Detection of Rare β‐Thalassemia Mutations by Denaturing Gradient Gel Electrophoresis Among Indians

HEMOGLOBIN

Vol 28 No 1 pp 15ndash24 2004

ORIGINAL ARTICLE

Detection of Rare bb-Thalassemia Mutations by DenaturingGradient Gel Electrophoresis Among Indians

Ajit C Gorakshakar Supriya P Phanasgaonkar Anita H NadkarniRoshan B Colah and Dipika Mohanty

Indian Council of Medical Research (ICMR) Institute of Immunohaematology

Mumbai India

ABSTRACT

We report four rare b-thalassemia (thal) mutations viz AATAAAAACAAA

[polyadenylation (poly A) site mutation] IVS-II-745 (CG) codon 121 (GT) and

IVS-II-1 (GA) detected by denaturing gradient gel electrophoresis (DGGE) among

Indians Of these the poly A site mutation has been found in combination with

deletional db-thal in one case and with the IVS-1-5 (GC) mutation in another Two

DGGE patterns corresponding to the same IVS-II-1 (GA) mutation were seen in

one family Framework (FW) analyses in family studies have shown that the poly A

site mutation is associated with FW-1 while both the codon 121 (GT) and IVS-II-1

(GA) mutations are associated with FW-2 Denaturing gradient gel electrophoresis

facilitates the screening of rare b-thal mutations in the diverse Indian population with

its many ethnic groups covering a vast geographic territory

Key Words b-Thalassemia (thal) Rare mutations Denaturing gradient gel

electrophoresis (DGGE) India

Correspondence Dr Roshan B Colah Indian Council of Medical Research (ICMR) Institute of

Immunohaematology 13th Floor New Multistoreyed Building King Edward Memorial Hospital

Campus Parel Mumbai 400 012 India Fax +91(0)22-2-413-85-21 E-mail mohanty

bom5vsnlnetin

15

DOI 101081HEM-120028883 0363-0269 (Print) 1532-432X (Online)

Copyright D 2004 by Marcel Dekker Inc wwwdekkercom

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INTRODUCTION

b-Thalassemia (thal) is one of the major inherited disorders affecting a large

number of individuals in India The prevalence of heterozygotes ranges from 3ndash15 in

different caste groups (1) At the molecular level this disorder is extremely

heterogeneous as more than 200 mutations causing b-thal have been identified

worldwide (2) A total of 45 mutations have so far been reported from the Indian

subcontinent (3ndash7) This spectrum is continuously being expanded by the identifica-

tion of novel or rare mutations There is a need for a versatile technique that can

handle identification of known and unknown mutations in this large and heteroge-

neous population

Today several polymerase chain reaction (PCR)-based protocols are available to

identify mutations (8) The denaturing gradient gel electrophoresis (DGGE) technology

suits this diagnostic need in India Here we report the usefulness of DGGE in the

identification of four rare b-thal mutations among Indians

CASE HISTORIES

Case 1 NC a 2-year-old boy from Andhra Pradesh was referred for definite

diagnosis of b-thal He was diagnosed at the age of 15 years and had maintained his

hemoglobin (Hb) level at 80ndash85 gdL without any blood transfusions

Case 2 VG a 4-year-old girl from Gujrat was referred for molecular analysis She

was from a remote area and was brought to the hospital only when she was severely

anemic with a Hb level of 45 gdL at 3 years of age and was transfused

Case 3 VS a 32-year-old male from Tamilnadu whose wife was referred for

prenatal diagnosis Their first child was still-born Their second baby was diagnosed to

have b-thal major at the age of 4 months she required blood transfusions every month

until her death at the age of 2 years

Case 4 SP a 27-year-old female from Karnataka was referred for prenatal

diagnosis Her first baby was diagnosed to have b-thal major at the age of 11 months

Since that time she was regularly transfused till she died at the age of 17 months

