Rare Abnormal Hemoglobin Variants - Biochemistry

Includes description of clinically relevant hemoglobin variants and diagnostic modalities

1. Examples of rare hemoglobin variants

2. Abnormal
Hb
Knownhemoglobinvariants: ≥ 1342 (05/2019)
•αchain variants
•βchain variants
•γchain variants
•δchain variants
Others: double mutation, deletion, insertion, hybrids
Consequence of these mutations:
increased or decreased oxygen affinity for the hemoglobin
unstable hemoglobin
many of these variants are completely asymptomatic

3. Profiles of some rare beta variants on
Capillarys/Minicap

4. Variants of β-globin chain, heterozygous state
Presence of one additional peak on
electrophoresis

5. Also Known as Hopkins-I; Jenkins; N-Memphis; Kenwood
Hematology Normal in heterozygote
Agarose
Electrophoresis
This Hb isone of the fastestmovingbeta chain
variants
Function studiesNormal oxygen affinity.
Occurrence Found in numerous Black families in the SE-USA.
Quantity in the hetrozygotes44-54%; found in
combination with HbC and also HbS
HbN-Baltimore
b95 Lys→Glu

6. HbN-Baltimore
Hb A
Hb F
Hb A2
HbN-Baltimore
b95 Lys→Glu

7. Also Known as
Korle-Bu
Hematology Normal in heterozygote
Agarose
Electrophoresis
Moves like Hb S
No separation at acidic pH
Function studiesDecreased oxygen affinity
Occurrence Found in a black families from Ghana, the Ivory
Coast, Costa Rica, USA, Mexico….
Found in combination with HbS or HbC
Homozygote is clinically normal
HbG-Accra
b73 Asp→Asn

8. Hb A
Hb Korle-bu
Hb A2
Z(D)
α2β2*
b73 Asp→Asn
HbG-Accra

10. Also Known as
Hematology Normal in heterozygote (36-45% of total Hb)
Agarose
Electrophoresis
Moves about as HbS
Function studiesNot reported
Occurrence Ethnicbackground: Iranian, Italian, Jamaican,
Pakistani
Observedin combinationwithHbS and beta Thal
HbD-Iran
β22 Glu→Gln

11. 55
HbA
HbD-Iran
HbA2
55,2
42,9
1,9
D-Punjab(208)D-Iran
(200)
Zone D
HeterozygousA/D-Iran (overlaidwithA/D-Punjab)
Hb D-Iran variant migrates in zone D in the position X= 200 +/-1
Hb D-Punjab which migrates in the same zone but in a different position
–208 +/-1

12. Profiles of somerare alpha variantson
Capillarys/Minicap

13. Hb J Mexico (α54 Gln→Glu)
Also Known asJ-Paris, Uppsala
HematologyNo hematological abnormalities in the heterozygote
Agarose
Electrophoresis
Moves faster than HbA at alkaline buffer
Function studiesNot reported
OccurrenceFoundin Mexican, Swedish, Algerian, Indian
familiesand a black familyin northAfrica.
Heterozygous: 20-25%
Homozygous: 55%
Foundin combinationwithbeta thal, alpha thal, with
Hb S

14. Heterozygote A/J Mexico
a
54 Gln→Glu
Hb J Mexico
Hb A
Hb A2
Hb J-A2

15. Also Known asSinai; Sealy; L-Ferrara; Michigan-I; Michigan-II
HematologyNormal in heterozygote
Agarose
Electrophoresis
Moves like Hb S
Moves between Hb S and Hb C at acidic pH
Function studiesNot reported
OccurrenceFound in Askenazijewsand italianfamilies
Hb Hasharon (α47 Asp→His)

16. a
47 Asp→His
Hb Hasharon
Hb A
Hb A2
Hb F
Hb Hasharon
Hb Ha-A2

17. Also Known as
HematologyNormal in heterozygote
Agarose
Electrophoresis
Moves to the position of Hb S atbothalkalineand
acidicpH
Function studiesOxygen affinity about normal.
OccurrenceFound in families from Algeria, Iran, Lebanon, Saudi
Arabia, etc…
Quantity in heterozygotes12-15%; red cells with Hb
Setif“sickle” due to intracellular crystallization of
insoluble Hb
Hb Setif (α94 Asp→Tyr)

