15.38% remained uncharacterised.8 In the University Malaya Medical Center (UMMC), 17 P-globin defects are rapidly characterised using the amplification refractory mutation system (ARMS) and gap-polymerase chain reaction (PCR): initiation codon for translation (T-G), 229 (A-G), 228 (A-G), CAP +1 (A-C), CD8/9 (+G), CD15 (G-A), CD17 (A-T), CD19 (A-G), HbE (G-A), IVS1-1 (G-T), IVS1-5 (G-C), CD41/42 (–TTCT), CD71/72 (+A), IVS2-654 (C-T), poly A (A-G), the 100 kb

ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia DOI: 10.1080/00313020600922538

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c(AcdP)u-deletion and the 45 kb Filipino deletion.8,10 The common P-globin gene mutations responsible for Pthalassaemia in the different Malaysian ethnic groups have been identified.11,12 However, there is a paucity of data on the types of rare P-globin gene mutations and this paper represents the first such report. The identification of the rare P-globin defects in the different ethnic groups in Malaysia is necessary because of the country’s multi-racial population and high frequency of inter-racial marriages. Knowledge of the common and rare P-globin mutations will allow the establishment of more specific and costeffective molecular characterisation and prenatal diagnosis programs for each different ethnic group.

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Patient samples The study included 150 Chinese, 22 Malay and 1 Indian P-thalassaemia carriers, in addition to two Chinese, two Malay and one Indian Pthalassaemia major patients. The five P-major patients are transfusiondependent and one P-globin mutation in each patient was confirmed using ARMS while the other P-mutation was uncharacterised. In the case of the 173 P-thalassaemia carriers, DNA amplification using ARMS and gapPCR detected the P-globin gene mutations in 169 patients. The P-defects in the remaining four P-thalassemia carriers were uncharacterised. This study was approved by the Medical Ethics Committee of the University Malaya Medical Center in accordance with the Declaration of Helsinki. Verbal and written consent were obtained from the patients prior to blood sample collection. DNA extraction Peripheral blood samples (5 mL) anti-coagulated with EDTA were collected. DNA was extracted from leucocytes using proteinase K and sodium dodecyl-sulfate (SDS) digestion at 37uC. The DNA solution was then purified using phenol/chloroform/isoamyl alcohol extractions. Purified DNA was solubilised in double-distilled water and stored at 270uC. DNA amplification studies using ARMS and gap-PCR The P-globin mutations determined using ARMS are at the initiation codon for translation (T-G), 229 (A-G), 228 (A-G), CAP +1 (A-C), CD8/ 9 (+G), CD15 (G-A), CD17 (A-T), CD19 (A-G), HbE (G-A), IVS1-1 (GT), IVS1-5 (G-C), CD41/42 (-TTCT), CD71/72 (+A), IVS2-654 (C-T) and poly A (A-G). The primer sequences to detect the fifteen P-globin mutations were paired with common ARMS primers to amplify each Pmutation as a specific molecular weight product.1,8,13 Beta-thalassaemia can also be a result of large deletions along the P-globin gene complex. Beta-thalassaemia caused by a large 45 kb Filipino deletion and a 100kb Gc(AcdP)u Chinese -specific deletion were confirmed by DNA amplification across the deleted regions of the P-globin gene complex (gap-PCR) using specific primers that flank the deleted sequences.14,15 Patient DNA where the P-globin mutations could not be characterized using ARMS and gap-PCR was then processed for genomic sequencing in the P-globin gene. Genomic sequencing A specific 904-bp fragment of the P-globin gene consisting of the promoter region, exon 1, intervening sequence (IVS) 1, exon 2 and the 59 IVS2 regions was first amplified. Amplification was carried out using two primers: B1, 59-GCTTACCAAGCTGTGAT TCCAA-39 (direct) at nucleotide positions 2294 to 2272, and primer B16, 59TCATTCGTCTGTTTCCCATTCTAAAC-39 (reverse) at positions +609 to +574, both relative to the Cap site of the P-globin gene. DNA amplification was carried out in a 25 mL reaction mixture containing 1.25 U Taq DNA Polymerase (Invitrogen Life Technologies, USA), 250 mM of

