In order to succeed, your desire for success should be greater than your fear of failure.”

– Bill Cosby

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The present study aims to understand the role of DNA repair, apoptosis as well as MDM2

gene in the origin and progression of acute leukemia. Primary acute leukemia cases

comprising of both AML and ALL as well as age and sex matched health controls were

recruited. These cases were collected from Nizams Institute of Medical Sciences (NIMS),

Hyderabad and Mehdi Nawaj Jung (MNJ) hospital and regional cancer center, Hyderabad

the two major cancer hospitals in the state where patients belonging to different

socioeconomic status were being treated. 307 Age and sex matched healthy individuals

without familial histories of cancers were recruited as controls.

A total of 446 acute leukemia cases (225 AML and 221 ALL) were collected by visiting

NIMS and MNJ hospitals on alternative days for a period of 2yrs (from 2009-2011). About

35% (80 AML and 80 ALL cases) of the samples included in the study had been collected

by one of our previous scholars who worked on acute leukemia. This study was approved

by the Institutional ethics committees of MNJ and Osmania University. The patients with

confirmed diagnosis of acute leukemia and those who had provided written informed

consent for testing on DNA were recruited for the present study. Only primary denovo

acute leukemia cases were included in the study, secondary and therapy related cases were

excluded.

A personal interview was performed by investigators to collect epidemiology data using a

structured questionnaire which consists of demographic characteristics (age, gender,

occupation, habits and habitat etc.). Clinical data (baseline characteristics at diagnosis like

Hb%, WBC count, platelet count, bone marrow blast%, complete remission rate (CR) and

disease free survival (DFS) time in months) was collected from tumor registries with the

help of a medical oncologist.

5 ml of blood sample was drawn from each patient into an EDTA vaccutainer which was

used for isolation of DNA and RNA. Extracted DNA and RNA samples were stored at -

200c and were used for genotyping and expression analysis of MDM2.

Epidemiological variables:

In the present study, majority of AML patients were found to be in the age group of >30yrs

(44.9%) indicating AML is the disease of elderly while ALL occurred more frequently

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among individuals aged <20yrs (72.9%) indicating that ALL is the disease of younger

people. There was no deviation in the sex ratio of AML patients (1.10:1.0). However, ALL

had shown a significant male preponderance with a sex ratio of 3.33:1.0. In general, 92.0%

of AML and 88.7% of ALL belonged to non-vegetarian group. High frequency of AML

patients were from rural area (65.3%). The frequency of smokers was found to be elevated

in AML (16.9%) group compared to ALL (8.1%) and controls (12.6%). There was an

increase in the frequency of AML patients belonging to laborer community (43.1%),

whereas 69.2% of ALL patients belonged to students community. 8.0% of AML cases and

13.6% of ALL cases had shown the parental consanguinity. About 6.7% of AML cases and

7.7% of ALL patients had reported familial incidence of cancers.

Clinical variables

In the present study, mean age at diagnosis of AML and ALL patients was found to be

30.88±14.23yrs and 16.89±12.14yrs respectively. Anemia (<9.0g/dl), high blast percentage

(>50%), severe leucocytosis (>50,000/cumm), mild to moderate thrombocytopenia

(<100,000/ cumm) and elevated LDH level (>500 IU/L) were observed in both AML and

ALL cases. Mean Disease free survival rate (DFS) was found to be significantly reduced in

AML (10.06±15.03 in months) compared to ALL (22.52±25.42 in months) cases

indicating that AML is more progressive disease.

58.8% of AML patients achieved complete remission which was significantly lower than

that of ALL (81.9%). M2 subtype was more frequent (46.3%) in AML followed by M3

(17.4%) and M4 (16.4%). In ALL, the L2 subtype was the most frequent (86.0%).

In AML, age at diagnosis did not show any significant correlation with clinical variables

whereas in ALL, age at diagnosis had shown a positive correlation with WBC count

(r=0.134, p<0.05) and Hb levels (r=0.170, p<0.05). WBC count was positively correlated

with blast%, LDH level and negatively correlated with platelet count in both AML (blast%

r=0.292, p<0.01; LDH levels r=0.194, p<0.05; Platelet counts r=-0.197, p<0.01) and ALL

(blast% r=0.373, p<0.01; LDH levels r=0.508, p<0.01; Platelet counts r=-0.279, p<0.01).

Blast% had shown a positive correlation with LDH level in AML (LDH r=0.228, p<0.01)

and ALL (LDH r=0.300, p<0.01). Further, in case of ALL blast% had a negative

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correlation with platelet count (r=-0.178, p<0.01) and Hb levels (r=-0.231, p<0.01). A

positive correlation was observed between platelet count and Hb level in both AML (Hb

levels r=0.174, p<0.01) and ALL (Hb levels r=0.269, p<0.01) cases. However, platelet

count was negatively correlated with LDH (r=-0.196, p<0.05) and DFS (r=-0.152, p<0.05)

in ALL patients. With respect to complete remission (CR) rate, a highly positive

correlation was observed between CR and DFS among AML (r=0.470, p<0.01) and ALL

(r=0.265, p<0.01) cases.

The present study had shown a significant regression of CR on WBC count (B=2.528E-

006, p=0.000), LDH (B=0.000, p=0.004) and DFS (M) (B=0.011, p=0.000) in AML. In

case of ALL, CR (B=0.002, p=0.026) had shown a significant regression on DFS

(B=26.024, p=0.026) and vice versa.

DNA REPAIR PATHWAY

Base excision repair pathway (BER) gene

XRCC1 gene polymorphisms

XRCC1 gene is located on chromosome 19q13.2, comprises of 17 exons and encodes 63

amino acid protein. XRCC1 has no known enzymatic activity but participates as a scaffold

protein to interact and coordinate the activities of other proteins involved in both single-

strand break repair and base excision repair activities. In the present study, we have

evaluated the role of two SNPs in the development of acute leukemia. They are

a) XRCC1 Arg194Trp polymorphism

XRCC1 polymorphism in exon 6 at 22 nucleotide position (C>T) leads to change of amino

acid from arginine (Arg) to tryptophan (Trp). Presence of Trp allele was found to be

associated with decreased ability to repair DNA damage.

In the present study, XRCC1 Arg194Trp polymorphism was not significantly associated

with acute leukemia (AML and ALL) development. Gender, area of living, age at onset

and smoking habits of the patients were not associated with XRCC1 Arg194Trp

polymorphism in AML and ALL. However, XRCC1 194Arg/Trp genotype frequency was

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elevated in AML females (22.8%) compared to males (14.7%) and no such trend was

observed in ALL. When diet of the probands was considered, the frequencies of XRCC1

194Arg/Trp heterozygote and Trp allele were increased in non-vegetarian (19.4%, 13.0%)

group of AML compared to vegetarians (6.2%, 3.0%) although differences were

insignificant.

