Salud

Artículo arbitrado

Identification of novel point mutations in

c-kit gene from Leukemia cases: a study

from Lucknow, Uttar Pradesh, India

Identificación de nuevas mutaciones puntuales en el gen

c-kit en casos de Leucemia: un estudio realizado en

Lucknow, Uttar Pradesh, India

SYED RIZWAN HUSSAIN , AMNA SIDDIQUI , JAVIER VARGAS-MEDRANO , HENA NAQVI ,

2,4

JONATHON MOHL , FARZANA MAHDI AND FAHIM AHMAD

Recibido: Octubre17, 2011

Aceptado: Enero 18, 2012

Abstract

Resumen

The c-kit gene is a receptor tyrosine kinase (RTK) class III that

is expressed in early hematopoietic progenitor cells. Aberrantly

activated RTK and related downstream signaling partners have

been reported as key elements in the molecular pathogenesis

of several malignancies. Within the c-kit gene exon-11 is the

most frequent site for mutations in different kinds of tumours.

Mutations in c-kit gene may enhance or interfere with the ability

of c-kit receptor to initiate the intracellular pathways resulting in

cell proliferation. Therefore, we aimed to screen the mutations

in c-kit gene at exon-8 and -11 in malignant Leukemias. Ninety

Leukemia cases were studied and analyzed by mutation-

specific PCR-SSCP followed by DNA sequencing. Twenty point

mutations were detected in eightAML (acute myeloid Leukemia)

cases within exon-11 which includes Tyr568Ser, Ile571Thr,

Thr574Pro, Gln575His, Tyr578Pro, Asp579His, His580Gln,

Arg586Thr, Asn587Asp and Arg588Met. The substitutions

Lys550Asn, Ile571Leu and Trp582Ser were observed in two

independent cases and four novel point mutations at codons

Ile563Lys, Val569Leu, Tyr570Ser, and Pro577Ser. Further, six

point mutations were detected at exon-8 in six cases (four AML

and two CML cases), comprising three novel mutations

Asn423Asp, Gln448Thr, and Gln448His. The point mutations

Thr417Asp, Tyr418Phe, and Leu421His were observed, but

were detected only in three cases. These observations suggest

that mutations in c-kit gene might represent a useful molecular

genetic marker in Leukemia and incidence of mutation at exon-

El gen c-kit, que codifica para un receptor tirosina quinasa (RTK)

de clase III, se expresa en las primeras células progenitoras

hematopoyéticas. La activación de este RTK y su vía de

señalización se encuentran involucradas en la patogénesis

molecular de varias enfermedades. La mutación del gen c-kit en el

exón 11 es una de las mutaciones más frecuentemente reportadas

en diferente tipos de tumores. Mutaciones en c-kit podrían

incrementar o interferir con la habilidad del receptor c-kit para

iniciar la activación de cascadas de señalización intracelulares

responsables en la proliferación celular. Por estas razones,

estudiamos las mutaciones del gen c-kit en el exon 8 y 11 en

casos con Leucemias. Noventa casos de Leucemia en la India

fueron estudiados mediante PCR SSCP, seguida por secuenciación

de DNA. Veinte mutaciones puntuales fueron detectadas en el

exon 11 en tan solo ocho de los casos conAML (leucemia mieloide

aguda), entre las que encontraron las mutaciones Tyr568Ser,

Ile571Thr, Thr574Pro, Gln575His, Tyr578Pro, Asp579His,

His580Gln, Arg586Thr, Asn587Asp y Arg588Met. Las

sustituciones Lys550Asn, Ile571Leu y Trp582Ser fueron

observadas en tan solo dos casos. Además, cuatro nuevas

mutaciones para los codones Ile563Lys, Val569Leu, Tyr570Ser,

y Pro577Ser se observaron en este estudio. En el exon 8, seis

mutaciones puntuales fueron observadas y en seis de los casos

(

cuatro en AML y dos en CML) encontramos tres nuevas

mutaciones Asn423Asp, Gln448Thr y Gln448His. Sin embargo,

las mutaciones puntuales Thr417Asp, Tyr418Phe y Leu421His

fueron observadas en varias ocasiones, pero en tan solo tres de

los casos estudiados. Estas observaciones sugieren que las

mutaciones en c-kit podrían representar un marcador genético

para Leucemia. La incidencia en la mutación del exon 8 y 11 es

elevada y podría estar relacionada con la patogénesis de la AML.

and -11 is high and might be involve in pathogenesis of AML.

Palabras clave: c-kit, exon-8 and -11, Leukemia, mutation,

SSCP-PAGE.

Palabras clave: c-kit, exones 8 y 11, Leucemia, mutación, SSCP

PAGE.

________________________________

Department of Biochemistry, Era's Lucknow Medical College and Hospital, Lucknow, India.

