Rapid Diagnosis of P-Thalassemia Mutations in Chinese by Naturally and Amplified Created Restriction Sites

By Jan-Gowth Chang, Pao-Huei Chen, Shyh-Shin Chiou, Long-Shyong Lee, Liuh-l Perng, and Ta-Chih Liu

We developed a rapid and simple method to diagnose the molecular defects of p-thalassemia in Chinese patients. This method involves the selective amplification of a DNA frag- ment from human p globin gene with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. To detect the 4-nucleotide deletion of codon 41-42, we introduced a single mismatch nucleotide into the 3‘ end of the upstream primer to create an artificial Taq I restriction site. With a similar approach, an artificial Rsa I site was generated to detect the nucleotide 654 mutation (C + T) of IVS-2, an Alu I restriction site was created to detect the codon 17 mutation (A -+ T), and EcoRl restriction site was

-THALASSEMIA is a heterogenous group of an inher- ited disease characterized by an abnormality of P

globin production.’ More than 90 types of mutation have been reported.2 In general, each population has a different group of mutations, consisting of a few very common ones and a variable number of rare ones.3 In the Chinese, the spectra of mutation are: (1) frameshift codon 41 and 42 (-TCTT); (2) the C + T substitution at position 654 of intron 2 (IVS-2); (3) the nonsense mutation A + T at codon 17; (4) the mutation at position -28 (A + G), position -29 (A + G), and position -30 (T + C) of the promoter region; ( 5 ) the G + T mutation at position 1 of intron 1 (IVS-1); (6) the frameshift codon 14 and 15 (+G); (7) the G + C mutation at position 5 of IVS-1; (8) the frameshift codon 71 and 72 (+A) and the frameshift codon 71 (+T); (9) the frameshift codon 27 and 28 (+C); (10) the nonsense mutation G -+ T at codon 43; (11) a 4-nucleotide (nt) deletion (-AAAC) at position +40 from the cap site; and (12) the initial codon mutation (ATG -+ AGG).4-’3 The first four mutations account for 91% of P-thalassemia in South China.

The study of these genetic mutations has been based on polymerase chain reaction (PCR) and allele-specific oligo- nucleotide hybridization patterns. These methods require

P

From the Department of Molecular Medicine and Clinical Pathol- ogy, Taipei Municipal Jen-Ai Hospital and Taipei Institute of Pathol- ogy, Taipei, Taiwan; and the Departments of Pediatrics and Intemal Medicine, Kaohsiung Medical College, Kaohsiung, Taiwan.

Submitted December 9, 1991; accepted June 19, 1992. Supported by grants from the National Science Council of the

Republic of China and Taipei Institute of Pathology (NSC-79-0412-B- 196-02, NSC-81-0412-B-I96-01, and TIP-I).

Address reprint requests to Jan-Gowth Chang, MD, Department of Molecular Medicine and Clinical Pathology, Taipei Municipal Jen-Ai Hospital, IO, Section 4, Jen-Ai Road, Taipei, Taiwan.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

0 1992 by The American Society of Hematology. 0006-4971 I92 l8008-0022$3.00/ 0

created for the -28 mutation (A - G), a Rsa I restriction site was created for the nucleotide 5 mutation (G + C) of IVS-1, and a Spe I restriction site was created to distinguish the codon 71 (+T) and codon 71/72 (+A) mutations from a normal sequence. The other eight rare mutations that occur in the genes of the Chinese people naturally create or abolish restriction sites. Using this kind of approach, we are able to provide a simple, rapid, accurate, and nonradioactive method to detect the genetic defects of p-thalassemia in the Chinese population. It should be used not only for routine screening but also for prenatal diagnosis. 0 1992 by The American Society of Hematology.

radioisotope or immunochemical detection, which is incon- venient for clinical use. Another approach is the use of simple PCR followed by the restrictive enzyme digestion and visualization of the fragments after electrophoresis in agarose gel. However, this process has its difficulties be- cause not all the mutations have created or abolished an enzyme cutting site. To overcome this problem, we used artificial base substitution to create restriction sites after PCR. The created restriction sites are immediately adja- cent to loci for point mutations and enable us to distinguish the normal from mutated genes on the basis of restriction analysis of the appropriate enzyme.

MATERIALS AND METHODS

DNA isolated from peripheral blood samples of 60 Chinese patients with p-thalassemia minor or major was analyzed. Of those patients, 45 patients with P-thalassemia minor had been previously analyzed using allele-specific oligonucleotide hybridization or di- rect sequen~ing.’~-’~

The strategy of the characterization of the mutations is shown in Table 1. The PCR products with artificial base substitutions near position -28, codon 17, codon 41-42, and nt 654 of IVS-2 are generated by the means of a terminal 3’ base alteration (Figs lA, 2A, 3A, and 4A).