The genotype distribution among clinical variables like WBC count, Hb level, LDH level

and complete remission rates in acute leukemia patients (AML and ALL) did not show

association with XRCC1 Arg194Trp polymorphism. However, XRCC1 194Arg/Trp

heterozygotes frequency (16.8%) was significantly decreased in AML patients with severe

thrombocytopenia.

A significant reduction in mean WBC count (20550.00±25752.42/cumm), Blast%

(42.50±31.12%) and increase in mean DFS (16.60±27.58 in months) were observed in

AML cases with XRCC1 194Trp/Trp genotype. Similarly, reduction in mean WBC count

(36625.00±32637.85/cumm), Blast% (44.00±35.37%) and LDH (660.00±160.62IU/L)

were observed in ALL with respect to XRCC1 194Trp/Trp genotype.

Survival analysis had shown that DFS rates were elevated in both AML (18 months) and

ALL (28 months) with XRCC1 194Trp/Trp genotype, although this association was found

to be insignificant. These results suggested that XRCC1 Arg194Trp polymorphism was not

associated with the development of acute leukemia. However, the reduction in WBC, blast

% and simultaneous increase in DFS might suggest that Trp/Trp genotype may be

associated with better prognosis in AML. In conclusion, defective DNA repair capacity

associated with Trp/Trp genotype might offer chemosensitivity in transformed cells

resulting in better survival rates.

b) XRCC1 Arg399Gln polymorphism

Polymorphism at exon10 results in substitution of arginine (Arg) to glutamine (Gln) at 399

codon position results in diminishing repair capacity due to reduced affinity with

interacting proteins. In the present study, XRCC1 Arg399Gln polymorphism was not

associated with the susceptibility to acute leukemia. With respect to epidemiological

variables gender, diet, smoking habit of the patients were not associated with XRCC1

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Arg399Gln polymorphism. However, there was two-fold increased risk of AML for

carriers of Gln/Gln genotype (OR=2.50; 95%CI=1.04-5.96; p=0.05) living in urban area.

Similar but insignificant trend was observed in ALL with respect to XRCC1 399Gln allele.

Stratification of genotype distribution with respect to clinical variables had indicated two

fold increased risk of AML for Arg/Gln genotype in the age group of 20-30yrs (OR=2.01;

95%CI=1.01-3.99; p=0.05). While genotype association with age at onset was not

observed in ALL. Among WBC classes, significant association between Gln allele and

leukopenia was found in ALL patients (37.50%). No such association was observed in

AML.

AML patients with homozygous Gln/Gln genotype had shown risk for severe

thrombocytopenia (OR=3.55; 95%CI=0.94-13.44; p=0.06), while no such association was

observed in ALL. XRCC1 Arg399Gln genotype distribution was not deviated with respect

to Hb level among AML and ALL patients. There was an increasing trend in the frequency

of 399Arg/Gln genotype (43.2%) and Gln allele (38.42%) among AML patients with

elevated LDH level (>200IU/L). No significant association was observed with respect to

complete remission rates and XRCC1 Arg399Gln polymorphism among AML and ALL

patients.

Increase in mean WBC count (71703.57±88687.0/cumm) and reduction in mean platelet

count (60553.57±80434.16/cumm) were observed in AML patients with Gln/Gln genotype

compared to those with Arg/Arg genotype. In case of ALL, WBC count

(29620.00±39096.62/cumm), blast% (53.70±35.28%) and LDH levels

(416.47±141.769IU/L) were reduced and mean DFS (31.15±31.52 in months) was found to

be elevated among XRCC1 399Gln/Gln genotype carriers compared to those with Arg/Arg

genotype.

There was no significant association observed between XRCC1 Arg399Gln polymorphism

and DFS rates in AML & ALL patients who have achieved complete remission after initial

induction therapy. However, Kaplan Meir analysis had shown that DFS rates were elevated

in AML (15 months) and ALL (20 months) patients with XRCC1 399Gln/Gln genotype

compared to other genotypes. The data on clinical variables indicated that presence of Gln

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allele with reduced repair capacity might failed to repair damage caused by chemotherapy

and undergo apoptosis leading to elimination of tumor cells and increased DFS.

XRCC1 (Arg194Trp and Arg399Gln) HAPLOTYPE ANALYSIS

In the present study, no significant association was observed between combined effect of

two XRCC1 polymorphisms (Arg194Trp and Arg399Gln) and acute leukemia

development. However, Trp-Gln haplotype frequency was slightly elevated in ALL cases

(0.028%) compared to controls (0.012%). This association was attributed to the presence

of two mutant alleles at 194Trp and 399Gln positions in a haplotype group. Both these

mutant alleles had deficient repair capacities leading to increased risk of disease

susceptibility due to accumulation of mutations as a result of reduced DNA repair in cells

exposed to carcinogens.

Further, a slight reduction in Trp-Arg haplotype frequency was observed in ALL cases

(0.100%) compared to controls (0.117%) indicating that presence of at least one wild type

allele at 399Arg position might offer protection against ALL susceptibility due to its

normal DNA repair capacity. No such trend was observed in AML cases.

Nucleotide Excision Repair Pathway (NER) gene

XPD Lys751Gln polymorphism:

The human XPD protein is one of the ten subunits of transcription factor IIH (TFIIH), a

protein factor required for transcription initiation by RNA polymerase II. XPD has helicase

activity which is essential during NER pathway. XPD is located on chromosome 19q13.3

and contains 23 exons. XPD Lys751Gln polymorphism arises as a result of A to C

transversion at 35931 nucleotide position. The variant allele (Gln) at codon 751 completely

changes the electronic configuration of the amino acid. This is a major change located in

the important domain of interaction between XPD protein and its helicase activator, p44

protein inside the TFIIH complex. Presence of Gln allele at 751 was shown to be

associated with reduced DNA repair capacity. In the present study, no significant

association was found between acute leukemia and XPD Lys751Gln polymorphism.