Center of Excellence for Infectious Diseases, Biomedical Sciences Department, Texas Tech University Health Science Center and

Paul L. Foster School of Medicine, El Paso, TX, U.S.A. 79905

Department of Anestheology, Texas Tech University, El Paso, TX, U.S.A.

Author for correspondence: fahim.ahmad@ttuhsc.edu.

• Vol. VI, No. 1 • Enero-Abril 2012 •

SYED RIZWAN HUSSAIN, AMNA SIDDIQUI, JAVIER VARGAS-MEDRANO, HENA NAQVI, JONATHON MOHL, FARZANA MAHDI AND FAHIM AHMAD:

Identification of novel point mutations in c-kit gene from Leukemia cases: a study from Lucknow, Uttar Pradesh, India

Introduction

eukemia is classified based on the presence of specific cytogenetic abnormalities as well

as the French-American-British (FAB) classification of the leukemic cells (Rowley, 1973). A

mutation on c-kit gene, a member of the receptor tyrosine kinase (RTK) family type III, is the

most frequently occurring genetic aberration in acute myeloid leukemia (AML). A number of

observations also suggest a role for c-kit that is important for the development of a range of cells

including hematopoietic cells in leukaemogenesis (Reilly, 2002).

High expression of c-kit in AML (60%-80%

higher than control) has been reported (Reuss-

Borst et al., 1994; Cole et al., 1996) and point

mutations in c-kit gene have been identified in

Harri et al., 2005). Until now, no study has

reported the frequency and prevalence of

mutations in exon-8 and -11 of c-kit gene in

Leukemia patients from northern India. In this

study we have screened the mutation status of

exon-8 and -11 of c-kit gene in malignant

Leukemias (Acute Myeloid Leukemia, Acute

Lymphoblastic Leukemia, Chronic Myeloid

Leukemia and Chronic Lymphocytic Leukemia)

and further explored whether the c-kit gene

mutations were valuable as malignant markers

in Leukemia.

3.4-45.0% ofAMLcases (Higuchi et al., 2002).

However, many of these studies looked for

mutations in c-kit gen only at coding sequence

region. It is known that c-kit is a Leukemia proto-

oncogene and activating c-kit mutations are likely

to contribute in the development of Leukemia in

humans (Smith et al., 2004; Piloto et al., 2006;

Gao et al., 2011; Marcucci et al., 2011). The

activation sphere of the receptor has resulted in

the constitutive c-kit kinase activity and c-kit

receptors harboring such mutations when

introduced into mammalian cells downstream

signaling pathways lead to factor-independent

growth in vitro and leukemogenesis in vivo (Ihle

et al., 1995; Gao et al., 2011). The c-kit gene is

a member of the class III tyrosine kinase receptor

family that includes the platelet-derived growth

factor receptors (PDGFRs) (Ullrich et al., 1990;

Matthews et al., 1991; Martín-Broto et al., 2010).

Class III receptor tyrosine kinases (RTKs) share

sequence homology and have an overall similar

structure with five immunoglobulin-like repeats

in the extracellular domain, a single

transmembrane domain (TM), a juxtamembrane

domain (JM), two intracellular tyrosine kinase

domains (TK1 and TK2) divided by a kinase

insert domain (KI), and a C-terminal domain

Material and Methods

Subjects. The study group included 90

cases of Leukemia, from the Department of

Pathology at Era’s Lucknow Medical College and

Hospital, and from other hospitals and

pathologies situated in and around the city of

Lucknow, Uttar Pradesh, in northern India.

Ethical approval was obtained from the

Institutional Ethical Committee of Era’s Lucknow

Medical and Hospital, Lucknow, Uttar Pradesh,

India. In addition, clinical data was also recorded.

The blood or bone marrow samples were

stained by Leishman stain method and the

cases were classified, according to the FAB

criteria (Bennett et al., 1976). From the 90

Leukemia patients, 60 (66.7%) samples were

withAcute Myeloid Leukemia (AML), 10 (11.1%)

samples with Acute Lymphoblastic Leukemia

(ALL), 10 (11.1%) samples with Chronic Myeloid

Leukemia (CML) and 10 (11.1%) samples with

Chronic Lymphocytic Leukemia (CLL). The

demographic profile of patients can be finding

at supplementary table 1, as well as for controls

(supplementary table 2).

(

Yarden and Ullrich, 1988). The genomic locus

encoding the c-kit gene receptor has 21 exons,

ranging 100-300 base pairs (bp) (Abu-Duhier et

al., 2001). The c-kit gene mutations in exon-11

are reported in gastrointestinal stromal tumors,

human solid tumors and human germ cell

tumors (Qingsheng et al., 1999; Hou et al., 2004;

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SYED RIZWAN HUSSAIN, AMNA SIDDIQUI, JAVIER VARGAS-MEDRANO, HENA NAQVI, JONATHON MOHL, FARZANA MAHDI AND FAHIM AHMAD:

Identification of novel point mutations in c-kit gene from Leukemia cases: a study from Lucknow, Uttar Pradesh, India

Sample collection and DNA extraction.

