**4. Discussion**

310 Advances in Hematopoietic Stem Cell Research

For the MMP-9 C-1562T polymorphism, significant difference was obtained with CT

For the GNB3 C825T polymorphism, we didn't observe any difference between the 2 groups

This let us consider that there's no association between GNB3 C825T polymorphism and the

For the group of healthy PBSC donors, and with respect to our classification according to mobilization failure (<3x106 CD34/kg within 4 apheresis days), we have found an important association of SDF1-3'A distribution with higher mobilization yield of hematopoietic stem cells

Besides, we have observed a similar increase in the SDF1-3'G allele in the intermediate to poor mobilizers' subgroup reaching a statistical significance (p=0.035; OR=1.25; table 4). Similarly, the association was already observed when comparing the genotypic frequencies

The AA genotype was absent in the poor mobilizer subgroup, then was highly increased in

While, the GG genotype was more represented in the poor mobilizers and the differences

Healthy allogenic PBSC Donors

32 0,55 5 0,208 *0,001§* 12,6 [2,407-65,953] 26 0,45 19 0,792 *0,035§* 1,25 [1,045-1,495]

3 0,10 7 0,583 *0,001§* 0,079 [0,015-0,415]

6 0,20 0 0 *0,035§* 1,25 [1,045-1,495]

Abbreviations: OR, odds ratio; af, allele frequency; gf, genotype frequency; CI, confidence interval (CI=95%); Corrected *p* value; NS, not significant; \*, for SDF-1 polymorphism; \*\* for MMP-9 polymorphism, for healthy allogenic PBPC donors: Good mobilizers (>3X106 CD34/kg), Poor

Table 5. Allele and genotype frequencies of *SDF-1,* and *MMP-9* polymorphisms in

Good mobilizers Poor mobilizers *P* OR CI

genotypes between the two groups (p=0.004; OR= 0.297; CI (95%) [0.125-0.703]).

CD34+ reaching a higher statistical significance (p=0.001; OR=12.6; table 5).

the other subgroup reaching a statistical significance (p=0.035; OR=1.25).

of poor and good mobilizers.

between the two subgroups.

capacity of mobilization of hematopoietic stem cells.

were significant too (p=0.001; OR=0.079; table 4).

N af N af

N gf N gf

N' af N' af

N' gf N' gf

mobilizers (<3x106CD34/kg)

20 0,69 5 0,417 *NS* 

7 0,17 6 0,23 *0,633 (NS)* 33 0,82 20 0,77 *NS*

13 0,65 7 0,54 *NS* 7 0,35 6 0,46 *NS* 0 0 0 0 *NS*

mobilized peripheral blood of healthy allogenic PBSC donors

In the present study, we investigated the effect of polymorphisms in the genes SDF-1, GNB3 and MMP-9 on the outcome of mobilization of peripheral blood stem cells for autologous transplantation by using a PCR-RFLP analysis.

We observed a significant association for SDF-1 and MMP-9 polymorphisms exclusively in patients with MM, NHL and Hodgkin's disease suggesting that these polymorphisms are fair candidate gene variants to these 3 hematological diseases.

In fact, Association of these polymorphisms to cancer has been previously reported by many investigators [De Oliveira KB et al, 2009; Rabkin CS et al, 1999].

Our results were in agreement with other studies suggesting that SDF1-3'A polymorphism is a genetic determinant of NHL [Gabriela Gonçavales de Olivera Cavassin et al, 2004]. Furthermore; as the SDF1-3'A polymorphism is situated in the mRNAs of 3'UTR region (untranslated region) which has been identified as an important regulator of the mRNA transcript, as well as the translated product [Catia Andreassi and Antonella Riccio, 2004; Marilyn Kozak, 2004; Gavin S. Wilkie et al, 2003].

The second polymorphism studied encoded for MMP-9, J. Arai et al, have reported that SDF-1 mRNAs abundantly expressed in stromal cells from the lymph nodes of patients with malignant lymphoma, so that 3'A carriers NHL are good candidates for presenting proliferation of neoplasic cells in the lymph nodes since that SDF-1 variant is associated with an increase of SDF-1 levels [J. Arai et al, 2000; Gabriela Gonçavales de Olivera Cavassin et al, 2004].