However, a borderline significant association was observed between ALL and XPD

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Lys751Gln (35931A>C) polymorphism, where, Lys/Lys+Lys/Gln genotype frequency was

increased in disease group (92.2%) under recessive model of inheritance compared to

controls (88.1%). The frequencies of XPD 751 Lys/Gln genotype and Gln allele were

significantly elevated in AML males (46.1%, 35.22%) compared to females (33.0%,

28.16%). No significant association was found between XPD Lys751Gln (35931A>C)

polymorphism and diet or area of living of the probands. XPD 35931 Lys/Gln (50.0%)

genotype and Gln (40.79%) allele frequencies were significantly elevated in AML patients

with smoking habit (p=0.056) under dominant model. No such trend was observed in case

of ALL smokers.

The clinical variables such as age at onset, Hb, LDH levels did not show any significant

association with XPD Lys751Gln (35931A>C) polymorphism in acute leukemia cases

(AML and ALL). However, Lys/Gln genotype frequency was significantly elevated in

AML (57.1%) and ALL (87.5%) patients who had leukopenia and conferred 2 fold

increased risk of thrombocytopenia in ALL patients (OR=2.23; 95% CI=1.07-4.62;

P=0.031). No such association was found in AML patients. 66.7% of ALL patients with

Lys/Gln genotype had failed to achieve complete remission.

There was no significant deviation in mean values of different clinical variables such as

age at onset, WBC count, Platelet count, Blast%, Hb level and LDH level among different

genotype groups, whereas mean DFS was reduced in both AML (9.38±12.930 in months)

and ALL (21.99±22.909 in months) cases with Lys/Gln genotype compared to Gln/Gln

genotype (AML: 14.85± 19.66, ALL: 28.06 ±31.57 in months).

Survival analysis in CR+ve AML patients with respect to XPD Lys751Gln (35931A>C)

polymorphism had revealed that both Lys/Lys and Lys/Gln genotype had lower DFS of 10

months and 11months respectively than Gln/Gln (18 months) genotype. In ALL cases, no

such trend was observed. Our results suggest that there was no significant association of

XPD Lys751Gln (35931A>C) polymorphism with the risk of AML and ALL. However,

Lys/Gln genotype seems to influence CR rates in ALL and DFS rate in both AML and

ALL.

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Homologous recombination repair pathway (HRR) genes:

RAD51 -135G>C polymorphism

In the present study, the association of RAD51 -135G>C polymorphism with acute

leukemia was analyzed. The RAD51 -135G>C is a promoter SNP located at 5’

untranslated region (5’UTR) which rises due to transversion of G to C nucleotide. Presence

of -135C allele had shown to enhance promoter activity and over expression of RAD51

gene. RAD51 -135CC genotype frequency was significantly elevated in AML as compared

to controls (Adjusted OR=2.55; 95% CI: 1.09-5.99). However, a similar but an

insignificant trend was observed in ALL.

RAD51 -135CC genotype had conferred 6 fold increased risk to ALL in females

(OR=5.98, 95% CI= 1.66-21.58, p=0.011), no such trend was found in AML. A significant

association of RAD51-135CC genotype was observed with AML patients living in rural

area (p=0.05) under recessive model of inheritance. No association was found between

RAD51 -135G>C polymorphism and area of living in ALL group. Smoking habit of the

patients did not show any association with RAD51 -135G>C polymorphism in both AML

and ALL. There was no significant association found between age at onset, WBC count,

Hb levels, LDH levels and CR response and RAD51 -135G>C polymorphism in both

AML and ALL. However, AML patients with GC genotype had three fold increased risk

of severe thrombocytopenia (OR=3.76, 95%CI=1.05-13.43, p=0.04). On contrary, ALL

patients with GC genotype showed reduced risk for moderate thrombocytopenia (50000-

1.0Lakh/cumm).

A significantly reduced mean DFS was observed in both AML (5.33±5.788 in months) and

ALL (17.27± 20.37 in months) cases with RAD51 -135CC genotype. Further, survival

analysis had shown that CR+ve patients with CC genotype had reduced median survival

rates in AML (7 months) and ALL (12 months) compared to other genotypes. Our results

suggested that RAD51 -135G>C polymorphism might confer the risk to develop AML due

to increased error prone repair in leukemiogenesis. Rural area of living might enhance the

risk to AML as confounding factor in the presence of RAD51 -135CC genotype. In ALL,

females with RAD51 -135CC genotype had 6 fold increased risk to develop disease

although female sex was not associated with ALL. RAD51 -135CC genotype might also

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influence rate of DFS in both AML and ALL due to acquired resistance to apoptosis after

induction therapy.

XRCC3 Thr241Met polymorphism

X-ray repair cross-complementing protein group 3 (XRCC3) is a RAD51 paralog, which

participates in the repair of DNA double-strand breaks (DSBs) through homologous

recombination repair pathway (HRR). XRCC3 has a crucial role in stabilizing Rad51

protein and loads Rad51 on the DNA strands during HRR, this function is mediated along

with XRCC2. XRCC3 Thr241 Met, a codon polymorphism in exon 7 is frequently studied

in association with various cancers including acute myeloid leukemia. This polymorphism

arises due to substitution of cytosine (C) by thymine (T) at 18067 nucleotide position.

Presence of Met genotype at codon 241 had shown to be associated with reduced DNA

repair capacity.

In the present study, XRCC3 241Met/Met genotype was not associated with risk to

develop acute leukemia (AML and ALL). XRCC3 241Met/Met genotype frequency was

elevated in female AML patients (5.0%) compared to male patients (1.7%). While in ALL

cases, Thr/Met heterozygote frequency was found to be slightly increased in females

(37.2%) than males (31.1%). Diet, area of living and smoking habits of the patients were

not associated with XRCC3 Thr241Met polymorphism in both AML and ALL. With

respect to age at onset, XRCC3 241 Thr/Met heterozygote frequency was found to increase

2 fold risk for early onset of AML (<20yrs) (OR=2.067, 95%CI=0.97-4.38, p=0.059)

whereas in ALL cases, no significant trend was observed.

Distribution of genotype data with respect to clinical variables had shown that Thr/Met

genotype and Met allele frequencies were associated with leucopenia (<4000/cumm), mild

thrombocytopenia (50000-1.0Lakhs/cumm) and anemia (<9.0g/dl) in AML patients. While

in case of ALL, Thr/Met genotype and Met allele frequencies were elevated in patients

with increased LDH levels. There was no significant association found between complete

remission (CR) and XRCC3 Thr241Met polymorphism in AML. The frequencies of

XRCC3 241Thr/Met genotype (33.7%) and Met (17.98%) allele were increased in ALL

CR positive cases. Mean age at onset (24.67±18.448yrs), WBC count

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(71066.67±79185.942/cumm) and blast% (80.00±5.00%) were significantly increased in

ALL cases with XRCC3 241Met/Met genotype. There was no variation with respect to

mean clinical variables in AML patients. However, mean DFS was found to be reduced in

both AML (4.20±4.087 in months) and ALL (18.67±16.921 in months) in carriers of

XRCC3 241Met/Met variant genotype.