Specimen was collected from 90 routinely-

processed unstained bone marrow slides and

blood diagnosed as Leukemia. Patients were

from the Department of Pathology at Era’s

Lucknow Medical College and Hospital, and

from other hospitals and pathologies situated

in and around the city of Lucknow, Uttar

Pradesh, India. Finally, samples were stored

Amplifications were done using a MJ Mini

Thermocycler (Bio-Rad, UK). The cycling

conditions were adjusted from the procedure

proposed by Tian et al. (1999). Briefly,

denaturation was at 94°C for 40 seconds,

followed by annealing at 56°C for 30 seconds,

and extension at 72°C for 30 seconds, repeated

for 30 cycles followed by a final extension step at

72°C for 8 minutes. Single-strand conformational

polymorphism (SSCP) analysis was performed

according to Orita et al. (1989) with few

at -20 C. Genomic DNA was extracted

according to Moskaluk et al. (1997) with little

modifications.

modifications. Samples were denatured at 94 C

for 8 minutes and immediately snap-cooled. Fifty

Polymerase Chain Reaction and Single-

Strand Conformational Polymorphism (PCR

SSCP). Polymerase Chain Reaction (PCR)

was performed in a 25 l of 1X PCR reaction

containing 200 ng of template DNA, 10 pmol

of each primer (forward and reverse

primers), 10 mmol/L of dNTPs and 0.3 units

of Taq DNA polymerase (Fermentas,

Germany). Forward and reverse primers for

exon-8 were 5’-GGCCATTTCTGTTTTCCTGT-3’

and 5’-TCTGCTCAGTTCCTGGACAA-3’

respectively. Both were designed and

customized by entering the sequence from

exon-8 into the JustBio.com server.

Forward and reverse primers for exon-11



l of amplified PCR product were loaded along

with 20 l of stop dye in a 10% polyacrylamide

gel. The gel was run in pre-cooled 2X buffer at

4°C, for 12 hours at 150 volts. The DNA in the gel

was stained after separation by electrophoresis

using a silver stain. Electrophoresis mobility shift

in single stranded or double stranded DNA from

patients was detected and compared with DNA

from wild-type controls (Fig. 1).

DNA Sequencing. Amplicons were

sequenced using an automated sequencer, ABI

3730XL DNA Analyzer (Applied Biosystems,

Foster city, California, USA) and analyzed using

FinchTV Software. DNA mutations were

reconfirmed by sequencing the amplicons in

both directions and in independent second

samples. The sequence was analysed using the

BioEdit software from JustBio.

'-ATTATTAAAAGGTGATCTATTTTTC-3' and

'-ACTGTTATGTGTACCCAAAAAG-3'

respectively, were proposed by Qingsheng

Tian et al. (1999).

Figure 1. SSCP-PAGE analysis showing electrophoresis mobility shift on native page. DNA control

was loaded in lane 1 and DNA from cases in 2, 3, 4, 5, and 6th lane (no shift in case 29 was observed,

however, there were shifts in cases: 09, 13, 20, and 27).

Control

Case 09

Case 13

Case 20

Case 27

Case 29

• Vol. VI, No. 1 • Enero-Abril 2012 •

SYED RIZWAN HUSSAIN, AMNA SIDDIQUI, JAVIER VARGAS-MEDRANO, HENA NAQVI, JONATHON MOHL, FARZANA MAHDI AND FAHIM AHMAD:

Identification of novel point mutations in c-kit gene from Leukemia cases: a study from Lucknow, Uttar Pradesh, India

shown in supplementary tables 1 and 2. Out of

Results

90 Leukemia cases, 80 samples were found to

Out of 90 Leukemia cases 51 (56.7%) were

male and 39 (43.3%) were female with age

ranging from 2-65 years. The mean age of

cases is 38.25 years with a SD ± 6.21 (mean

age of male cases was 38.60 years, SD ± 6.27

and mean age of female cases was 37.79 years,

SD ± 6.15). The cases were classified

according to the FAB criteria (Moskaluk et al.,

have mutations by a shift in DNA position on

SSCP-PAGE with respect to DNA from healthy

donors (Fig. 1). A total of 17 point mutations for

c-kit gene at exon-11 were found in this

investigation and only in eight cases with AML

(Table 1, Fig. 2 and 5). In addition, six point

mutations for c-kit gene at exon-8 for six AML

and CML cases were detected by our

experiments (Table 2, Fig. 3 and 4). After

comparison to previous reported findings, as is

shown in tables 3 and 4, c-kit point mutations at

exon-11 for codons Ile563Lys, Val569Leu,

Tyr570Ser, and Pro577Ser, and at exon-8 for

codonsAsn423Asp, Gln448Thr, and Gln448His

are describe here for the first time.

