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Molecular Characterization of Glucose-6-Phosphate Dehydrogenase (G6PD)

Deficiency by Natural and Amplification Created Restriction Sites: Five
Mutations Account for Most G6PD Deficiency Cases in Taiwan
By Jan-Gowth Chang, Shyh-Shin Chiou, Liuh-l Perng, Ta-Chend Chen, Ta-Chih Liu,
Long-Shyong Lee, Pao-Huei Chen, and Tang K. Tang

We have developed a rapid and simple method to diagnose 94) were C to Tmutation at nt 1024,1.1% (1 of 94) was G to T
the molecular defects of glucose-6-phosphate dehydroge- mutation at nt 392, and 1.1% (1 of 94) was G to A mutation at
nase (GGPD) deficiency in Chinese in Taiwan. This method nt 487. These results show that the former five mutations
involves the selective amplification of a DNA fragment from account for more than 90% of GGPD deficiency cases in
human GGPD gene with specific oligonucleotide primers Taiwan. Aside from showing that G to T change at nt 1376 is
followed by digestion with restriction enzymes that recog- the most common mutation, our research indicates that nt
nize artificially created or naturally occurring restriction 493 mutation is a frequent mutation among Chinese in
sites. Ninety-four Chinese males with GGPD deficiency were Taiwan. We compared GGPD activity among different muta-
studied. The results show that 50% (47 of 94) were G to T tions, without discovering significant differences between
mutation at nucleotide (nt) 1376,21.3% (20 of 94) were G to A them.
mutation at nt 1388,7.4% (7 of 94) were A to G mutation at nt o 1992by The American Society of Hematology.
493.7.4% (7 of 94) were A to G mutation at nt 95,4.2% (4 of

G LUCOSE-6-PHOSPHATE dehydrogenase (G6PD) is

an X chromosome-linked enzyme, the first key en-
zyme of the pentose phosphate pathway. It plays a major
primer design and restriction enzyme analysis are shown in Table 1.
Our main purpose was to design an accurate and affordable
method of creating a restriction site in mutations that did not
role in the production of ribose-5-phosphate and the originally have a natural restriction site because the enzyme for
natural restriction site is too expensive. For the 1376 G to T
generation of nicotinamide-adenine-dinucleotide phos-
mutation, which normally does not generate any restriction site,
phate (reduced form) (NADPH) in the hexose monophos- the mutant T at nucleotide (nt) 1376 with the artificial C (nt 1377)
phate shunt. NADPH is required for a variety of biosynthe- introduced by 3’ mutagenesis primer, created a CTCGAG restric-
sis functions as well as detoxification of free radicals and tion site of enzyme Xho I. However, normal CGCGAG sequences
peroxides within cells. Because mature erythrocytes lack disrupt the restriction site. Using the same method, a Nde I
the citric acid cycle, and hexose monophosphate shunt is restriction site was generated to detect G to A mutation at nt 1388,
the only process to generate NADPH, G6PD deficiency is and aMlu I restriction site for A to G mutation at nt 95. For G to T
often associated with chronic drug- or food-induced hemo- mutation at nt 392, a C at nt 398 was introduced to create a BstEII
lytic anemia and neonatal jaundice.’P2 restriction site in the unaffected person (Table 1). G to A mutation
at nt 487, A to G mutation at nt 493, and C to T mutation at nt 1024
Currently, more than 400 G6PD variants have been
create A h I, Ava 11, and Mbo I1 restriction sites, respectively.
described and at least 30 biochemical variants were identi- However, there are two additional Alu I restriction sites at nt 487
fied in Mainland China.3,4Our research, in conjunction with area and an additionalAva I1 site at nt 493 area. To overcome these
that of Chiu et a1 shows that there are at least seven problems, we destroyed the normal A h I sites and Ava I1 site by
different types of mutations in Chinese.s.6 For the detection using mutagenesis primers (Table 1).The PCR was performed as
of known mutations, we used traditional methods of poly- described: but with modification of the annealing temperature
merase chain reaction (PCR) and allele-specific oligonucle- according to the Tm of the primer pairs respectively. The amplified
otide hybridization.’ These methods require radioisotope products were digested with appropriate restriction enzymes fol-
or they rely on immunochemical detection, which is incon- lowed by electrophoresis on a 3.5% agarose gel. Some cases were
venient in clinical use. Another approach simply uses PCR confirmed by direct sequencing or allele-specific probe hybridiza-
followed by restriction enzyme digestion. However, this
process has limitations in that not all of the mutations can
~ ~ ~