For the A + G mutation at position -28, the mutant G at position -28 together with the artificial T at position -25 create a GAAlTC site for the EcoRI restriction enzyme, while the normal AAAlTC sequence disrupts this target sequence.

The A + T substitution at codon 17 creates a new Mae I restriction site, but the enzyme (Mae I) is expensive and inconve- nient to use. For this mutation, we introduce an artificial A to codon 16 of the upstream primer, with the mutant base of T at codon 17, so that an AGCT site for the restriction enzyme ofAlu I is created.

For the 4-nt deletion of codon 41-42, the artificial C at codon 41, together with the 4-base deletion, creates a TCGA site for the restriction enzyme Taq I, while the normal TCCT disrupts this target sequence.

For the nt 654 mutation ( C + T ) of IVS-2, the mutant T at position 654, together with the artificial C at position 656, creates a GTAC site for the restriction enzyme of h a I, while the normal GCAC disrupts this target sequence.

For the nt 5 mutation (G-C) of IVS-1, the normal G at position 5, together with the artificial G at position 8 (antisense

2092 Blood, Vol80, No 8 (October 15). 1992: pp 2092-2096

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RAPID DIAGNOSIS OF O-THALASSEMIA IN MUTATIONS 2093

Table 1. List of Restriction Enzymes That Distinguish Between Normal and All Reported Mutants in Chinese

Restriction Enzyme

Mutation Position Natural Created Ifor mutant) ~

A - G - 28 A+G - 29 T - A - 30 -AAAC ATG - AGG Initial codon +G Codon 14/15 AAG - TAG Codon 17 +C Codon 27/28 G-T IVS-1 nt 1 G+C IVS-1 nt 5 -TClT Codon 41 I42 GAG + TAG Codon 43 +T Codon 71 +A Codon71172 C+T IVS-2 nt 654

+40 + +43 from cap

- EcoRl Nla 111 - BSP 1236 I - Ode I - EcoRll - EcoRll - Mae I Alu i Nla IV - BSPM I -

- Rsa I (for normal) - Taq I

Abolish Hinfl - - Spe I (for normal)

Mse I Spe I (for normal) - Rsa I

strand), creates a GTAC site (downstream primer, 5'-CITAAAC- CTGTCTTGTAACCTTGGTA-3') for the restriction enzyme of Rra I, while the mutant CTAG disrupts this target sequence. The upstream primer for this mutation is 5'-GCAACCTCAAACAGA- CACCA-3'.

A

0 m ., k I n7. y

enad M 8 .I"

-7 3. 3 '

Fig 1. (A) The strategy for diagnosis of the position -28 mutation (A + 0) of the promoter area. The normal DNA sequences adjacent t o position -28, the mutagenesis primer (downstream primer), and the upstream primer are shown. The sequences of amplified product and the site for the restriction enzyme EcoRl are indicated. The theory of the strategy is the same in each of the following figures. (6) The results of PCR product digested by EcoRI. The molecular weight marker in lane M is the pGem marker. Lane 1 contains undigested amplified DNA. Lane 6 is a heterozygote mutation, whereas lanes 2,3, 4, and 5 are normal cases. In each of the figures, M represents the pGem marker. Similarly, lane 1 represents the undigested PCR product. (C) The results of direct sequencing of heterozygote (case 6, left panel) and normal cases (case 5, right panel). Position -28 is denoted by an arrow.

U Ir

Fig 2. (A) The strategy for diagnosis of codon 17 mutation (A + l). The Alu I restriction site is underlined. (6) The results of PCR product digested by Alu 1. Lane 2 is a heterozygote and lane 3 is a homozygote mutation. Lane 4 is a normal case. (C) The results of direct sequencing of normal, (case4, left panel), heterozygote (case 2, middle panel), and homozygote (case 3, right panel) cases. The codon 17 is denoted by a bracket.

For the mutations of codon 71 (+T) and codon 71/72 (+A), we substitute the TIT sequences of codon 71 to ACT artificially, which, together with the normal sequence of codon 72 (AGT), will create a ACTAGT site for Spe 1. The mutant ACTAAGT or ACTTAGT disrupts this target sequence (Fig SA). These two mutations are further distinguished by Mse 1 digestion of the regular PCR product.

