Rev. Bras. Hematol. Hemoter. vol.37 número3

w w w . r b h h . o r g

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Case

Report

Compound

heterozygous

state

of

␤

-thalassemia

with

IVS1-5

(G

→

C)

mutation

and

Indian

deletion-inversion

G

␥

(A

␥␦␤

)

0

-thalassemia

in

eastern

India

Snehadhini

Dehury

_,

_Prasanta

_Purohit

_,

_Satyabrata

_Meher,

_Kishalaya

_Das,

_Siris

_Patel

VeerSurendraSaiMedicalCollege,Burla,India

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Articlehistory:

Received21October2014 Accepted11December2014 Availableonline12May2015

Introduction

Ithasrecentlybeenestimatedthateachyear,morethanseven millionbabies worldwideare born witheitheracongenital abnormalityor ageneticdisease.1 _{Hemoglobinopathiesare}

thecommonestautosomalhereditarydisordersandpresenta majorpublichealthprobleminIndia.Theoverallprevalence ofthe␤-thalassemiatraitis2.78%butthisvariesfrom1.48 to3.64%indifferentstatesofIndia2_{comparedtothecarrier}

frequencyinBrazil(1%).3_{Recentlywereportedthatthecarrier}

frequencyofthe␤-thalassemiagenewiththeIVS1-5(G→C)

mutation in western districts of Odisha, India is 3.75%.4,5

In India, the IVS1-5 (G→C) mutation isthe mostcommon

␤-thalassemiamutation.However,theIVS1-5(G→C)mutation

alongwithothermutations,includingIVS1-1(G→T),Cd41/42

(-TCTT),Cd 8/9and a619 basepair deletion, accounts for >90%ofmutationscausing␤-thalassemia.InBrazil,theCd39 (C→T)mutationisthemostprevalentcauseof␤-thalassemia

∗ _{Correspondingauthorat:Qr.3R/27,DoctorsColony,768017Burla,Dist.Sambalpur,Odisha,India.}

E-mailaddress:[email protected](S.Patel).

1 _{Bothauthorssharefirstauthorship.}

followedbytheIVSI-6(T→C),andIVSI-110(G→A)mutations.6

Amapshowingzonaldistributionof␤-thalassemiamutations inIndiaisdepictedinFigure1.

TheVeerSurendraSaiMedicalCollegeandHospital,Burla, Odisha isatertiary carereferral hospitalcateringfor west-ernOdishaaswellaseasterndistrictsofChhattisgarhstate. Under acomprehensivesicklecellcareprogram,wescreen outpatientandhospitalizedcasesintheInstituteforSickle CellDiseaseandotherhemoglobinopathiesattheSickleCell ClinicandMolecularBiologyLaboratory.

Compound heterozygotes for ␤-thalassemia and struc-turalhemoglobin(Hb)variantsusuallypresentwithasevere form of the disease. Here, we report a case, a com-pound heterozygotefor the␤-thalassemiamutationIVS1-5 (G→C)andIndiandeletion-inversionG␥(A␥␦␤)0_-thalassemia

(HbVarID-1038) from thedistrictofBargarhinthestateof Odisha,India.ThemolecularstructureoftheIndian deletion-inversion[G␥(A␥␦␤)0-thal]iscausedbyamajorrearrangement

http://dx.doi.org/10.1016/j.bjhh.2014.12.002

North India

West India

Central India

East India

South India

IVSI-5(G>C) IVSI-1(G>T) Codon 8/9(+G) Codon 30(G>C) Codon 5(–CT) –88(C>T) –90(C>T) IVSI-1(G>A) –28(A>G)

619-bp deletion Codon 41/42(–TCTT) Codon 15(G>C) Cap site +1(A>C) Codon 16(–C) Codon 15(–T) Poly A site(T>C) IVSII-837(T>G) Others/unknown

Figure1–MapofIndiadepictingthezonaldistributionof␤-thalassemiamutations.

includinganinversionofthesequencebetweenthe3′_endof

theA␥geneandtheIVS-IIregionofthe␤-globingene,followed bytwodeletions(total8.5kb)oftheflankingDNAsequence. ThisisthefirstcasereportofsuchacasefromeasternIndia.

