The diagnosis of thalassemia

Transcript

The diagnosis of
thalassemia (α(α and β-)heterozygotes
β )hetero ygotes
Main features; the value of MCH/MCV, HbA2 and HbF.
iron deficiency;a-thalassemia; β+δ-thalassemia; a diagnostic flowchart
Drssa Cristina Passarello
U.O.C. di Ematologia II con Talassemia
A O O R “Riuniti
A.O.O.R.
Riuniti Villa Sofia Cervello”
Cervello - Palermo,
Palermo Italia
Si ringrazia per il sostegno alla Ricerca
1
U. O. C. Ematologia II A.O.“VillaSofiaCervello”
Hemoglobinopathies are the only hereditary diseases in which it
is possible identify healthy carriers with blood tests (screening
of the first level) rather than molecular analysis.
U.O.C. Ematologia II
The diagnosis of thalassemia heterozygotes
2
HEMOGLOBINOPATHIES HEREDITARY
¾ MICROCYTHEMIA or THALASSEMIA
1) lack of or reduction in the synthesis of the corresponding globin chain.
2)) altered amino acid sequence
q
with the p
production of highly
g y unstable g
globin
chains and / or low affinity for other chains.
Cause: UNBALANCED ratios of the normal bio-synthetic α/β.
¾ HEMOGLOBIN VARIANTS
1) synthesis of a normal amount of the corresponding globin chain.
2)) altered amino acid sequence
q
((HbS,, HbC,, HbD ... ... ..))
Cause: an alteration of the normal physiology of the globin chain produced.
PHENOTYPIC CLASSIFICATION OF HEMOGLOBINOPATHIES
Typical Phenotypes
the typical parameters of α− , β− or δ- thalassemia or variant hemoglobin carrier’ phenotype are present
Atypical Phenotypes
the typical parameters of α,
α β
β−thalassemia
thalassemia or variant hemoglobin carrier
carrier’ phenotype are not present
Alpha-Thalassemia
Phenotypic
classification
Functional genes
Hematologic
status
Hemoglobin
status
α+ Thal
almost normal
Normal
αo Thal
altereted
Normal
altereted
Presence of
Hb H
HbH (β4) desease
F t l Hydrops
Fetal
H d
with Hb Bart’s (γ4)
This condition is
not compatible with life
Molecular cause of α Thalassemia
Deletions
more frequent given the structure of the cluster α
May involve a single gene alpha (α+ thal)
May involve both alpha genes or regulatory area α-MRE (α° thal)
Point Mutations
Most involte the α2 gene, according to its dominant
expression (374/314);
cause α+ / α+/° thal
¾ Defects in Maturation of pre-mRNA;
¾ Defects in mRNA translation;
¾ Defects that cause instability of the globin chain;
¾ Variants.
α+
α°/+
RBC
HB
HCT
MCV
MCH
MCHC
RDW
HbA2
RBC
HB
HCT
MCV
MCH
MCHC
RDW
HbA2
4.94
13.7
40.8
80.6
27.1
33.5
13.1
2.9%
5.14
13.7
41.8
79.5
26.7
32.7
13.9
2.7%
α-3.7/αα
α-4.2/αα
5.45
13.5
40 9
40.9
74.9
24.8
33 1
33.1
14.5
2.3%
6.07
15.2
46 4
46.4
76.4
25.0
32 8
32.8
15.5
2.5%
-(AC)α3.7 II/αα
αS.A.α/αα
5.55
13.5