Case 5 NK a 32-year-old Sheikh Muslim female from Karnataka was referred for

prenatal diagnosis She had a consanguinous marriage Her first baby was diagnosed

with b-thal major at the age of 1 year and was on a regular blood transfusion regimen

every month

MATERIALS AND METHODS

Peripheral blood (8ndash10 mL) was collected from all family members of the index

cases For the diagnosis of b-thal the red cell indices were measured on an automated

blood cell counter (Erma-PC-608 Erma Inc Tokyo Japan) the Hb A2 level was

estimated by elution after cellulose acetate electrophoresis and Hb F by the Singer et

al (9) alkali denaturation method DNA was extracted using the standard phenol-

chloroform extraction procedure (10)

16 Gorakshakar et al

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Ta

ble

1

Hem

ato

log

ical

inv

esti

gat

ion

san

dD

GG

Ean

aly

sis

of

two

fam

ilie

sca

rry

ing

the

po

lyA

(T

C)

mu

tati

on

Par

amet

ers

NC

fam

ily

VG

fam

ily

I-1

I-2

II-1

II-2

Fat

her

Mo

ther

Pro

ban

d

Sex

ndashA

ge

Mndash

36

Fndash

34

Fndash

10

Mndash

2M

ndash3

5F

ndash2

7F

ndash4

RB

C(1

01

2L

)6

39

50

15

46

47

65

36

51

13

94

Hb

(gd

L)

15

71

23

12

19

01

40

14

41

01

MC

V(f

L)

69

77

23

69

86

61

73

37

85

70

6

MC

H(p

g)

24

62

46

22

21

89

26

12

82

25

6

Hb

A2

()

28

41

41

26

58

33

44

Hb

F(

)1

97

06

27

72

60

90

26

9

Fra

gm

ents

wit

h

aty

pic

alD

GG

E

pat

tern

s

ndashF

FF

BF

B+

F

Mu

tati

on

sdb

-th

alp

oly

A(T

C)

po

lyA

(T

C)

db-t

hal

+

po

lyA

(T

C)

IVS

-I-5

(G

C)

po

lyA

(T

C)

IVS

-I-5

(G

C)

+

po

lyA

(T

C)

Fra

mew

ork

2db-

thal

13

a1

21

db-

thal

3a

3a

12

13

a

DGGE for Rare Indian bb-Thal Mutations 17

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The DGGE strategy of Ghanem et al (11) further detailed by Gorakshakar et al

(12) was used to analyze the b-globin gene segments The b-globin gene FW was

explored by analyzing the G fragment

The DNA segments exhibiting atypical DGGE profiles were sequenced either by

the Sanger et al (13) dideoxy chain termination method using the Sequenase Version

20 Kit [United States Biochemical (USB) Cleveland OH USA] or on the ABI

PRISMk 310 automatic DNA sequencer (Applied BioSystems Foster City CA USA)

The sequence of the primers used for sequencing were the same as those used for

DGGE analysis but without the lsquoGC Clamprsquo (11)

RESULTS

Table 1 shows the hematological investigations of the two families with the poly A

site mutation All members showing this mutation had low RBC indices and normal or

marginally elevated Hb A2 levels One of the parents in the family of NC showed

reduced indices and raised Hb F suggesting among other possibilities the presence of

db-thal By DGGE analysis the poly A mutation was localized to fragment F In this

family FW analysis appeared incompatible due to the presence of deletional db-thal

Only the normal allele in the father and the mutant allele in the proband are visualized

Figure 1 A) The DGGE patterns of fragment F showing the presence of the AATAAAAACAAA mutation and deletional db-thal in the NC family Lane 1 normal control (a single

band is seen due to the absence of a polymorphic site in this fragment) lane 2 (I-1) father

lanes 3 (I-2) and 5 (II-1) heterozygotes for the mutations lane 4 (II-2) homozygous child

B) DNA sequencing of fragment F on the ABI PRISMk 310 DNA sequencer (Applied

BioSystems) using a reverse primer for identification of the poly A site mutation


