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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. Acknowledgements. Dr Veronica Lam Department of Biochemistry HKU Dr W Y Au Department of Medicine QMH. Metabolic role of G6PD.

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G6PD deficiency in Hong Kong: diversity and clinical relevance


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g6pd deficiency in hong kong diversity and clinical relevance

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

acknowledgements
Acknowledgements
  • Dr Veronica Lam
    • Department of Biochemistry HKU
  • Dr W Y Au
    • Department of Medicine QMH
metabolic role of g6pd
Metabolic role of G6PD
  • Detoxification of H2O2
    • glutathione
    • catalase
  • Cell growth
    • redox regulation
    • J Biol Chem 273: 10609 - 17, 1998
prevalence of g6pd deficiency on neonatal screening in hk
Prevalence of G6PD deficiency on neonatal screening in HK

Males 4.47 % (n = 223,696)

Females 0.27 % (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 33 - 35.

manifestations of g6pd deficiency
Manifestations of G6PD deficiency
  • Drug-induced haemolytic anaemia
  • Infection-induced haemolysis
  • Favism
  • Neonatal jaundice
  • Chronic ‘non-spherocytic’ haemolytic anaemia (CNSHA)
g6pd deficiency and nnj in males asian perspective
G6PD deficiency and NNJ in males: Asian Perspective

LocationPrevalence ofPrevalence of NNJPrevalence of G6PD

G6PD deficiencyamong G6PD deficientdeficiency among subjectsNNJ patients

China/HK 3.6 % NA 15 - 30 %

Thailand 7.5 % 30 % 30 - 60 %

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.

diagnosis of g6pd deficiency
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: 6.35 - 10.33 IU/gHb
  • Review of blood film during acute haemolytic episode
distribution of g6pd gene mutations
Distribution of G6PD gene mutations
  • G6PD gene:
  • 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
classification of g6pd variants
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 (10 - 60% of normal activity)
  • Class IV very mild enzyme deficiency or almost normal enzyme activity (> 60% normal activity and no clinical problem)
the types of mutation that cause g6pd deficiency
The types of mutation that cause G6PD deficiency

Type of mutationNumber

Single missense 111

Double or triple missense 8

Small in frame deletions 8

Splice site 3’ intron 10 1

(G6PD Vansdorf)

Nonsense (female heterozygote) 1

(G6PD Georgia 1284 CA)

Total 129

Note: Maternally transmitted severe G6PD deficiency is embryonic lethal.

Longo L et al. The EMBO journal 16: 4229 – 4239, 2002

g6pd structure
G6PD structure

Dimeric structure

slide14
Distribution of G6PD mutations in South ChinaBased on Zuo L, Chen E, Du CS et al, Blood 76: 51a, 1990 (suppl)

MutantNumber (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

distribution of g6pd mutations in taiwan huang c s et al am j hematol 51 19 25 1996
Distribution of G6PD mutations in TaiwanHuang C-S et al. Am J Hematol 51: 19 - 25, 1996

Prevalence based on neonatal screening

Sex No. screenedNo. deficient%

Male 4,277 112 2.6

Female 3,771 50 1.3

distribution of g6pd mutations in taiwan huang c s et al am j hematol 51 19 25 19961
Distribution of G6PD mutations in TaiwanHuang C-S et al. Am J Hematol 51: 19 - 25, 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%

g6pd deficiency in chinese males
G6PD deficiency in Chinese males
  • Case accrued from 1996 - 2002
  • 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%)
g6pd deficiency in chinese males1
G6PD deficiency in Chinese males

Variant NumberG6PD activity (IU/gHb)

