Mutational and promoter hypermethylation status of FHIT gene in breast cancer patients of Kashmir

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ARTICLE IN PRESSModel

UT 10993 1–8

Mutation Research xxx (2010) xxx–xxx

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /molmutCommuni ty address : www.e lsev ier .com/ locate /mutres

utational and promoter hypermethylation status of FHIT gene in breast canceratients of Kashmir

idda Syeeda,b,d, Syed Akhtar Husainb,d, A. Syed Sameera,b,issar A. Chowdhrib,c, Mushtaq A. Siddiqia,b,∗

Department of Immunology and Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences, Soura, Srinagar 190011, Kashmir, IndiaDepartment of Clinical Biochemistry, Sher-I-Kashmir Institute of Medical Sciences, Soura, Srinagar 190011, Kashmir, IndiaDepartment of General Surgery, Sher-I-Kashmir Institute of Medical Sciences, Soura, Srinagar 190011, Kashmir, IndiaDepartment of Biosciences, Jamia Millia Islamia, New Delhi, India

r t i c l e i n f o

rticle history:eceived 3 September 2010eceived in revised form 2 November 2010ccepted 12 November 2010vailable online xxx

eywords:HIT

a b s t r a c t

Objective: Fragile histidine triad (FHIT) gene located at chromosome 3p14.2 is a putative tumor suppressorgene involved in the pathogenesis of breast cancer. Both genetic and epigenetic alterations in FHIT havebeen implicated in breast carcinoma. In the present study, our main aim was to study the impact of thesetwo kinds of alterations of FHIT gene in breast cancer patients of Kashmir.Methods: We screened a total of 130 breast cancer patients of Kashmir by PCR-SSCP followed by directsequencing and methylation specific PCR.Results: Mutational screening of FHIT gene revealed significant amount of mutations [40.7% (53/130)] in

reast cancerashmirutationypermethylation

five hot spot exons (exons 5–9), FHIT promoter was found to be hypermethylated in 59 of 130 [45.3%]breast cancer patients in our population.Conclusion: In the present study we have shown a significant association between the mutational andhypermethylation profile of FHIT gene. Hence, we provide the first evidence to our knowledge that thesignificant association of FHIT mutation and hypermethylation leads to the complete inactivation of FHITgene in patients with breast cancer. Silencing of the FHIT gene by promoter hypermethylation occurs in

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breast carcinomas, espec

. Introduction

Breast cancer is the most frequent neoplasm affecting women allver the world and is third most common malignancy in the world1] with more than 1 million women diagnosed with breast can-er each year [2]. Breast cancer has been associated with a varietyf risk factors [3] both genetic and epigenetic changes [4]. Envi-onmental carcinogens have been shown to damage DNA at activeragile sites by disrupting surveillance, which has been shown toe tumorigenic [5,6]. Development of human breast cancer arisesrom genetic alterations that drive the transformation of normal

ammary epithelial cells into highly malignant derivatives [7].

Please cite this article in press as: N. Syeed, et al., Mutational and promotKashmir, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2010), doi:10.1016/j

The FHIT gene has been identified in the fragile locus, FRA3B, atp14.2 of human chromosome 3 [8], and was recently identified ascandidate tumor suppressor gene [9]. FHIT gene is a member of theistidine triad gene family, encoding a protein similar to the yeast

∗ Corresponding author at: Department of Immunology and Molecular Medicine,her-I-Kashmir Institute of Medical Sciences, Soura, Srinagar 190011, Kashmir,ndia. Tel.: +91 194 2401013x2262; mobile: +91 9419767768; fax: +91 194 2403470.

E-mail address: [email protected] (M.A. Siddiqi).

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027-5107/$ – see front matter © 2010 Published by Elsevier B.V.oi:10.1016/j.mrfmmm.2010.11.001

hose with the significant amount of mutations.© 2010 Published by Elsevier B.V.

diadenosine oligophosphates (ApnA) hydrolase, which are intra-cellular and extracellular signaling molecules involved in cellulardifferentiation and apoptosis.