1997) as acute myeloid leukemia (AML) (n=60),

ALL (n=10), CML (n=10), CLL (n=10). The details

of clinical feature and demographic profile are

Table 1. c-kit gene point mutations at exon-11 in Leukemia cases.

Case

Leukemia Type

Nucleotide

Codon

AML

TAT  TCT

TGG  TCA

AGG  ATG

Tyr568Ser

Trp582Ser

Arg588Met

After our findings, where we found point

mutations around the protein, it was important

to address where in the protein these mutation

where located in order to determine the

possible implication(s) of these mutations in

protein function. Therefore, we analyzed the

protein sequence (reference number for c-kit

protein is P10721) using the UnitPro Knowledge

Base server. Mutations for exon-11 are located

between positions 546-976 bp and are in a

cytoplasmic domain. Mutation Tyr568Ser is

located in a metal binding site, specifically, a

magnesium binding site. In addition, the Tyr

residue at this position is normally

autophosphorylated by autocatalysis (Price et

al., 1997; Mol et al., 2003; Sun et al., 2009;

Zadjali et al., 2011). Moreover, mutations

Val569Leu and Tyr570Ser are located in a

domain that interacts with phosphotyrosine-

binding proteins, and residue Tyr570 is

AML

ATA  CTA

AAA  AAC

Ile571Leu

Lys550Asn

AML

TAC  TCC

CCT  TCC

TAT  CCT

GAT  CAT

CAC  CAA

TGG  TCA

AAC  GAC

Tyr570Ser

Pro577Ser

Tyr578Pro

Asp579His

His580Gln

Trp582Ser

Asn587Asp

AML

GTT  CTT

Val569Leu

ATA  AAA

ATA  ACA

ACA  CCA

CAA  CAC

AGA  ACA

Ile563Lys

Ile571Thr

Thr574Pro

Gln575His

Arg586Thr

Figure 2. Amino acid sequences of the exon-11 of c-kit gene. The sequence starts at codon 550

and ends at 591. The wild-type sequence is shown above. Seventeen point mutations in c-kit gene

at exon-11 are highlighted in grey colour. Case number is indicated at the left column.

•

Vol. VI, No. 1 • Enero-Abril 2012 •

SYED RIZWAN HUSSAIN, AMNA SIDDIQUI, JAVIER VARGAS-MEDRANO, HENA NAQVI, JONATHON MOHL, FARZANA MAHDI AND FAHIM AHMAD:

Identification of novel point mutations in c-kit gene from Leukemia cases: a study from Lucknow, Uttar Pradesh, India

Table 2. c-kit gene point mutations at exon-8 in Leukemia cases.

normally autophosphorylated by autocatalysis.

All point mutations in exon-8 are located in an

Case

Leukemia Type

Nucleotide

Codon

Ig-like C2-type 5 domain which is located in

position 413-507 bp and is part of the

extracellular portion of the protein (residues 26-

AML

AAT  GAT

Asn423Asp

AML

CML

CAG  CAC

ACT  GAT

CAG  ACA

TAC  TTC

CTC  CAC

Gln448His

Thr417Asp

Gln448Thr

Tyr418Phe

Leu421His

27). According to this information it seems

most probably, that mutations in exon-11 will

produce the worse alterations to the normal

function of c-kit, because these mutations are

located in places for autophosphorylation and

magnesium binding sites. However, mutations

at exon-8 may be affected ligand binding.

Figure 3. Amino acid sequences of the exon-8 of c-kit gene. The sequence starts at codon

12 and ends at 448. The wild-type sequence is shown above. Six point mutations in c-kit gene at

exon-8 are shown in gray colour. Case number is indicated at the left column.

Figure 4. Mutations found during the sequencing

Discussion

analysis of c-kit at exon-8. Point mutations AG, CA,

AC, GA, GC, AT, and TA (resulting in the amino-

acid substitutions Asn423Asp, Gln448Thr, Gln448His,

Thr417Asp, Tyr418Phe, and Leu421His).

To the best of our knowledge, this study is

the first done from in and around the city of

Lucknow, Uttar Pradesh, northern India. Here

we report mutations in exon-8 and exon-11 of c-

kit gene in Leukemia patients. Previous

molecular studies inAsian populations (Chinese,

Korean, and Japanese) have revealed several

mutations in exon-11 in various types of tumours

(

Hou et al., 2004; Choe et al., 2006; Taniguchi et

al., 1999; Kim et al., 2004). Mutations in exons-

, -13 and -17 of c-kit gene are less frequently

detected than in exon-11. These are considered

rare in gastrointestinal stromal tumors with a

reported frequency of less than 10%, but are

seen more commonly in hematopoietic

malignancies and germ cell neoplasms (Lux et

al., 2000; Lasota et al., 2000; Lasota et al.,

008). In gastrointestinal stromal tumors, 65–

2% of tumors are reported to harbor kit-

activating mutations, the majority of which are

localized to the juxtamembrane region involving

exon-11 (Lasota et al., 1999; Rubin et al., 2001).

