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The 6th Biennial Meeting of the Human Variome Project
Consortium Global Globin 2020
PavingtheWayforPrevention
ofHemoglobinopathies
inEgyptParis, 30 May-3 June 2016 Ghada El-Kamah, Prof. Clinical Genetics,
Coordinator HBD Clinic & Team.
• Hereditary blood disorders (HBD) are chronic genetic disorders where the affected person suffers ill health all through his life
2
•They have a strong socioeconomic and emotional burden on the governmental and family levels
3
HBD
Hemoglobinopathies
The fall in thalassemia major birth rate through several national thalassemia
prevention programs
THALASSEMI
• PREVENTION
5
PRENATAL DIAGNOSIS CONVENTIONAL COUNSELING
Life long intervention that is sometimes in itself harmful Limitation of availabilities of therapy
Mild Moderate Severe
Age of onset > 6yrs 2-6 yrs < 2yrs
Hb level (g/dl) > 8 5-8 < 5
Splenomegaly (cm) 2-4 4-10 >10
+± NoGrowth retardation
+± NoFacial ± skeletal changes
± ±
NoSplenectomy
>126-12OccasionalTransfusion rate /yr
< 3yrs 3-6 yrs> 6yrs Age of first transfusion
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Mutation Our study
IVSI,6 26%IVSI,1 18,8%
IVSI,110 15,9%IVSII,745 4,57%
COD 27 4%
IVSII,1 3,9%IVSII,848 3,57%COD 39 2,78%
COD 5 2,58%IVSI,5 1,19%
COD 28 1,19%Codon 44 0,99%
-87 0,99%COD 4 0,39%
-29 0,39%
COD 9 0,39%COD 37 0,19%
COD 15 0,19%-101 0,19%
COD 3 0,19%-31 0,19%
HbC 0,19%Hb knossoss 0,4%
Egypt Lebanon Gaza Strip
Elucidatethegene.cbackgroundofβ-thalassemiaallelesinEgyp.ansthroughdeterminingtherestric.onfragmentlengthpolymorphism(RFLP)-haplotypeinβ-thalassemiachromosomebyPCR-basedmethodcombinedwithfamilylinkagestudy.
Linkageanalysiscouldhelpindetec.ngfragmentsneededforsequencing.
11
Allele frequency
1st fragment
2nd fragment
3rd fragment
4th fragment
5th fragment
6th fragment
7th fragment
_ 56% 65% 83% 76% 49% 41% 41%
+ 44% 35% 17% 24% 51% 59% 59%
Allele frequency
1st fragment
2nd fragment
3rd fragment
4th fragment
5th fragment
6th fragment
7th fragment
_ 53% 63% 79% 98% 34% 43% 54%
+ 47% 37% 21% 2% 66% 57% 46%
Haplotype of 100 beta-thalassemia cases
Haplotype of 100 controls
Mutation Haplotypes
IVS 1.6
(- + + - - - -) 25% (- + - - - + +) 23.3% (+ - - - + - + )11.7%
(40% several different haplotypes)
IVSI.110(+ - - - - + + ) 50% (+ - - - -+ - )25% (- - - - - - - )25%
IVSI.I
(+ - - - - + - )16.6% (- + + - - ++ ) 11.11% (- - - - + + +) 11.11%
(the rest were linked to several haplotypes)
IVSII.745(+ - - - - - + ) 50% (+ - - - - + - ) 25% (- + - - - - +) 25%
Codon 6(+ - - - + + + ) 50% (- + - - + + + )25% (- - - - - + - ) 25%
Codon28GCC>TCCAlanine>Serine
Codon38,TGG>TGA, Tryptophan>StopCodon,homomuta:on
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!IVS!2&16,!C!>!G.!
!
Normal!! Mutated!!
NormalMutated
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Codon49,CTG>CCGLeucine>Proline
Codon106CTG>CGCleucine>Arginine
HeteroCodon107CTG>CTGLeu>Val
Normal
Heterodele:oncodon47(-c)
Heteromuta.onGCC>TCCNormalsequence
Heteromuta:on-29
Heteromuta:onofH64R(CAT>CGT)NormalSequence
LCR
A wide array of abnormalities, underlie different phenotypes and help in their identification
ß-globin locus
Comparison between the clinical outcomes among different globin gene mutations
was performed to evaluate the predictive power of genetic determinants on the
phenotypic severity of patients with beta-thalassemia.