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Ta

ble

2

Hem

ato

log

ical

inv

esti

gat

ion

san

dD

GG

Ean

aly

sis

of

ind

ivid

ual

and

fam

ilie

sw

ith

rare

b-th

alas

sem

iam

uta

tio

ns

VS

SP

fam

ily

NK

fam

ily

I-1

I-2

II-1

I-1

I-2

II-2

II-1

Sex

ndashA

ge

Mndash

2M

ndash3

0F

ndash2

7F

ndash1

31

2M

ndash4

0F

ndash3

2F

ndash8

Mndash

11

RB

C(1

01

2L

)6

28

62

25

57

56

06

52

52

85

74

18

Hb

(gd

L)

12

81

20

10

91

26

13

11

03

10

14

7

MC

V(f

L)

51

06

53

66

67

05

60

45

93

51

97

89

MC

H(p

g)

20

41

93

19

62

25

20

11

95

17

62

61

Hb

A2

()

62

62

61

30

54

61

51

38

Hb

F(

)0

80

30

90

61

40

63

61

12

Fra

gm

ents

wit

hat

yp

ical

DG

GE

pat

tern

s

DE

EE

CC

CC

Mu

tati

on

sIV

S-I

I-7

45

(C

G)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)a

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)a

Fra

mew

ork

nd

2

22

22

21

22

32

32

2

No

te

nd

=

no

td

efin

ed

aH

om

ozy

go

te


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Figure 2 Analysis of fragment D showing the presence of the IVS-II-745 (CG) mutation

Lane 1 heterozygous IVS-II-745 (CG) (IVS-II-666 CT) lanes 2 and 3 normal control

(IVS-II-666 CT) lane 4 normal control (IVS-II 666 TT)

Figure 3 Analysis of fragment E showing the presence of the codon 121 (GT) mutation in the

SP family Lane 1 normal control lane 2 (II-1) codon 121 (GT) homozygote lane 3 (II-2)

chorionic villi sample (CVS) codon 121 (GT) homozygote lane 4 (I-1) codon 121 (GT)

heterozygote lane 5 (I-2) codon 121 (GT) heterozygote


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in the DGGE profile (Fig 1) The proband of the VG family carried both the poly A

and IVS-I-5 (GC) mutations providing atypical DGGE profiles for fragment B [IVS-

I-5 (GC)] and fragment F (poly A mutation) The poly A mutation was found in

association with FW-1 in both families

Table 2 summarizes the hematological and molecular data of two families and one

case presenting other rare mutations Heterozygotes for the IVS-II-745 (CG) codon

121 (GT) and IVS-II-1 (GA) mutations showed reduced red cell indices and raised

Hb A2 levels Framework analyses showed that the codon 121 (GT) and IVS-II-1

(GA) mutations were linked to FW-2

Figure 2 reveals the atypical DGGE pattern of proband VS seen in fragment D

This fragment contains one polymorphic site viz IVS-II-666 (CT) Thus there would

be three possible patterns in normal individuals Two of these patterns are seen in

the figure

Figure 3 depicts the atypical DGGE profile of fragment E seen in the SP family

There is no polymorphism in this fragment From the DGGE patterns it is clear that

both the parents carry the same mutation Based on the patterns in the homozygous

Figure 4 Analysis of fragment C showing two DGGE patterns of the IVS-II-1 (GA) mutation

in the NK family Lane 1 (II-1) IVS-II-1 (GA) homozygote lane 2 (I-1) IVS-II-1 (GA)

heterozygote (IVS-II-16 CC) lane 3 (I-2) IVS-II-1 (GA) heterozygote (IVS-II-16 CG) lane 4

normal control (IVS-II-16 CG) lane 5 (II-2) IVS-II-1 (GA) heterozygote (IVS-II-16 CG) lane

6 (II-3) CVS IVS-II-1 (GA) heterozygote (IVS-II-16 CC) lane 7 (II-3) normal control (IVS-

II-16 CG)


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child and the parents we had earlier offered prenatal diagnosis to this family without

characterizing the mutation (14)