• Vol. VI, No. 1 • Enero-Abril 2012 •

SYED RIZWAN HUSSAIN, AMNA SIDDIQUI, JAVIER VARGAS-MEDRANO, HENA NAQVI, JONATHON MOHL, FARZANA MAHDI AND FAHIM AHMAD:

Identification of novel point mutations in c-kit gene from Leukemia cases: a study from Lucknow, Uttar Pradesh, India

Figure 5. Mutations found during the sequencing analysis of c-kit gen at exon-11. Point mutations TA, GC, AC,

CT, TC, CA, GA, AG, and GT (resulting in the amino-acid substitution Ile563Lys, Val569Leu, Tyr570Ser, Pro577Ser,

Tyr568Ser, Ile571Thr, Thr574Pro, Gln575His, Tyr578Pro, Asp579His, His580Gln, Trp582Ser, Arg586Thr, Asn587Asp, Arg588Met,

Lys550Asn, and Ile571Leu).

codons in 576–590 bp have been described by

Antonescu et al. (2003). These include frame

deletions of one, to several codons (typically

involving codons 557–560 bp); point mutations

and internal tandem duplications (typically

involving the 30 end). We found heterogeneous

point mutations, some of which have been

reported and some are new for AML. In eight

leukemia cases we found 17 point mutations.

Lys550Asn and Ile571Leu point mutations have

already been reported by Yasuoka et al. (2003).

Mutations in codon 582 reported by Tae et al.

(2004) (Kim et al., 2004) were for Trp582Tyr and

Trp582His, whereas Hou et al. (2004) reported

Trp582Try and Trp582Gln. However, we have

detected different substitution in the same codon

where tryptophan is replaced by serine

(

Trp582Ser) in two independent Leukemia

cases. Mutations at codons Tyr568Asp,

Ile571Leu, Thr574Tyr, Gln575Ile, Tyr578Phe,

Asp579Gln,Asp579Pro, His580Leu, His580Tyr,

His580Pro, Arg586Trp, Arg586Ile, Arg586Phe,

Arg586Asp, Asn587Glu,Asn587Pro,Asn587His

The majority of exon-11 mutations are clustered

within the classic hotspot region of the codon 5

end involving codons in 550–560 bp, however,

a second hot spot at the codon 3 end involving

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Vol. VI, No. 1 • Enero-Abril 2012 •

SYED RIZWAN HUSSAIN, AMNA SIDDIQUI, JAVIER VARGAS-MEDRANO, HENA NAQVI, JONATHON MOHL, FARZANA MAHDI AND FAHIM AHMAD:

Identification of novel point mutations in c-kit gene from Leukemia cases: a study from Lucknow, Uttar Pradesh, India

and Arg588Phe, Arg588Tyr, Arg588Lys have

already been reported (Yasuoka et al., 2003;

Hou et al., 2004; Kim et al., 2004; Choe et al.,

the literature before (Table 3). All the mutations

detected in exon-11, lie between codons 550-

591. For a comparison purposes, we arrayed

our findings with the ones found in the literature,

see Table 3. From this table, it is easy to

determine which point mutations are novel for

the field.

2006).

As we aimed, we analyzed exon-11 of c-kit

gene in order to detect point mutations in patients

with Leukemia. In our analysis, we were able to

determine new amino acids substitutions

derived from the point mutations that we

detected in exon-11 and we found: Tyr568Ser,

Ile571Thr, Thr574Pro, Gln575His, Tyr578Pro,

Asp579His, His580Gln,Arg586Thr,Asn587Asp,

andArg588Met (Table 1). From these mutations,

we are reporting here, 4 novel mutations:

Ile563Lys, Val569Leu, Tyr570Ser, and

Pro577Ser which have never been reported in

On the other hand, we analyzed exon-8 of

c-kit gene and we were able to find 6 point

mutations (Table 2) in Leukemia cases:

Thr417Val, Tyr418Arg, and Leu421Gly were

previously reported by Taniguchi et al. (1999),

and Kohl et al. (2005). In addition, we also

detected substitutions: Thr417Asp, Tyr418Phe,

and Leu421His which are novel for the field.

Moreover, point mutations in codons 423 and

448 of exon-8 have not been reported for any

Table 3. Comparison between mutations detected in our study

or described already for c-kit gene at the exon-11.

type of Leukemia. However, it was remarkable

to find out that these point mutations produced

the following new substitutions: Asn423Asp,

Gln448Thr, and Gln448His (Table 4).

