1. G6PD deficiency in Hong Kong: diversity and clinical relevance
Dr Edmond S K Ma
Division of Haematology
Department of Pathology
The University of Hong Kong

2. Acknowledgements Dr Veronica Lam Dr W Y Au
Department of Biochemistry HKU
Dr W Y Au
Department of Medicine QMH

3. Metabolic role of G6PD Detoxification of H2O2 Cell growth glutathione
catalase
Cell growth
redox regulation
J Biol Chem 273: , 1998

4. Prevalence of G6PD deficiency on neonatal screening in HK
Males % (n = 223,696)
Females % (n = 208,457)
Data from Lo KK et al: Neonatal screening for G6PD deficiency in Hong Kong. In Lam STS, Pang CCD (eds): Neonatal and Perinatal Screening - the Asian Perspective, CUHK press, 1996, pp

5. Manifestations of G6PD deficiency
Drug-induced haemolytic anaemia
Infection-induced haemolysis
Favism
Neonatal jaundice
Chronic ‘non-spherocytic’ haemolytic anaemia (CNSHA)

6. G6PD deficiency and NNJ in males: Asian Perspective
Location Prevalence of Prevalence of NNJ Prevalence of G6PD
G6PD deficiency among G6PD deficient deficiency among subjects NNJ patients
China/HK 3.6 % NA %
Thailand 7.5 % 30 % %
Malaysia/ 1.3 % 20 % NA
Singapore
References:
1. Lai HC, Lai MPY, Leung KS. J Clin Pathol 21: 44, 1968.
2. Lu TC, Wei H, Blackwell RQ. Pediatrics 37: 994, 1966.
3. Flatz G, Sringam S, Premyothin C, Penbharkkul S, Ketusingh R, Chulajata R. Arch Dis Child 38: 566, 1963.
4. Phornphutkul C, Whitaker JA, Worathumrong N. Clin Pediatr 8: 275, 1969.
5. Vella F. Experientia 17: 181, 1961.
6. Lie-Injo LE, Virjk HK, Lim PW, Lie AK, Ganesan J. Acta Haematol 58: 152, 1977.

7. Diagnosis of G6PD deficiency
Screening test: Fluorescent spot test
G6PD assay
based on reduction of NADP as measured spectrophotometrically at 340 nm when haemolysate is incubated with G6P
caveats: reticulocytosis and recent blood transfusion
reference range: IU/gHb
Review of blood film during acute haemolytic episode

8. G6PD haemolysis

9. Distribution of G6PD gene mutations
Maps to Xq28
Spans 18 kb and consists of 13 exons (first exon is non-coding)
Active enzyme: either 2 or 4 identical subunits, each 59 kDa
Primary sequence of 515 amino acid

10. Classification of G6PD variants
Most of the 127 different mutations identified to-date are classified from Class I to Class IV (WHO classification) according to the severity / type of clinical manifestations:
Class I severe enzyme deficiency resulting in chronic non-spherocytic haemolytic anaemia (CNSHA)
Class II severe enzyme deficiency with (< 10% of the normal activity)
Class III mild to moderate enzyme deficiency ( % of normal activity)
Class IV very mild enzyme deficiency or almost normal enzyme activity (> 60% normal activity and no clinical problem)

11. The types of mutation that cause G6PD deficiency
Type of mutation Number
Single missense
Double or triple missense 8
Small in frame deletions 8
Splice site 3’ intron
(G6PD Vansdorf)
Nonsense (female heterozygote) 1
(G6PD Georgia 1284 CA)
Total
Note: Maternally transmitted severe G6PD deficiency is embryonic lethal.
Longo L et al. The EMBO journal 16: 4229 – 4239, 2002

12. G6PD mutations: relationship to structural domains

13. G6PD structure
Dimeric structure

14. Distribution of G6PD mutations in South China Based on Zuo L, Chen E, Du CS et al, Blood 76: 51a, 1990 (suppl)
Mutant Number (total n =20)
Canton 1376 GT 10
Kaiping (Anant) 1388 GA 5
Gaohe 95 AG 2
Viangchan 871 GA 2
Fushan 1004 CA 1

15. Prevalence based on neonatal screening
Distribution of G6PD mutations in Taiwan Huang C-S et al. Am J Hematol 51: , 1996
Prevalence based on neonatal screening
Sex No. screened No. deficient %
Male 4,
Female 3,