41.3
74.0
24.2
32.6
14.2
2.7%
5.65
14.4
43.7
77.3
25.5
33.0
14.1
2.6%
αNcoIα/αα
α°
αHphIα/αα
RBC
HB
HCT
MCV
MCH
MCHC
RDW
HbA2
5.59
12.3
38 5
38.5
68.8
22.1
32 1
32.1
14.2
2.7%
-- MED/αα
6.39
13.3
40 4
40.4
63.0
20.7
32 9
32.9
13.8
2.4%
-- 20.5/αα
5.43
12.0
36 9
36.9
68.0
22.0
32 4
32.4
15.4
2.6%
-- CAL/αα
−α 3.7I/αα
RBC
Hb
HCT
MCV
MCH
MCHC
RDW
HbA2
4.97
13.1
3.
37.7
77.9
26 5
26.5
33.5
13.7
2.7%
66.23
23
14.6
45.1
72.3
23.4
32.4
14.4
2.5%
α3.7 Ι/α3.7 Ι
RBC
Hb
HCT
MCV
MCH
MCHC
RDW
HbA2
5.45
13.5
40.9
74.9
24 8
24.8
33.1
14.5
2.3%
‐(AC)α3.7 II/αα
RBC
Hb
HCT
MCV
MCH
MCHC
RDW
HbA2
HbH
3.6
3
6
7.2
24.4
67.7
20.1
29.7
29.6
1.7
16.3
3
~16
‐(AC)α3.7 II/ ‐(AC)α3.7 II
ALPHA-GLOBIN GENE MUTATIONS IN THE SICILIAN POPULATION
Deletion
Point α−thal mutations
α−globin
g
Variants
−α3.7/αα
αNcoIα/αα
αOxfordα/αα
Cd 15
−α−(AC)3.7II/αα
ααNcoI/αα
αAgrinioα/αα
Cd 29
−α4.2/αα
ααIVS I nt 115/αα
ααIwate/αα
Cd 87
αHphIα/αα
αIns A Cd59-60α/αα
αSunPraireα/αα
Cd 130
−α20.5/αα
αα Fr(-C) Cd 109/αα
αSetifα/αα
Cd 94
α−−Cal /αα
αSaudiArabiaα/αα
ααCharolles/αα
Cd 103
α−−SEA /αα
C
ld α/αα
αContaldo
Cd 103
α−−FIL /αα
ααBernaldaα/αα
Cd 119
−α−−Μed/αα
ααUtrecht/αα
Cd 129
3 7/αα
ααα3.7
αPolicorοα/αα
Cd 124
ααS.G.Jato/αα
Cd 31
ααReading/αα
Cd 48
ααNEW/αα
Cd 49 / Cd72 / Cd 81 / Cd 83
αNEWα/αα
Cd 51
αJ-Norfolkα/αα
Cd 57
αTruidenα/αα
Cd 68
αStanleyvilleIIα/αα
Cd 78
αEtobicokeα/αα
Cd 84
αCSα/αα
Cd 142
αIcariaα/αα
Cd 142
αSouthernItalyα/αα
Cd 26+Cd130
ααα4.2/αα
TYPICAL PHENOTYPE OF β-THALASSEMIA TRAIT
¾ Microcytosis
i
i ( reduced
d d values
l
off MCV
C ed
d MCH
C )
¾
Increased levels of HbA2
β+
β°
absence of beta-chains
Marked microcytosis (MCV < 65 fl)
High value of HbA2 ((>5.0%)
5.0%)
modest presence of beta-chains
Marked microcytosis (MCV 65-68 fl)
High value of HbA2 (4.5
(4.5-5.0%)
5.0%)
β++
β+++
low presence of beta chains
mild microcytosis (MCV 68-75 fl)
HbA2 borderline or slightly above the
normal levels (3.5-4.5% or >5,5%)
low presence of beta chains
slight microcytosis or normal (MCV >78-80 fl)
HbA2 borderline (3,3-3,9%)
β-Thalassemia:
In most cases,
cases first-level analysis is used to define the status of a healthy carrier
Si ringrazia
g
per
p il sostegno
g alla Ricerca
10
RBC
5.60
5.46
4.96
HB
13.8
13.9
12.8
HCT
41.4
42.9
41.4
MCV
61.5
64.9
70.5
MCH
22.1
20.1
22.6
MCHC
32.1
34.1
32.0
RDW
13.4
13.4
13.4
HbA2
5.4%
4.9%
4.0%
β0‐carrier
β+‐carrier
β++‐carrier
Presumptive Diagnosis of:
Molecular causes of β-Thalassemia
Point Mutations
Deletions
¾ Transcription of the gene defects;
¾ Defects in Maturation of pre‐mRNA;
¾ Defects in mRNA Translation;
¾ Defects that cause instability of the globin chain.
ATYPICAL PHENOTYPES