Mutation with different

substitution

Existing

Reported

Mutations

Novel

Mutations

Not reported

substitution

Our Result)

References

Reported

Substitution

Table 4. Novel mutations detected during our study or mutations

described already for c-kit gene at exon-8.

(

ATA  AAA Ile563Lys

GTT  CTT Val569Leu

TAC  TCC Tyr570Ser

CCT  TCC Pro577Ser

Mutation with different

substitution

Novel

mutations

Mutations

Not reported

substitution

Our result)

References

Reported

substitution

(

TAT  TCT

Tyr568Ser

Tyr568Asp

Taniguchi et al. (1999)

AAT  GAT

CAG  ACA

CAG  CAC

Asn423Asp

Gln448Thr

Gln448His

ATA  ACA

ACA  CCA

CAA  CAC

TAT  CCT

GAT  CAT

Ile571Thr

Ile571Leu

Thr574Tyr

Gln575Ile

Tyr578Phe

Choe et al. (2006)

Hou et al. (2004)

Kim et al. (2004)

Thr574Pro

Gln575His

Tyr578Pro

Asp579His

Kohl et al. (2005)

Gari et al. (1999)

ACT  GAT

TAC  TTC

CTC  CAC

Thr417Asp

Tyr418Phe

Leu421His

Thr417Val

Tyr418Arg

Leu421Gly

Asp579Gln

Asp579Pro

Kim et al. (2004)

Kohl et al. (2005)

Gari et al. (1999)

CAC  CAA

TGG  TCA

AGA  ACA

His580Gln

Trp582Ser

Arg586Thr

His580Leu

His580Tyr

His580Pro

Hou et al. (2004)

Kim et al. (2004)

Kohl et al. (2005)

Gari et al. (1999)

Trp582Tyr

Trp582His

Trp582Gln

Hou et al. (2004)

Kim et al. (2004)

From all point mutations detected it seems

that the residue that was more replaced was

isoleucine following by tyrosine. However,

because point mutations in the c-kit protein were

located in extra- and cytoplasmic-domains, we

thought that maybe these mutations were

affecting hydrophobicity of these domains.

Indeed, mutations Arg588Met (exon-11) and

Tyr418Phe (exon-8) were substitutions where

a hydrolytic residue was replaced by a

Arg586Trp

Arg586Ile

Arg586Phe

Arg586Asp

Hou et al. (2004)

Kim et al. (2004)

AAC  GAC

AGG  ATG

Asn587Asp

Arg588Met

Asn587Glu

Asn587Pro

Asn587His

Hou et al. (2004)

Kim et al. (2004)

Arg588Phe

Arg588Tyr

Arg588Lys

Hou et al. (2004)

Kim et al. (2004)

AAA  AAC

ATA  CTA

Lys550Asn Taniguchi et al. (1999)

Ile571Leu Choe et al. (2006)

• Vol. VI, No. 1 • Enero-Abril 2012 •

SYED RIZWAN HUSSAIN, AMNA SIDDIQUI, JAVIER VARGAS-MEDRANO, HENA NAQVI, JONATHON MOHL, FARZANA MAHDI AND FAHIM AHMAD:

Identification of novel point mutations in c-kit gene from Leukemia cases: a study from Lucknow, Uttar Pradesh, India

hydrophobic residue. However, most of the

mutations involved a substitution in a

hydrophobic residue for a hydrophobic or

conversely. Physiologically, it seems that

mutations in exon-11 are possibly more relevant

regarding c-kit protein function. Interestingly 3

tyrosines, 1 threonine and 2 lysines residues

were substituted it, recall that tyrosines and

threonines are phosphorylation targets and

lysine is an ubiquitination and sumoylation target.

in altering the biochemistry of c-kit protein,

because point mutations at Tyr568Ser,

Val569Leu, and Tyr570Ser are in places for

autophosphorylation or magnesium binding

which are crucial steps in signaling from ligand

binding. However, this is something that needs

to be elucidated by additional experiments. On

the other hand, mutations in exon-8 may also

be involved in ligand binding. From them,

mutation Tyr568Ser is located in a magnesium

binding site. In addition, the Tyr residue at this

position is normally autophosphorylated by

autocatalysis (Price et al., 1997; Mol et al., 2003;

Sun et al., 2009; Zadjali et al., 2011). Moreover,

mutations Val569Leu and Tyr570Ser are located

in a domain that interacts with phosphotyrosine-

binding proteins, and residue Tyr570 is normally

autophosphorylated by autocatalysis.

Normally, these types of residues in

membrane proteins are target for post-

translational modifications and mutations in

them may change the way of how proteins

function (Miranda et al., 2007; Vargas-Medrano

et al., 2011). In contrast, for exon-11, 4 serines,

threonines and 1 lysine were detected as the

end residue product from a point mutation.