Kaplan-Meier analysis revealed that CR+ve AML patients with the XRCC3 241Met/Met

genotype had shorter DFS than those with other genotypes (Thr/Thr and Thr/Met) (median

survival 5 months vs 9 and 18 months, log-rank p= 0.152). While no specific trend in DFS

was observed in ALL with respect to XRCC3 241 Met/Met genotype. These results

indicate that XRCC3 Thr241Met polymorphism was not associated with the development

of acute leukemia but XRCC3 241Met/Met genotype might influence the survival rates of

leukemic patients.

INTRINSIC APOPTOTIC PATHWAY

BCL2 -938C>A polymorphism

BCL2 is an anti-apoptotic gene located on chromosome 18q21.3, consists of 3 exons and 2

promoters with different functional properties. P2 is a second promoter located 1400-bp

upstream of the translation initiation site and decreases the activity of the P1 promoter,

thus functions as a negative regulatory element. A P2 promoter SNP (-938C>A) is the

most commonly studied SNP in various cancers. This polymorphism has got a functional

significance where -938C allele was found to be associated with increased P2 activity and

binding of nuclear proteins compared with the A allele. As P2 is a negative regulator of P1,

reduced expression of the Bcl-2 protein was associated with the C allele in comparison

with the A allele. In the present study, BCL2 -938C>A polymorphism was significantly

associated with both AML (p=0.015) and ALL (p<0.0001). The level of significance was

found to be greater in ALL compared to AML under different models of inheritance. The

stratification of genotype distribution with respect to epidemiological variables revealed

that AA genotype conferred 3 fold increased risk of AML in females under codominant

model (Adjusted OR=3.00; 95% CI: 1.29-7.01; p=0.024). However, similar but

insignificant trend was found in distribution of BCL2 -938C>A polymorphism with respect

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to gender of the probands in ALL where AA genotype frequency was increased in females

(9.8%) compared to male (6.0%) cases. Further, no significant association of this

polymorphism was observed with age at onset, diet, area of living, smoking habits of the

probands in both AML and ALL.

The genotypic distribution with respect to clinical variables had shown that BCL2 -938AA

genotype frequency was significantly elevated in AML patients who had leucopenia

(<4000/cumm) (26.7%) and in ALL patients (12.3%) with mild leukocytosis. With respect

to platelet count, CA genotype was significantly elevated in AML (p=0.04) patients with

mild thrombocytopenia and ALL patients with severe thrombocytosis (35.5%). Further,

BCL2 -938 A allele frequency was increased in both AML (22.16%) and ALL (22.73%)

patients with LDH level >200IU/L. No significant association was observed between

BCL2 -938C>A polymorphism with respect to complete remission rate in both AML and

ALL. There was no significant deviation in mean age at onset, platelet count, blast%, Hb

level with respect to BCL2 -938C>A polymorphism in both AML and ALL, while the

mean WBC count (72189.33±102118.50/cumm) was elevated in AML cases with BCL2 -

938AA genotype and mean LDH level (1144.00±1060.45IU/L) was increased in ALL with

AA genotype.

Survival analysis had revealed that patients with CA genotype had reduced median DFS of

9 months compared to other genotypes in AML though the deviation is not significant. No

significant association was observed with respect to survival analysis in case of ALL

patients. In the present study, BCL2 -938C>A polymorphism was significantly associated

with the development of acute leukemia. Females with CA genotype had high risk of acute

leukemia (AML and ALL). Further, BCL2 -938CA genotype may not influence either

clinical outcome or survival rate of AML and ALL patients.

BAX -248G>A polymorphism:

Bax is a pro-apoptotic protein, known as bcl2 like protein 4. It was the first identified pro-apoptotic

protein member of Bcl2 protein family. Apoptotic regulator BAX promotes apoptosis by binding to

and antagonizing the Bcl2 protein. BAX gene is located on chromosome 19q13.3-q13.4. A

promoter polymorphism -248G>A at 5’untranslated region (5'-UTR) of BAX was found to

be associated with reduced expression. In the present study, BAX -248GG genotype and G

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allele frequencies were significantly elevated in AML (p=0.039) and ALL (p=0.011) cases

as compared to control group, whereas BAX -248A allele was found to be associated with

reduced risk of both AML (p=0.04) and ALL (p=0.008). Since GG genotype was found to

be associated with reduced BAX gene expression, significant elevation of GG genotype

frequency in acute leukemia compared with controls indicates reduction in apoptosis which

leads to survival advantage of malignant cells and leukemiogenesis.

BAX -248GG genotype and G allele frequencies were increased in AML females (92.2%,

96.12%) and those on non vegetarian diet (89.1%, 94.53%) but statistical significance was

not observed. In case of ALL, no significant trend was observed between BAX -248G>C

polymorphism and gender or diet of the probands. Further, the frequency of BAX -248GG

genotype was elevated in both AML (90.9%) and ALL (92.9%) patients belonging to rural

area. Lack of association between smoking habit of the acute leukemia patients (AML &

ALL) and BAX -248G>A polymorphism was observed. BAX -284G>A polymorphism did

not show association with gender, age at onset, diet, habit and area of living in both AML

& ALL group.

With respect to clinical variables, 3 fold risk of leukocytosis was observed among AML

(OR=3.40; 95% CI= 0.91-12.71; p=0.07) and ALL (OR=3.01; 95% CI=0.90-10.09;

p=0.07) patients who carry BAX -248GA genotype. Mild thrombocytopenia was

associated with heterozygotes (GA) in both AML and ALL groups. Hb and LDH levels did

not show any association with BAX -248G>C polymorphism in both AML and ALL.

A 6 six fold increased risk of complete remission failure among AML patients with respect

to GG genotype was observed indicating that GG genotype was associated with poor

response to first induction therapy. No such association was observed in case of ALL

patients.

The mean WBC count and DFS were increased, and platelet count was decreased among

AML patients with -248GA genotype. In case of ALL, mean WBC counts were decreased

and DFS was considerably elevated. Survival analysis showed that CR+ve patients with

GA genotype had elevated median DFS months (AML=18months; ALL=33 months)

though the log rank test is not significant. Thus, GG genotype of BAX-248 SNP was

associated with acute leukemia development as well as with CR-ve AML cases and with

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reduced DFS among AML and ALL. These results indicate that GG genotype is a marker

for poor prognosis in acute leukemia.