DNA methylation has also been shown to contribute to car-cinogenesis; hypermethylation of regulatory regions of manytumor suppressor genes has been correlated with decreased geneexpression [10,11]. In previous studies, FHIT gene promoter hyper-methylation has been correlated with loss of gene expression inoesophageal, lung, breast, prostate, bladder, cervical, and oral can-cers [12–17]. A recent study [18] has shown that methylationprofiles in familial breast cancer can be defined by the mutationalstatus that is different from the intrinsic subtypes.

FHIT, through binding or hydrolyzing Ap3A, influences theAp4A/Ap3A ratio, which is known to cause changes in cell prolif-eration or to induce apoptosis [9,23]. However, controversy existswith regard to a tumor suppressor role for FHIT [24,25]. Breast car-cinoma emerges through a multistep process of transformation

er hypermethylation status of FHIT gene in breast cancer patients of.mrfmmm.2010.11.001

of normal epithelium through hyperplasia, atypical hyperplasia, 55

and carcinoma in situ [26]. Loss of expression of a tumor sup- 56

pressor gene is also an important step in tumor progression from 57

premalignant, to in situ, to invasive carcinoma. Because a loss of 58

heterozygosity of 3p loci has been frequently observed in various 59

dx.doi.org/10.1016/j.mrfmmm.2010.11.001


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mailto:[email protected]


ARTICLE IN PRESSG Model

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3. Results 139

Our study comprised of 130 breast cancer patients, revealed that 140

out of 130 cases 53 [40.7%] were mutated in one or more hot spot 141

Fig. 1. SSCP analysis of FHIT gene exons

ancers including breast cancer [27–32] recently, abnormal tran-cription of the FHIT gene has been reported in human breast cancerell lines and in primary breast cancers, showing that about 30% ofrimary breast cancers exhibited abnormalities in FHIT transcripts33].

It further seems to be necessary to examine not only the fre-uency of abnormalities of FHIT transcription in primary breastancers but also the association of the abnormalities with clinico-athological characteristics of cancer, which will be of great help

n characterizing the role of the FHIT gene in breast carcinogenesis.Therefore we devised this study to analyze the frequency of FHIT

berrations – both mutations and hypermethylation in our ethnicashmir Population and also to correlate the FHIT gene status witharious clinicopathological characteristics.

. Materials

.1. Patients and tumor tissue procurement

A cohort of 130 randomly selected breast cancer patients admitted to the Sher--Kashmir Institute of Medical Sciences was included in the study. All patientsncluded in the study were both male and female, with the histopathological diag-osis of the breast done under Institutional histopathology department.

Tissue samples consisting of tumor and adjacent normal were collected directlyrom Operation Theater of Department of General Surgery. Only histopathologi-al confirmed tumor tissues were included in the study. Peripheral blood samplesere also collected from the patients before the surgical resection of the tumor.

he study was approved by the Sher-I-Kashmir Institute of Medical Sciences Ethicalommittee.

.2. DNA isolation

Genomic DNA was extracted from tissue samples and peripheral blood samplessing DNA Extraction Kit (Qiagen, USA). The quality of the resulting genomic DNAas stringently assessed by low percentage agarose gel electrophoresis followed byV spectrophotometer.

.3. Conventional PCR-SSCP

In our study we screened the mutations in the open reading frame of theHIT gene, i.e., exons 5–9. Primer designs of these exons (5th to 9th) were takenrom earlier studied reports [35,36]. The DNA samples were amplified for targetegions/exons by conventional PCR. Each PCR reaction was performed in a 50 �lnal volume containing 20–100 ng of genomic DNA, 10 mmol/l Tris–HCl, 10 pmol/lf each primer, 200 �mol/l of each dNTP, and 1 U of Taq DNA polymerase (Genei,ndia). PCR was performed in a thermal cycler (Biorad icycler) using different pro-rams for five exons. Both positive and negative controls were used in each PCReaction. Negative controls were without DNA template to exclude non-specificmplification. The PCR products were run on 1.8% agarose gel and analyzed underV illuminator.

