Agnieszka Dzikiewicz-Krawczyk

The importance of making ends meet: Mutations in genes and altered expression of proteins of the MRN complex and cancer

The MRN protein complex, consisting of MRE1, RAD50 and NBS1, plays a crucial role in sensing DNA double-strand breaks (DSBs), and it is involved in cell cycle control. This makes the MRN complex an important guard of genome stability. Hypomorphic mutations in NBS1 result in the Nijmegen breakage syndrome (NBS), which is characterized by, among other things, an increased predisposition to malignancies, especially leukemia/lymphoma. Relatives of NBS patients carrying heterozygous mutations are also more prone to cancer development. This review summarizes several studies searching for associations between heterozygous mutations in NBS1, MRE11, and RAD50 and cancer and examining the levels of expression of proteins coded by these genes in tumor tissues. The results indicate that both decreased and increased expression of NBS1 may contribute to tumorigenesis, whereas overexpressed RAD50 has an anti-tumoric effect. MRE11 and RAD50 are also affected in tumors with microsatellite instability. However, the outcomes of association studies, which concerned primarily lymphomas/leukemias and breast cancer, were inconclusive. Heterozygous NBS1 mutations and molecular variants 657del5, I171V, R215W and E185Q were most commonly analyzed. Among these, an association with cancer was found most frequently for 657del5 (in leukemia/lymphoma and breast cancer) and I171V (in leukemia, breast, head and neck and colorectal cancers); however, other studies gave contradictory results. For other NBS1 as well as MRE11 and RAD50 variants, too little data were available to assess their role in cancer risk. Overall, the results suggest that heterozygous MRN complex mutations and molecular variants may contribute only to a limited fraction of tumors. This may be caused by several factors: various frequencies of the variants in specific populations, different criteria used for selection of control groups, possible effects of environmental factors, and potential interactions with variants of other low-risk genes. These issues, as well as the impact of the alterations on protein function, need to be addressed in future studies.

Polymorphisms in microRNA target sites modulate risk of lymphoblastic and myeloid leukemias and affect microRNA binding

BackgroundMicroRNA dysregulation is a common event in leukemia. Polymorphisms in microRNA-binding sites (miRSNPs) in target genes may alter the strength of microRNA interaction with target transcripts thereby affecting protein levels. In this study we aimed at identifying miRSNPs associated with leukemia risk and assessing impact of these miRSNPs on miRNA binding to target transcripts.MethodsWe analyzed with specialized algorithms the 3′ untranslated regions of 137 leukemia-associated genes and identified 111 putative miRSNPs, of which 10 were chosen for further investigation. We genotyped patients with acute myeloid leukemia (AML, n = 87), chronic myeloid leukemia (CML, n = 140), childhood acute lymphoblastic leukemia (ALL, n = 101) and healthy controls (n = 471). Association between SNPs and leukemia risk was calculated by estimating odds ratios in the multivariate logistic regression analysis. For miRSNPs that were associated with leukemia risk we performed luciferase reporter assays to examine whether they influence miRNA binding.ResultsHere we show that variant alleles of TLX1 _rs2742038 and ETV6 _rs1573613 were associated with increased risk of childhood ALL (OR (95% CI) = 3.97 (1.43-11.02) and 1.9 (1.16-3.11), respectively), while PML _rs9479 was associated with decreased ALL risk (OR = 0.55 (0.36-0.86). In adult myeloid leukemias we found significant associations between the variant allele of PML _rs9479 and decreased AML risk (OR = 0.61 (0.38-0.97), and between variant alleles of IRF8 _ rs10514611 and ARHGAP26 _rs187729 and increased CML risk (OR = 2.4 (1.12-5.15) and 1.63 (1.07-2.47), respectively). Moreover, we observed a significant trend for an increasing ALL and CML risk with the growing number of risk genotypes with OR = 13.91 (4.38-44.11) for carriers of ≥3 risk genotypes in ALL and OR = 4.9 (1.27-18.85) for carriers of 2 risk genotypes in CML. Luciferase reporter assays revealed that the C allele of ARHGAP26 _rs187729 creates an illegitimate binding site for miR-18a-3p, while the A allele of PML _rs9479 enhances binding of miR-510-5p and the C allele of ETV6 _rs1573613 weakens binding of miR-34c-5p and miR-449b-5p.ConclusionsOur study implicates that microRNA-binding site polymorphisms modulate leukemia risk by interfering with the miRNA-mediated regulation. Our findings underscore the significance of variability in 3′ untranslated regions in leukemia.