Caspase 9 Single Nucleotide Polymorphisms:

CASP9, an initiator caspase activated by the release of cytochrome c from mitochondria

activates downstream CASP3 and CASP7 during intrinsic apoptotic pathway. CASP9

gene, located on chromosome l at 1p36.21, is a member of the caspase (cysteine aspartate

protease) family of proteins. The human CASP 9 gene spans over 35kb region and contains

9 exons and 8 introns. Single nucleotide polymorphisms in the CASP9 gene were found to

be associated with various cancers due to their reduced expression. In the present study, we

have evaluated the role of three promoter SNPs and one codon SNP in the development of

acute leukemia.

i) Caspase 9-1263 A>G polymorphism

Promoter polymorphism at -1263 position results in A to G transition had shown to create

additional transcription factor (TF) binding site (simian virus-40 protein 1(SP-1)-binding

Site). The presence of a tissue-specific TF that may bind at the specific site and act as a

transcriptional repressor or, presumably, tissue-specific modifications of the TF could alter

its affinity to the binding site or to other interacting TFs, which could result in a change of

balance of the basic transcription complex towards reduced transcription.

In the present study CASP9 -1263A>G polymorphism was significantly associated with

acute leukemia development. The frequency of -1263AG/GG genotype was found to be

significantly elevated in AML patients under dominant model (Adjusted OR = 1.54; 95%

CI: 1.20-2.33; p= 0.04). While in case of ALL, -1263AG genotype was significantly

associated with ALL risk under codominant (Adjusted OR =1.72; 95% CI: 1.04-2.86;

p=0.008). No significant association was observed between age at onset, sex, diet and area

of living with CASP9 -1263A>G polymorphism in AML and ALL. Whereas frequencies

of -1263GG genotype (p=0.049) and G allele (p=0.04) were significantly elevated in ALL

smokers under recessive model. No such significant trend was observed in AML smokers.

The genotype distribution with respect to clinical variables like Platelet count, Hb level

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and LDH level could not reveal any significant association with CASP9 -1263A>G

polymorphism in AML patients. However, CASP9 -1263 GG genotype (p=0.02) and G

allele (p=0.02) frequencies were reduced in ALL patients with severe leukocytosis. No

such association was found in AML patients with respect to WBC count.

50.0% of AML patients and 68.2% of ALL patients who failed to achieve complete

remission induction therapy had -1263AG genotype.

Significant deviation was not observed with respect to mean clinical variables such as age

at onset, WBC count, Platelet count, Blast%, Hb level and LDH level among different

genotype groups in AML and ALL. There was significant reduction in mean DFS in both

AML (6.56±8.82 in months) and ALL (16.02±19.03 in months) cases with GG genotype.

Further, these results were confirmed by survival analysis in CR+ve patients where AML

or ALL patients with GG genotype had a significantly lower DFS of 7 months (log rank

p=0.05) and 14 months (log rank p=0.02) respectively compared to other genotypes.

The present study concludes that CASP9 -1263G allele might confer the risk to develop

AML due to reduced tissue specific expression in hematopoietic tissue that leads to failure

of apoptosis in malignant cells resulting in leukemiogenesis. In ALL, CASP9 -1263AG

genotype was associated with disease development. Further, CASP9 -1263GG genotype

was found to influence the remission rate and DFS in both AML and ALL due to acquired

apoptotic resistance that might provide survival advantage to leukemic cells to progression

of the disease. Thus CASP9 -1263A>G polymorphism might be a marker for poor

prognosis of acute leukemia.

ii) Caspase 9 -712C>T polymorphism

The CASP9 -712T variant allele had been shown to have reduced promoter activity as C to

T transition eliminates Krox-20, NF-1 and ETF-binding sites leading to reduced apoptosis.

In the present study, CASP9 -712 CT &TT genotypes and T allele frequencies were

significantly elevated in AML (p=<0.001) cases under dominant (p=<0.001) and over-

dominant (p=<0.0001) model of inheritance. Significant increase of CT genotype and T

allele frequencies were also observed in ALL (p=0.04) cases. With respect to gender of the

probands, frequencies of CASP9 -712CT/TT genotype (p=0.047) and T (p=0.05) allele

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were significantly elevated in female AML patients. Similar but an insignificant trend was

observed in ALL.

There was an increasing trend of -712TT genotype (12.6%) and T allele (36.0%)

frequencies among non-vegetarian group of AML and ALL patients. Further, ALL patients

from rural area had significantly elevated frequencies of TT genotype (10.5%) and T allele

(35.0%) as compared to those from urban group (2.2%; 25.0%). AML patients who were

smokers had reduced TT genotype (p=0.038) and T allele (p=0.03) frequencies compared

to non-smokers. CASP9 -712TT genotype (13.0%; 15.1%) and T allele (42.39%; 37.67%)

frequencies were significantly elevated among early age at onset group (<30yrs) in AML

respectively.

Further, TT genotype was associated with severe leukocytosis and thrombocytopenia in

AML patients, while there was no significant association observed between CASP9 -

712C>T polymorphism and ALL with respect to clinical variables. There was no

association between CASP9 -712C>T polymorphism with Hb levels, LDH levels and the

rate of complete remission failure in both AML and ALL cases.

Significant increase in mean WBC count, LDH level and reduction in platelet count were

observed in AML patients with TT genotype. However, mean DFS was significantly

reduced in AML patients with CT (6.99±8.214 in months) and TT (8.19±9.13 in months)

genotype. No correlation was observed with respect to clinical variables among ALL

patients. In ALL group, patients with TT (10.36±10.77 in months) genotype had

significantly reduced DFS. Further, these results were replicated in survival analysis of

CR+ve cases of AML and ALL with respect to CASP9 -712C>T polymorphism, where

median DFS in AML was significantly reduced with respect to CT genotype (9 months)

compared to other genotypes. In case of ALL, reduced DFS was found with respect to TT

genotype (10 months). This association was found to be significant in both AML (log rank

p=0.022) and ALL (log rank p= 0.003). Our results suggested that the CASP9 -712T allele

might be considered as a risk allele for development of acute leukemia due to reduced

apoptosis that might lead to escape of malignant cells from cell surveillance mechanism

and develop leukemia. Further, epidemiological risk factors like non vegetarian diet in

AML and rural area of living in ALL might enhance the risk of disease development as

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confounding factors in the presence of CAS9 -712T allele. CASP9 -712TT genotype might

influence the treatment out come in AML and DFS rate in acute leukemia (AML and

ALL). Thus, CASP9 -712TT genotype also can be considered as prognostic marker.

iii) Caspase9 -293_ -275delCGTGAGGTCAGTGCGGGGA (-293del) polymorphism

CASP9 -293del polymorphism was hypothesized to alter the expression of CASP9 gene

and might contribute to cancer susceptibility. In the present study, CASP9 -293del

polymorphism was not associated with AML. However, CASP9 -293(+/+) genotype was

significantly associated with ALL (χ2p=0.04) susceptibility. Further, CASP9 -293(-/-)

genotype frequency was significantly reduced in ALL under recessive model (Adjusted

χ2p=0.03). No significant association was found between CASP9 -293del polymorphism

and all epidemiological variables in AML & ALL. However, frequencies of del-/-

genotype (Adjusted χ2 p=0.013) and del- allele (χ2 p=0.01) were significantly elevated in

ALL smokers, whereas this trend was not observed in AML patients.

