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Table 3 Mutations of the HBB gene detected by the HRM-PCR assay and GRACE-PCR

From: Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR)

Genotype

Common name

Sequencing or Gap-PCR

Sample set 1

Sample set 2

Primer set used for detection

Phenotype

(n = 410)

(n = 342)

(n = 68)

1. HBB:c.-151C > T

-101 C > T

2

1

1a

H1

β+ thalassaemia trait

2. HBB:c.-138C > A

-88 C > A

5

3

2a

H1

β+ thalassaemia trait

3. HBB:c.-138C > A; HBB:c.92 + 5G > C

-88 C > A; IVS1-5 G > C

1

1

0

H1 + H3

β thalassaemia major

4. HBB:c.-121C > T

-71C > T

1

1

0

H1

β+ thalassaemia trait

5. HBB:c.17_18delCTb

Codon 5-CT

13

12

1

H2

βo thalassaemia trait

6. HBB:c.17_18delCT; HBB:c.92 + 5G > Cb

Codon 5 -CT; IVS1-5 G > C

1

1

0

H2 + H3

β thalassaemia major

7. HBB:c.19G > A

Hb C

7

5

2

H2

Hb C trait

8. HBB:c.19G > A; HBB:c.20A > T

Hb C; Hb S

1

0

1a

H2

Hb SC disease

9. HBB:c.20A > T heterozygoteb

Hb S

24

21

3

H2

Hb S trait

10. HBB:c.20A > T homozygoteb

Hb S

18

18

0

H2

Hb S disease

11. HBB:c.20A > T;HBB:c.92 + 5G > Cb

Hb S; IVS1-5 G > C

3

3

0

H2 + H3

Hb S disease

12. HBB:c.20A > T; HBB:c.83-22_95delb

Hb S; 25 bp deletion

2

2

0

H2 + H4

Hb S disease

13. HBB:c.20A > T; HBB:c.176C > A

Hb S; Hb Sheffield

1

1

0

H2 + H5

Hb S + Hb Sheffield

14. HBB:c.20A > T; HBB:c.364G > C

Hb S; Hb D

2

2

0

H2 + H7

Hb SD disease

15. HBB:c.25_26delAAb

Codon 8 -AA

3

3

0

H2

βo thalassaemia trait

16. HBB:c.27_28insGb

Codon 8/9 + G

5

4

1a

H2

βo thalassaemia trait

17. HBB:c.33C > A; HBB:c.51delC

Codon 10 C > A; Codon 16 -C

1

1

0

H2 + H3

β thalassaemia major

18. HBB:c.47G > Ab

Codon 15 G > A

6

4

2a

H2

βo thalassaemia trait

19. HBB:c.67G > T

Codon 22 G > T

2

2

0

H3

βo thalassaemia trait

20. HBB:c.79G > A

Hb E

7

5

2

H3

Hb E trait

21. HBB:c.79G > A; HBB:c.126_129delCTTT

Hb E; Codon 41/42 -CTTT

2

1

1

H3 + H4 + H5

Hb E/βo thalassaemia

22. HBB:c.92G > C; HBB:c.-92C > G

Hb Monroe

3

1

2a

H1 + H3

βo thalassaemia trait

23. HBB:c.92 + 1G > Ab

IVS1-1 G > A

16

12

4

H3

βo thalassaemia trait

24. HBB:c.92 + 5G > Cb

IVS1-5 G > C

96

82

14

H3

β+ thalassaemia trait

25. HBB:c.92 + 6 T > Cb

IVS1-6 T > C

7

6

1a

H3

β+ thalassaemia trait

26. HBB:c.92 + 6 T > C; HBB:c.93-21_96delb

IVS1-6 T > C; 25 bp deletion

1

1

0

H3 + H4

β thalassaemia major

27. HBB:c.93-21_96delb

25 bp deletion

35

27

8

H4

βo thalassaemia trait

28. HBB:c.93-21G > Ab

IVS1-110 G > A

18

11

7

H4

β+ thalassaemia trait

29. HBB:c.112delTb

Codon 36/37 -T

1

1

0

H4

βo thalassaemia trait

30. HBB:c.114G > A

Codon 37 G > A

1

1

0

H4

βo thalassaemia trait

31. HBB:c.118C > Tb

Codon 39 C > T

16

13

3

H4

βo thalassaemia trait

32. HBB:c.126_129delCTTTb

Codon 41/42 -CTTT

2

2

0

H4 + H5

βo thalassaemia trait

33. HBB:c.135delCb

Codon 44 -C

14

10

4

H4 + H5

βo thalassaemia trait

34. HBB:c.140G > A

Hb K-Ibadan

1

1

0

H4 + H5

Hb K-Ibadan

35. HBB:c.157G > C

Hb Summer Hill

1

1

0

H5

Hb Summer Hill

36. HBB:c.251delG

Codon 82/83 -G

6

6

0

H6

βo thalassaemia trait

37. HBB:c.315 + 1G > Ab

IVS2-1 G > A

14

10

4

H6

βo thalassaemia trait

38. HBB:c.316-106G > Cb

IVS2-745 G > C

1

1

0

H8

β+ thalassaemia trait

39. HBB:c.316-3C > A

IVS2-848 C > A

1

1

0

H8 + H9

β+ thalassaemia trait

40. HBB:c.321_322insG

Codon 105/106 + G

1

1

0

H8 + H9

βo thalassaemia trait

41. HBB:c.364G > C

Hb D Los Angeles

14

14

0

H9

Hb D trait

42. HBB:c.316-149_*342delinsAAGTAGA

619 bp deletion (hetero)

1

1

0

G2

βo thalassaemia trait

43. HBB:c.316-149_*342delinsAAGTAGA

619 bp deletion (homo)

1

1

0

G2

β thalassaemia major

44. NG_000007.3:g.66258_184734del118477

Filipino deletion

3

2

1

G1 + G2

βo thalassaemia trait

False positive by HRM

 

-

1

0

1

Not applicable

No mutation detected

 

49

44

4

-

Not applicable

  1. Samples were analysed by HRM-PCR either after (Sample set 1; n = 342) or prior to (Sample set 2; n = 68) sequencing of the HBB gene. HRM-PCR correctly identified all samples with pathological sequence variations (n = 355). Furthermore, it was possible to correctly assign a genotype based on the HRM-PCR data (Sample set 2) for all but 10 samples (indicated by aa). GRACE-PCR correctly identified two known cases with 619 bp deletions and also identified a further three cases with deletions of both the promoter and third exon. Gap-PCR identified the 116 kb deletion for these three cases. The commercial β-Globin StripAssay used in our laboratory for the detection of mutations of the HBB gene would be able to resolve 23 (indicated by ab) of the 44 abnormal genotypes detected