Mean  SE Range

1388 (Kaiping) 43 0.90  0.23 0 - 8.6

1376 (Canton) 40 0.43  0.09 0 - 3.3

95 (Gaohe) 14 0.60  0.19 0 - 2.36

871 (Viangchan) 9 0.42  0.12 0.02 - 1.03

392 (Chinese-4) 7 1.05  0.19 0.18 - 1.82

1360 (Union) 4 0.22  0.13 0.045 - 0.6

1024 (Chinese-5) 2 1.95 (mean) 1.5 & 2.4

indications for g6pd screen males
Indications for G6PD screen: males

IndicationNumber

Haematological disorder 33

Routine 28

Jaundice and/or anaemia 25

BMT donor 13

Neonatal jaundice 11

Cerebral palsy, dyskinesia/dystonia 6

Known history of G6PD deficiency 3

Unknown 20

g6pd deficiency in chinese females
G6PD deficiency in Chinese females
  • Case accrued from 1996 - 2002
  • 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 392 1 (2.5%)
    • Unknown 4 (9%)
    • Poor DNA quality 3 (7%)
g6pd deficiency in heterozygous females
G6PD deficiency in heterozygous females

Variant NumberG6PD activity (IU/gHb)

Mean  SE Range

1376 (Canton) 16 3.19  0.82 0.29 - 9.3

1388 (Kaiping) 7 4.94  0.48 3.05 - 6.37

871 (Viangchan) 5 2.88  0.77 0.29 - 4.98

95 (Gaohe) 5 5.20  1.89 0.6 - 12.1

indications for g6pd screen females
Indications for G6PD screen: females

IndicationNumber

Routine 14

Haematological disorder 11

Jaundice and/or anaemia 5

Neonatal jaundice 1

Cerebral palsy, dyskinesia/dystonia 1

Known history of G6PD deficiency 1

Unknown 9

conclusions
Conclusions
  • Spectrum of G6PD variants are similar to Chinese elsewhere
    • 3 commonest variants Canton, Kaiping and Gaohe accounts for 70 - 80% 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
conclusions1
Conclusions
  • Female heterozygotes
    • Range of enzyme activity?
    • Normal enzyme level does not exclude heterozygosity
  • Males hemizygotes
    • G6PD Chinese-4 and Chinese-5 appear to show higher activity
g6pd haemolysis in female
F/61

Complained of fever, chills and rigors

Took Chinese herbs

Prescribed nitrofurantoin by GP

Admitted for jaundice

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: 6.35 - 10.33 IU/gHb)

Urine culture: E. coli

USG liver: no cholangitic changes or gallstones

G6PD haemolysis in female
g6pd haemolysis in female1
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
g6pd deficiency in females
G6PD deficiency in females
  • Compound heterozygous or homozygous
  • XO
  • Clonal haemopoiesis
  • Extreme Lyonization
slide35

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

g6pd deficiency in elderly females
G6PD deficiency in elderly females
  • 132 elderly females screened for G6PD deficiency
    • Median age: 80 years (range: 71 - 101)
    • G6PD deficiency = 7 (5.3%)
      • Median enzyme activity = 1.57 IU/gHb (range 0.51 - 4.73)
      • G6PD variant:
        • Canton = 2; Kaiping = 2; Goahe = 2;
        • Canton + 871 = 1
  • 160 female BMT donors
    • Median age: 32 years (range: 15 - 58)
    • No G6PD deficiency identified
prevalence of g6pd deficiency on neonatal screening in hk1
Prevalence of G6PD deficiency on neonatal screening in HK

Males 4.47 % (n = 223,696)

Females 0.27 % (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 33 - 35.

hardy weinberg law
Hardy-Weinberg Law
  • Take
    • 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
humara assay
HUMARA assay
  • Conventional
    • 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
slide41

First exon

HUMARA assay and HUMARA-MSP

slide42

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

slide43

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

slide44

Elderly female

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/(88.4+11.6x1.4)=82.3%

*corrected for stuttering of B allele into A area

XE169 >95% digested

Humara A and B alleles

assumptions
Assumptions
  • In females with G6PD deficiency, the over-presented allele is the mutant
  • X-inactivation pattern in leucocytes parallel that of erythroid cells
point of note
Point of note
  • Absence of Class I mutants
    • Implies no acquired skewing due to somatic cell selection, typically seen in dyskeratosis congenita
humara study results
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
effect of lyonization on g6pd level
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
discussion
Discussion
  • Awareness of G6PD deficiency in elderly females
    • related to acquired skewing of lyonization with age
    • screening as for males?