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

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•They have a strong socioeconomic and emotional burden on the governmental and family levels

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HBD

Hemoglobinopathies

The fall in thalassemia major birth rate through several national thalassemia

prevention programs

THALASSEMI

• PREVENTION

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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.

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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

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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.

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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.

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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

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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.

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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