-1- HbA2 borderline with normocytosis or microcytosis
-22 Microcytosis
Mi
t i with
ith normall levels
l l off HbA2 edd HbF
-33 Microcytosis with very high level of HbA2
-4- Increased levels of HbF
Si ringrazia
i
i per il sostegno alla
ll Ri
Ricerca
15
ATYPICAL PHENOTYPES
-2- Microcytosis with normal levels of HbA2 ed HbF
-44 Increased levels of HbF
Si ringrazia
i
ll Ri
Ricerca
16
HBA2 BORDERLINE WITH NORMOCYTOSIS OR MICROCYTOSIS
Causes:
• Silent mutations in the β-globin gene
p
p allelic structure
• Presence of triple-alpha
• β-globin variants
• Co-inheritance
Co inheritance of β mutations and δ mutations
• Co-inheritance of β mutations and α mutations
• Other: Hyperthyroidism
Use of anti-HIV retroviral drugs (AZT)
Dosage of the HbA2 value
Si ringrazia
g
per
p il sostegno
g alla Ricerca
17
Borderline HbA2 is not a rare event and it should be more
investigate,
g , specially
p
y in p
presence of reduced MCV value and if p
partner
is an healty carrier of β−thalassemia.
18
22,9%
,
19
(MCV<80)
(MCV≥80)
20
21
HbA2
genotype
cases
NEG/ IVSI nt 6
Means
Dev.St.
24
3,69
0,14
β-prom (-92/-101)
20
3,69
0,12
NEG/ IVSI nt6
24
3,69
0,14
βo +δ Cd27
11
3 50
3,50
0 17
0,17
NEG/ IVSI nt6
24
3,69
0,14
ααα/αα
15
3,33
0,13
NEG/ IVSI nt6
24
3,69
0,14
αα/−α
12
3.15
0.10
βo +δ Cd27
11
3,50
0,17
ααα/αα
15
3,33
0,13
βo +δ Cd27
11
3,50
0,17
β−prom (-92/-101)
20
3,69
0,12
ααα/αα
15
3,33
0,13
β-prom (-92/-101)
20
3,69
0,12
MCV
t student
p value
0.062
0,95
3.67
0,0009
7.94
0,00001
12 10
12.10
0 00001
0.00001
2.64
3.73
8.07
0,014
0.0008
0,00001
Means
Dev.St.
69,18
2,58
83.51
4.76
69,18
2,58
61 53
61,53
2 91
2,91
69,18
2,58
85,76
4,06
69,18
2,58
75.42
6.72
61,53
2,91
85,76
4,06
61,53
2,91
83.51
4.76
85,76
4,06
83.51
4.76
t student
p value
12.70
0,00001
7.83
0,00001
15.64
0,00001
4 04
4.04
0 0003
0.0003
16.85
0,00001
13.89
0,00001
1.47
0,15
¾ β−Thalassemia
¾ α−Thalassemia
¾ δ−Τhalassemia
DECREASED HbA2
23
Molecular causes of δ-Thalassemia
Deletions
Point Mutations
¾ Transcription
T
i ti off th
the gene d
defects;
f t
¾ Defects in Maturation of pre‐mRNA;
¾ Defects in mRNA Translation;
¾ Defects that cause instability of the globin chain
chain.
24
INTERACTIONS BETWEEN BETA AND DELTA MUTATIONS
β
α
=
MCV, MCH
δ
HbA2
δ+β°
RBC
HB
HCT
MCV
MCH
MCHC
RDW
A2
F
6.01
6 01
12.2
37.1
61.8
61 8
20.2
32.7
14.3
14 3
3.5%
1.3%
βCd30/β
δCd27/δ
HbA2 Yialousa
6.10
6
10
12.3
37.3
61 2
61.2
20.0
32.7
14 3
14.3
5.6%
0.6%
βCd30/β
55.54
54
11.2
36.1
59 6
59.6
20.2
32.0
15 1
15.1
3.5%
0.5%
βCd39/β
5.58
5
58
11.9
37.3
62 0
62.0
21.3
31.9
13 9
13.9
5.2%
2.6%
βCd39/β
δCd27/δ
HbA2 Yialousa
δ+β+
RBC
HB
HCT
MCV
MCH
MCHC
RDW
A2
F
5.81
5 81
12.1
37.0
64.0
64 0
20.9
32.8
14.8
4.8%
<0.5%