Figure 4 shows atypical DGGE patterns observed in the NK family In this

fragment there is one polymorphic site (IVS-II-16 CG) From the DGGE pattern of

the homozygous child it is clear that both parents must have the same mutation

Nevertheless both of them reveal different DGGE patterns

DISCUSSION

We have detected four rare b-thal mutations among Indians viz the

AATAAAAACAAA poly A codon 121 (GT) IVS-II-1 (GA) and IVS-II-745

(CG)) by DGGE Of these the IVS-II-745 (CG) mutation is being reported for the

first time among Indians

The poly A site mutation AATAAAAACAAA was reported earlier in

American Blacks (15) This TC substitution in the conserved sequence extends

mRNA transcription by about 900 nucleotides beyond the cleavage site This novel

mRNA is seen at a lower level in addition to a small amount of normally cleaved and

polyadenylated RNA Therefore in both the families the clinical presentation of the

index cases was relatively mild

In the IVS-II-1 (GA) mutation normal splicing is disturbed due to alteration at

the junction of the donor splicing site (GT) resulting in b0-thal This mutation has been

reported earlier on two occasions among Indians (1617) As reported earlier (18) we

found this mutation to be linked to FW-2 IVS-II-1 (GA) is a very common mutation

in the Middle East region (19)

Codon 121 (GT) is a rare mutation in exon 3 initially reported in French Polish

and Japanese individuals (20ndash22) In our study it was detected in both the partners of

a non consanguineous marriage (Fig 3)This mutation was linked to FW-2 as in the

Japanese case (22) In the IVS-II-745 mutation the CG substitution generates a new

internal splice site

The entire b-globin gene in all our cases was scanned by DGGE The atypical

DGGE patterns were detected in various fragments as shown in the figures

Subsequently DNA sequencing of only those fragments (about 200ndash400 bp long)

was carried out to identify the mutation This is an important advantage of DGGE

where instead of sequencing the entire b-globin gene targeted sequencing can rapidly

be carried out

Of these five cases we offered prenatal diagnosis of b-thal to the SP and NK

families on the basis of their DGGE patterns but without characterizing the mutation

(14) This is another advantage of DGGE In the NK family it can be seen that

the two DGGE patterns of the parents correspond to the same mutation This was

confirmed by the pattern of the homozygous child The DGGE patterns in hetero-

zygotes change according to the FW of the wild type chromosome present (14)

Therefore a comprehensive catalogue of various DGGE patterns corresponding to

different mutations must be useful and can be achieved by in vitro generation of

heteroduplexes with normal DNA and known homozygous FWs The present study

further illustrates the molecular heterogeneity of b-thal in the extremely heterogeneous

Indian population


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Although the mutations reported here are not new they are unusual among Indians

The novelty of this paper is the applicability of DGGE to detect mutated DNA

fragments in the local situation In spite of DGGE being a laborious method it

would be useful particularly for those laboratories that do not have the resources to

invest in an automated DNA sequencer or have access to a centralized DNA

sequencing facility

REFERENCES

1 In Sood SK Madan N Colah R Sharma S Apte SV eds Collaborative Study on

Thalassemiamdashan ICMR Task Force Study New Delhi Indian Council of Medical

Research 1993 12ndash14

2 Huisman THJ Carver MFH The b- and d-thalassemia repository (Ninth edition

Part I) Hemoglobin 1998 22(2)169ndash1953 Colah RB Gorakshakar AC Thalassemias molecular genetics in antenatal

diagnosis In Lokeshwar MR Marwaha RK Sha N eds Recent Advances in

Pediatrics New Delhi Jaypee Brothers 2000 22ndash45

4 Agarwal S Hattori Y Agarwal SS Identification of a novel frameshift b-

thalassemia mutation in an Asian Indian Clin Genet 2000 57(4)311ndash3125 Agarwal S Pradhan M Gupta UR Sarwai S Agarwal SS Geographic and ethnic

distribution of b-thalassemia mutations in Uttar Pradesh India Hemoglobin 200024(2)89ndash97

6 Shaji RV Srivastava A Krishnamoorthy R Chandy M Co-existence of a novel b-

globin gene deletion (codons 81ndash87) with the codon 30 (GC) mutation in an

Indian patient with b0-thalassemia Hemoglobin 2002 26(3)237ndash2437 Nadkarni A Sakaguchi T Takkaku H Gorakshakar AC Phanasgaonkar S Colah