Difference in phenotypes corresponding to ß++/ ß ++ genotype
23.5%
52.9%
23.5%
mild severe
Difference in phenotype corresponding to ß0/ ß0 genotype
12
24
64
mild moderate severe
This confusion required the studying of genetic modifiers
group Beta globin mutation Alpha-globin mutation XmnI polymorph severe Late onset Thal. intermedia
I ß++/ ß ++
IVSI,6/IVSI,6 IVSI,6/IVSI,6 (10 cases) IVSI,6/IVSI,6 (2cases) -87/-87 IVSI,6/-87
-α3.7/-α3.7 αα/αα unchar. αα/αα αα/αα
-/- -/- -/- -/- -/-
0 0 0 0 0
0 0 0 0 0
1 10 2 1 1
II ß++/ ß +
IVSI,6/IVSI,110 IVSI,6/IVSI,110 (9 cases) IVSI,6/IVSI,110 IVSI,6/IVSI,110
Unchar. αα/αα -α3.7/-α3.7 -α3.7/αα
-/- -/-
+/- -/-
1 6 0 0
0 0 0 0
0 3 1 1
III ß+/ ß +
IVSI,110/IVSI,110 (7cases) IVSI,110/IVSI,110 IVSI,110/IVSII,745 IVSII,848/IVSII,848 (3cases) IVSI,110/IVSII,848 IVSI,110/IVSII,848
αα/αα -α3.7/-α 3.7 αα/αα αα/αα αα/ααα anti 3.7 αα/αα
-/- -/- -/- -/- -/- -/-
4 1 0 0 1 0
1 0 1 1 0 0
2 0 0 2 0 1
IV ß0/ ß 0
IVSI,1/IVSI,1 (7cases) COD5/COD5 COD39/COD39 (2cases)
αα/αα αα/αα αα/αα
-/- -/- -/-
7 1 1
0 0 0
0 0 1
V ß0/ ß ++
IVSI,6/IVSI,1 Unchar. +/- 1 0 0
• Thus facing the challenge of prenatal diagnosis
• A new study comparing phenotypes among family members carrying the same mutations was conducted
Family No. proband consang No. of affected members genotype Phenotypic score
1 M +ve 3 ß 0/ ß 0Severe Severe Severe
2 M +ve 2 ß ++ /ß ++ Moderate Moderate
3 M -ve 2 ß 0/? Moderate Moderate
4 F +ve 2 ß ++/? Mild Mild
5 M -ve 2 ß 0/ ß 0 Moderate Moderate
6 M +ve 2 ß ++ /ß + Severe Severe
7 M +ve 2 ß 0/ ß 0 Severe Severe
8 M +ve 2 ß ++ /ß ++ Moderate Moderate
9 M +ve 3 ß +/ ß +Severe Severe Severe
10 M +ve 2 ß ++ /ß + Mild Mild
Genotype/phenotype among 20 Betathalassemia families
Genotype/phenotype among 20 Betathalassemia families (cont)
11 F -ve 2 ß +/ ß + Moderate Moderate
12 M +ve 3 ß +/ ß +Moderate Moderate Moderate
13 F -ve 2 ß 0/ ß + Moderate Moderate
14 F -ve 3 ß +/ ß +Moderate Moderate Moderate
15 M +ve 2 ?/? Moderate Moderate
16 M +ve 2 ß 0/ ß 0 Severe Severe
17 F -ve 2 ß +/ ß 0 Severe Severe
18 M +ve 2 ß +/ ß + Severe Severe
19 F +ve 2 ß +/ ß + Severe Severe
20 F -ve 3 ß 0/ ß 0Severe Severe Severe
Other services includes
- Introduction of video counseling - Simplified book for the patients and family members - Booklets for the general practitioners about the genetic background of beta thalassemia - Participating in a book about beta-thalassemia
ParametersHealth subjects (n=20) β-thalassemia patients (n=56) P value
Median Range (min-max) Median Range (min-max)
Neoptrin (mmol/L) 9 6.0-12.8 19 10.0-33.0 0.000*
IL-4 (pg/ml) 25,5 8.0-59.0 18 9.0-80.0 0,439
IL-6 (pg/ml) 40,5 25.0-72.0 51,5 21.0-120.0 0.033*