Pathology (2006), 38(5), October

each dNTP, 20 pmol oligonucleotide primers, 1.5 mM magnesium chloride and 106 PCR buffer. The cycling conditions involved initial denaturation at 95uC for 5 min followed by 35 cycles of denaturation at 95uC for 30 s, annealing at 61uC for 30 s with an extension at 68uC for 1 min with a final extension at 72uC for 3 min. The 904 bp amplified product was purified from excess primers and PCR reagents using the High Pure PCR Purification Kit (Boehringer Mannheim, Germany). The purified PCR products were then electrophoresed in 1.5 % agarose to ensure that the specific 904 bp amplified DNA was isolated. Genomic sequencing was carried out with primer B1 using the ABI PRISM Bigdye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Applied Biosystems Division, USA). The sequencing conditions were initial denaturation at 96uC for 1 min, followed by denaturation at 96uC for 30 s; annealing at 53uC for 5 s; extension at 60uC for 4 min (25 cycles). In addition, another sequencing primer B2: 59-ACCTCACCCTGTGGA-39 (nucleotide positions 2110 to 292 relative to the cap site) was used to detect P-globin mutations further downstream that could not be analysed using primer B1. Confirmation of the P-globin gene mutations obtained by genomic sequencing was carried out using reverse sequencing with primers B16 and B11: 59-TGATACCAACCTGCCCAGG-39 (nucleotide positions +151 to +133). The sequenced products were purified using ethanol/sodium acetate precipitation in micro-centrifuge tubes and electrophoresed on the ABI PRISM 377 Sequencer.

RESULTS Figure 1 shows the PCR products electrophoresed on a 1.5% agarose gel after ARMS and gap-PCR. Fifteen mutations in the P-globin gene can be detected using ARMS and each individual P-mutation is amplified as a distinct fragment with a specific molecular weight (Fig. 1, upper gel lanes 3–11; lower gel lanes 15–20). Each ARMS reaction is carried out with simultaneous amplification (in the same reaction tube) of an internal control (861 bp fragment) which confirms the success of the PCR. The 45 kb Filipino deletion was amplified as a 376 bp Filipino-deletion specific fragment (Fig. 1, lower gel lane 21). The 100 kb Gc(AcdP)u-deletion present in Malaysian Chinese patients was amplified as a 508 bp G A c( cdP)u-deletion specific fragment (Fig. 1, lower gel lane 22). Using the ARMS and gap-PCR, the P-globin gene mutations were characterized in 98% (151/154) of the Chinese, 81% (21/26) of the Malay and 67% (2/3) of the Indian patients (Table 1). The ARMS detected one mutation in each of the five P-major patients (Table 2). The second mutant P-allele in each of the P-major patients could not be identified by ARMS or gap-PCR. Similarly, ARMS and gap-PCR were unable to determine the Pmutations in DNA from four P-thalassaaemia carriers. Genomic sequencing of the 904 bp fragment of the P-globin gene detected the nine uncharacterised P-defects in these patients (Table 2). Four rare P-globin gene mutations and one haemoglobin variant were identified by genomic sequencing of DNA from the Malay patients. Beta-mutations at IVS2-1 (G-A), IVS1-1 (G-A), CD16 (2C), 286 (C-G) and Haemoglobin South Florida (CD1, G-A) were confirmed in DNA from the P-thalassaemia major and carrier patients (Table 2). The rare haemoglobin variant, Hb South Florida (GTGATG), was confirmed in a P-thalassaemia patient who also possessed the IVS1-1 (G-A) mutation.