18. Hb Setif
a
94 Asp→Tyr
Hb A
Hb F
Hb A2
Hb Setif
Hb Se-F
Hb Se-A2

19. HbG Philadelphia (α68 Asn→Lys)
Also Known as G-Knoxville, G-Bristol, G-Azakouli
D-Baltimore, D-St Louis, D-Washington
Hematology No hematological abnormalities in the heterozygote
Agarose
Electrophoresis
Moves to the position of Hb S
Same migration as Hb A at acidic pH
Function studiesNormal
Occurrence Most common a chain variant in Blacks, also
present in north Italy and Sardinia and in a few
Chinese families
Homozygote: 100% G-Philadelphia :
-distinct microcytosis
-hypochromia

20. α

21. Hb G-Philadelphia associated with a-thal
α
α
OR
XX
X
X
An elevatedpercentageof an alpha variant ( > 20 –25%)
canindicatethe presenceof a-thalassemia;
To confirmby CBC

22. Alpha variant (in Zone 12) : HbJ-Meerut
X1 = major fraction
of the variant
X2 = minorfraction
of the variant
Zone 12 Zone D

23. Rare variants with other mutation types

24. Hb Lepore
bet dchains recombination by crossing over.
Alk. buffer: Decrease of the total charge → Migra?on slowed
down like Hb S on agarose gel, more anodic than S on Capillarys
Heterozygous form:
HbLeporefraction: 5 –15 %
Clinical signs of minor bthalassemia
Homozygous form:
Hb Leporefraction: about 30 %
Clinical signs of homozygous
bthalassemia
Frequency –localization:
Lepore(-Boston –Washington):Found mainly in
Italian families; it has also been observed in
families from Rumania, Australia, Mexico (There is
2 other HbLeporemore rare:Lepore-Hollandia
and Lepore-Baltimore )
Characterization:
GgAgdb
GgAgdb
GgAgdb b
Chromosomes 11

25. Hb Lepore
Hb A2
Hb A
Hb F
Hb Lepore
Z(D)

26. Hb
Constant Spring
Abnormality of the alpha globin chain :
 Gene amutation producing a longer globin chain (28-31
additional amino acids at the Ct extremity)
 Bad association with beta globin chains
 Symptoms of a minor alpha-thalassemia
 Homozygote: 6% <

27. Hb
Constant Spring
witha thalassemia
Hb Constant Spring

28. Complete detection of Hb
Constant Spring
on Sebia capillary electrophoresis
Liao C et al.Hemoglobin,34 (2); 2010: 175-178

29. SEBIA Hemoglobinmeta-analysis
HbConstant Spring

30. Examples of advanced variant cases

31. Rare variants co-migrating with HbA
and unstable variants

32. Hb Camperdown (b104 Arg→Ser)
Also Known as
HematologyNormal in heterozygote
Agarose
Electrophoresis
Moves faster than Hb A
At acidic pH, moves between Hb A and Hb F, close
to Hb F
Function studiesNormal,
Occurrence Quantity in the heterozygote about 50%

33. Hb Camperdown
A
Camp.
+
Anh.
Car.
A
2
N
Alc.
N
Ac.
A
1
A
0
Camp.
A
0
A
1
b
104 Arg→Ser
Hb A + Camperdown
Hb A2

34. Presenceof a peakin zone 15: isitHbH?
26-yrsoldwoman
↓ HbA2
But no microcytosis
or anemia
It isNOT a-thalassemia;
Presenceof a rare hemoglobinvariant in zone 15
HbI-Texas
HbI-Texas isan alpha variant witha major formmigratingin zone 15 and
a minorformprobablyhiddenin HbA explainingwhyHbA2 isdecreased
Zone 15 =
zone for HbH
HbA2
zone

35. AlsoKnownasHbI-Burlington,HbI-Philadelphia,HbI-Skamania,
HbI
HematologyNormal
Occurrence Foundinaustralian,black,
indian
,japaneseand
caucasianfamilies
HeterozygoteHbX24-28%(majorform)
HbI-Texas
(Alpha16 (A14)Lys->Glu)

36. Be carefulabout variant migration behaviorbetweentechniques
HbShaareZedek
(in HbF position)
HbShaareZedek
(in zone 15)
Variant II (Bio-Rad) Capillarys 2 (SEBIA)
Check hematologicalparametersand HbA2 to avoid
misdiagnosiswithbeta or alpha-thalassemiacontext
alpha2 or alpha1 56(E5) Lys->Glu
Zone 15

37. Also Known as San Francisco (Pacific); Ube-1
Hematology Mild hemolytic anemia in the heterozygote;
reticulocytosis; Heinz body formation
Agarose
Electrophoresis
Moves as a multiple Hbcomponent between HbA
and HbA2 at alkaline pH
Function studiesIncreased oxygen affinity
Occurrence Found in various racial and ethnic groups; It is the
most common unstable Hb.
Quantity in the heterozygote not accurately
determined
Hb Köln (b98 Val→Met)