hs-CRP (mg/L) 3,6 3.6-4.4 4,2 3.6-14.0 0.005*
TNF-α (pg/mL) 37,5 16.0-70.0 49,5 28.0-100.0 0.004*
IgA (g/L) 1,7 0.9-2.4 2,3 0.7-5.7 0.004*
IgM (g/L) 1,1 0.6-1.8 1,2 0.6-2.5 0,372
IgG (g/L) 10,1 7.2-12.0 14,5 6.0-20.0 0.000*
IgG1 (g/L) 6.400 5,100-7,900 9.300 2,614-10,900 0.000*
IgG2 (g/L) 4.750 3,000-5,500 5.450 2,500-6,600 0.033*
IgG3 (g/L) 470 146.0-1,320.0 879 143.0-1,950.0 0.000*
IgG4 (g/L) 137 58.0-380.0 249,5 58.0-815.0
*Statistically significant (p<0.05)IL-4: Interleukin-4, IL-6: Interleukin-6, TNF-α: Tumor necrosis factor-alpha, hs-CRP: High sensitive C-reactive may be due to inflammation which occurs with frequent blood transfusion and is associated with elevation in hs-CRP, the cytokines IL-6 and TNF-α and the immunoglobulins IgA, IgG and subclasses of IgG. This elevation of neopterin was not affected by sex or age
Comparison of serum parameters between patients with β-thalassemia and healthy subjects.
QualityofLifeAssessmentinBeta-thalassemiaPa:entsinEgypt.2014
64pa.entswithhomozygousβ-Thalassemia.32malesand32femaleswithameanageof59.48monthsThirty-sixcaseswerebloodtransfusiondependent(TD)while28werenottransfusion-dependent(NTD).Thirty-four pa.ents came from urban areas and 30 came fromruralareas.Resultsindicatedasignificantassocia.onbetweenlowerlevelofmaternaleduca.on,andchronicbloodtransfusionwithlowerphysicalandemo.onalQoLscores.
Correlation between physical role and different characteristicsPatient's characteristics p-value
Gender 0,345Age 0,354Blood transfusion 0.004*Origin (urban, rural) 0,77Mother's educational level 0.000*
*statistically significant results≤0.05..
Correlation between emotional role and different characteristicsPatient's characteristics p-value
Gender 0,239Age 0,343
Blood transfusion 0,276
Origin (urban, rural) 0,142Mother's educational level 0.006*
*statistically significant results ≤0.05.
Thecorrela:onoftheQoLscoreswithdemographic,clinicalandsocialcharacteris:cs
Mean scores of participants for the 8 indicies of the SF-36 test
Indicies of SF-36 test Mean scores of NTD patients (SI)
Mean scores of TD patients (SD)
t-test P-value
Physical functioning 70.41 (22.12) 47.44 (22.15) 0.000*
Physical role 63.61 (34.77) 28.76 (35.92) 0.000*
Emotional role 62.29 (34.28) 60.69 (41.74) 0,87
Vitality 52.32 (22.34) 38.75 (18.41) 0.01*
well being 66.85 (12.52) 63.33 (16.07) 0,34
Social functioning 71.30 (22.90) 60.26 (20.99) 0.049*
Pain 68.14 (24.51) 61.08 (24.57) 0,258
General health 55.98 (26.36) 41.72 (22.77) 0.024** Statistically significant
Thetotalscoreofthese8categoriesrangesbetween0and100,withdesigna.onsofweak(<20),bad(21-40),good(41-60),
verygood(61-80),andexcellent(>81)(Montazertetal.,2005).
AnalysisofdatawasperformedusingSPSS18forWindows.