For the remaining eight rare genetic mutations of the Chinese, which naturally create or abolish a specific restriction site, three pairs of primers were used for PCR. For position -29 (A + G )

- htaq.M.1. p r l r r I*--ACCCllCGACCUCAGGK-l

3' 5 # ... ... ... . I I P m F d - r l :;t::;:::::*/

No-1 5 * P N X C K C G A - 1' N o Taq I .It.

Cr.aC.d Taq I SIC. Mutant 5*--ACCCllCGA-- . I *

- c i.

Fig 3. (A) Schematic diagram of the codon 41-42 (-4 nt) frameshift assay. The restriction site of T89 I is shown. (6) The results of PCR product digested by Tag 1. Lane 3 is a heterozygote and lane 4 is a homozygote mutation, while lane 2 is a normal case. (C) The direct sequencing data of case 2 (left panel, normal), case 3 (middle panel, heterozygote), and case 4 (right panel, homozygote). The codons 40 and 41 are denoted by brackets. The site of the 4-nt deletion is denoted by an arrow.

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2094 CHANG ET AL

A I n - 2 n C 6 I 4 C -B T

654

Fig 4. D e t d o n of nt 654 mutation (C-T) of IVS-2. (A) The sequences of normal DNA adjacent to nt 654, of mutagenesis primer (downstream), and of upstream primer are shown. The Rsa I restric- tion site is underlined. (B) The results of PCR products digested by Rsa 1. Lanes 2, 5, and 6 are heterozygote mutations and lane 8 is a homozygote mutation, while lanes 3, 4, and 7 are normal cases. (C) The direct sequencing data of case 7 (left panel, normal), case 5 (middle panel, heterozygote), and case 8 (right panel, homozygote). The nucleotide 654 is indicated by an arrow.

and position -30 (T + A), the upstream primer is 5'-ATCACITA- GACCTCACCCTGTGGAGCCA-3' and the downstream primer is 5'-GAACACAGTTGTGTCAGAAG-3'. For the 4-base dele- tion (-AAAC) of position +40 to +43 from the cap sites, the

I f b I T

0

F

U T G A

b T G

C

1. ;*

Fig 5. Detection of codon 71 (+T) and codon 71/72 (+A) muta- tions. (A) The sequences of normal DNA adjacent to codon 71/72, of mutagenesis primer (upstream), and of downstream primer are shown. The created Spe I restriction site is underlined. (6) The results of PCR products digested by Spe 1. Lane 2 is a case of heterozygote of codon 71/72 (+A) mutation, while lane 3 is a normal case. (C) The direct sequencing data of case 3 (left panel, normal) and case 2 (right panel, heterozygote). Codon 71 is denoted by a bracket.

mutation of the initial codon (ATG 4 AGG), the addition of G at codon 14/15 ( + G ) , the addition of C at codon 27/28 (C), and the mutation of nt 1 of IVS-1 (G + T), the primer sets are: upstream primer, 5'-CTGGGCATAAAAGTCAGGG-3'; and downstream primer, 5'-GGCAGAGAGAGTCAGTGCCTA-3'. For the muta- tion of codon 43 (GAG +TAG) and codon 71/72 (+A). the primer sets are: upstream primer, S'-CATGTGGAGACAGAGAA- GAC-3'; and downstream primer, 5'-TCATTCGTCTGTITC- CCATTCTAAAC-3' (Table 1).

All the mutants can be distinguished from the normal allele on the basis of enzyme digestion of the PCR product, with changes in fragment sizes detected in the 3% agarose gel electrophoresis.

The DNA amplification was performed as described,I5 with some modification. Genomic DNA (0.5 kg) was mixed with 100 ng of each primer and 200 kmol/L of each dNTP in 100 KL reaction buffer containing 10 mmol/L Tris-HCI (pH 8.3), 50 mmol/L KCI, 1.5 mmol/L MgCI. and 0.01% gelatin. The mixture was heated to 94°C for 10 minutes for strand separation and then 2.5 U of Taq polymerase (Promega, Madison, WI) was added. The PCR pro- grams were the same for all primer pairs; the procedure was denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 2 minutes.