De Oliveira KB et al, when studying distribution of SDF1-3'A polymorphism have reported also a significant difference in genotype distribution between NHL patients (GG: 51.4%; GA: 47.1%; AA: 1.5%) compared to healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%). Whereas, they didn't find any significant differences in genotypes distributions with breast cancer and Hodgkin's lymphoma [De Oliveira KB et al, 2009].

Moreover, previous reports on AIDS related non-Hodgkin's lymphoma (NHL) demonstrated that the CXCL12–3'A chemokine variant was associated with approximate doubling of the NHL risk in heterozygotes and an approximately fourfold increase in homozygotes [Rabkin CS et al, 1999; A Zafiropoulos et al, 2004]. Hence, this might let us suggest the possible role of such variant in the pathogenesis of NHL.

In this present work, we did not find a significant association between SDF1-3'A polymorphism and our group of patients with AML, this could be due to the lower number of patients (15 patients).

However, Dommange et al, have reported the implication of SDF1-3'A polymorphism in the clinical representation of acute myeloid leukemia in 86 patients with AML, as an association between this polymorphism and the risk of tissue infiltration by malignant cell was established by an increased release of the blast from the bone marrow in the blood in the SDF1-3'A carriers suggesting that this SDF-1 variant is associated with clinical representation of AML [A Zafiropoulos et al, 2004].

MMP-9 is a zinc-dependent proteinase, which is involved in numerous physiological and pathological processes. In the present study, we reported the distribution of the functional

Distribution of SDF1-3'A, GNB3 C825T and MMP-9 C-1562T Polymorphisms

hard-to-mobilize patients [Benboubker L et al, 2001; Patrick J Stiff, 1999].

NHL groups.

(p=0.009).

polymorphism.

are needed.

[Bogunia-Kubik K et al, 2009].

compared to GG homozygotes [Patrick J Stiff, 1999].

in HSC CD34+ from Peripheral Blood of Patients with Hematological Malignancies 313

In fact, when analyzing the distribution of the two functional polymorphisms SDF-1 G801A and MMP-9 C-1562T considering the two groups of "good" and "poor" mobilizers, we've found an association only with SDF1-3'A polymorphism. While no association with capacity of mobilization was observed with GNB3 C825T and MMP-9 C-1562T polymorphisms.

When observing the distribution of the two polymorphisms not only when considering the mobilization capacity but also in relation to each studied disease enrolled in this work we've found that the good mobilizer group was mainly composed of MM patients. Whereas the poor mobilizer group contains Hodgkin's disease who are considered in previous studies as

The fact that multiple myeloma patients mobilized better PBSC CD34+ (peripheral blood stem cells) than the others groups seem to be related to their ulterior chemotherapy (dexamethasone + thalidomide) and didn't receive any radiation therapy unlike the HD and

In the good mobilizer group composed of patients needing fewer apheresis than the other group, genotypes frequencies for the GG,GA, AA represented respectively 30.6%, 58% and 11.4%, and corresponded respectively to 45.5%, 47% and 7.6% in the poor mobilizer's group, and significant differences were found for GG genotype (p=0.007) and for A allele

This confirms on the one hand that the SDF1-3'A allele was associated with good mobilizing capacity not only in the group of patients but for instance in the group of healthy allogenic PBSC donors (see table 5). Thus, our results regarding patients undergoing autologous transplantation of haematopoietic stem cells concur with those reported by Benboubker et al

Moreover this deduction is already found in the group of healthy allogenic transplantation donors as it was reported in the present study and by Bogunia-Kubik K et al who have suggested that the SDF1-3'A allele was associated with a higher yield of CD34+ cells from healthy donors of PBPC for allogeneic haematopoietic SCT (stem cell transplantation)

Recent studies by the same group underlined an association of the SDF1-3'A allele with faster granulocyte and platelet recovery after transplantation. Therefore they suggested that the SDF-1 gene polymorphism could be a useful tool of prognostic value for recipients of autologous haematopoietic stem cells [A. Gieryng et al, 2010]. The allelic variant SDF1-3'A is a result of the SNP rs1801157, which is located in a highly demethylated area of the 3'UTR region. This SNP confers a G to A transition in the nucleotide position 801, resulting in a loss of a methylation site, which could affect the methylating effect of G-CSF [Nagler A et al, 2004], and leading to a more decreased SDF-1 expression in healthy individuals carrying the

So, it's of interest to investigate either this variant have major influence on the circulating

Further studies examining how these three polymorphisms interact with disease risk factors

levels of SDF-1 and its mRNA expression, one of our future's interests.