create or abolish a restriction site. To overcome this From the Departments of Molecular Medicine and Clinical Pathol-
ogy, Pediattic, Taipei Municipal Jan-Ai Hospital, and Taipei Institute
problem, we used artificial base substitution to create
of Pathology, Taipei, Taiwan; the Departments of Intemal Medicine
restriction sites after PCR. The created restriction sites are and Pediatrics, Knohsiung Medical College, Kaohsiung; and the
near or immediately adjacent to loci of point mutations and Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
enable us to distinguish the normal from mutated genes on Submitted November 22, 1991; accepted May I , 1992.
the basis of restriction analysis with the appropriate en- Supported in part by research Grant No. NSC 80-0412-8-196-03
zyme. from the National Science Council, and an Institutional grant from
Academia Sinica, Republic of China.
Address reprint requests to Jan-Gowth Chang, MD, Depament of
Molecular Medicine and Clinical Pathology, Taipei Municipal Jen-Ai
Subjects. Ninety-four unrelated male subjects, who were admit- Hospital, No. IO, Section 4, Jen-Ai Rd, Taipei, Taiwan, Republic of
ted to Taipei Municipal Jen-Ai Hospital and Kaohsiung Medical China.
College Hospital, had G6PD deficiency diagnosed by Sigma Diag- The publication costs of this article were defrayed in part by page
nostic Kit with the method described by the manufacturer (Sigma, charge payment. Zbis article must therefore be hereby marked
St Louis, MO). “advertisement” in accordance with 18 U.S.C.section I734 solely to
DNA amplificationand reshictwn enzyme analysis. Total genomic indicate this fact.
DNA was isolated from peripheral blood leukocytes of the affected 0 1992 by The American Society of Hematology.
subjects as previously described.’ The strategy of oligonucleotide 0006-4971192 /8004-OO28$3.00/0