38. Hb Köln
UnstableHbwithmultiple fractions
b
98 Val→Met
Hb A+Hb Köln
Hb Köln
Hb Köln
Hb A2+Hb Köln

39. Additionalinformation about variant peaksin
Phoresis≥9.15

40. *
(star/asterisk)-variantpeakishiddenorpartiallyhiddenundera
normalpeak(HbAorHbA2):
#
(hash)-variantsshowingseveralpeaks(alphachainvariants,unstable
variants):
Additional information about variant peaks in
Phoresis≥9.15

41. !!
(doubleexclamationpoint)-riskofazoneshiftforararevariant
migratingataboundarybetweentwozones(+/-1pointfromthe
boundary)
Additional information about variant peaks in
Phoresis≥9.15
WHY?
Ifthepercentageofapeakisunusual(co-existenceofthalassemia,
transfusion)ashiftofmigrationposition(+/-1point)canoccur

!!
sign–onlyforvariantsthatcanchangezoneincaseofa1-
pointshift

42. Combination of alpha, beta and
alpha + beta variants

43. Compound heterozygote : 2 bvariants
α
β
d
g
α2d2: HbA2
α2g2: HbF
α2β2*
+
no
HbA
+
α2β2*

44. Mix the samplewithnormal control (1V + 1V) and
re-analyzeto obtainthe identification zones
HbAor Variant ?

45. Analysis of the sample
mixed withnormal control
(qualitative analysis, used
onlyfor zone
identification)
Compound heterozygoteS/Hope?
(the presenceof HbSneedsto be
confirmedby a complementarytest)
2
nd
peakisin
zone S
1st peakisin
zone 10

46. Confirmation of HbS by a complementarymethod
Compound heterozygoteS/Hope?
•HbSconfirmed
•On HPLC, elutiontime if the second
variant (1.39)
iscompatible withHbHope

47. Double heterozygote: avariant + bvariant
α
β
d
α2β2: HbA
α2d2: HbA2
α2*β2
+
+
α2*d2
* gchainsare not shown, to avoidover-complexity
+
α2β2*
+
α*2β2*

48. When the profile as «too many» peaks, the presence of an alpha variant
can be suspected
α2β2: HbA
α2d2: HbA2
α2*d2
α2*β2
Presence of 2 peaks can be explained by an alpha –variant. But there are
still 2 more peaks presence of another variant!
Zone S Zone A2Zone EPatient 1

49. Double heterozygote: avariant + bvariant
α2β2: HbA
α2β2*
α2d2:
HbA2
α2*β2*
α2*d2α2*β2
Patient 1

50. α2β2: HbA
α2d2: HbA2
α2*d2
α2*β2 ?

51. Double heterozygote: avariant + bvariant
α2β2: HbA
α2β2*
α2d2: HbA2
α2*β2*
α2*d2
α2*β2
HbS + G-Philadelphia
Similarpercentage for HB G-Philadelphia and HbS becauseof
association of G-Philadelphia withα-thalassemia

52. Compound heterozygote: 2 αvariants
α
β
d
α2d2: HbA2
α2*β2
+
α2β2: HbA
* gchainsare not shown, to avoidover-complexity
α2*d2
α2*d2
+
α2*β2
+
+

53. Compound heterozygote : 2 αvariants
1st alpha
variant
1st alpha
variant
2nd alpha
variant
2nd alpha
variant

54. Mother: Heterozygote A/Q-India
Q-India alpha variant: α64 Asp→His
Hb A: 79.7%
Hb Q-India: 17.5%
HbA2: 2.3%
Hb ‘’Q-India-A2’’: 0.5%
Case of a familywithalpha and beta variants
α2*β2 : Q-India
(major fraction)
α2*d2: Q-India-A2
(minor fraction)

55. Father: Heterozygote A/D Punjab
D-Punjab beta variant: b121 Glu→Gln
Case of a familywithalpha and beta variants
Hb A:56.4%
Hb D-Punjab: 40.4%
HbA2: 3.2%
α2β2* : Hb D-Punjab

56. Child: he has both alpha and beta mutations => Heterozygote A/D-
Punjab with Q-India
Case of a familywithalpha and beta variants
HbA:47.1%
HbF: 1.9%
HbD-Punjab+ HbQ-India: 40.9%
AbnormalHb+ HbA2: 9.6%
Zone 1: 0.5%
α2*d2:
Q-India-A2
α2*β2 :
Q-India
α2*β2*
α2β2*
(D-Punjab)
+
HbQ-Indiaco-migratewithHbD-Punjab