16. Distribution of G6PD mutations in Taiwan Huang C-S et al
Distribution of G6PD mutations in Taiwan Huang C-S et al. Am J Hematol 51: , 1996
Variant Males (n = 102) Females (n = 43)
Canton 1376 GT 50% 44%
Kaiping 1388 GA 16.1% 18%
‘Chinese-3’ 493 AG 8% 12%
‘Chinese-5’ 1024 CT 6.2% 6%
Gaohe 95 AG 5.4% 6%
‘Chinese-4’ 392 GT 1.8%
Mahidol 487 GA 1.8%
Viangchan 871 GA 0.9%
Union 1360 CT 0.9%

17. G6PD variants in Malaysian Chinese Ainoon et al, Human Mutation 14: 352, 1999

18. G6PD variants in Malaysian Malays Ainoon et al, Human Mutation 21: 101, 2003

19. G6PD deficiency in Chinese males
Case accrued from
Based on dubious or abnormal FST
Mutation detection: ARMS  sequencing
Among 139 samples collected
G6PD Kaiping (1388) 46 (33%)
G6PD Canton (1376) 40 (29%)
G6PD Goahe (95) 14 (10%)
G6PD Viangchan (871) 9 (6.5%)
G6PD Chinese-4 (392) 7 (5%)
G6PD Union (1360) 4 (3%)
G6PD Chinese-5 (1024) 2 (1.5%)
Unknown 9 (6.5%)
Poor DNA quality 8 (5.5%)

20. G6PD deficiency in Chinese males
Variant Number G6PD activity (IU/gHb)
Mean  SE Range
1388 (Kaiping) 
1376 (Canton) 
95 (Gaohe) 
871 (Viangchan) 
392 (Chinese-4) 
1360 (Union) 
1024 (Chinese-5) (mean) 1.5 & 2.4

21. Indications for G6PD screen: males
Indication Number
Haematological disorder 33
Routine
Jaundice and/or anaemia 25
BMT donor
Neonatal jaundice 11
Cerebral palsy, dyskinesia/dystonia 6
Known history of G6PD deficiency 3
Unknown

24. G6PD deficiency in Chinese females
Case accrued from
Based on dubious or abnormal FST
Among 42 samples collected
Heterozygous for G6PD Canton (1376) 16 (38%)
Heterozygous for G6PD Kaiping (1388) 7 (17%)
Heterozygous for G6PD Viangchan (871) 5 (12%)
Heterozygous for G6PD Goahe (95) 5 (12%)
Homozygous for G6PD Canton (1376) 1 (2.5%)
Compound heterozygous for 1376 and (2.5%)
Unknown (9%)
Poor DNA quality 3 (7%)

25. G6PD deficiency in heterozygous females
Variant Number G6PD activity (IU/gHb)
Mean  SE Range
1376 (Canton) 
1388 (Kaiping) 
871 (Viangchan) 
95 (Gaohe) 

26. Indications for G6PD screen: females
Indication Number
Routine
Haematological disorder 11
Jaundice and/or anaemia 5
Neonatal jaundice 1
Cerebral palsy, dyskinesia/dystonia 1
Known history of G6PD deficiency 1
Unknown 9

29. Conclusions Spectrum of G6PD variants are similar to Chinese elsewhere
3 commonest variants Canton, Kaiping and Gaohe accounts for % of cases
No class I variants encountered
G6PD Chinese-3 or Taipei (493 A G) while 9.3% in Taiwan, not seen in HK
Most diagnosed on routine screening, few (6) presented as haemolysis

30. Conclusions Female heterozygotes Males hemizygotes
Range of enzyme activity?
Normal enzyme level does not exclude heterozygosity
Males hemizygotes
G6PD Chinese-4 and Chinese-5 appear to show higher activity

31. G6PD haemolysis in female
Hb 11.4 g/dL (no retic %)
WBC 17.1 X 109/L
Plt 193 X 109/L
Bilirubin 155 mmol/L (unconjugated)
Haptoglobin <0.05 g/L
Methaemalbumin 0.1 mg/dL
Direct Coombs’ test negative
Hb pattern normal
G6PD: FST abnormal; assay 1.67 IU/gHb
(normal range: IU/gHb)
Urine culture: E. coli
USG liver: no cholangitic changes or gallstones
F/61
Complained of fever, chills and rigors
Took Chinese herbs
Prescribed nitrofurantoin by GP
Admitted for jaundice

