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

• 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

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

• Drug-induced haemolytic anaemia

• Infection-induced haemolysis

• Favism

• Neonatal jaundice

• Chronic ‘non-spherocytic’ haemolytic anaemia (CNSHA)

G6PD deficiency and NNJ in males: Asian Perspective

Location Prevalence of Prevalence of NNJ Prevalence of G6PD

G6PD deficiency among G6PD deficient deficiency 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

• 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

G6PD haemolysis

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

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

Type of mutation Number

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 CA)

Total 129

Note: Maternally transmitted severe G6PD deficiency is embryonic lethal. Longo L et al. The EMBO journal 16: 4229 – 4239, 2002

G6PD mutations: relationship to structural domains

G6PD structure

Dimeric structure

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

Mutant Number (total n =20)

Canton 1376 GT 10

Kaiping (Anant) 1388 GA 5

Gaohe 95 AG 2

Viangchan 871 GA 2

Fushan 1004 CA 1

Distribution of G6PD mutations in TaiwanHuang C-S et al. Am J Hematol 51: 19 - 25, 1996

Prevalence based on neonatal screening

Sex No. screened No. deficient %

Male 4,277 112 2.6

Female 3,771 50 1.3

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 GT 50% 44%

Kaiping 1388 GA 16.1% 18%

‘Chinese-3’ 493 AG 8% 12%

‘Chinese-5’ 1024 CT 6.2% 6%

Gaohe 95 AG 5.4% 6%

‘Chinese-4’ 392 GT 1.8%

Mahidol 487 GA 1.8%

Viangchan 871 GA 0.9%

Union 1360 CT 0.9%

G6PD variants in Malaysian ChineseAinoon et al, Human Mutation 14: 352, 1999

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

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 males

Variant Number G6PD 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

Indication Number

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

• 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

Variant Number G6PD 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

Indication Number

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

• 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

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 mol/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

Acute haemolysis in G6PD deficiency

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

• Compound heterozygous or homozygous

• XO

• Clonal haemopoiesis

• Extreme Lyonization

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

• 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 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

• 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

• 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

HUMARA assay and HUMARA-MSP

First exon

Normal young female

Pre Hpa-II

Ratio areas* A/A+B=52.3%

Post Hpa-II

Ratio areas A/A+B = 45.3%

Digestion completion control Dde1 93% digested

A

A

B

B

Humara A and B alleles heterozgyous polymorphic repeats

*note stuttering

CML case

Pre-Hpa-II A/A+B=53.5%

Post-Hpa-II

A/A+B = 8.3%

B

B

A

A

Dde1 94% digested Humara A and B alleles

Elderly female

Pre-Hpa-II * A/A+B=59.0%

i.e. :overamplification of A allele by 1.4 times

Post-Hpa-II* A/A+B = 88.4%

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

A

A B

B

XE169 >95% digested Humara A and B alleles*corrected for stuttering of B allele into A area

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

• Absence of Class I mutants– Implies no acquired skewing due to

somatic cell selection, typically seen in dyskeratosis congenita

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

• 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

• Awareness of G6PD deficiency in elderly females– related to acquired skewing of lyonization

with age– screening as for males?