MMP-9 polymorphism -1567 C/T in the promoter region of the MMP-9 gene in group of patients with some haematological malignancies as well as in patients undergoing stem cell mobilization.

Then, we observed that the T allele was highly associated to the susceptibility to the four diseases studied (table 3). We have to investigate either this variant have major influence on the circulating levels of MMP-9.

Concerning the group of MM, we observed a significant association in all allelic and genotypic frequencies of SDF1-3'A polymorphism with statistical differences when compared to control. Hence, as increased angiogenesis was related to the pathogenesis of MM, and because SDF-1 chemokine induces increased VEGF production, which is responsible for an angiogenic activity [Florence Dommange et al, 2006], we hypothesize that the SDF1-3'A polymorphism might increase SDF-1 protein which would have a role in developing angiogenesis and in the pathogenesis of the disease.

On the other hand, frequent distribution of the SDF-1 3'A allele in multiple myeloma patients confirms the implication of SDF-1 in hematopoietic stem cells. This logical consequence of the widely distribution of SDF-1 3'A allele proving that multiple myeloma patient's could be considered as good mobilizers.

For the GNB3 polymorphism we've observed that the TT genotype and the T allele frequencies are more frequent especially in patients with MM (0.72 for Tallele frequency) and NHL (0.45 for Tallele frequency) compared to healthy donors of PBSC ( peripheral blood stem cells) (Table 3) which is far from the others populations [Maggie C.Y et al, 2004] . Then, suggesting the possible relation with these diseases.

Maggie et al when studying the ethnic differences in the linkage disequilibrium and distribution of single-nucleotide polymorphisms in 35 candidate genes for cardiovascular diseases have reported that the frequency of the T allele of GNB3 polymorphism in Chinese population is about 0.545. Then, such frequency is far from those of the French and of the Spanish population (0.329 and 0.359) and more closer to our result in Tunisian population [Yair Gazitt & Cagla Akai, 2004].

When interesting to the capacity of mobilization which was largely demonstrated to vary from a subject to another, several studies have focused on such phenomena and have reported that 10–30% of patients with hematological malignancies fail to mobilize PBSC [Ingrid G. Winkler & Jean-Pierre Levesque, 2006] and either a small proportion of normal donors (1–5%) fail to mobilize sufficient CD34+ cells.

Besides, many reports suggest that numerous factors are related to poorer mobilization including age, gender, type of growth factor, dose of the growth factor and in the autologous setting patient's diagnosis, chemotherapy regimen and number of previous chemotherapy cycles or radiation [Sugrue MW et al, 2001].

In our study we were interested in the possible implication of some genetic factors in mobilization and as we've found an association with the SDF-1 3'A variant only, then we supposed that this polymorphism is the only predictor of mobilization capacity of PBSC CD34+.

MMP-9 polymorphism -1567 C/T in the promoter region of the MMP-9 gene in group of patients with some haematological malignancies as well as in patients undergoing stem cell

Then, we observed that the T allele was highly associated to the susceptibility to the four diseases studied (table 3). We have to investigate either this variant have major influence on

Concerning the group of MM, we observed a significant association in all allelic and genotypic frequencies of SDF1-3'A polymorphism with statistical differences when compared to control. Hence, as increased angiogenesis was related to the pathogenesis of MM, and because SDF-1 chemokine induces increased VEGF production, which is responsible for an angiogenic activity [Florence Dommange et al, 2006], we hypothesize that the SDF1-3'A polymorphism might increase SDF-1 protein which would have a role in

On the other hand, frequent distribution of the SDF-1 3'A allele in multiple myeloma patients confirms the implication of SDF-1 in hematopoietic stem cells. This logical consequence of the widely distribution of SDF-1 3'A allele proving that multiple myeloma

For the GNB3 polymorphism we've observed that the TT genotype and the T allele frequencies are more frequent especially in patients with MM (0.72 for Tallele frequency) and NHL (0.45 for Tallele frequency) compared to healthy donors of PBSC ( peripheral blood stem cells) (Table 3) which is far from the others populations [Maggie C.Y et al, 2004] .