Importantly, threonine and serine residues are

phosphorylation targets and lysine residue is a

ubiquitination and sumoylation target (Miranda

et al., 2007; Vargas-Medrano et al., 2011).

The identification of novel mutations in c-kit

in patients withAMLnot only provides new insight

into the pathogenesis of this disease, but also

may serve to provide a means of confirming a

diagnosis and assessing prognosis for

developing new intervention strategies. The

incidence of mutations at exon-8 and -11 is high

and might be involved in pathogenesis of AML.

The mutations described here are

recommended as prognostic markers in the

northern Indian population. However, we do not

discard the idea that these mutations could be

found in other populations around the world.

These changes may affect the normal

phosphorylation and ubiquitination maps for c-

kit protein which can modify the way of how c-

kit functions. However, experimental data for

these hypotheses need to be first generated in

order to determine if mutations described here

have a significant effect on the c-kit protein activity.

Conclusions

In summary, this study is the first to report

the presence of c-kit gene mutations in

Leukemia cases in northern India. Mutations in

exon-8 and -11 may be involved in c-kit over

expression in Leukemia. Four novel mutations

at codons Ile563Lys, Val569Leu, Tyr570Ser, and

Pro577Ser in exon-11 and three novel mutations

at codons Asn423Asp, Gln448Thr, and

Gln448His in exon-8 c-kit gene might be useful

as molecular genetic markers for Leukemia.

Future studies in a larger group may be required

to determine the prognostic implications and

how these mutations are related with

progression and pathogenesis of myeloid

malignancy. Based on our in silico analysis, only

mutations in exon-11 seem to play a crucial role

Acknowledgments

This study was supported by the Intramural

Grant from the Era’s Lucknow Medical College

and Hospital, Lucknow, India.

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SYED RIZWAN HUSSAIN, AMNA SIDDIQUI, JAVIER VARGAS-MEDRANO, HENA NAQVI, JONATHON MOHL, FARZANA MAHDI AND FAHIM AHMAD:

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Supplementary table 1. Demographic profile of patients

Variables

AML (n = 60)

4 (56.7%) /

ALL (n = 10)

CML (n = 10)

CLL (n = 10)

06 (60.0%) /

04 (40.0%)

05 (50.0%) /

05 (50.0%)

06 (60.0%) /

04 (40.0%)

M (%)/ F (%)

26 (43.3%)

Age range

2-65

25-47

33-56

30-56

Mean (±SD)

Clinical features

36.43 (± 6.08)

35.70 (± 6.29)

43.70 (± 6.96)

46.30 (± 7.17)

WBC count cells/

μl/ cumm

5000 - 60000

20000 - 40000

L1/ L2 (n = 10)

25000 - 450000

18000 - 35000

CLL (n = 10)

FAB

M (n = 10),

CML Chronic

M (n = 15),

phase (n = 10)

M (n = 15),

M (n = 04),

M (n = 08) and

M (n = 08)

Supplementary table 2. Demographic profile of controls

Variables

M (%)/ F (%)

Normal Healthy (n = 100)

8 (56.7%) /

42 (43.3%)

Age range

2-65

Mean (±SD)

36.43 (± 6.08)

All morphological features

normal and < 5% blast cells

Clinical features

WBC count cells/ μl/

4300- 10800

cumm

Cite this article as follows:

Hussain, S. R., A. Siddiqui, J. Vargas-Medrano, H. Naqvi, J. Mohl, F. Mahdi and F. Ahmad. 2012.

Identification of novel point mutations in c-kit gene from Leukemia cases: a study from Lucknow, Uttar

Pradesh, India. TECNOCIENCIA Chihuahua 6(1): 22-32.

•

Vol. VI, No. 1 • Enero-Abril 2012 •

SYED RIZWAN HUSSAIN, AMNA SIDDIQUI, JAVIER VARGAS-MEDRANO, HENA NAQVI, JONATHON MOHL, FARZANA MAHDI AND FAHIM AHMAD:

Identification of novel point mutations in c-kit gene from Leukemia cases: a study from Lucknow, Uttar Pradesh, India

Resumes of the author and co-authors

SYED RIZWAN HUSSAIN. He was born in Bokaro Steel City India on 11 August 1980. He did his Bachelor’s degree in CBZ (Chemistry,

Biology and Zoology) and Master’s degree in Biomedical Sciences from India. He is pursuing his Ph.D. in Human Cancer Genetics

from University of Baba Saheb Bhim Rao Ambedkar University India. He has a diverse area of research, such as Human Cancer

Genetics, Molecular Diagnosis, Medical Clinical Biochemistry and Male Infertility. Currently, Syed Rizwan is a Research Assistant at

Department of Biotechnology, Era’s Lucknow Medical College and Hospital, India.

