Microsoft PowerPoint - Diagnosis of Genetic Hb DisordersDIAGNOSIS OF GENETIC HAEMOGLOBIN DISORDERS
Maj Gen (R) Suhaib Ahmed, HI (M) MBBS; MCPS; FCPS (Pak); PhD (London)
Genetics Resource Centre (GRC)
g
Deletion Non-deletion
With α-chain Hb variants With β-chain Hb variants
Sickling Disorders: With β-Thalassaemia With α-chain Hb variants With β-chain Hb variants
Other Structural Variants (Hb-E, C, D etc.): With β-Thalassaemia
With β-Thalassaemia
β With α-Thalassaemia
δβ-Thalassaemia: (δβ)o -Thalassaemia (Aγδβ)o -Thalassaemia
Altered Affinity Haemoglobins:
Deletion (δβ)o -HPFH
Non-deletion Linked to β-globin gene cluster Unlinked to β-globin gene cluster
Investigations for a Genetic Investigations for a Genetic Haemoglobin Disorder
Complete Blood Counts
History and Examination
Complete Blood Counts
Other related Investigations
Electrophoresis
Size of the molecule Charge on the g molecule Pore size of the
dimedium Voltage and Current Buffer composition and Buffer composition and concentration
Hb-Electrophoresis
Cellulose Acetate Membrane C ll lCellogel Agarose Gel Agar GelAgar Gel Polyacrylamide Gel
Cellulose Acetate MembraneCellulose Acetate Membrane Hb-Electrophoresis (pH 8.7)
Slow moving Hb-A2/E/C
Fast moving Hb-H/J/Barts
By Chromatography Elution after Electrophoresis Densitometry??Densitometry??
For Hb-A2Hb A2
Miscellaneous Investigations
Sickling Test Heinz Body Test Test for Unstable Haemoglobins Test for Methaemoglobin
β-Thalassaemia Trait
Typical β-thalassaemia Trait 120
160
140
120
100
80
MCH
30.028.026.024.022.020.018.016.0
80
60
40
20
0
MCH
0.36% Saline
PCR: Cap+1 or –88 mutation
Sex TRBC Hb MCV MCH Mutation Sex TRBC Hb MCV MCH Mutation
F 4.77 13.3 83.0 27.9 Cap+1 M 6.57 12.9 71.8 19.6 IVSI-5
F 4.47 11.5 81.3 25.2 Cap+1 M 7.50 13.6 54.5 18.1 Fr 41-42
F 4.18 10.9 82.8 26.1 Cap+1 M 6.78 13.1 62.1 19.3 IVSI-5
M 4.96 14.9 85.9 30.1 Cap+1 M 6.93 14.2 62.8 20.5 Del 619
F 4.06 12.1 85.2 29.8 Cap+1 M 6.21 12.0 59.7 19.3 Del 619
F 5.41 13.4 76.0 24.8 Cap+1 F 5.35 10.3 58.5 19.3 Cd 15
F 4.05 11.2 86.4 27.7 Cap+1 M 6.73 13.3 61.1 19.8 Cd 5
F 4.17 11.3 80.6 27.1 Cap+1 F 5.16 9.9 55.7 19.2 IVSI-1
M 4.83 14.2 87.6 28.8 Cap+1 M 6.76 15.8 65.0 23.4 IVSI-5
F 4.19 11.2 80.7 26.7 Cap+1 F 5.15 11.3 66.0 21.9 IVSI-5
F 3.80 9.9 77.9 26.1 Cap+1 M 6.80 12.6 60.7 18.5 IVSI-5
F 3 37 9 9 87 5 29 4 Cap+1 F 5 50 10 7 53 0 19 5 IVSI 5F 3.37 9.9 87.5 29.4 Cap+1 F 5.50 10.7 53.0 19.5 IVSI-5
M 6.05 15.4 82.6 25.5 Cap+1 M 7.79 14.5 61.0 18.6 Cd 30
F 4.15 10.0 76.9 24.1 Cap+1 F 4.92 9.8 68.3 19.9 Cd 30
Co-existing α andg β-thalassaemia Trait
Approximately 2% of β-thalassaemia carriers have co-existing α-thalassaemia t ittrait Red cell indices are normalized
b dHb-A2 is increased PCR may be required
β-Thalassaemia Trait and β Thalassaemia Trait and Interacting Structural Hb Variants
Hb-D/β-thalassaemia Trait
Hb-A2: 5.1% PCR: Fr 8-9
RDW in Thalassaemia
40
50
20
30
40
0
10
32 or less 32.1 to 36 36.1 to 40 40.1 to 44 44.1 to 48 48.1 to 52 52.1 to 56 56.1 or more
Thalassaemia trait Iron deficiency anaemia
Thalassaemia trait 10 35 44 10 1 0 0 0
Iron deficiency anaemia 0 0 2 18 22 12 4 5
Screening Strategy for Thalassaemia Carriers
One tube osmotic fragility Negative
Positive Spouse normal