The high-quality amplified products were assessed with SSCP analysis. For thisssessment, 8 �l of each PCR product was added 2 �l of loading solution. The tubeas immediately heat-denatured at 95 ◦C for 5 min, then cooled on ice for 5 min, and

t 4 ◦C for 5 min, respectively, and kept there or on ice until a sample was loaded


nto the gel. 4 �l of each processed PCR product were electrophoresed on 1× MDETM

els. The products were run at 600 V for 10–12 h at room temperature in 1× TBEuffer solution (89 mM tris-base, 89 mM boric acid, 2 mM EDTA, and pH 8.0). Theels were stained using a non-radioactive silver staining method and results wereisualised and photographed with an image analyzer, as shown in Fig. 1. Sampleshat showed one or two band-shifts separated from the wild-type bands were iden-

owing mobility shifts in tumor sample.

tified as SSCP positive. All the samples that contained mutations were subjected tothe SSCP analysis procedure at least twice to rule out contamination.

2.4. Sequencing

The samples which showed variant band-shifts in SSCP were re-amplified forsequencing. The PCR products were gel-extracted using a Gel Extraction Kit (Qiagen,USA) and then sent for direct DNA sequencing. DNA sequencing was carried out atMacrogen Inc., Korea. To minimize the sequencing artifacts by PCR, products fromat least two different PCRs were sequenced using forward and reverse primers.

2.5. Methylation-specific polymerase chain reaction

Genomic DNA isolated from tumor and adjacent normal tissues by the proto-col described above was bisulphite modified by using commercial kit (MethylationDirect Kit, Zymo Research) according to manufacturer’s instructions. The modi-fied DNA were amplified by using methylation/unmethylation specific primers asreported earlier [37], 3 �l of bisulphite modified DNA was used in the PCR mixcontaining 1× PCR buffer, 200 �mol/l of each dNTP and 2 U Amp gold Taq DNApolymerase, 0.4 �M of primer and were amplified using the following reaction con-ditions: initial denaturation at 95 ◦C for 5 min followed by 38 cycles at 95 ◦C 50 s,62 ◦C 50 s, 72 ◦C 1 min, with the final incubation at 72 ◦C for 7 min. Ten-�l of the PCRproducts were loaded onto 6% denaturing polyacyrlamide gels and stained usingethidium bromide and visualised under UV illuminator, the PCR generated a 74 bpproduct both for methylated and unmethylated, as shown in Fig. 2.

2.6. Statistical analysis

Pearson’s two proportions test was used to compare the determined FHIT genemutational and methylational status with various clinical parameters. Differenceswith P > 0.05 were accepted as statistically not significant. Calculations were doneusing SPSS for Windows, version 11.5 (SPSS, Chicago, IL, USA).


Fig. 2. Methylation status of p16 and FHIT genes in normal and tumor tissues fromthe same patient (74 bp). Lane marked N: normal breast tissue, T: tumor tissue. Lanemarked M: methylated, lane marked U: unmethylated. Lane L: 100 bp molecularladder. Where T1 represents Bca3 and N1 is its adjacent normal tissue, T2 representsBca7 and N2 is its adjacent normal tissue. T3 represents Bca3.


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ARTICLEModel

UT 10993 1–8

N. Syeed et al. / Mutation

xons of FHIT gene, and hypermethylation of the promoter regionf FHIT gene was found in 45.3% [59 of 130] of breast cancer casesn our population.

.1. Mutational analysis of FHIT in breast carcinomas

Mutational screening of the five hot spot exons (exons 5–9)f FHIT gene revealed significant amount of mutations [40.7%53/130)] in our population. In the module of direct sequencing, weere able to detect 53 mutations out of which 26 were missense,

3 were frameshifts, 7 were nonsense, and 7 were silent mutations,n addition to these we were also able to detect 10 novel mutations,s shown in Fig. 3.

Among the 26 missense mutations, there were six missenseutations present in exon-5 (three in codon 34, two in codon 24,

ne in codon 30), five in exon-6 (two in codon 82, two in codon7 and one in codon 74), four in exon-7 (two in codon 90 and two

n codon 89), eleven in exon-8 (two in codon 112, two in codon16 and one in codon 98, 103, 115, 102, 107, 110, 113) and none inxon-9.