βIVSI nt110/β
5.64
5
64
11.6
37.0
65 6
65.6
20.6
31.4
14.5
3.5%
0.9%
6.05
6
05
12.4
39.6
65 0
65.0
20.6
31.5
13.2
3.2%
0.0%
6.62
6
62
13.1
41.5
62 7
62.7
19.9
31.7
14.5
3.1%
0.5%
βIVSI nt 110/β
δCd27/δ
HbA2 Yialousa
RBC
HB
HCT
MCV
MCH
MCHC
C C
RDW
A2
F
Sw
βIVSI nt110/β
5.81
12.1
37.0
64.0
20.9
32.8
3 .8
14.8
4.8%
<0
<0.5%
5%
0
6.44
13.4
41.4
64.0 =
20.9
32.5
3
.5
14.6
3.1%
<0 5%
<0.5%
2.7%
δCd16/δ
HbA2’ o HbB2
INTERACTION BETWEEN BETA AND ALPHA
MUTATIONS
β
α
Reducing the imbalance between the chains α/β
MCV
= HbA2
RBC
HB
HCT
MCV
MCH
MCHC
RDW
A2
F
5.27
11.9
11 9
37.6
71.3
22.5
31.6
14.0
4.1%
0.9%
βIVSI nt 6/β
4.26
11 6
11.6
34.6
81.2
27.1
33.4
14.3
4.0% =
0.7%
βIVSI nt 6/β
3 7/αα
α3.7
/
5.10
13.2
42.6
84.0
27.0
32.2
12.5
4.2%
0.9%
βIVSI nt 6/β
=
4.13
11.9
35.1
84.9
28.7
33.9
12.7
4.0%
1.9%
β-101/β
4.77
12 9
12.9
35.3
82.3
28.5
31.5
12.9
4.1%
1.5%
β-92/β
αHpHIα/αα
/
HYPERTHYROIDISM
RBC
4.69
5.12
Hb
13 0
13.0
14 5
14.5
Ht
39.0
45.7
MCV
84.0
89.0
MCH
27 9
27.9
28 5
28.5
RDW
17.0
12.7
HbA2
3.70
3.00
HbF
<1
<1
β-gene
β/β
α−gene
α
gene
αα/αα
δ-gene
β/β
αα/αα
NEG
NEG
Post therapy
31
ATYPICAL PHENOTYPES
Si ringrazia
i
ll Ri
Ricerca
32
MICROCYTOSIS WITH NORMAL LEVELS OF HBA2 AND HBF
•
Reduced values of MCV and MCH
•
HbA2 ≤3.2%, HbF ≤ 2%
(< 80 fl, e < 26 pg)
Causes:
• α° thalassemia ( two alpha genes mutated)
• α+/° thalassemia ( one alpha gene alterated)
• α+ thalassemia (one alpha gene alterated )
• β mild or slight mutations in eterozygosis with δ mutations
• Hemoglobin Variants
• Iron deficiency
• Age
33
δ+β++
RBC
HB
HCT
MCV
MCH
MCHC
RDW
A2
F
5.27
11.9
37.6
71.3
71 3
22.5
31.6
14.0
14 0
4.1%
0.9%
βIVSI nt 6/β
5.24
12.3
38.5
73 5
73.5
23.5
31.9
14 0
14.0
2.6%
0.0%
6.25
14.2
44.3
70 9
70.9
22.7
32.1
13 5
13.5
2.8%
0.5%
βIVSI nt 6/β
5.02
13.4
36.9
73 5
73.5
24.8
33.8
13 3
13.3
2.6%
0.5%
αNcoIα/αα
δCd27/δ
HbA2 Yialousa
RBC
HB
HCT
MCV
MCH
MCHC
RDW
A2
F
βIVSI nt110/β
5.81
12.1
37.0
64.0
20.9
20 9
32.8
14.8
4 8%
4.8%
<0.5%
5.65
11.9
34.7
61.4
19 5
19.5
31.7
15.3
2 4%
2.4%
1.0%
6.39
13.3
40.4
63.0
20 7
20.7
32.9
13.8
2 4%
2.4%
<0.5%
βIVSI nt110/β
α-20.5/αα
δCd142/δ
HbA2 Fitrzoy
ATYPICAL PHENOTYPES
Si ringrazia
i
ll Ri
Ricerca
36
MICROCYTOSIS WITH VERY HIGH LEVEL OF HBA2
•
Reduced values of MCV ed MCH ((< 80 fl,, e < 26 pg)
•
HbA2 > 6.0%
Causes:
o Hb Lepore
o Hemoglobin Variants : Hb E (Cd 26 Glu →Lys)
oβ
β−cluster
cluster deletions
Recommendation: Careful evaluation of hemoglobin and haematological status
Hb
11-15
11 15
Hb
12
12-15
15
Hb
11-14
11 14
MCV
70.0
MCV
75.0
MCV
65-70
HbA2
28 – 35 %
HbA2
7 0 – 10 %
7.0
HbF
1.0-2.0 %
HbF
2.0-4.0 %
HbA2
HbF
10 – 15 %
1.0-2.0 %
Hb-Lepore
Hb-E
del. 1393
38
ATYPICAL PHENOTYPES