MicroRNA polymorphisms as markers of risk, prognosis and treatment response in hematological malignancies

MicroRNA dysregulation is a common event in hematological malignancies. Apart from genomic and epigenetic alterations, miRNA networks may be disturbed by polymorphisms in the miRNA regulatory pathway (miRSNPs). In this review we provide an overview of three categories of miRSNPs: (1) SNPs in genes involved in miRNA biogenesis and processing; (2) SNPs in miRNA genes; and (3) SNPs in miRNA binding sites in target genes and discuss their potential role as markers of disease risk, prognosis and treatment response in hematological cancers. Although so far only the tip of the iceberg has been touched, studies of polymorphisms in the miRNA regulatory pathways have already provided some clues for the mechanisms of miRNA dysregulation in cancer and open new perspectives in the management of hematological malignancies.

MicroRNA-binding site polymorphisms in hematological malignancies

Dysregulation of microRNA networks has been implicated in hematological malignancies. One of the reasons for disturbed miRNA-mediated regulation are polymorphisms in miRNA-binding sites (miRSNPs), which alter the strength of miRNA interaction with target transcripts. In the recent years the first findings of miRSNPs associated with risk and prognosis in hematological malignancies have been reported. From the studies described in this review miRSNPs not only emerge as novel markers of risk and prognosis but can also lead to better understanding of the role of miRNAs in regulating gene expression in health and disease.

Cumulative effect of IFIH1 variants and increased gene expression associated with type 1 diabetes

AIMS IFIH1 (Interferon Induced with Helicase C domain 1) gene encodes a sensor of double-stranded RNA, which initiates antiviral activity. Recent studies have indicated the association of rare and common IFIH1 variants with type 1 diabetes mellitus (T1D). The aim of this study was to investigate whether polymorphisms in the IFIH1 locus are a risk factor for T1D in Caucasian patients from Poland. METHODS We genotyped 514 T1D patients and 713 healthy control individuals for rs3747517, rs1990760, rs2111485 and rs13422767 variants. Cumulative genetic risk score (CGRS) was calculated using unweighted and weighted approaches. We also examined the expression of IFIH1 gene in a cohort of 90 T1D patients. RESULTS All studied polymorphisms showed significant association with type 1 diabetes. The risk alleles G of rs3747517, rs2111485, rs13422767 and A of rs1990760 were observed more frequently in T1D group with P values and allelic odds ratio OR (95%CI) < 0.0001, 1.742 (1.428-2.126); 0.001, 1.336 (1.125-1.588); < 0.0001, 1.799 (1.416-2.285); 0.0005, 1.359 (1.144-1.616), respectively. The risk for type 1 diabetes increased with the growing number of the risk alleles. OR (95%CI) for carriers of ≥ 6 risk alleles reached 2.387 (1.552-3.670) for unweighted CGRS and 3.132 (1.928-5.089) for weighted CGRS. Furthermore, IFIH1 gene expression levels in unstimulated peripheral blood mononuclear cells of T1D patients were significantly higher compared to healthy individuals (mean ± SEM mRNA copy number 163.8 ± 15.7 vs. 117.8 ± 7.2; P = 0.046). CONCLUSIONS This study confirms the association of the IFIH1 locus with susceptibility to T1D in the Polish population. The cumulative effect of rs3747517, rs1990760, rs2111485 and rs13422767 variants on type 1 diabetes risk was observed.