Among the 13 frameshift mutations, there were six frameshiftutations in exon-5 (three in codon 20, two in codon 30 and one in

odon 31), four in exon-6 (two in codon 74 and two in codon 69),ne in exon-7 (one in codon 90), none in exon-8 and two in exon-9both in codon 146).

Among the 7 nonsense mutations, no nonsense mutation wereresent in exon-5, three were present in exon-6 (three in codon4), two were present in exon-7 (one in codon 93 and one in codon0), two were present in exon-8 (both in codon 103) and none wereresent in exon-9.

Among the 7 silent mutations, three silent mutations wereresent in exon-5 (three in codon 11), none were present in exon-6nd exon-7, three were present in exon-8 (three in codon 98), oneas present in exon-9 (one in codon 135).

The mutational analysis revealed that most of the mutationsccurred in exon 8 followed by exons 5–7, and very few muta-ions were found in exon-9. Most of the missense mutations wereresent in exon-8, a good number of frameshift and silent muta-ions were present in exon-5, large number of nonsense mutationsas present in exon-6, and most of the silent mutations wereresent in exon-8 as shown in Table 1.

.2. Frequency of FHIT hypermethylation in breast carcinomas

In the present study a total of 130 breast cancer patients weretudied for the methylational status of FHIT. Methylational analy-is of the breast tumors revealed that 45.3% (59/130) of FHIT geneere methylated. FHIT gene was also observed to be methylated in

ome adjacent normal tissues. Using statistical analysis, we exam-ned methylation with regard to clinicopathological parameters ofhese cancer patients.

.3. Clinicopathological association

In the present study, breast tumors carrying the mutations weretatistically significant with as menopausal status, nodal status,istopathological grade and breast involved. However there waso significant association with age, sex, and smoking status.

Breast tumors that were found to be hypermethylated forHIT gene were significantly associated with parameters like age,enopausal status, smoking status, nodal status and tumor stage.


.4. FHIT mutation and hypermethylation status: two hit

In 30 cancer samples there were both – inactivation of FHITumor suppressor gene by mutation and epigenetic silencing

PRESSrch xxx (2010) xxx–xxx 3

by promoter hypermethylation, leading to complete inacti-vation of FHIT, suggesting the involvement of both for theinactivation of FHIT that leads to tumor development and progres-sion.

4. Discussion

Breast cancer is one of the leading causes of death in femalesall over the world [1]. A number of alterations that occur at thegenetic level involving oncogenes, tumor suppressor genes as wellas DNA repair genes all of which are responsible for the devel-opment of breast carcinoma. But the complete inactivation of thetumor suppressor gene is highly affected by mutational and epige-netic silencing.

FHIT gene is a candidate tumor suppressor gene, since its dis-covery in 1996, alterations of the FHIT gene have been frequentlyobserved in a variety of tumors [8,38–41]. It has been postulatedthat in some cancers, including breast cancer, the alterations inthe FHIT gene represent an early event in carcinogenesis [42–45]whereas in other cancers aberrant FHIT transcripts occur at laterstages of cancer development [46].

4.1. Effect of the mutations of FHIT on breast cancer genetics

Present study evaluated the reported hot spot region of FHITgene (exon-5 to exon-9). A total of 40.7% tumors were found tobe mutated. The exon-8 which contains the histidine triad domainessential for anti cancer activity of FHIT protein housed most of themutations as has been reported earlier [47], followed by exon-7,exon-5 and exon-6. Exon-9 exhibited least number of mutations asis also reported in earlier studies [33]. The significant feature of thestudy is the seven nonsense mutations, that lead to the truncationof FHIT protein hence the reduced expression level of FHIT; when itsprotein is translated from transcripts lacking the non-coding exons3 or 4 [48].

