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