Blood, VOI 80, NO4 (August 15), 1992: pp 1079-1082 1079

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Table 1. The Primer Sequences, RestrictionEnzyme, and Restriction rcstriction sitcs arc indicatcd. Figurc 2 shows thc digcstion
Fragment Sizes of the Mutation in G6PD Deficiency pattcrn of PCR products by rcstriction cnzymc. Thc sizc
Size of changc aftcr digcstion is indicatcd in Tablc I . Thc band
Mutation Primer Sequence Enzyme Fragment would not hc visualizcd in thc 3% agarosc gcl if thc sizc
G -+ T (nt 1376) 5'P: 5'-ACGTGAAGCTC- Xho I (C) 213 bp (N) wcrc lcss than SO bp. For G to T mutation of nt 1376, a
CCTGACGC-3' 213-bp fragmcnt was undigcstcd in normal allclc, but 192-
3'P: 5'-TGAAAATACGC- and 21-bp fragmcnts wcrc formcd in mutants aftcr digcs-
G A (nt 1388) 5'P: the same as for nt
tion by Xlio 1. For G to A mutation of nt 1388. thc amplificd
227-bp fragment rcmaincd undigcstcd by Nde I in normal
allclc. Howcvcr, thc products would bc digcstcd to a 205-bp
3'P: 5'-GTGCAG-
fragmcnt in mutants. Similarly, an undigcstcd 260-bp frag-
-CATA-3' mcnt in normal. and 233-bp fragment in mutant. was found
A -.G (nt 95) 5'P: 5'-CGITCACAAG- Mlu aftcr digcstion with M h I in thc A to G mutation of nt OS.
GAGTGATITG-3' For G t o T mutation of nt 392, thc PCR products of normal
3'P: 5'CGATGCACCCAT- allclc wcrc digcstcd to a 182-bp fragmcnt aftcr digcstion
GATGATGAATACG-3' with RsrEII, whcrcas thc 202-bp products rcmaincd uncut
G -.T (nt 392) 5'P: 5'-GGACTCAAA- 8stE II (C) 20,182 bp (N) in mutants. For mutations of A to G at nt 493, G t o A at nt
GAGAGGGGCTG-3' 487, and C to T at nt 1024, thc PCR products wcrc digcstcd
3'P: 5'-AGAGGCGGITG- 202 bp (M) b y h n II,Alir 1. and M h ) 11, rcspcctivcly. Thcrc arc a 86-bp
A G (nt 493) 5'P: 5'-GCGTCTGAATG-
Av8 II 142 bp (N)
and a 56-bp fragmcnt in nt 493 mutation, and 142-bp in
normal allclc. In nt 487 mutation, two fragmcnts (82 and 60
3'P: C'-AGCCGGTCAGZ 86,56 bp (MI
bp) in normal allclc and thrcc fragmcnts (thc mixcd 62- and
60-bp bands and a 20-bp band) in mutant. In nt 1024
G A (nt 487) 5'P: the same as for nt
Alu I 82,60 bp (N) mutation. thcrc is a 187-bp fragmcnt in normal allclc and
two fragmcnts (150 and 37 bp) in mutant.
3'P: the same as for nt 62,20,60 bp Thc clinical data and rcsults of mutations analysis of 94
- 493
C T (nt 1024) 5'P: 5'-GTCAAGGTGT-
M60 II
187 bp (N)
unrclatcd G6PD-dcficicncy paticnts arc shown in Tablc 2.
Thc rcsults show that 50% (47 of 94) wcrc nt 1376
mutation. 2 1 Y 6 (20 of 94) wcrc nt 1388 mutation, scvcn
3'P: 5'-CATCCCACCTCT- 150.37 bp (MI
cascs wcrc nt 493 mutation, anothcr scvcn caws wcrc nt 95
mutation. four cascs wcrc nt 1024 mutation. onc casc was nt
Abbreviations: P, primer; C, created site: N, normal; M, mutant. 392 mutation, and onc CBSC was nt 487 mutation. Thcrc arc
Underline = mutagenesisbase. no significant diffcrcnccs bctwccn thcsc scvcn mutations
and thcir G6PD activity.
tion of the PCR products. and these cases were used as positive or
negative control."
RESULTS G6PD dcficicncy affccts approximatcly 3% of thc Chi-
Thc rcsults of dircct scqucncing of PCR products of sitc ncsc in Taiwan and is thc main causc of nconatal hypcrbil-
mutagcncsis of thc two most common mutations in Chincsc irubincmia and drug-induccd hcmolytic ancmia.l".ll Al-
arc shown in Fig 1. Thc mutagcncsis bascs and thc though thcrc arc morc than 30 biochemical variants and

A CLNA 1376 G +T B CCNA 1388 G +A


Fig 1. (A) The result of direct sequencing of PCR

product of G to T mutation at nt 1376 with site
mutagenesis. An arrowhead indicates the created
restriction site of Xho 1. The arrows denote the
mutagenesis base of nt 1377 and the mutation base
of nt 1376. (E) The result of direct sequencing of G to
A mutation of nt 1388. The created restrictionsite of
Nde I is indicatedby an arrowhead, and the mutagen-
esis base of nt 1392 and mutation base of nt 1388 are
indicatedby arrows.
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+ +
. . . - - -
Ir Y \ Y \
" H

Fig 2. The results of PCR products digested with


Xho I is for the 1376 G -
restrictionenzymes for their different mutations. The
T mutation, Nde I for the
- e
1388 G

- -
A mutation, Mlu I for the 95 A
mutation, BstE II for the 392 G T mutation, M60 II

2 22 -

mutation, and Alu I for the 487 G -

for the 1024 C T mutation, Av8 II for the 493 A
A mutation. M
represents the pGem marker, and the N and M in the

upper portion of the figure indicate the normal and

mutant allele, respectively.