Case

report

An18-year-oldfemalepatient(height147cmandweight40kg) belongingtothe‘Chasa’castewasadmittedtothe Depart-mentofMedicineofVeerSurendraSaiMedicalCollegeand Hospital,Burla,Odisha,Indiawithsevereanemia,but with nohistoryofanyothermedicalcomplaints.HerHblevelwas 2g/dLatthetimeofadmissiontothehospital.Aftera trans-fusionofthreebloodunits,theHblevelreached7.8g/dL.The erythrocytesedimentationratewas12mm/h.Other parame-tersofbloodindicesafterthebloodtransfusionwerewhite bloodcellcount(WBC):10.9×109cells/L;redbloodcells(RBC):

3.35×106/␮L;hematocrit(HCT):23.9%;meancorpuscular vol-ume (MCV): 71.7fL; mean cell hemoglobin (MCH): 20.6pg; meancellhemoglobinconcentration(MCHC):29.0g/dL;and

platelets (PLT): 149×109/L. Peripheral blood smear

exam-ination showed microcytic, hypochromic anemia, marked anisopoikilocytosis, many microcytes, few macrocytes and fragmented RBCs. Stainingby the Kleihauer–Betke method demonstrated pancellular distribution of fetal hemoglobin (HbF).Ultrasonographyexaminationrevealedhepatomegaly (16.5cm)andsplenomegaly(14.5cm).AnX-rayofchest(P-A view)andX-rayofbothhipjoints(A-Pview)showedno abnor-malities.

Automated high-performance liquid chromatography usingthe␤-ThalassemiaShortProgramonBio-RadVariant-II systemshowedvariousfractionsofHbwitharaisedlevelof HbF(84.6%)and anHb A2 concentrationof6.9%(Figure2).

Parents’ studiesrevealed that hermother hada highHb F level (13%)whereasherfatherhadhigh HbA2 level (5.2%).

All hematological parameters of the case and herparents are showninTable1. Screeningforthecommonmolecular determinants of raisedHb F, the Indian deletion-inversion G␥(A␥␦␤)0_{-thalassemia and hereditary persistence of fetal}

0 0.0 7.5 15.0 22.5 30.0 37.5

%

45.0

*Values outside of expected ranges F Concentration = 84.6* % A2 Concentration = 6.9* %

Peak name Peak

area Calibrated

area %

Rotention time (min) Area %

Analysis comments:

Total area: 1,380,884

3963 1126284 142459 108178 0.75

1.16 2.49 3.64 P1

F Ao A2

0.3 -10.3

-84.6 * -6.9 *

1 2 3

2.49

0.75

1.16

F

A2

3.44

Time (min.)

4 5 6

Figure2–Highperformanceliquidchromatograminthecaseofcompoundheterozygousstatefor␤-thalassemiawith IVS1-5(G→C)mutationandIndiandeletion-inversionG␥(A␥␦␤)0thalassemia(chromatogramoneyearafterhospitalization

atfollow-up).

Common Indian ␤-thalassemia mutations were confirmed by multiplex amplification refractory mutation system (ARMS)-PCR using a previously described protocol.4

More-over, alpha globin gene deletions (␣−3.7 _{and ␣}−4.2_{) were}

investigated by gap-PCR.8 _{The DNA study showed father}

asheterozygousforthe IVS1-5(G→C)mutationandmother

as a G␥(A␥␦␤)0_{-thalassemia carrier. The gel picture for}

Indiandeletion-inversionG␥(A␥␦␤)0-thalassemiaisshownin

Figure3.Alpha-thalassemiawasnotobservedinanyofthem.

Writteninformedconsentwasobtainedfromthepatient alongwithherparentsandthestudywasapprovedby Institu-tionalEthicsCommitteeofVeerSurendraSaiMedicalCollege andHospital,Burla,Odisha,India.

