1 / 53

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

iolani
Download Presentation

G6PD deficiency in Hong Kong: diversity and clinical relevance

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 • Department of Biochemistry HKU • Dr W Y Au • Department of Medicine QMH

  3. Metabolic role of G6PD • Detoxification of H2O2 • glutathione • catalase • Cell growth • redox regulation • J Biol Chem 273: 10609 - 17, 1998

  4. 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.

  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 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.

  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: 6.35 - 10.33 IU/gHb • Review of blood film during acute haemolytic episode

  8. G6PD haemolysis

  9. 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

  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 (10 - 60% 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 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

  12. G6PD mutations: relationship to structural domains

  13. G6PD structure Dimeric structure

  14. 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

  15. 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

  16. 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%

  17. G6PD variants in Malaysian ChineseAinoon 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 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%)

  20. 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

  21. 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

  22. 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%)

  23. 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

  24. 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

  25. 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

  26. 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

  27. 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

  28. Acute haemolysis in G6PD deficiency

  29. 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

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

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

  32. 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

  33. 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.

  34. 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

  35. 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

  36. First exon HUMARA assay and HUMARA-MSP

  37. 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

  38. 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

  39. 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

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

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

  42. 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

  43. 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

More Related