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Abstract Cryptic deletions at chromosome 6q are common cytogenetic abnormalities in T-cell lymphoblastic leukemia/lymphoma (T-LBL), but the target genes have not been formally identified. Our results build on detection of specific chromosomal losses in a mouse model of γ-radiation-induced T-LBLs and provide interesting clues for new putative susceptibility genes in a region orthologous to human 6q15–6q16.3. Among these, Epha7 emerges as a bona fide candidate tumor suppressor gene because it is inactivated in practically all the T-LBLs analyzed (100% in mouse and 95.23% in human).

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We provide evidence showing that Epha7 downregulation may occur, at least in part, by loss of heterozygosity (19.35% in mouse and 12.5% in human) or promoter hypermethylation (51.61% in mouse and 43.75% in human) or a combination of both mechanisms (12.90% in mouse and 6.25% in human). These results indicate that EPHA7 might be considered a new tumor suppressor gene for 6q deletions in T-LBLs.

Notably, this gene is located in 6q16.1 proximal to GRIK2 and CASP8AP2, other candidate genes identified in this region. Thus, del6q seems to be a complex region where inactivation of multiple genes may cooperatively contribute to the onset of T-cell lymphomas. Candidate genes for 6q deletion in T-LBL. LOH study in γ-radiation-induced T-cell lymphomas evidences a common LOH region (TLSR5) between markers D4Mit105 and D4Mit214 (right) that is orthologous to human 6q15–16.1 (on the left). Putative candidate genes are indicated.

Gene content of minimal region of deletion and selection of candidate genes Gene scanning of the mouse LOH region, using the ENSEMBL ( ) and MGI ( ) mouse genome browsers, revealed the presence of 13 coding genes as potential targets of this deletion ( ). Although all these genes are expressed in the thymus, only five of them ( Manea, Pnrc1, Casp8ap2, Map3k7 and Epha7 ) showed significant downregulation in mouse T-LBLs induced by γ-irradiation, Epha7 being the one exhibiting the lowest expression levels ( is available at Carcinogenesis online). Given that Epha7 might be expressed as alternative transcripts with different functional properties ( ), we conduct a more detailed analysis of the gene expression patterns using different primers that specifically amplify the main alternative isoforms. We show here that control thymuses are able to express two different transcripts corresponding to the full length and the shorter soluble variant. In contrast, all T-cell lymphomas (31/31), including tumors without LOH, exhibited reduced expression of both isoforms (; is available at Carcinogenesis online). Quantitative real-time RT–PCR analysis of transcript variants of Epha7, in randomly selected mouse T-cell lymphomas, showing significant differences between control and tumors.

Methylation analysis of a CGI in the promoter region of the mouse Epha7 gene. Schematic depiction of the Epha7 CGI around the transcription start site (long black arrow) and methylation density in randomly selected mouse T-cell lymphomas.

Short vertical lines represent CpG dinucleotides. The presence of methylated (black square) or unmethylated cytosines (white square) is also indicated. Methylation density is indicated as the percentage of methylated CpG sites. From these observations, Epha7 would be downregulated by either LOH (6/31; 19.35%) or promoter hypermethylation (16/31; 51.61%) or a combination of both mechanisms (4/31; 12.90%) and emerges as the best candidate gene for TLSR5 deletion ( is available at Carcinogenesis online). Studies in human samples Similar to our results in murine lymphomas, we have found significant downregulation of EPHA7 in almost all the analyzed primary T-LBLs (20/21; 95.23%), but only a part of them exhibited allele deletion (3/22; 13.63%) (; is available at Carcinogenesis online). As occurred in mouse, EPHA7 exhibits a CGI region that was significantly hypermethylated in a subset of human T-LBLs (; is available at Carcinogenesis online). The methylation prevalence was 44.44% (8/18), and the methylation density ranged from 18.50 to 86%.

Thus, we conclude that inactivation of EPHA7 in human T-LBLs may occur exclusively by promoter hypermethylation (7/16; 43.75%), although in some tumors, it seems to occur by allele deletion (2/16; 12.5%) or by a combination of deletion and epigenetic events (1/16; 6.25%) ( is available at Carcinogenesis online). Methylation analysis of a CGI in the promoter methylation region of EPHA7 of thymuses from human fetuses (control) and randomly selected human T-LBLs. ( a ) Methylation-specific PCR for the EPHA7 gene in a representative sample human T-LBLs.

The presence of a PCR band under lanes M or U indicates methylated or unmethylated genes, respectively. In vitro methylated DNA (IVD) was used as a positive control for methylated DNA; Normal Lymphocytes (NL) was used as a positive control for unmethylated DNA. ( b ) Schematic depiction of the EPHA7 CGI around the transcription start site (long black arrow). Short vertical lines represent CpG dinucleotides. The presence of methylated (black square) or unmethylated cytosines (white square) is indicated. Methylation density is indicated as the percentage of methylated CpG sites.

Methylation analysis of a CGI in the promoter methylation region of EPHA7 of thymuses from human fetuses (control) and randomly selected human T-LBLs. ( a ) Methylation-specific PCR for the EPHA7 gene in a representative sample human T-LBLs. The presence of a PCR band under lanes M or U indicates methylated or unmethylated genes, respectively.

In vitro methylated DNA (IVD) was used as a positive control for methylated DNA; Normal Lymphocytes (NL) was used as a positive control for unmethylated DNA. ( b ) Schematic depiction of the EPHA7 CGI around the transcription start site (long black arrow).

Short vertical lines represent CpG dinucleotides. The presence of methylated (black square) or unmethylated cytosines (white square) is indicated. Methylation density is indicated as the percentage of methylated CpG sites. In addition to primary tumors, Jurkat cell line derived from T-ALL patients also exhibited promoter hypermethylation despite not having 6q deletions ( ). To confirm whether promoter DNA hypermethylation is involved in deregulating the expression of EPHA7, Jurkat cells were treated with 5-aza-2′deoxycytidine (a specific inhibitor of DNA methylation). Significant demethylation was observed after treatment with 5-aza-2′deoxycytidine ( ), which brings about EPHA7 upregulation ( ).

The methylation profile and the level of CGI methylation of EPHA7 in Jurkat cells are indicated in. Methylation analysis of EPHA7 in Jurkat cells. ( a ) Methylation-specific PCR analysis. Semi-quantitative RT–PCR reveals that treatment with the demethylating agent 5-aza-2′-deoxycytidine (AZA) is able to reactivate EPHA7 gene expression. ( c ) Schematic depiction of the EPHA7 CGI around the transcription start site (long black arrow) and methylation profile.

Short vertical lines represent CpG dinucleotides. The presence of methylated (black square) or unmethylated cytosines (white square) is indicated. Methylation density is indicated as the percentage of methylated CpG sites.

Abstract Iron deficiency constitutes a major public health problem in Morocco, mainly among women and children. The aim of our paper is to assess the efficacy of consumption of multiple micronutrients (MMN) fortified milk on iron status of Moroccan schoolchildren living in rural region. Children ( ), aged 7 to 9 y, were recruited from schools and divided into two groups: the nonfortified group (NFG) received daily a nonfortified Ultra-High-Temperature (UHT) milk and the fortified group received (FG) daily UHT milk fortified with multiple micronutrients including iron sulfate. Blood samples were collected at baseline (T0) and after 9 months (T9). Hemoglobin (Hb) was measured in situ by Hemocue device; ferritin and C Reactive Protein were assessed in serum using ELISA and nephelometry techniques, respectively. Results were considered significant when the value was.