With respect to age at onset, del+/+ genotype frequency (37.2%) was found to be

significantly elevated in early age at onset group (<20yrs) of AML cases. While in case of

ALL, del+/- heterozygote was found to have two fold increased risk of ALL in late age at

onset group (Adjusted OR =2.45, 95% CI: 1.00-5.98, p=0.05). The present study could not

reveal any significant association between CASP9 -293del polymorphism and clinical

variables such has WBC count, platelet count, Hb level and LDH levels. The presence of

del- allele in ALL patients was significantly associated with complete remission rates

failure (Adjusted χ2 p=0.04), while no such association was found in AML patients.

No significant deviation was observed with respect to mean clinical variables and CASP9

-293del polymorphism among AML and ALL. There was a significant reduction in mean

DFS in both AML (5.68±7.47 in months) and ALL (17.67±29.80 in months) cases with

del-/- genotype. Survival analysis had further confirmed the association of CASP9 -293 del

polymorphism with reduced DFS among AML and ALL cases. A significant lower median

DFS was observed in AML (7 months) and ALL (14 months) patients with del-/- genotype.

However, this association was found to be significant only in AML (log rank p=0.020) but

not in ALL (log rank p=0.44).

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In conclusion our results suggest that CASP9 -293del polymorphism was not associated

with AML development but del+/+ genotype was found to increase the risk of ALL and

del-/- genotype conferred protection against ALL. In ALL, del-/- genotype was found to

reduce CR rate and DFS. CASP9 -293del-/- genotype was found to significantly reduced

DFS rate in both AML and ALL.

iv) Caspase9 Q221R(Ex5+ 32A>G) polymorphism

CASP9 Q221R Ex5+32A >G polymorphism results from A to G transition at exon5

leading to the substitution of glutamine by arginine at codon 221(Q221R), might alter the

CASP9 protein activity. In the present study, frequencies of QQ genotype (Adjusted χ2

p=0.02) and Q allele (χ2 p<0.001) were significantly elevated in ALL cases compared to

controls. CASP9 Q221R (Ex5+32A>G) polymorphism was not associated with AML

susceptibility.

The CASP9 Q221R (Ex5+32A>G) genotype distribution in AML and ALL with respect to

epidemiological as well as clinical variables did not show any association in both AML

and ALL. There was no significant association found between complete remission (CR)

rate and CASP9 Q221R (Ex5+32A>G) polymorphism in AML. While, CASP9 221RR

genotype frequency was found to be significantly elevated in ALL CR-ve cases under

recessive model of inheritance (Adjusted χ2p=0.04).

Mean clinical variables like age at onset, WBC count, platelet count, Blast%, HB level and

LDH level were not associated with CASP9 Q221R (Ex5+32A>G) polymorphism in AML

and ALL. But reduced DFS was observed in both AML (7.13±9.48 in months) and ALL

(17.02±26.37 in months) patients with RR genotype. Further, survival analysis showed that

RR genotype had reduced median DFS among AML (7 months) and ALL (12 months)

patients who had achieved complete remission rates after initial induction therapy though

the log rank test was insignificant.

In conclusion, our results reveal that CASP9 221QQ genotype was associated with ALL

development but not with AML. While, CASP9 221RR genotype was found to be

associated with CR failure and reduced DFS in ALL. In AML, 221RR genotype was found

to influence the DFS rates.

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CASP9 HAPLOTYPE ANALYSIS

In the present study, A-del--C-R haplotype had borderline significant reduced risk of AML

with an adjusted OR of 0.45 (95% CI, 0.18–1.10, p=0.08). No such trend was observed in

case of ALL haplotype groups. This reduced risk association in AML was attributed due to

the presence of both wild type alleles -1263A and -712C which might have a protective

effect against the development of AML. Further, three CASP9 haplotypes, A-del+-T-Q, G-

del+-C-Q and G-del+-T-Q were found to be significantly associated with increased AML

risk by 1.94-(95% CI: 1.12-3.37, p=0.019), 2.17-(95% CI: 1.08-4.36, p=0.03) and 12.79

folds (95% CI: 1.06-154.59, p= 0.046), respectively. As it was evident in our study that

presence of two risk alleles at CASP9 -1263G and CASP9 -712T position in a haplotype

group had an increased risk of AML by 12 folds and even a presence of single allele G at

CASP9 -1263position and T allele at CASP9 -712 position in individual haplotypes also

increases 2 fold risk.

In case of ALL, the two CASP9 haplotype groups G-del--T-R and G-del+-T-R were found

to be associated with 3.11-(95% CI: 1.80-5.38, p<0.001) and 5.57 folds (95% CI: 1.14-

27.10, p= 0.046) increased risk of ALL development. Thus, three risk alleles at CASP9 -

1263G, CASP9 -712T and CASP9 221R positions in the haplotypes G-del--T-R and G-

del+-T-R had conferred 3 and 5 fold risk. However, only G-del--T-R haplotype could

confer the risk after adjusting for covariates as this haplotype group had all four risk allele

combination.

A possible reason for association of observed haplotype groups with acute leukemia risk

might be due to lower expression of CASP9 gene, as an initiator CASP plays an important

role in the apoptosome-driven apoptosis pathway which is essential for eliminating

mutated or transformed cells from the body.