2158 Three Dimensional Speckle Tracking Echocardiography (3D-STE) for Early Detection of Subtle Myocardial Deformation Dysfunction in Thalassemic PatientsThalassemia and Globin Gene RegulationProgram: Oral and Poster AbstractsSession: 112. Thalassemia and Globin Gene Regulation: Poster IISunday, December 6, 2015, 6:00 PM-8:00 PMHall A, Level 2 (Orange County Convention Center)
In asymptomatic thalassemia patients with preserved left ventricular global systolic function 3D-STE derived strain can detect early subtle myocardial dysfunction. The observed subtle myocardial deformation dysfunction is not related to the extent of myocardial iron deposition.
32
Ten families with Sickle Cell Anemia mutation in codon 6 (A>T) HbS.
Two patients with sickle-beta thalassemia are found to be having compound
heterozygous mutation in two different alleles (Codon6/ IVSI.6) and (Codon6/
IVSII.745).
Two amniotic fluid (AF) samples from 2 pregnant mothers with previous sickle
cell anemia sibs, revealed heterozygous (A>T) mutation in codon 6 in both
cases.
Carrier detection was offered in families with positive family history enabling
for proper genetic counseling.
Sickle Cell Anemia
Gender Age of onset Hb HBS% Rate of transfusion
M 7m 3gm/dl 25 /2 weeks
M 16m 4gm/dl 40once at onset & another at age of 2yrs
F S/B
27m 6.8gm/dl 72 never
Glucose 6 phosphate dehydrogenase deficiency
G6PD deficiency is the most common enzyme disorder in humans.
It is prevalent in the Mediterranean region, the Middle East, Africa, and South Asia.
G6PD deficiency causes neonatal hyperbillirubinemia, acute hemolytic anaemia and in severe cases fatal chronic non-spherocytic haemolytic anaemia.
Although X-linked diseases are usually thought to affect males only, homozygous females represent 10% G6PD deficient population.
About 10% heterozygous females are G6PD deficient probably due to X-chromosome inactivation.
35
Within about 130 million births/year, about 4.5 million newborns are at risk of neonatal jaundice and acute hemolytic crisis.
Area % Deficiency Year Study
Cairo 42% of males 2013 El-Deen et al.
Different urban & rural areas
14.4% 21.2% of males 2010 Abdel Fattah et al.
Mansoura 11.4 2006 Settin et al.
Tanta (Delta) 12 1996 Hosini et al.
(from Settin et al., 2006)
Middle Delta 11 1992 Shebl et al.
(from Settin et al., 2006)
Alexandria 9.8 1986
Hammad et al. (from Settin et al., 2006)
In Egypt G6PD among Jaundiced neonates
36
Misleading Enzyme assay during hemolytic attacks & missed neonatal cases necessitate further analysis
PhD thesis in 2014, through RFLP analysis including 50 cases two Mediterranean mutations were detected in 32%
Molecular investigation was performed for 24 families (28 cases). Mediterranean variant 563CT transition represented 35.7 % only i.e. 10 patients, 5 of which were heterozygous females, and the other five were 3 hemizygous males and two homozygous
Exons3and4ofG6PDgeneshowingtheAfrican(A-)variant(c.202G/A
Exons3and4ofG6PDgeneshowingtheAfricanc.376A/G
Therapeutic options for HBD are limited and it is much more important to control further
spread of the disease-causing gene by screening when tests are
available.
Pilot study for screening for β-thalassemia in Egypt: LC MS/MS spectrometry as a cost effective method.
Genetic Modifiers in Beta-Thalassemia & Hemophilia-A: A Possible Cause for Altered Therapy.
Optimization of use of nanoparticles biosensors as an innovative diagnostic method of HBD.
41
Mohamad Ghandour Heba Ahmad
Ghada El-Kamah
Maha Eid Dalia Abdeen
Khalda Amr Eman Bayoumy Rabab Khairat Eman Rabee
Noha El Taweel
Khaled Gaber Mona Kamal Ahmad Ibrahim
Rehab Mosaad abeer Ramadan
Naglaa Kholousy Iman Helwa
Assem MetwalyHanan Afiffi
Manal Michelle
Collaborators NRC STDF
Phoebe
Thank You