Table 2. The Clinical Data and Results of Mutations Analysis

finding is in accordancc with Chiu ct al." In studying thc
GGPD Activity rclationship bctwccn thc mutations and thc G6PD activity.
lIU/g Hbl
Case wc did not find any mcaningful diffcrencc among these
Mutation No. Range Mean
scven mutations.

G -.T (nt 1376)
G A (nt 1388)
1.65 2
1.74 2
Thcrc arc scvcral mcthods to dctcct the known point
mutations and small dclctions, such as allele-spccific hybrid-
A -.G (nt 95)
A G (nt 493)
2.51 2
1.57 2
~ ~ ' scqucncing of thc PCR products,l7 ligasc-
i ~ a t i o n , direct
mcdiatc allclc dctcction,Ix and clcavagc mismatch dctcc-
C -.T (nt 1024)
G A (nt 487)
G -.T (nt 392)
1.68 2 0.24
tion.'".'" In this study, wc uscd thc natural and amplification-
crcatcd rcstriction sites to molecularly characterize the
G6PD dcficicncy. We found this is a vcry uscful method not
Unknown 7 0.1-6.5 only to dctcct thc genetic lcsion of G6PD deficiency, but
Normal 51 8.0-1 1.2 9.61 2 1.63 also to provide a screening mcthod for further investigation
of new mutations. Similar stratcgics havc bccn uscd to
diagnosc PKU mutations," p-thalasscmia in Mcditcrranc-
seven mutations havc bccn reported, no dcfinitc gcnc am,'? rus oncogcnc mutations,:' G6PD variant in the
frequency has bccn studicd until In this rcport. Middlc East,?4cystic fibrosis, and rctinitis pigmcntosa.2sAll
wc find that five mutations account for morc than 90% of of thc studies show that thc approach providcs a simplc.
cases of G6PD dcficicncy in Taiwan. This condition is rapid, and accurate screening method. Our experience has
similar to P-thalasscmia in that four mutations arc vcry confirmcd this for us and we rccommcnd its continued use.
common, whereas several mutations arc rarc.15Jf1Thc G to
T mutation at nt 1376 (50%) and G to A mutation at nt 1388 ACKNOWLEDGMENT
(21.3%) have morc than 70% occurrcncc rate, and the A to We thank R.L. Wong and R.S. Yang for technical assistance and
G mutation at nt 493 is spccific to Chincsc in Taiwan. This I.C. Lu and S.Y. Sun for typing.

I. Arese P, De Flora A: Pathophysiology of hemolysis in 9. Liu TC, Lin SF, Chen TP, Liu HW, Chang JG: Mutation
glucose-6-phosphate dehydrogenase deficiency. Semin Hematol analysis of the ras gene in myelocytic leukemia by polymerase chain
27:l. 1990 reaction and oligonucleotide probes. J Formosan Med Assoc
2. Beutler E: Glucose-6-phosphate dehydrogenase deficiency. N 90:825. 1991
Engl J Med 324:169, 1991 10. Chen SH, Lin KS, Lee TY, Chen CL: Glucose-6-phosphate
3. Beutler E: The genetics of glucose-&phosphate dehydroge- dehydrogenase deficiency and neonatal hyperbilirubinemia in Chi-
nase deficiency. Semin Hematol27:137. 1990 nese. Acta Paediatr Sin 27:286, 1986
4. Du CS. Xu YK, Hua XY. Wu OL, Liu LB: Glucose-6- 11. Lee TC, Shih LY, Huang PC, Lin CC. Blackwell BN.
phosphate dehydrogenase variants and their frequency in Guang- Blackwell RQ, Hsia DYY: Glucose-6-phosphate dehydrogenase
dong, China. Hum Genet 80:385, 1988 deficiency in Taiwan. Am J Hum Genet 15:126. 1962
5. Tang TK. Huang CS. Huang MJ, Tam KR. Tang C-JC: 12. Vulliamy TJ, Wanachiwanawin W, Mason PJ. Luzzatto L
Diverse point mutations result in glucose-6-phosphate (G6PD) G6PD Mahidol, a common deficient variant in South East Asia is
polymorphism in Taiwan. Blood 79:2135,1992 caused by a (163) glycine to serine mutation. Nucleic Acids Res
6. Chiu DTY. Zuo L. Chen E, Chao LT, Louie E, Lubin BH. Du 17:5868. 1989
CS: DNA sequence abnormalities in Chinese glucose-6-phosphate 13. Stevens DJ, Wanachiwanawin W. Mason PJ. Vulliamy TJ,
dehydrogenase variants. Blood 78:252a, 1991 (abstr. suppl 1) Luzzatto L G6PD Canton. a common deficient variant in South
7. Lee HH, Chang JG, Lee LS, Lin ST, Ko TM. Choo KB: East Asia caused by a 459 Arg to Leu mutation. Nucleic Acids Res
Detection of p-globin gene mutations by polymerase chain reac- 18:7190, 1990
tion. Proc Natl Sci Counc Repub China [B] 15:97, 1991 14. Chiu DTY, Zuo L, Chen E, Chao L. Louie E, Lubin B, Liu
8. Liu TC, Chang JG,Lin CP, Lee LS. Lin SF, Liu HW. Chen TZ, Du CS: Two commonly occurring nucleotide base substitutions
TP: Detection of p-globin gene from single hairs. Kao Hsiung J in Chinese G6PD variants. Biochem Biophys Res Commun 180:
Med Sci 6: 18 1, 1990 988,1991
From www.bloodjournal.org by guest on May 21, 2019. For personal use only.