MDM2 -309T>G polymorphism

MDM2 is a cellular proto-oncogene, acts as a negative regulator of TP53. MDM2 regulates

p53 through direct binding to the N-terminal end of p53 that inhibits the transcriptional

activation function of p53 and through E3 ubiquitin ligase activity, Mdm2 targets p53 for

modification and subsequent degradation through the 26S proteasome. MDM2 gene is

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located at chromosome 12q 14.3-q15 and spans around 33 kb in length. It consists of 12

exons with two promoter elements P1 and P2. The P1 promoter controls basal expression

of MDM2, and is situated upstream of the first exon of the MDM2 gene. The P2 promoter,

located in the first intron is highly regulated and responsible for inducible expression of

MDM2. The MDM2 -309T<G promoter polymorphism was present in the P2 promoter

element. The presence of G allele at -309(T>G) position increases the affinity of the

transcription factor sp1 to the MDM2 promoter and enhances the expression of MDM2

RNA and MDM2 protein, thereby leading to attenuation of the p53 stress response. In the

present study, MDM2 -309GG genotype and G allele frequencies were significantly

elevated in both AML (p=<0.0001) and ALL (p=0.009) cases compared to controls. No

significant association was found between MDM2 -309T>G polymorphism and gender of

the probands in acute leukemia (AML and ALL). However, MDM2 -309TG heterozygote

frequency was found to be slightly elevated in AML female patients (39.0%) compared to

males (29.7%) and no such trend was observed in ALL patients. With respect to diet of the

probands, -309GG genotype and G allele frequencies were significantly elevated in

vegetarian group of AML patients compared to non-vegetarians under recessive model of

inheritance (p=0.008) and conferred reduced risk for non vegetarians carriers of -309G

allele (OR=0.28; 95% CI: 0.12-0.69; p=0.006). Lack of association was observed in ALL

with respect to diet. Further, MDM2 -309GG genotype had conferred 2 fold increased

risk to AML in urban patients compared to rural patients (Adjusted OR=2.50, 95%

CI=1.15-5.42, p=0.034). While in case of ALL, MDM2 -309TG heterozygote frequency

was significantly elevated in rural patients (Adjusted p=0.021). Smoking did not show

significant association with MDM2 -309T>G polymorphism in both AML and ALL.

The genotype distribution with respect to clinical variables had shown that age at diagnosis

was not significantly associated with MDM2 -309T>G polymorphism in acute leukemia

(AML & ALL), although G allele frequency was elevated among late age at onset (>30yrs)

group of AML patients (62.5%) compared to early age at onset (<20yrs=55.32%). Further,

G allele was found to be significantly associated with leucopenia (p=0.02), severe

thrombocytopenia (p=0.04) and elevated LDH level (>200IU/L) (p=0.04) in AML. No

such association was observed among ALL patients.

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With respect to complete remission rate, MDM2 -309GG genotype and G allele

frequencies were found to be significantly elevated in CR+ve AML cases (49.0%; 62.02%)

compared to CR-ve cases (33.8%; 52.21%). There was no such trend observed in ALL

with respect to MDM2 -309T>G polymorphism and complete remission rate. Mean

platelet counts were reduced in AML patients and DFS was found to be significantly

elevated in AML (13.83±19.09 in months) and ALL (27.31±32.24 in months) patients with

GG genotype. Further, these results were supported by survival analysis where CR+ve

patients with MDM2 -309GG genotype were found to have significantly increased median

survival rate of 16 months in AML group and 25 months in ALL group. The log rank test

was found to be significant in both AML (p=0.052) and ALL (p=0.050).

Our results suggest that presence of MDM2 -309GG genotype might increase the risk of

acute leukemia development due to inhibition of TP53 tumor suppressor activity and

resultant genomic instability. Interestingly, MDM2 -309GG genotype was also associated

with increase in CR rate, elevated DFS, hence can be considered as a good prognostic

marker. MDM2 -309GG genotype might be good predictive marker in AML development

and influence the DFS rate in both AML and ALL.

Real-time analysis of MDM2 mRNA level in Acute myeloid leukemia

The effect of MDM2-309T>G polymorphism on expression of MDM2 mRNA levels in 30

primary acute myeloid leukemia cases had shown that MDM2 GG genotype carriers (0.698

± 0.979 (n = 15), p=0.003) had significantly increased MDM2 mRNA level compared to

TT (0.127 ± 0.194; n=5) and TG (0.274 ± 0.479; n=9) genotype carriers. When MDM2

mRNA expression levels were compared with respect to sex of the probands, female AML

cases (0.739 ± 0.965; n=16) had shown significantly elevated mRNA expression level

compared to male cases (0.134 ± 0.208; n=13).

Further, expression data was stratified with respect to baseline clinical characteristics like

age at onset, WBC count, Platelet count, blast%, Hb level, LDH level and complete

remission (CR) rate after first induction treatment. Age at onset did not show any

association with MDM2 mRNA expression level. Although, WBC count, platelet count,

blast%, Hb and LDH levels did not show any significant association with MDM2 mRNA

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expression, an increasing trend of MDM2 expression level was observed in patients with

severe leukocytosis, thrombocytopenia and those with >50% blast.

With respect to complete remission rate, MDM2 mRNA levels were significantly elevated

in CR+ve cases (0.587 ± 0.984; n=14; p=0.0036) compared to CR-ve patients (0.121 ±

0.166; n=5). Since MDM2 is negative regulator of TP53 gene which is a guardian of the

genome, the overexpression of MDM2 may lead to cell vulnerability to chemotherapy in

absence of regulation of cellular machinery.

In conclusion, our results suggest that MDM2 overexpression among GG carriers might

increase risk to AML susceptibility. AML patients with high MDM2 mRNA expression at

diagnosis tend to show elevated CR rate after first induction therapy.

COX Regression Analysis

To test the hypothesis that SNP studied as well as clinical variables included in the present

study has an independent effect as a prognostic factor, Univariate Cox regression analysis

was carried out. In the present study, male AML patients were found to be significantly a

favorable group for elevated DFS (HR=0.615; 95%CI=0.404-0.936; p=0.023) compared to

female AML patients. AML patients with >1000IU/L of LDH level (HR=2.685;

95%CI=1.08-6.677; p=0.034) had shown to have HR=2.685 indicating increased risk for

relapse and shortened DFS. While in case of ALL patients, severe leucopenia (HR=1.697;

95%CI=1.172-2.457; p=0.005) and abnormal LDH level (HR=4.493; 95%CI=1.509-

13.372; p=0.007) at diagnosis were appeared to elevate risk of reduced DFS and relapse

rate.

Cox regression analysis of SNPs with respect to DFS had revealed that CASP9 -712CT

and CASP9 -293del-/- genotypes were found to have HR of 1.91 and 1.88 which indicated

that these genotypes have risk to relapse and subsequent reduced DFS in AML. Further, a

borderline hazardous risk for reduced DFS was also found with respect to RAD51 -135CT

genotype (HR=2.06; 95% CI=0.88-4.83; p=0.096) in AML. In case of ALL, CASP9 -

1263GG and CASP9 -712TT genotypes had demonstrated significantly increased

hazardous risk ratios of 1.576 and 1.153 respectively, which indicated influence of these

genotypes on the increased relapse rate and reduced DFS.