32. Acute haemolysis in G6PD deficiency

33. G6PD haemolysis in female
Clinical progress
Fever and neutrophilia down with levofloxacin
Jaundice subsided
Heterozygous carrier of G6PD Canton
Family study
Son (M/29): G6PD enzyme level 0.28 IU/gHb
Confirmed G6PD Canton
Daughter (F/31): G6PD enzyme level 5.5 IU/gHb
Confirmed G6PD Canton heterozygote

34. G6PD deficiency in females
Compound heterozygous or homozygous XO
Clonal haemopoiesis
Extreme Lyonization

35. Distribution of X-inactivation pattern from peripheral blood of normal females in 3 age groups (Gale RE et al, BJH 98: 512-9, 1997)

36. G6PD deficiency in elderly females
132 elderly females screened for G6PD deficiency
Median age: 80 years (range: )
G6PD deficiency = 7 (5.3%)
Median enzyme activity = 1.57 IU/gHb (range )
G6PD variant:
Canton = 2; Kaiping = 2; Goahe = 2;
Canton = 1
160 female BMT donors
Median age: 32 years (range: )
No G6PD deficiency identified

37. Prevalence of G6PD deficiency on neonatal screening in HK
Males % (n = 223,696)
Females % (n = 208,457)
Data from Lo KK et al: Neonatal screening for G6PD deficiency in Hong Kong. In Lam STS, Pang CCD (eds): Neonatal and Perinatal Screening - the Asian Perspective, CUHK press, 1996, pp

38. Hardy-Weinberg Law Take Proportion of females
gene frequency of normal allele (p) = 95.53%
gene frequency of mutant allele (q) = 4.47%
Proportion of females
homozygous normal (p2) = 91.26%
heterozygous (2pq) = 8.54%
homozygous mutant (q2) = 0.2%
note: prevalence in female = 0.27% on neonatal screening

40. HUMARA assay Conventional HUMARA methylation specific PCR (MSP)
DNA amplification by primers targeting CAG repeats with or without digestion with HhaI or HpaII (methylation sensitive restriction enzyme)
HUMARA methylation specific PCR (MSP)
Chemical modification by sodium bisulfite
unmethylated cytosine  uracil
methylated cytosine unchanged
PCR amplification
Gel electrophoresis and product detection

41. HUMARA assay and HUMARA-MSP
First exon
HUMARA assay and HUMARA-MSP

42. Normal young female Pre Hpa-II Ratio areas* A/A+B=52.3% A B
Post Hpa-II
Ratio areas A/A+B = 45.3% A B
Digestion completion control Dde1 93% digested
Humara A and B alleles heterozgyous polymorphic repeats
*note stuttering

43. CML case A B Pre-Hpa-II A/A+B=53.5% B Post-Hpa-II A/A+B = 8.3% A
Dde1 94% digested
Humara A and B alleles

44. Elderly female A B A B Pre-Hpa-II * A/A+B=59.0%
i.e. :overamplification of A allele by 1.4 times A B
Post-Hpa-II* A/A+B = 88.4%
Corrected for overamplification = 88.4/( x1.4)=82.3%
*corrected for stuttering of B allele into A area
XE169 >95% digested
Humara A and B alleles

45. Assumptions
In females with G6PD deficiency, the over-presented allele is the mutant
X-inactivation pattern in leucocytes parallel that of erythroid cells

46. Point of note Absence of Class I mutants
Implies no acquired skewing due to somatic cell selection, typically seen in dyskeratosis congenita

47. HUMARA study: results Lyonization becomes increasingly skewed with age
77.9% (62-97%) skewing in 9 elderly subjects
60.4% (52-95%) skewing in offsprings
62.3% (51-76%) in 20 young female controls

49. Effect of lyonization on G6PD level
In female heterozygote carriers, the G6PD level correlated with age
G6PD level showed close correlation with that predicted from degree of lyonization
Expected G6PD activity = % normal allele x 0.38 IU/gHb + % mutant allel x 9.8 IU/gHb

52. Discussion Awareness of G6PD deficiency in elderly females
related to acquired skewing of lyonization with age
screening as for males?