15. Chang JG, Liu TC, Lin CP, Chen PH, Lee LS: Molecular 21. Eiken HG, Odland E, Boman H, Skjelkvale L, Engebretsen
basis of P thalassemia minor in Taiwan. Blood 76:57a, 1990 (abstr, LF, Apold J: Application of natural and amplification created
SUPPI 1) restriction sites for the diagnosis of PKU mutations. Nucleic Acids
16. Kazazian HH Jr, Boehm CD: Molecular basis and prenatal Res 19:1427,1991
diagnosis of f3 thalassemia. Blood 72:1107,1988 22. Lindeman R, Hu SP, Valpato F, Trent RJ: Polymerase chain
17. Chang JG, Lee LS, Lin CP, Chen PH, Chen CP: Rapid reaction mutagenesis enabling rapid non-radioactive detection of
diagnosis of a-thalassemia-1 of Southeast Asia type and hydrops common P-thalassemia mutations in Mediterraneans. Br J Haema-
fetalis by polymerase chain reaction. Blood 78853,1991 to1 78:100,1991
23. Haliassos A, Chomel JC, Tesson L, Baudis M, Kruh J,
18. Barany F: Genetic disease detection and DNA amplification Kaplan JC, Kitzis A Modification of enzymatically amplified DNA
using cloned thermostable ligase. Proc Natl Acad Sci USA 88:189, for the detection of point mutations. Nucleic Acids Res 17:3606,
1991 1989
19. Kaufman DL, Ramesh V, McClatchey AI, Menkes JH, 24. Kurdi-Haidar B, Mason PJ, Berrebi A, Ankra-Badu G,
Tabin AJ: Detection of point mutations associated with genetic AI-Ali A, Oppenheim A, Luzzatto L Origin and spread of the
disease by an exon scanning technique. Genomics 8:656,1990 glucose-6-phosphate dehydrogenase variant (G6PD-Mediterra-
20. Cotton RG, Rodrigues NR, Campbell RD: Reactivity of nean) in the Middle East. Am J Hum Genet 47:1013,1990
cytosine and thymine in single-base-pair mismatches with hydroxy- 25. Sorscher EJ, Huang Z: Diagnosis of genetic disease by
lamine and osmium tetroxide and its application to the study of primer-specified restriction map modification, with application to
mutations. Proc Natl Acad Sci USA 854397,1988 cystic fibrosis and retinitis pigmentosa. Lancet 3371115,1991
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1992 80: 1079-1082

Molecular characterization of glucose-6-phosphate dehydrogenase

(G6PD) deficiency by natural and amplification created restriction
sites: five mutations account for most G6PD deficiency cases in
JG Chang, SS Chiou, LI Perng, TC Chen, TC Liu, LS Lee, PH Chen and TK Tang

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