Statistically significant association of FHIT gene mutations withcertain clinico-epidemiological characteristics, such as menopausalstatus, nodal status, histopathological grade and breast involvedwas observed as shown in Table 2. Significant association betweenFHIT mutation and pre-menopause stage suggests breast canceris more strongly associated with risk in premenopausal women,although the difference between pre- and post-menopausalwomen has not been reported to be statistically significant [49].Correlation of FHIT gene mutation with lymph node involve-ment has been found significant, suggesting its role in thebreast cancer metastases. To date axillary lymph node sta-tus is the best prognostic indicator in breast cancer patients[50,51]. Further, significant association of FHIT gene mutationwith histopathological grade has been found in the present study,suggesting that a distinctive mechanism is responsible to inac-tivate the tumor suppressor function of FHIT gene. Althoughwe found significant association of FHIT gene and the breastinvolved, but cannot assign any relevant reason about this asso-ciation.

4.2. Effect of FHIT hypermethylation related to breast cancerdevelopment

Aberrant promoter methylation changes that occur duringthe development of cancer are associated with transcriptionalrepression and loss of function of the gene by interrupting the


binding of proteins involved in transcription activator complex 255

[52]. 256

Hypermethylation of the FHIT promoter was detected in 59 of 257

the 130 (45.3%) cases examined. The presence of high percent- 258

age of hypermethylation in our case reveals that methylation is 259



MUT 10993 1–8


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ig. 3. Partial electropherograms representing the mutant (shown by arrows) (abond 8.


ne of the major determinants in tumor progression because inajority of the cases, expression of the genes in these breast

ancer cases correlates with hypermethylation of the promoterequences. The rate of hypermethylation is higher than the

ong with its normal sequence (below) of FHIT gene: (a) exons 5 and 6; (b) exons 7


percentage of mutation which suggests that FHIT hypermethy- 264

lation is a more common event in breast carcinoma. Promoter 265

aberrant methylation of FHIT is an important mechanism for 266

inactivation of this tumor suppressor gene in mammary tumori- 267



MUT 10993 1–8

N. Syeed et al. / Mutation Research xxx (2010) xxx–xxx 5

Table 1Details of FHIT mutations in exons 5–9 of breast cancer patients from Kashmir valley.Q5

Exon involved Codon number Base changeg Amino acid change Effecth

Exon 5 34 GGA > AGA Gly > Arg Missense11 AAG > AAA Lys > Lys Silent20 GAA > GAGC INS G Frameshift24 GCT > CCT Ala > Pro Missense11 AAG > AAA Lys > Lys Silent30 CCT > CAT Pro > His Missense30 CCT > CCTA INS A Frameshift34 GGA > AGA Gly > Arg Missense31 GTG > GTAG INS A Frameshift34 GGA > AGA Gly > Arg Missense20 GAA > GAGC INS G Frameshift24 GCT > CCT Ala > Pro Missense20 GAA > GAGC INS G Frameshift11 AAG > AAA Lys > Lys Silent30 CCT > CCTA INS A Frameshift

Exon 6 64 AGA > TGA Arg > Stop Nonsense82 ATG > AAG Met > Lys Missense74 CAT > CATA INS A Frameshift77 TCT > CCT Ser > Pro Missense69 GTG > GTAG INS A Frameshift64 AGA > TGA Arg > Stop Nonsense74 CAT > CAG His > Gln Missense69 GTG > GTAG INS A Frameshift77 TCT > CCT Ser > Pro Missense64 AGA > TGA Arg > Stop Nonsense82 ATG > AAG Met > Lys Missense74 CAT > CATA INS A Frameshift

Exon 7 90 CAG > CAT Gln > His Missense93 AAG > TAG Lys > Stop Nonsense89 GGA > AGA Gly > Arg Missense90 CAG > TAG Gln > Stop Nonsense90 CAG > CAT Gln > His Missense89 GGA > AGA Gly > Arg Missense90 CAG > CAAG INS A Frameshift

Exon 8 116 GAG > GGG Glu > Gly Missense98 CAT > CAC His > Lys Missense

103 AAG > AAA Arg > Lys Missense98 CAT > CAC His > His Silent

115 GAG > GTG Glu > Val Missense102 AGG > AAG Arg > Lys Missense103 AAG > TAG Lys > Stop Nonsense107 TTT > TCT Phe > Ser Missense