RB Mohanty D Kiyama R A novel b0-thalassemia mutation at codon 55 (A) and

a rare 17 bp deletion at codons 126ndash131 in the Indian population Hemoglobin

2002 26(1)41ndash478 Gorakshakar AC Progress in technology for mutation detection in hematological

disorders ICMR Bull 2000 30(1)1ndash109 Singer K Chernoff AI Singer L Studies of abnormal hemoglobins I Their

demonstration in sickle cell anemia and other hematologic disorders by means of

alkali denaturation Blood 1951 6413ndash42810 Old JM Higgs DR Gene analysis In Weatherall DJ ed The Thalassemias

Methods in Hematology Vol 6 Edinburgh Churchill Livingstone 1982 74ndash

102

11 Ghanem N Girodon E Vidaud M Martin J Fanen P Plassa F Goossens M A

comprehensive scanning method for rapid detection of b-globin mutations and

polymorphisms Hum Mutat 1992 1(3)229ndash23912 Gorakshakar AC Pawar AR Nadkarni AH Lu CY Krishnamoorthy R Besmond

C Colah RB Potential of denaturing gradient gel electrophoresis for scanning b-

thalassemia mutations in India Am J Hematol 1999 61(1)120ndash12513 Sanger F Nicklen S Coulson AR DNA sequencing with chain termination

inhibitors Proc Natl Acad Sci U S A 1977 74(12)5463ndash546914 Orkin SH Cheng TC Antonarakis SE Kazazian HH Jr Thalassemia due to a


Hem

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mutation in the cleavage-polyadenylation signal of the human b-globin gene

EMBO J 1985 4(2)453ndash45615 Varawalla NY Old JM Sarkar R Venkatesan R Weatherall DJ The spectrum of

b-thalassaemia mutations on the Indian subcontinent the basis for prenatal

diagnosis Br J Haematol 1991 78(2)242ndash24716 Vaz FEE Thakur CB Banerjee MK Gangal SG Distribution of b-thalassemia

mutations in the Indian population referred to a diagnostic center Hemoglobin

2000 24(3)181ndash19417 Varawalla NY Fitches AC Old JM Analysis of b-globin gene haplotypes in Asian

Indians origin and spread of b-thalassemia on the Indian subcontinent Hum Genet

1992 90(4)443ndash44918 Adekile AD Historical and anthropological correlates of bS haplotypes and a- and

b-thalassemia alleles in the Arabian peninsula Hemoglobin 1997 21(3)281ndash29619 Kazazian HH Jr Orkin SH Boehm CD Goff SC Wong C Dowling CE

Newburger PE Knowlton RG Brown V Donis-Keller H Characterization of a

spontaneous mutation to a b-thalassemia allele Am J Hum Genet 1986 38(6)860ndash867

20 Fei YJ Stoming TA Kutlar A Huisman THJ Stamatoyannopoulas G One form of

inclusion body b-thalassemia is due to a GAATAA mutation at codon 121 of the

b-globin chain Blood 1989 73(4)1075ndash107621 Yamamoto Ku Yamamoto Ki Hattori Y Yamashiro Y Hoshitani M Morishita M

Ohba Y Katahira H Karasawa M Omine M Narukiyo T Hirabayashi K

Miyawaki S Two b-thalassemia mutations in Japan codon 121(GAA TAA) and

IVS-I-130 (GC) Hemoglobin 1992 16(4)295ndash30122 Gorakshakar AC Pawar AR Nadkarni AH Pawar AR Desai SN Colah RB