Discussion

␤+-Thalassemiaisthesecondmostcommon hemoglobinopa-thy in our clinic’s population. We have reported earlier the clinical and molecular characteristics of G␥(A␥␦␤)0

-thalassemia in heterozygous as well as in compound

heterozygousstateswithHbS.InHbS/G␥(A␥␦␤)0_-thalassemia

cases, the patients had repeated painful crises along with histories of blood transfusions.9 _{Indian deletion-inversion}

G␥(A␥␦␤)0_{-thalassemia in the heterozygous form has been}

reported in western India in the state of Maharashtra.10

Recently, Pandey et al. described seven cases with Indian deletion-inversion G␥(A␥␦␤)0_{-thalassemia, of which three}

cases were co-inheritedwith ␤-thalassemia withthe IVS1-5(G→C)mutation.11Thesethreecaseshadlowvaluesofboth

HbA2(3.2%,2.8%,and2.7%)andHbF(21.3%,10.7%and20.3%)

comparedtothe6.9%and84.6%ofHbA2andHbF,

respec-tivelyinthecurrentcase.Theirthreecasesweretransfusion dependentandshowedmoderateanemia.Ourcaseisthefirst caseofcompoundheterozygotestateof␤-thalassemiawith IVS1-5(G→C)mutationandG␥(A␥␦␤)0_{-thalassemiawithmild}

A1 A2

861bp

285bp

1195bp

665bp

371bp 327bp A3 A4 A5 A6 A7 B1 B2 B3 B4 B5 B6 B7

I

I L M F M F L I M F

Figure3–Agarosegelelectrophoresisforthedetectionof␤-thalassemiawithIVS1-5(G→C)mutation(ARMSPCR)and

Indiandeletion-inversionG␥(A␥␦␤)0_{thalassemia(GAP-PCR).*I:index;M:mother;F:father;L:ladder.Detectionof}

␤-thalassemiawithIVS1-5(G→C)mutationbyARMSPCR(LineA1,LineA4andLineA6fornormal␤-globingene,andLine

A2,LineA5andLineA7forIVS1-5[G→C]mutation).DetectionofIndiandeletion-inversionG␥(A␥␦␤)0_{thalassemiaby} GAP-PCR(LineB1,LineB2andLineB3forBreakpoint-A,andLineB4,LineB5andLineB6forBreakpoint-B).

Hb Flevel (84.6%). Our observation ofhomogeneous Fcell distributionandanelevatedHbFlevelinperipheralblood, isinagreementtothe reportofWayeet al.inan African-American with compound heterozygosity of a Black form of(A␥␦␤)0_{-thalassemia and the}

−29 (A→G) ␤+-thalassemia mutation.12_{Weonlyinvestigatedtwotypesof␣-thalassemia}

(␣−3.7_and␣−4.2_{),themostprevalentinIndia.Anotherformof}

␣-thalassemiamaybeafactorformilderclinicalpresentation ofthepatient.

Table1–Hematologicalinvestigationsinthecaseof

compoundheterozygousstatefor␤-thalassemiawith

IVS1-5(G→C)mutationandIndiandeletion-Inversion

G␥(A␥␦␤)0_{thalassemiawithherparents.}

Parameters Index(follow-up) Oneyearafter hospitalization

Father Mother

Completebloodcount

WBC(103_{/␮L) 13 13.9 9.2}

RBC(106_{/␮L) 4.81 3.87 3.67} Hemoglobin(g/dL) 8.0 8.3 7.8

HCT(%) 32.3 30.4 29.7

MCV(fL) 67.2 78.6 80.9

MCH(pg) 16.6 21.4 21.3

MCHC(g/dL) 24.8 27.3 26.3

Platelets(103_{/␮L) 125 312 188}

FractionsofHbvariantsbyHPLC

HbA0(%) 10.3 83 76.2

HbA2(%) 6.9 5.2 2.8

HbF(%) 84.6 0.3 13

WBC:white blood cells; RBC: redblood cells; HCT: hematocrit; MCV: mean cell volume; MCH: mean cell hemoglobin; MCHC: meancellhemoglobinconcentration;HPLC:high-performance liq-uidchromatography.

The Indian subcontinent has a heterogeneous

popula-tionwithdifferenthemoglobinopathies.TheseHbdisorders shouldbeincludedintheprenataldiagnosisofpatientswith

severe or mild thalassemia. The characterization of these

hemoglobinopathies willfacilitateapreventionandcontrol programofhemoglobinopathiesincludingthalassemiainthis region.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest

Acknowledgments

Thisstudy wassupportedbyresearchfundingfrom Indian

CouncilofMedicalResearch(ICMR),NewDelhi,Departmentof ScienceandTechnology(DST),NewDelhi,andNationalHealth

Mission(NHM),Odisha.

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