Maggie et al when studying the ethnic differences in the linkage disequilibrium and distribution of single-nucleotide polymorphisms in 35 candidate genes for cardiovascular diseases have reported that the frequency of the T allele of GNB3 polymorphism in Chinese population is about 0.545. Then, such frequency is far from those of the French and of the Spanish population (0.329 and 0.359) and more closer to our result in Tunisian population

When interesting to the capacity of mobilization which was largely demonstrated to vary from a subject to another, several studies have focused on such phenomena and have reported that 10–30% of patients with hematological malignancies fail to mobilize PBSC [Ingrid G. Winkler & Jean-Pierre Levesque, 2006] and either a small proportion of normal

Besides, many reports suggest that numerous factors are related to poorer mobilization including age, gender, type of growth factor, dose of the growth factor and in the autologous setting patient's diagnosis, chemotherapy regimen and number of previous

In our study we were interested in the possible implication of some genetic factors in mobilization and as we've found an association with the SDF-1 3'A variant only, then we supposed that this polymorphism is the only predictor of mobilization capacity of PBSC

developing angiogenesis and in the pathogenesis of the disease.

patient's could be considered as good mobilizers.

Then, suggesting the possible relation with these diseases.

donors (1–5%) fail to mobilize sufficient CD34+ cells.

chemotherapy cycles or radiation [Sugrue MW et al, 2001].

mobilization.

the circulating levels of MMP-9.

[Yair Gazitt & Cagla Akai, 2004].

CD34+.

In fact, when analyzing the distribution of the two functional polymorphisms SDF-1 G801A and MMP-9 C-1562T considering the two groups of "good" and "poor" mobilizers, we've found an association only with SDF1-3'A polymorphism. While no association with capacity of mobilization was observed with GNB3 C825T and MMP-9 C-1562T polymorphisms.

When observing the distribution of the two polymorphisms not only when considering the mobilization capacity but also in relation to each studied disease enrolled in this work we've found that the good mobilizer group was mainly composed of MM patients. Whereas the poor mobilizer group contains Hodgkin's disease who are considered in previous studies as hard-to-mobilize patients [Benboubker L et al, 2001; Patrick J Stiff, 1999].

The fact that multiple myeloma patients mobilized better PBSC CD34+ (peripheral blood stem cells) than the others groups seem to be related to their ulterior chemotherapy (dexamethasone + thalidomide) and didn't receive any radiation therapy unlike the HD and NHL groups.

In the good mobilizer group composed of patients needing fewer apheresis than the other group, genotypes frequencies for the GG,GA, AA represented respectively 30.6%, 58% and 11.4%, and corresponded respectively to 45.5%, 47% and 7.6% in the poor mobilizer's group, and significant differences were found for GG genotype (p=0.007) and for A allele (p=0.009).

This confirms on the one hand that the SDF1-3'A allele was associated with good mobilizing capacity not only in the group of patients but for instance in the group of healthy allogenic PBSC donors (see table 5). Thus, our results regarding patients undergoing autologous transplantation of haematopoietic stem cells concur with those reported by Benboubker et al [Bogunia-Kubik K et al, 2009].

Moreover this deduction is already found in the group of healthy allogenic transplantation donors as it was reported in the present study and by Bogunia-Kubik K et al who have suggested that the SDF1-3'A allele was associated with a higher yield of CD34+ cells from healthy donors of PBPC for allogeneic haematopoietic SCT (stem cell transplantation) compared to GG homozygotes [Patrick J Stiff, 1999].

Recent studies by the same group underlined an association of the SDF1-3'A allele with faster granulocyte and platelet recovery after transplantation. Therefore they suggested that the SDF-1 gene polymorphism could be a useful tool of prognostic value for recipients of autologous haematopoietic stem cells [A. Gieryng et al, 2010]. The allelic variant SDF1-3'A is a result of the SNP rs1801157, which is located in a highly demethylated area of the 3'UTR region. This SNP confers a G to A transition in the nucleotide position 801, resulting in a loss of a methylation site, which could affect the methylating effect of G-CSF [Nagler A et al, 2004], and leading to a more decreased SDF-1 expression in healthy individuals carrying the polymorphism.

So, it's of interest to investigate either this variant have major influence on the circulating levels of SDF-1 and its mRNA expression, one of our future's interests.

Further studies examining how these three polymorphisms interact with disease risk factors are needed.

Distribution of SDF1-3'A, GNB3 C825T and MMP-9 C-1562T Polymorphisms

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Interestingly, the possible implication of others genes involved of homing and migration process of CD34+ cells and for instance VCAM-1 to higher or lower mobilization yield of PBPC might emphasize new strategies for poor mobilizers subjects and lead to the identication of new biomarkers and/or therapeutic targets.