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DISCUSSION

Fig. 1 Agarose gel electrophoresis of PCR products after DNA amplification using the amplification refractory mutation system (ARMS) and gap-PCR. Upper gel lane 1, 100 bp ladder; lane 2: normal, 861 bp internal control; lane 3, IVS2-654 P-band (828 bp); lane 4, 861 bp internal control and 228 P-band (624 bp); lane 5: 861 bp internal control and CD19 P-band (487 bp); lane 6, 861 bp internal control and CD41/42 P-band (443 bp); lane 7, 861 bp internal control and poly A P-band (392 bp); lane 8, 861 bp internal control and IVS1-5 P-band (285 bp); lane 9, 861 bp internal control and IVS1-1 P-band (281 bp); lane 10, 861 bp internal control and CD71/72 P-band (242 bp); lane 11, 861 bp internal control and CD17 P-band (240 bp); lane 12, PCR control where no water was added. Lower gel lane 13, 100 bp ladder; lane 14, normal; 861 bp internal control; lane 15, 861 bp internal control and -29 P-band (625 bp); lane 16, 861 bp internal control and CAP (+1) P-band (594 bp); lane 17, 861 bp internal control and initiation codon mutant P-band (544 bp); lane 18, 861 bp internal control and CD15 P-band (499 bp); lane 19, 861 bp internal control and CD26 P-band (457 bp); lane 20, 861 bp internal control and CD8/9 P-band (214 bp); lane 21, 376 bp Filipino-deletion specific fragment and 482 bp undeleted P-globin fragment; lane 22, 508 bp Gc(AcdP)u-deletion specific fragment and 682 bp undeleted P-globin fragment.

TABLE 1 P-globin gene mutations identified in Malay, Chinese and Indian P-thalassaemia patients in Malaysia using ARMS and gap-PCR

P-mutations 228 (A-G) CD15 (G-A) CD17 (A-T) CD19 (A-G) IVS1-1 (G-T) IVS1-5 (G-C) CD41/42 (-CTTT) CD71-72 (+A) IVS2-654 (C-T) Poly A (A-G) HbE (G-A) 100 kb Gc(AcdP)u-deletion 45 kb Filipino deletion Unidentified Total number of alleles studied

Chinese (%)

Malay (%)

Indian (%)

14 (9.1) 1 (33.3) 13 (8.4 ) 1 (0.65 ) 65 (42.2) 5 (3.3 ) 49 (32) 2 (1.3) 2 (1.3) 3 (1.9) 154

2 2 7 3

(7.7 ) (7.7 ) (27 ) (11.5 )

2 (7.7) 5 (19.2)

1 (33.3)

5 (19.2) 26

1 (33.3) 3

The two rare mutations confirmed in Chinese patients were CD43 (G-T) and CD27/28 (+C). Genomic sequencing of DNA from an Indian P-thalassaemia major patient revealed the P-globin mutation at 288 (C-T) (Table 2).

The spectrum of P-thalassaemia alleles has been determined in a wide variety of population groups. A small number of ethnic/population group-specific alleles account for about 90% of the P-thalassaemia genes while a larger number of rarer alleles have been observed in each ethnic group that account for the remaining thalassaemia genes.16 In the Malaysian Chinese, four P-globin gene mutations at 228, CD17, CD41/42 and IVS2-654 were responsible for 91.7% of P-thalassaemia in this ethnic group (Table 1). Two mutations alone at CD41/42 and IVS2-654 accounted for 74% of the P-thalassaemia genes. In the Malaysian Malay patients, six P-globin mutations at CD19, IVS1-1 (G-T), IVS1-5 (G-C), CD41/42, poly A and CD26 (HbE) were responsible for 81% of P-thalassaemia (Table 1). Betathalassaemia is rare in Malaysian Indians and previously reported mutations were at 288 and IVS1-5 (G-C).6 The Pglobin mutations detected in the Indian patients in this study using ARMS were CD15 and CD26 (HbE). Using genomic sequencing, seven rare P-globin mutations and one rare haemoglobin variant previously not described in the Malaysian population were confirmed. In the Malaysian Malay patients, the rare P-mutation at IVS21 (G-A) was observed in a compound heterozygous Pthalassaemia major patient who also had the P-mutation at IVSI-1 (G-T) (a common mutation in Malays). IVS2-1 (G-A) has been reported in Mediterraneans, Tunisians and American Negroes,17–19 and in Israel, Turkish and Middle Eastern populations.20 The rare P-mutation at IVS1-1 (G-A), previously reported in Mediterranean,21 East European, Turkish, Italian, Middle Eastern and Egyptian populations,20,22 was confirmed in a Malaysian Malay patient in combination with HbE, while CD16 (2C) and 286 (C-G) were observed in two Malay P-thalassaemia carriers (Table 2). CD16 has been confirmed in Lebanese,16 Asian Indian4 and Singaporean Malay populations,20 while 286 has been reported in Asian Indian, Thai and Lebanese populations.16,23 The rare haemoglobin variant, Hb South Florida, with a base substitution of GTG to ATG at codon 1 of the Pglobin gene,20 was confirmed in a Malay P-thalassaemia carrier who was also found to possess the rare P-mutation at IVS1-1 (G-A) (Table 2). Hb South Florida involves an amino acid substitution of methionine for the NH2terminal valine of the P-globin chain. The initiator methionine is retained on the mutant polypoptide and there is partial acetylation at the NH2 terminus.24 Hb South Florida has only been previously reported once in four members of a Caucasian family.24 The quantity of Hb South Florida reported in the patient was 40% of the total haemoglobin whereby about 20% of the variant was acetylated.24 Hb South Florida does not cause any significant clinical problems in patients25 and this may account for the P-thalassaemia carrier trait in the Malay patient in this study even though she was compound heterozygous for IVS1-1 (G-A) and Hb South Florida. CD43 (G-T) was confirmed in a compound heterozygous state with CD41/42 (-TTCT) in two unrelated Malaysian Chinese P-thalassaemia major patients. CD43 (G-T) results in a non-sense mutation as it changes a GAG codon (glutamic acid) to a TAG stop codon, resulting in a new termination codon at codon 43. This leads to an absence of