RESULTS

The results of the restriction map change of P-thalas- semia after restriction enzyme digestion are shown in Figs lB, 2B, 3B, 4B, and 5B. For the position -28 mutation (A + G) of the promotor area, a 292-bp fragment was amplified by the appropriate primer pairs, and a new EcoRI restriction site was created in the PCR product of the mutant allele. The PCR product was digested by EcoRI and a 292-bp band identical to the undigested product was noted in the normal chromosome. However, there was a 267-bp band for the mutant case (Fig 1B). The normal and mutant cases were also confirmed by direct sequencing (Fig

For the codon 17 mutation (A + T), a 170-bp fragment was amplified by the PCR. After digestion of the PCR products by restriction enzyme A h I, a 170-bp fragment identical to the undigested product was shown in the normal allele, but a 147-bp band was found in the mutant case (Fig 2B). Direct sequencing of normal, heterozygote, and homozygote is shown in Fig 2C.

For the 4-nt deletion of codon 41-42, the size of the PCR product was 334 bp. The fragment was 334 bp in the undigested product and normal allele digested by Tuq I, but 306 bp in the mutant digested with Tuq I (Fig 3B). The cases of normal, heterozygote, and homozygote were con- firmed by direct sequencing (Fig 3C).

For nt 654 mutation (C + T) of IVS-2, digestion of PCR product by Rsa I showed a 233-bp fragment in the normal allele and a 198-bp fragment in the mutant allele (Fig 4B). Lanes 2,5, and 6 were heterozygote cases and lane 8 was a homozygote case, as confirmed by direct sequencing (Fig

For the mutations of codon 71 (+T) and codon 71/72 (+A), the PCR product was 241 bp in length. The PCR products were digested by Spe I. A 241-bp fragment

IC).

4C).

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RAPID DIAGNOSIS OF f3-THAIASSEMIA IN MUTATIONS 2095

A codon 71/72 +A B codon 27/28 +c

0 284 354 164 N- 0

n i IU. I

170 114 :84 418 n 204 I 150 64 I )U. I IU- I N1. I V

Fig 6. (A) The results of PCR product digested by Mae I for diagnosls of codon 71/72 (+A) mutation. b n e 2 is a heterozygote mutation, the 418-bp PCR product was digested to 354-bp, 204-bp, 150-bp, and 64-bp fragments. Lane 3 is a normal case, the PCR product was digested to 354-bp and 64-bp fragments. (B) The results of PCR product digested by NIa IV for diagnosis of the codon 27/28 (+C) mutation. Lane 2 is a heterozygote mutation; 170-bp and 114-bp fragments were noted. Lane 3 is a normal case.

identical to the uncut product was noted in the mutant and a 216-bp fragment was found in the normal allele (Fig 5B). The normal and mutant cases were confirmed by direct sequencing (Fig 5C).

In the nt 5 mutation of IVS-1 (G + C), the PCR product was 142 bp in length. After digestion of the PCR product by &u I, a 142-bp fragment, the same length as the undigested product, was noted in the mutant and the normal showed a 116-bp band (data not shown). For the other eight rare mutations of the Chinese, their PCR products were di- gested by their respective specific restriction enzymes and analyzed in comparison with the normal restriction map. The results of two of these eight mutations are shown in Fig

REFERE 1. Bunn HF, Forget BG: .Hemoglobin: Molecular, Genetic, and

Clinical Aspect. Philadelphia, PA, Saunders, 1986 2. Kazazian HH Jr: The thalassemia syndromes: Molecular basis

and prenatal diagnosis in 1990. Semin Hematol27:209,1990 3. Kazazian HH Jr, Boehm CD: Molecular basis and prenatal

diagnosis of p-thalassemia. Blood 721 107, 1988 4. Kazazian HH Jr, Dowling CE, Waber PG, Huang S, Lo

WHY: The spectrum of @-thalassemia genes in China and South- east Asia. Blood 68:964,1986

5. Lam VMS, Xie SS, Tam JWO, Woo YK, Gu Y L A new single nucleotide change at the initiation codon (ATG 4 AGG) identi- fied in amplified genomic DNA in a Chinese @-thalassemic patient. Blood 75:1207,1990

6. Liu JZ, Gao QS, Jiang Z, Liang CC, Yang KB, Wu GY, Long GF. Li Q, Zhang J, Deng B, Wang R X Studies of 8-thalassemia mutations in families living in three provinces in Southern China. Hemoglobin 13585,1989 7. Antonarakis SE, Kang J, Lam VMS, Tam JWO, Li AMC

Molecular characterization of @-thalassemia intermedia in South China. Br J Haematol70357,1988

8. Chan V, Chan TK, Chehab FF, Todd D Distribution of @-thalassemia mutations in South China and their association with haplotypes. Am J Hum Genet 41:678,1987

6. Comparing the data with our previous studies,13J4 we found this study was in agreement with previous data (data not shown).