Mohanty D Prenatal diagnosis of b-thalassemia among Indians using denaturing

gradient gel electrophoresis Hemoglobin 1997 21(5)421ndash435

Received July 4 2003

Accepted July 21 2003


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onal

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onl

y

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ORDER REPRINTS

INTRODUCTION

b-Thalassemia (thal) is one of the major inherited disorders affecting a large

number of individuals in India The prevalence of heterozygotes ranges from 3ndash15 in

different caste groups (1) At the molecular level this disorder is extremely

heterogeneous as more than 200 mutations causing b-thal have been identified

worldwide (2) A total of 45 mutations have so far been reported from the Indian

subcontinent (3ndash7) This spectrum is continuously being expanded by the identifica-

tion of novel or rare mutations There is a need for a versatile technique that can

handle identification of known and unknown mutations in this large and heteroge-

neous population

Today several polymerase chain reaction (PCR)-based protocols are available to

identify mutations (8) The denaturing gradient gel electrophoresis (DGGE) technology

suits this diagnostic need in India Here we report the usefulness of DGGE in the

identification of four rare b-thal mutations among Indians

CASE HISTORIES

Case 1 NC a 2-year-old boy from Andhra Pradesh was referred for definite

diagnosis of b-thal He was diagnosed at the age of 15 years and had maintained his

hemoglobin (Hb) level at 80ndash85 gdL without any blood transfusions

Case 2 VG a 4-year-old girl from Gujrat was referred for molecular analysis She

was from a remote area and was brought to the hospital only when she was severely

anemic with a Hb level of 45 gdL at 3 years of age and was transfused

Case 3 VS a 32-year-old male from Tamilnadu whose wife was referred for

prenatal diagnosis Their first child was still-born Their second baby was diagnosed to

have b-thal major at the age of 4 months she required blood transfusions every month

until her death at the age of 2 years

Case 4 SP a 27-year-old female from Karnataka was referred for prenatal

diagnosis Her first baby was diagnosed to have b-thal major at the age of 11 months

Since that time she was regularly transfused till she died at the age of 17 months

Case 5 NK a 32-year-old Sheikh Muslim female from Karnataka was referred for

prenatal diagnosis She had a consanguinous marriage Her first baby was diagnosed

with b-thal major at the age of 1 year and was on a regular blood transfusion regimen

every month

MATERIALS AND METHODS

Peripheral blood (8ndash10 mL) was collected from all family members of the index

cases For the diagnosis of b-thal the red cell indices were measured on an automated

blood cell counter (Erma-PC-608 Erma Inc Tokyo Japan) the Hb A2 level was

estimated by elution after cellulose acetate electrophoresis and Hb F by the Singer et

al (9) alkali denaturation method DNA was extracted using the standard phenol-

chloroform extraction procedure (10)


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Ta

ble

1

Hem

ato

log

ical

inv

esti

gat

ion

san

dD

GG

Ean

aly

sis

of

two

fam

ilie

sca

rry

ing

the

po

lyA

(T

C)

mu

tati

on

Par

amet

ers

NC

fam

ily

VG

fam

ily

I-1

I-2

II-1

II-2

Fat

her

Mo

ther

Pro

ban

d

Sex

ndashA

ge

Mndash

36

Fndash

34

Fndash

10

Mndash

2M

ndash3

5F

ndash2

7F

ndash4

RB

C(1

01

2L

)6

39

50

15

46

47

65

36

51

13

94

Hb

(gd

L)

15

71

23

12

19

01

40

14

41

01

MC

V(f

L)

69

77

23

69

86

61

73

37

85

70

6

MC

H(p

g)

24

62

46

22

21

89

26

12

82

25

6

Hb

A2

()

28

41

41

26

58

33

44

Hb

F(

)1

97

06

27

72

60

90

26

9

Fra

gm

ents

wit

h

aty

pic

alD

GG

E

pat

tern

s

ndashF

FF

BF

B+

F

Mu

tati

on

sdb

-th

alp

oly

A(T

C)

po

lyA

(T

C)

db-t

hal

+

po

lyA

(T

C)

IVS

-I-5

(G

C)

po

lyA

(T

C)

IVS

-I-5

(G

C)

+

po

lyA

(T

C)