Si ringrazia
i
ll Ri
Ricerca
39
INCREASED LEVEL OF HBF
• Normal HbA2 levels, Increased HbF levels (2% - 30%)
Phenotypic classification
¾ “ Thalassemic mutations” with reduced MCV and MCH values and heterocellular
HbF distribution.
di t ib ti
¾
“ Hereditary persistence of HbF (HPFH)” with normal MCV and MCH values
and pancellular HbF distribution.
distribution
Gγ AγHPFH
Morfologia eritrociti
Gγ Aγ(δβ0)talassemia
normal
reduced
MCH
almost normal
reduced
HbF
15 30%
15-30%
4 18%
4-18%
pancellular
heterocellular
Distribuzione HbF
δ-β SICILIAN DELETION
RBC
5.50
Hb
12.5
MCV 65
65.0
0
MCH
22.5
RDW 12.5
12 5
Hb A2 2.8%
Hb F 11.1%
11 1%
• There are about
130 known point mutations of γγ
globin genes
• About
Ab t 20 affected
ff t d the
th γglobin gene promoter resulting
in increased
HbF
♀pregnant
RBC
HB
HCT
MCV
MCH
MCHC
RDW
A2
F
Gγ
γ-158
4.24
13.0
35.6
83 9
83.9
30.7
36 6
36.6
13.6
3.3%
6.8%
/N
CONCLUSIONS
• The first-level protocols are designed to obtain a reliable
diagnosis, which is essentially ”presumptive”.
presumptive .
• The first level screening should be distinguished from a
definitive diagnosis because its purpose is just to
provide indications through the use of simple biochemical
tests.
tests
• A definitive diagnosis
g
in most cases,, requires
q
DNA or pprotein
analysis.
44
Flow Chart for thalassemia screening
MCV fl
≥80
≥80
≥80
<80*
HbA2 %
<2
2-3,3
≥3,4
<2
δ thal Normal
carrier
β Thal or
ααα/αα
carrier
Molecolar
analysis
α thal
with δ thal
carrier
Molecolar
analysis
<80*
<80*
2-3,3
≥3,4
α thal or
β + δ thal
carrier
β Thal
carrier
Molecolar
analysis
* withouth iron deficency
45
CONCLUSIONS
¾β-Thalassemia::
g allows to define the healthy
y carrier state in
Screening
most cases (typical phenotypes). In other cases it is necessary to
resort to second-level investigations.
¾α-Talassemia:
There is no specific screening tests for healthy carriers of α
αthalassemia, which often remains a diagnosis of exclusion. Second
Level tests are needed to confirm the diagnosis.
¾Hb Variants:
in presence of an abnormal hemoglobin,
hemoglobin the results obtained by firstfirst
level testing remains a presumptive data. The interaction with the
laboratory of the II level is essential.

The diagnosis of thalassemia

Transcript

Documenti analoghi

riccioni rossella - Azienda USL6 Livorno

Prof. A.Maggio - Azienda Ospedaliera Ospedali Riuniti Villa Sofia

Bologna, Aemilia Hotel 1-3 ottobre 2012

Valutazione multicentrica dell`analizzatore Tosoh G8 per la

CARATTERISTICHE GENERALI

Linee guida per il referto ematologico

Diapositiva 1 - Cisl Lombardia

Il programma

Area Dipartimentale Riabilitazione e Continuità delle cure