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TABLE 2 Rare P-globin gene mutations in Malay, Chinese and Indian P-thalassaemia patients in Malaysia detected using genomic sequencing Patient

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P1 P2 P3 P4 P5 P6 P7 P8 P9

Ethnic group

P-thalassemia status

P-globin mutation (ARMS)

P-globin mutation (genomic sequencing)

Malay Malay Malay Malay Malay Chinese Chinese Chinese Indian

P-major P-major P-trait P-trait P-trait P-major P-major P-trait P-major

IVS1-1 (G-T) HBE (G-A) – – – CD41/42 (-TTCT) CD41/42 (-TTCT) – CD15 (G-A)

IVS2-1 (G-A) IVS1-1 (G-A) IVS1-1 (G-A)/Hb South Florida (CD1, G-A) CD16 (2C) 286 (C-G) CD43 (G-T) CD43 (G-T) CD27/28 (+C) 288 (C-T)

normal mRNA production and causes Pu-thalassaemia. The mutation has been previously reported in the Chinese from South China, Thailand and Singapore.26 The frameshift mutation at CD27/28 (+C) detected in a Chinese patient in this study has been observed mainly as a Chinese mutation.16,27,28 The only Indian P-thalassaemia patient in this study was confirmed with a P-mutation at 288 (C-T) with compound heterozygosity at CD15. The patient was of Sikh/Punjabi ancestry from Northern India. Both 288 and CD15 are rare P-globin mutations previously reported in the Kurdish, Asian Indian and America Negro populations.29–31 Molecular techniques that have been used for the characterisation of P-globin gene alleles include allelespecific oligonucleotide analysis, primer-specific amplification/ARMS, PCR: restriction enzyme analysis, gap-PCR and multiplex minisequencing.1,32 Genomic sequencing is commonly used to confirm P-globin mutations detected using other procedures, and its specialisation lies in the detection and confirmation of unknown mutations. In this study, we have identified seven rare P-mutations – CD16 (2C), CD27/28 (+C), CD43 (G-T), IVS1-1 (G-A), IVS2-1 (G-A), 286 (C-G), 288 (C-T) – and a Hb variant (Hb South Florida) in the Malaysian population. Out of these seven P-globin mutations, only Hb South Florida does not result in P-thalassemia major, and thus does not cause any significant clinical problems. Future work will now involve the development of simpler and more cost-effective PCRbased techniques to complement the existing ARMS and gap-PCR for the identification of the remaining six rare Pthalassaemia mutations. This will allow molecular characterisation and prenatal diagnosis of P-globin defects to be readily carried out in general hospitals, institutions and centres using protocols that involve equipment which is already available in the diagnostics laboratories.

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ACKNOWLEDGEMENTS This study was supported by the Malaysian Government IRPA R & D Grant (IRPA 06-0302-0717).

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Address for correspondence: Dr J. A. M. A. Tan, Department of Molecular Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia. E-mail: [email protected]

19. 20.

21.

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