Fra

mew

ork

2db-

thal

13

a1

21

db-

thal

3a

3a

12

13

a


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS










RESULTS














Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS

Ta

ble

2

Hem

ato

log

ical

inv

esti

gat

ion

san

dD

GG

Ean

aly

sis

of

ind

ivid

ual

and

fam

ilie

sw

ith

rare

b-th

alas

sem

iam

uta

tio

ns

VS

SP

fam

ily

NK

fam

ily

I-1

I-2

II-1

I-1

I-2

II-2

II-1

Sex

ndashA

ge

Mndash

2M

ndash3

0F

ndash2

7F

ndash1

31

2M

ndash4

0F

ndash3

2F

ndash8

Mndash

11

RB

C(1

01

2L

)6

28

62

25

57

56

06

52

52

85

74

18

Hb

(gd

L)

12

81

20

10

91

26

13

11

03

10

14

7

MC

V(f

L)

51

06

53

66

67

05

60

45

93

51

97

89

MC

H(p

g)

20

41

93

19

62

25

20

11

95

17

62

61

Hb

A2

()

62

62

61

30

54

61

51

38

Hb

F(

)0

80

30

90

61

40

63

61

12

Fra

gm

ents

wit

hat

yp

ical

DG

GE

pat

tern

s

DE

EE

CC

CC

Mu

tati

on

sIV

S-I

I-7

45

(C

G)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)a

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)a

Fra

mew

ork

nd

2

22

22

21

22

32

32

2

No

te

nd

=

no

td

efin

ed

aH

om

ozy

go

te


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS









Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS













the figure









II-16 CG)


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS







DISCUSSION


























be carried out











Indian population


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS






sequencing facility

REFERENCES





















102








Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS























Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y









Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS

Ta

ble

1

Hem

ato

log

ical

inv

esti

gat

ion

san

dD

GG

Ean

aly

sis

of

two

fam

ilie

sca

rry

ing

the

po

lyA

(T

C)

mu

tati

on

Par

amet

ers

NC

fam

ily

VG

fam

ily

I-1

I-2

II-1

II-2

Fat

her

Mo

ther

Pro

ban

d

Sex

ndashA

ge

Mndash

36

Fndash

34

Fndash

10

Mndash

2M

ndash3

5F

ndash2

7F

ndash4

RB

C(1

01

2L

)6

39

50

15

46

47

65

36

51

13

94

Hb

(gd

L)

15

71

23

12

19

01

40

14

41

01

MC

V(f

L)

69

77

23

69

86

61

73

37

85

70

6

MC

H(p

g)

24

62

46

22

21

89

26

12

82

25

6

Hb

A2

()

28

41

41

26

58

33

44

Hb

F(

)1

97

06

27

72

60

90

26

9

Fra

gm

ents

wit

h

aty

pic

alD

GG

E

pat

tern

s

ndashF

FF

BF

B+

F

Mu

tati

on

sdb

-th

alp

oly

A(T

C)

po

lyA

(T

C)

db-t

hal

+

po

lyA

(T

C)

IVS

-I-5

(G

C)

po

lyA

(T

C)

IVS

-I-5

(G

C)

+

po

lyA

(T

C)

Fra

mew

ork

2db-

thal

13

a1

21

db-

thal

3a

3a

12

13

a


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS










RESULTS














Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS

Ta

ble

2

Hem

ato

log

ical

inv

esti

gat

ion

san

dD

GG

Ean

aly

sis

of

ind

ivid

ual

and

fam

ilie

sw

ith

rare

b-th

alas

sem

iam

uta

tio

ns

VS

SP

fam

ily

NK

fam

ily

I-1

I-2

II-1

I-1

I-2

II-2

II-1

Sex

ndashA

ge

Mndash

2M

ndash3

0F

ndash2

7F

ndash1

31

2M

ndash4

0F

ndash3

2F

ndash8

Mndash

11

RB

C(1

01

2L

)6

28

62

25

57

56

06

52

52

85

74

18

Hb

(gd

L)

12

81

20

10

91

26

13

11

03

10

14

7

MC

V(f

L)

51

06

53

66

67

05

60

45

93

51

97

89

MC

H(p

g)

20

41

93

19

62

25

20

11

95

17

62

61

Hb

A2

()