98 CAT > CAC His > His Silent110 AAT > AAG Asn > Lys Missense116 GAG > GTG Glu > Val Missense103 AAG > TAG Lys > Stop Nonsense112 AGC > ACC Ser > Thr Missense

98 CAT > CAC His > His Silent113 ATC > ACC Ile > Thr Missense112 AGC > ACC Ser > Thr Missense

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Exon 9 146 TTT135 GCA146 TTT

enesis. Our results were similar to previously reported studies53,54]. We also found hypermethylation in some non-cancerousissues that might represent the appearance of premalignantesions. Thus, detection of FHIT hypermethylation using MSP

ight provide potential new molecular diagnostic markers ofreast carcinomas at an early stage during multistep carcinogene-is.

Clinicopathologically, aberrant FHIT expression has been asso-iated with pathogenesis and prognosis of various tumors8,34,55–61]. FHIT gene hypermethylation was found signifi-ant with some of the clinico-pathological parameters like age,


enopausal status, smoking status, nodal status and tumor stage.e found a significant association between FHIT gene hyperme-

hylation and age (>50 years) supported by enormous evidenceuggesting that some of the methylation changes may initi-

Del T FrameshiftAla > Ala SilentDel T Frameshift

ate in subpopulations of normal cells as a function of age andprogressively increase during carcinogenesis [62]. FHIT gene hyper-methylation was also found significant in pre-menopausal womensuggesting that the premenopausal women are at approximatelytwice risk, than the postmenopausal women, which has beenreported earlier that proliferation rate is somewhat lower andrelatively stable in post menopausal women [63–65]. Hyperme-thylation of the FHIT gene was associated with smoking variable;FHIT gene hypermethylation was significantly associated with eversmokers. As most of the female patients in our study originatedfrom far flung rural regions, where they have a tradition of Hukka


smoking (water pipe smoke) [66], which could be one of the possi- 294

ble reasons for hypermethylation. FHIT hypermethylation was also 295

found significantly associated with the lymph node involvement 296

and tumor stage (I and II). 297



MUT 10993 1–8


Table 2Effect of FHIT mutation and hypermethylation pattern in the breast cancer patients from Kashmir valley.

Variable TotalN = 130 (%)

MutantsM = 53 (%)

OR; P-value; CI(95%)

MethylatedN = 59 (%)

OR; P-value; CI(95%)

SexMales 7 (53.8%) 4 (57.1%) 2.01; 0.44; 0.43–9.39 2 0.46; 0.45; 0.08–2.48Females 123 (94.6%) 49 (39.8%) 57Age>50 13 (10.0%) 8 2.56; 0.13; 0.78–8.31 11 7.90; 0.003; 1.68–37.28≤50 117 (90.0%) 45 48DwellingRural 45 (34.6%) 38 21.7; 0.000; 8.10–58.15 37 13.24; 5.47; 5.36–32.75Urban 85 (65.4%) 15 22Smoking statusNever 93 (71.5%) 38 0.89; 0.83; 0.39–2.03 35 0.33; 0.006; 0.15–0.72Ever 37 (28.5%) 15 24Menopausal statusPre 94 (72.3%) 21 2.71; 0.01; 1.23–5.96 21 2.25; 0.04;1.02–4.96Post 94 (72.3%) 32 38Nodal statusInvolved 34 (26.2%) 25 6.74; 0.000; 2.79–16.26 23 3.48; 0.002; 1.52–7.98Not Involved 96 (73.8%) 28 36Breast involvedR 35 (26.9%) 33 61.87; 0.000;

13.66–280.1230 13.65; 1.38; 4.81–38.73

L 95 (73.1%) 20 29Tumor stageII (a + b) 72 (55.4%) 35 2.10; 0.04; 1.01–4.33 26 0.43; 0.02; 0.21–0.86III (a + b) + IV 58 (44.6%) 18 30Histopathological tumor gradePD 25 (96.0%) 18 5.14; 0.0005;

1.96–13.4713 1.38; 0.50; 0.57–3.33

MD + WD 58 + 47 (24.1%) 18 + 17 27 + 19

Table 3Statistical relation between promoter hypermethylation and mutation of FHIT gene.