AMNA SIDDIQUI. She was born in Jhansi India in October 9 1979. She did Bachelor’s degree in CBZ (Chemistry, Biology and Zoology)

and Master’s degree in Biotechnology from India. She completed her Ph.D. in Biotechnology from University of Bundelkhand India in

2010. She joined as a Sr. Manager in 2009 at Hindustan Bioenergy Ltd Lucknow India. She has diverse area of research such as

Molecular biology, Microbiology, Plant biology and Human genetics. Currently, Dr. Amna is a Postdoctoral-Research Associate at

Texas Tech University Health Science Center and Paul L. Foster School of Medicine. She got many scientific awards and fellowships

in India.

JAVIER VARGAS MEDRANO. He was born in Juarez City, Chih., México. From 1999-2005, Dr. Vargas attended college at the Autonomous

University of Juarez City. During his Bachelor's degree, Dr. Vargas was involved in the field of Toxicology, studying the effect of

pesticides on Ca -ATPases, and he became author of many publications and conferences. Later, he attended graduate school at

the University of Texas at El Paso, where he was involved in a research project studying the glycine transporter and its regulation,

and its possible role as a pharmacological target in the treatment of schizophrenia. During his doctoral studies, he was awarded

with two research assistantships funded by the National Institute of Health and Mental Health. Currently, Dr. Vargas is member of the

American Chemical Society and he is a Postdoctoral-Research Associate at Texas Tech University Health Science Center and Paul

L. Foster School of Medicine.

HENA NAQVI. She was born on 8 December 1982 at Gorakhpur India. She did her Bachelor’s degree in CBZ (Chemistry, Biology and

Zoology) and Master’s degree in Biotechnology from India. She is pursuing her Ph.D. in Molecular Medical Genetics from C.S.M.

Medical University, (Formerly-K.G. Medical University), Lucknow, India. She has worked as a junior lecturer in 2006-07 at Capital

College, Bangalore, India. She joined as a Research Assistant in 2008 at Era’s Lucknow Medical College and Hospital, Lucknow,

India. Her area of research is Molecular biology, Human genetics and Male infertility. Currently, Ms. Hena Naqvi is working as Women

Scientist A approved by Department of Science and Technology, Govt. of India at Era’s Lucknow Medical College and Hospital,

Lucknow, India.

JONATHON MOHL. He was born in St Louis, Missouri, United States (USA). He received a Bachelors of Science in Microbiology and

Biochemistry at Colorado State University in Fort Collins, Colorado in 2002. While attaining this degree, he worked in a Mycrobacterial

laboratory working on M. avium and M. avium ssp. paratuberculosis. He also worked as a teaching assistant in a molecular biology

laboratory. After moving to El Paso, Texas, USA, he received a Professional Master of Science degree in Bioinformatics in 2009 at

the University of Texas at El Paso. In the process of attaining his Master’s degree, he worked at a bioinformatics programmer on the

RNAVLab project and as a teaching assistant in the introductory bioinformatics courses. Currently, he is working in HIV research

lab at the Texas Tech University Health Sciences Center in El Paso as a research aid.

FARZANA MAHDI. He was born on 28 June 1966 at Lucknow, India. She did her Bachelor’s degree in CBZ (Chemistry, Biology and

Zoology) and a Master’s degree in Organic Chemistry from India. She completed her Ph.D. in Life Sciences from Kanpur University

1992. She joined as an Assistant Professor in 2000 at Era’s Lucknow Medical College and Hospital, Lucknow, India. She has diverse

area of research such as Free Radical Biology, Toxicology, Clinical Biochemistry, Molecular aspects of metal toxicity, Male infertility,

Human Cancer Genetics. Currently, Dr. Farzana is Life Member of Indian Society for Reproductive Biology and Comparative

Endocrinology and Association of Clinical Biochemists of India. She is also a Founder Member of Society for Free Radical Research

India. Currently, she is working as a Professor in Department of Biochemistry, Era’s Lucknow Medical College and Hospital,

Lucknow, India.

FAHIM AHMAD. He was born in Daltonganj India in 12 October 1976. He did a Bachelor’s and Master’s degrees in Biotechnology from

India. He completed his Ph.D. in Human Genetics from the University of Bundelkhand India in 2007. He joined as an Assistant

Professor in 2009 at Isabella Thoburn College Lucknow, India. Dr. Ahmad has diverse area of research such as Human genetics,

Molecular Virology and Cancer Genetics. Currently, Dr. Ahmad is member of Biotechnological Board in Jharkhand State and is a

Postdoctoral-Research Associate at Texas Tech University Health Science Center and Paul L. Foster School of Medicine. He got

many scientific awards and fellowships in India. His current project at Texas Tech University Health Sciences Center focuses on the

mechanisms of genetic resistance to HIV-1 infection funded by the National Institute of Health.

• Vol. VI, No. 1 • Enero-Abril 2012 •