62

62

61

30

54

61

51

38

Hb

F(

)0

80

30

90

61

40

63

61

12

Fra

gm

ents

wit

hat

yp

ical

DG

GE

pat

tern

s

DE

EE

CC

CC

Mu

tati

on

sIV

S-I

I-7

45

(C

G)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)a

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)a

Fra

mew

ork

nd

2

22

22

21

22

32

32

2

No

te

nd

=

no

td

efin

ed

aH

om

ozy

go

te


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS









Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS













the figure









II-16 CG)


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS







DISCUSSION


























be carried out











Indian population


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS






sequencing facility

REFERENCES





















102








Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS























Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y









Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS










RESULTS














Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS

Ta

ble

2

Hem

ato

log

ical

inv

esti

gat

ion

san

dD

GG

Ean

aly

sis

of

ind

ivid

ual

and

fam

ilie

sw

ith

rare

b-th

alas

sem

iam

uta

tio

ns

VS

SP

fam

ily

NK

fam

ily

I-1

I-2

II-1

I-1

I-2

II-2

II-1

Sex

ndashA

ge

Mndash

2M

ndash3

0F

ndash2

7F

ndash1

31

2M

ndash4

0F

ndash3

2F

ndash8

Mndash

11

RB

C(1

01

2L

)6

28

62

25

57

56

06

52

52

85

74

18

Hb

(gd

L)

12

81

20

10

91

26

13

11

03

10

14

7

MC

V(f

L)

51

06

53

66

67

05

60

45

93

51

97

89

MC

H(p

g)

20

41

93

19

62

25

20

11

95

17

62

61

Hb

A2

()

62

62

61

30

54

61

51

38

Hb

F(

)0

80

30

90

61

40

63

61

12

Fra

gm

ents

wit

hat

yp

ical

DG

GE

pat

tern

s

DE

EE

CC

CC

Mu

tati

on

sIV

S-I

I-7

45

(C

G)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)a

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)a

Fra

mew

ork

nd

2

22

22

21

22

32

32

2

No

te

nd

=

no

td

efin

ed

aH

om

ozy

go

te


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS









Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS













the figure









II-16 CG)


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS







DISCUSSION


























be carried out











Indian population


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS






sequencing facility

REFERENCES





















102








Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS























Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y









Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS

Ta

ble

2

Hem

ato

log

ical

inv

esti

gat

ion

san

dD

GG

Ean

aly

sis

of

ind

ivid

ual

and

fam

ilie

sw

ith

rare

b-th

alas

sem

iam

uta

tio

ns

VS

SP

fam

ily

NK

fam

ily

I-1

I-2

II-1

I-1

I-2

II-2

II-1

Sex

ndashA

ge

Mndash

2M

ndash3

0F

ndash2

7F

ndash1

31

2M

ndash4

0F

ndash3

2F

ndash8

Mndash

11

RB

C(1

01

2L

)6

28

62

25

57

56

06

52

52

85

74

18

Hb

(gd

L)

12

81

20

10

91

26

13

11

03

10

14

7

MC

V(f

L)

51

06

53

66

67

05

60

45

93

51

97

89

MC

H(p

g)

20

41

93

19

62

25

20

11

95

17

62

61

Hb

A2

()

62

62

61

30

54

61

51

38

Hb

F(

)0

80

30

90

61

40

63

61

12

Fra

gm

ents

wit

hat

yp

ical

DG

GE

pat

tern

s

DE

EE

CC

CC

Mu

tati

on

sIV

S-I

I-7

45

(C

G)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)

Co

do

n1

21

(G

T)a

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)

IVS

-II-

1

(G

A)a

Fra

mew

ork

nd

2

22

22

21

22

32

32

2

No

te

nd

=

no

td

efin

ed

aH

om

ozy

go

te


Hem

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bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS









Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS













the figure









II-16 CG)


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS







DISCUSSION


























be carried out











Indian population


Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS






sequencing facility

REFERENCES





















102








Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y

ORDER REPRINTS























Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

onl

y









Hem

oglo

bin

Dow

nloa

ded

from

info

rmah

ealth

care

com

by

CD

L-U

C D

avis

on

110

414

For

pers

onal

use

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REFERENCES





















102








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102








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102








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For

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Documents

Detection of Rare β‐Thalassemia Mutations by Denaturing Gradient Gel Electrophoresis Among Indians