FHIT promoter hypermethylation versus FHIT mutation

FHIT methylation OR; P-value; CI(95%)

UnmethylationN = 71

MethylationN = 59

FHIT mutation2

3

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TC

Wild: N = 77 48

Mutant: N = 53 23

.3. Two hits inactivate the tumor suppressor role of FHIT

The important feature of our study was the significant associa-ion of FHIT mutation with promoter hypermethylation, as shownn Table 3. In 30 samples, we found both FHIT mutation as wells FHIT promoter hypermethylation. It has been reported that oneit cannot result in the complete loss of function of the tumor sup-ressor gene FHIT, and therefore two hits are required for complete

nactivation [67], where hypermethylation has been considered asne of the hits [21,68]. It is also known that methylation can occurot as a primary event, but secondary to mutation [20,68] as has


een reported [69] that methylation of tumor suppressors cannotnitiate tumorigenesis, because in order to control the gene expres-ion and/or methylation the methylator phenotype itself requiresutations in genes that control them.

able 4orrelation of tumor grade versus mutation versus hypermethylation status.

FHIT gene status Tumor grade

Low grade II

Double hit (N = 30) 7Single hit – mutation only (N = 23) 12Single hit – methylation only (N = 29) 15

9 0.46; 0.04; 0.22–0.94

0

Among these 30 samples most of the mutations were truncat-ing mutations, and when these truncating mutations were coupledwith methylation, there are many possibilities that the methy-lated allele has actually harboured a somatic or germ line mutationwhich has predisposed the gene to methylation. We found tumorgrade statistically significant with mutational and hypermethyla-tion status both (Table 4), suggesting that both of these aberrations(mutation + hypermethylation) together are responsible for theprogression of breast cancer to higher grade.

According to Tomlinson et al. [70] “two hits” hypothesis, oneallele of a tumor suppressor can be inactivated by methylation


when the other harbors a mutation or allelic loss. Our results also fit 323

this model, revealing a strong association between FHIT promoter 324

hypermethylation and the mutation, suggesting that in breast car- 325

cinomas, one allele is lost by mutation, and the other is inactivated 326

P-value; �2

High grade III + IV

2311 0.04; 6.4114


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y aberrant methylation, with both events leading simultaneouslyo the biallelic inactivation and complete lack of function of theHIT gene. In addition, it is presumed that methylation usuallyccurs monoallelic, and complete loss of expression of FHIT genes a consequence of a combination of methylation and mutation13].

. Conclusion

FHIT gene, tumor suppressor gene is involved in breast carcino-enesis and in various other malignancies. We have studied theutational and hypermethylation profile of FHIT, which reveals

hat it is associated with breast cancer development and progres-ion. Our study reveals that the genetic and epigenetic aberrationsf FHIT gene lead to its complete inactivation, which leads to mam-ary carcinogenesis. The clinical significance of our findings should

e further evaluated in larger cohorts with longer follow-up tovaluate the effect of these findings on disease-free and overallurvival.

onflict of interest

The contributing authors have no financial or any non-financialompeting interests.

uthors’ contributions

Nidda Syeed formulated, designed and performed the lab workor the study. Akhtar Hussain supervised the analysis, A. Syedameer helped in the lab work. Nissar A. Chowdhri procured androvided the tumor samples for the study and Mushtaq A. Siddiqioordinated the study, revised the manuscript and entire work wasone under his supervision. All authors have read and approved thenal manuscript.

ncited references

[19,22].

cknowledgements

The authors gratefully acknowledge the Sher-I-Kashmir Insti-ute of Medical Sciences, Kashmir for providing funds for thisesearch work. The present research was not funded by any fund-ng agency. The collection of cancer samples used in this study wasupported by the Department of General Surgery, Sher-I-Kashmirnstitute of Medical Sciences.

The authors would like to thank all the breast cancer patientsho participated in the study who are responsible for the creation

nd maintenance of the entire group within which this study wasonducted but were not involved in the current paper.

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Mutational and promoter hypermethylation status of FHIT gene in breast cancer patients of Kashmir

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