DISCUSSION

In the genes of the Chinese, there are 15 or more mutations for p-thalassemia. Nine of these mutations create new restriction sites of various restriction enzymes and one mutation abolishes a normal restriction site; another five mutations neither create nor abolish restriction sites (Table 1). Traditionally, detection of these mutations is dependent on allele-specific hybridization or direct se- q~encing.4-l~ In this study, we devised a nonradioactive method by using selective amplification of a DNA fragment of p globin gene with specific oligonucleotide primers, followed by digestion of the amplified product by restriction enzymes that recognize artificially created or naturally occurring restriction sites. In comparison with our previous ~ t u d i e s , ~ ~ J ~ we find this method provides a rapid and simple procedure for identifying all the mutations found in the genes of the Chinese. The method is not only very useful for detecting mutations of p-thalassemia and prenatal diagno- sis in Chinese, but also for further investigation of new mutations.

The same strategy has been used to diagnose phenylketo- nuria (PKU),I6 p-thalassemia in Mediterranean~,~'rus onco- gene mutation,I8 (3-6-PD defi~iency,'~ cystic fibrosis, and retinitis pigmentosa.2n All the studies show that this kind of approach provides a simple, rapid, and accurate screening method and should be used routinely.

ACKNOWLEDGMENT We thank L.H. Yang, W.D. Su, and J.S. Yang for their excellent

technical assistance.

iNCES 9. Chan V, Chan TK, Kan YW, Todd D: A novel 8-thalassemia

frameshift mutation (codon 14/15) detectable by visualization of abnormal restriction fragment in amplified genomic DNA. Blood 721420,1988

10. Huang S, Waber PG. Dowling CE, Wong C, Antonarakis SE, Cai RL, Wang MQ, Lo WHY, Kazazian HH Jr: @-Thalassemia in China: A systemic molecular characterization of f3-thalassemia mutations. Hemoglobin 12621,1988

11. Zhang JZ, Cai SP. HE X, Lin HJ, Huang ZG, Chehab FF. Kan YW: Molecular basis of 8-thalassemia in South China. Strategy for DNA analysis. Hum Genet 7837,1988

12. Xu YH, Zhang ML, Ren JR, Huang SZ, Zeng YT: A novel Chinese @-thalassemia mutat ion4 bp (AAAC) deletion at posi- tion +40 to +43 from the cap site. Blood 7681a, 1990 (abstr, suppl 1)

13. Chang JG, Liu TC, Lin CP, Chen PH, Lee Ls: Molecular basis of @-thalassemia minor in Taiwan. Blood 7657a, 1990 (abstr,

14. Lee HH. Chang JG, Lee LS. Lin ST. KO TM, Choo KB: Detection of @ globin gene mutations by polymerase chain reac- tion. Proc Natl Sci Counc Repub China [B] 15:97,1991

SUPPI 1)

For personal use only.on March 25, 2018. by guest www.bloodjournal.orgFrom

2096 CHANG ET AL

15. Liu TC, Chang JG, Lin CP, Lee LS, Lin SF, Liu HW, Chen TP: Detection of @-globin gene from single hairs. Kao Hsiung J Med Sci 6:181,1990

16. Eiken HG, Odland E, Boman H, Skjelkvale L, Engebretsen LF, Apold J: Application of natural and amplification created restriction sites for the diagnosis of PKU mutations. Nucleic Acids Res 191427,1991

17. Lindeman R, Hu SP, Valpato F, Trent RJ: Polymerase chain reaction mutagenesis enabling rapid non-radioactive detection of common @-thalassemia in Mediterraneans. Br J Haematol78:100, 1991

18. Haliassos A, Chomel JC, Tesson L, Baudis M, Kruh J, Kaplan JC, Kitzis A: Modification of enzymatically amplified DNA for the detection of point mutation. Nucleic Acids Res 17:3606, 1989

19. Chang JG, Chiou SS, Perng LI, Chen TC, Liu TC, Lee LS, Chen PH, Tang T K Molecular characterization of G-6-PD defi- ciency by naturally and amplification created restriction sites- Five mutations account for most G-6-PD deficiency cases in Taiwan. Blood (in press)

20. Sorscher EJ, Huang Z Diagnosis of genetic disease by primer-specified restriction map modification, with application to cystic fibrosis and retinitis pigmentosa. Lancet 337:1115, 1991

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1992 80: 2092-2096  

JG Chang, PH Chen, SS Chiou, LS Lee, LI Perng and TC Liu naturally and amplified created restriction sitesRapid diagnosis of beta-thalassemia mutations in Chinese by 

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