MCV and MCH * MCV >75 fl or MCH >25 pg
Hb A ti ti
Hb-A2 estimation
Hb-A2 > 3.5% Hb-A2 3.0-3.4% or Hb-A2 <3% and Hb <9 g/dl
Hb-A2 < 3.0% and Hb >9 g/dl
PCR Negative
Untransfused (n=171)
Mean Range Mean Range
Hb-F (%) 95 35 - 97 31 0.5 - 97 < 0.001
Diagnosis of β-Thalassaemia Major in Diagnosis of β-Thalassaemia Major in Previously Transfused Patients
Hb 12 7 /dl Hb: 10.4 g/dl MCV: 64 fl MCH: 18 pg
Hb: 12.7 g/dl MCV: 66 fl MCH: 19 pg
Transfusion Dependent Anaemia ?? Hb: 6.7 g/dlg MCV: 76 fl MCH: 24 pg Hb-F: 3.5%
Thalassaemia Intermedia Mild/Silent alleles Co-incidental α-thalassaemia trait Co-incidental structural variants Xmn I pol morphismXmn-I polymorphism δβ-Thalassaemia
Thalassaemia Major
Thalassaemia Intermedia
Thalassaemia Minor
Splenomegaly ++++ ++,+++ +, 0
Jaundice + ++ 0
Hypochromia ++++ +++ ++
Microcytosis +++ ++ +
Stippling ++ + +
Hb-A2 1-8.7% <1-10.0% 3.5-8.0%
Thalassaemia Intermedia in Pakistan
Cause of Thalassaemia Intermedia:
n (%): Mean age: Intermedia:
Xmn-I +/+ genotype 14 36%) 6 years 13 years
β+-mutation 6 (15%) 3 years 8 years
β+-mutation and coincidental α-thalassaemia 6 (15%) 11¼ years 18 years
Unidentified thalassaemia mutation 2 (6%) 7½ years 12½ yearsUnidentified thalassaemia mutation 2 (6%) 7½ years 12½ years
Coincidental α-thalassaemia 11 (28%) 9½ years 13½ years
Total 39 7 years 14 years
(Ahmed 1998)
δβ Th l i I /D l G (A δβ)oδβ-Thalassaemia Inv/Del Gγ(Aγδβ)o
H5
H4H3
H2
α-Thalassaemia
Silent carrier: -α/αα α-Thalassaemia Trait: -α/-α or --/αα Hb-H Disease: --/-α Hydrops Fetalis: --/--
Diagnosis of α-Thalassaemia
PCR for α-Thalassaemia
P l Ch i R ti (PCR)Polymerase Chain Reaction (PCR)
Molec lar Methods in DiagnosisMolecular Methods in Diagnosis
Mutation Analysis Specific methods
• DGGEDGGE • SSCP
Prenatal Diagnosis Diagnosis in previously transfused patientsg p y p Silent thalassaemia alleles Distinction between structural variants Thalassaemia intermedia α-thalassaemiaα thalassaemia β-Thalassaemia carriers in certain situations Rare thalassaemiasRare thalassaemias Donor chimerism studies after SCT
β-Thalassaemia mutations in Pakistan
Primer ID:
Mutations Pooled:
AD-1 Fr 8-9 (+G) IVSI-5 (G-C) F 41 42 ( TTCT)
215 bp 285 bp 439 bFr 41-42 (-TTCT)
IVSI-1 (G-T) Del 619bp
439 bp 280 bp 242 bp
AD-2 Cd 5 (-CT) Fr 16 (-C) IVSI-1 (G-T) Cd 30 (G-C) Cd 30 (G-A) IVSII 1 (G A)
205 bp 238 bp 280 bp 280 bp 280 bp 634 bpIVSII-1 (G-A) 634 bp
AD-3 Cd 15 (G-A) Cap+1 (A-C)
500 bp 567 bp
p ( ) p
Termination of Pregnancy
Prenatal Diagnosis b ARMSPrenatal Diagnosis by ARMS
Mutation 1. Father 2. Mother 3. CVS 4. CVS 5. -ve
lNormal 6. CVS 7. CVS 8 +ve8. +ve 9. -ve 10. Blank
Misdiagnosis in PNDg (<0.5%)
Maternal Contamination in Fetal Sample PCR FailurePCR Failure Clerical Mistakes M i C i li k l iMeotic Crossover in linkage analysis
(Ahmed S. Submitted to Prenatal Diagnosis)
Donor Chimerism at D5S818 LocusDonor Chimerism at D5S818 Locus
AFIP
1. Allelic ladder (11, 12, 13, 14) 2 Recipient Pre Transplant (12 13)
AFIP
2. Recipient Pre Transplant (12, 13) 3. Donor (10, 12) 4. Recepient (Post Transplant) (10, 12)
1 2 3 4 5 6
AFIP
1. Father 2. Mother 3. Donor 4 Recipient pre transplant4. Recipient pre transplant 5. Recipient post transplant
Donor