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These results suggest that CASP9 promoter polymorphisms to be unfavourable prognostic

marker with reduced DFS time in both AML & ALL initial induction therapy.

Linkage Disequilibrium

Linkage Disequilibrium analysis helps in genes mapping for complex disease loci and

understanding of genome architecture. In the present study, a combination of SNP loci

CASP9 -293 del: CASP9 Q221R was found to be in a strong LD among controls

(D’>0.93) and in both AML (D’>0.89) & ALL (D’>0.89) cases as these SNPs are present

in the same gene and tend to inherit as a single block due to their physical vicinity. Further,

two more SNP combinations (CASP9 -1263A>G: CASP9 -293del and CASP9 -

1263A>G: CASP9 Q221R) of CASP9 gene had also demonstrated a moderate LD effect

in controls (D’>0.71; D’>0.74) and cases (AML D’>0.63; D’>0.60 & ALL D’>0.64;

D’>0.64) respectively. Thus these observations indicate that CASP9 SNPs are closely

linked with low recombination frequency due to their close physical vicinity.

Similarly, a combination of XRCC1 Arg194Trp: XRCC1 Arg399Gln SNP had shown

moderate to strong LD effect in controls (D’>0.74) and AML (D’>0.89). However low LD

score for the same loci was observed in ALL (D’<0.32) which might be due to reduced

minor allele frequency at one or other loci. Strong LD was observed between BAX -

248G>A: XRCC3 Thr241Met loci in AML (D’=1.0) cases and a mild LD score in ALL

(D’>0.53) compared to controls (D’<5) indicating the significant interaction between HRR

and pro-apoptotic pathways. SNP analysis based on single marker also indicated the

significant role of BAX gene in AML development and XRCC3 in clinical response of

AML and ALL.

Further, XRCC1 Arg194Trp: RAD51 -135G>C and Bax -248G>A: MDM2 -309T>G

loci combinations had shown moderate LD (D’>0.72) to mild LD scores (D’>0.59)

respectively in AML. While low LD scores were observed in controls (D’<0.10; D’<0.31)

and ALL cases (D’<0.07; D’<0.09). A mild LD effect was found between RAD51 -

135G>C: XRCC3 Thr241Met SNP loci in AML/ALL cases (AML D’>0.55; ALL

D’>0.44) but not in controls (D’<0.03). Thus, these results on LD explain that defective

HRR mechanism might increase the occurrence of DSBs and chromosomal aberrations in

cells exposed to various carcinogens. Further, failure of apoptosis in mutated cells can

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probably transform them into leukemic stem cells thereby promoting development of acute

myeloid leukemia.

In ALL, two SNP loci BAX -248G>A: RAD51 -135G>C (D’=1.0) and CASP9 -712C>T:

BAX -248G>A (D’=1.0) combinations were found to have highest LD score which was

not observed in AML and controls. Further, a mild LD score was observed between

CASP9 -1263A>G: BAX -248G>A in ALL (D’=0.44) while low LD scores were

observed in controls and AML. Interesting observations could be made in ALL, wherein

BAX -248G>A SNP was found to be the common SNP in all the three SNP loci

combinations showing strong to mild Linkage disequilibrium. Thus our results indicate

that failure of apoptosis in mutated cells due to decreased expression of pro apoptotic

genes like BAX might provide survival advantage and leukemiogenesis. Further, they can

also play a significant role in chemo resistance and disease progression.

In conclusion, our results reveal that both in AML and ALL BAX -248G>A SNP was in

LD either with XRCC3 Thr241Met or RAD51 -135G>C polymorphisms, which are

responsible for HRR pathway. Hence development of leukemia may be initiated due to

defective HRR resulting in high rate of mutations in cells leading to transformation. The

failure of apoptosis in these abnormal cells due to defective expression of pro-apoptosis

genes might promote leukemiogenesis.

Generalized Multi dimensionality reduction (GMDR) analyses:

GMDR analysis had revealed that CASP9 -712C>T is a highest predicting variable for

AML susceptibility. Further, best interaction models and dendrogram entropy analysis had

revealed an interesting observation where CASP9 -712C>T, MDM2 -309T>G SNPs are

commonly found in three, four and five best interaction models with a strong synergistic

interaction. XRCC3 Thr241Met & BAX -248G>A combinations had shown strong

redundancy in dendrogram analysis that indicates a strong correlation in AML

development.

Similarly, CASP9 -712 C>T a single locus model was found to be the highest predicting

marker in ALL risk. Thus, CASP9 -712C>T polymorphism might be a strong predictive

marker for acute leukemia. Further, best interaction models and dendrogram entropy

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analysis had revealed an interesting observation where CASP9 -712C>T, BCL2 -938C>A,

MDM2 -309T>G SNPs are commonly found in four and five best interaction models. Pro

apoptotic gene SNPs (BCL2-938C>A and BAX -248G>A) and initiator CASP9 SNPs

(CASP9 -712C>T and CASP9 Q221R) had shown strong correlation in ALL risk

indicating intrinsic apoptotic pathway might influence the ALL development. This

correlation was also confirmed by the strong LD observed between pro-apoptotic and

CASP9 SNPs.

A combination of BCL2 -938CA and CASP9 -712CT genotypes were found in all higher

order interaction models as a risk groups in ALL indicating significant of anti-apoptotic

and initiator caspase9 function in the development of ALL. CASP9 -712CC and CASP9 -

1263AA/AG/GG genotypes were present in all protective genotype groups indicating the

effective role of wild type genotypes in reducing ALL susceptibility.

In conclusion, DNA DSBs arising in HSC or HSPCS due to exposure to various agents if

repaired extensively by abnormal expression of HRR genes increases the possibility of

hyper recombination and malignant transformation. Further, the failure of apoptosis in

coordination with enhanced expression of negative regulator (MDM2) of TP53 might

confer survival advantage to transformed cells resulting in acute leukemia. These

deregulated cell surveillance mechanisms also play significant role in conferring resistance

to chemotherapy, thereby to reduced DFS rates in acute leukemia patients. The present

study recommended mandatory screening of AML and ALL patients for BCL2 and

Caspase9 genes in order to maintain the disease progression.

Words so innocent and powerless as they are, as standing in a dictionary, how potent for good and evil they become in the hands of one who knows how to combine them

--Nathaniel Hawthrone