Germline variants in MRE11/RAD50/NBN complex genes in childhood leukemia

BackgroundThe MRE11, RAD50, and NBN genes encode proteins of the MRE11-RAD50-NBN (MRN) complex involved in cellular response to DNA damage and the maintenance of genome stability. In our previous study we showed that the germline p.I171V mutation in NBN may be considered as a risk factor in the development of childhood acute lymphoblastic leukemia (ALL) and some specific haplotypes of that gene may be associated with childhood leukemia. These findings raise important questions about the role of mutations in others genes of the MRN complex in childhood leukemia. The aim of this study was to answer the question whether MRE11 and RAD50 alterations may be associated with childhood ALL or AML.MethodsWe estimated the frequency of constitutional mutations and polymorphisms in selected regions of MRE11, RAD50, and NBN in the group of 220 children diagnosed with childhood leukemias and controls (n=504/2200). The analysis was performed by specific amplification of region of interest by PCR and followed by multi-temperature single-strand conformation polymorphism (PCR-MSSCP) technique. We performed two molecular tests to examine any potential function of the detected the c.551+19G>A SNP in RAD50 gene. To our knowledge, this is the first analysis of the MRE11, RAD50 and NBN genes in childhood leukemia.ResultsThe frequency of either the AA genotype or A allele of RAD50_rs17166050 were significantly different in controls compared to leukemia group (ALL+AML) (p<0.0019 and p<0.0019, respectively). The cDNA analysis of AA or GA genotypes carriers has not revealed evidence of splicing abnormality of RAD50 pre-mRNA. We measured the allelic-specific expression of G and A alleles at c.551+19G>A and the statistically significant overexpression of the G allele has been observed. Additionally we confirmed the higher incidence of the p.I171V mutation in the leukemia group (7/220) than among controls (12/2400) (p<0.0001).ConclusionThe formerly reported sequence variants in the RAD50 and MRE11 gene may not constitute a risk factor of childhood ALL in Polish population. The RAD50_rs17166050 variant allele is linked to decreased ALL risk (p<0.0009, OR=0.6358 (95%CI: 0.4854-0.8327)). Despite the fact that there is no splicing abnormality in carriers of the variant allele but an excess of the G over the A allele was consistently observed. This data demonstrate that some specific alternations of the RAD50 gene may be associated with childhood ALL.

Impact of heterozygous c.657-661del, p.I171V and p.R215W mutations in NBN on nibrin functions

Nibrin, product of the NBN gene, together with MRE11 and RAD50 is involved in DNA double-strand breaks (DSBs) sensing and repair, induction of apoptosis and cell cycle control. Biallelic NBN mutations cause the Nijmegen breakage syndrome, a chromosomal instability disorder characterised by, among other things, radiosensitivity, immunodeficiency and an increased cancer risk. Several studies have shown an association of heterozygous c.657-661del, p.I171V and p.R215W mutations in the NBN gene with a variety of malignancies but the data are controversial. Little is known, however, whether and to what extent do these mutations in heterozygous state affect nibrin functions. We examined frequency of chromatid breaks, DSB repair, defects in S-phase checkpoint and radiosensitivity in X-ray-irradiated cells from control individuals, NBS patients and heterozygous carriers of the c.657-661del, p.I171V and p.R215W mutations. While cells homozygous for c.657-661del displayed a significantly increased number of chromatid breaks and residual γ-H2AX foci, as well as abrogation of the intra-S-phase checkpoint following irradiation, which resulted in increased radiosensitivity, cells with heterozygous c.657-661del, p.I171V and p.R215W mutations behaved similarly to control cells. Significant differences in the frequency of spontaneous and ionising radiation-induced chromatid breaks and the level of persistent γ-H2AX foci were observed when comparing control and mutant cells heterozygous for c.657-661del. However, it is still possible that heterozygous NBN mutations may contribute to cancer development.

Ten new ATM alterations in Polish patients with ataxia‐telangiectasia

Inherited biallelic mutations of the ATM gene are responsible for the development of ataxia telangiectasia (AT). The objective of the present study was to conduct molecular analysis of the ATM gene in a cohort of 24 Polish patients with ataxia‐telangiectasia with aim being to provide an updated mutational spectrum in Polish AT patients. As a result of molecular analysis, the status of recurrent mutation was confirmed and ten new ATM variants were detected. Application of MLPA analysis allowed the detection of large genomic deletion. Previously, this type of mutation had never been seen in our population. Finally, in silico analysis was carried out for newly detected ATM alterations. In addition, functional analysis was performed to evaluate the effects of intronic variants: c.3402+30_3402+32delATC.

ZDHHC11 and ZDHHC11B are critical novel components of the oncogenic MYC-miR-150-MYB network in Burkitt lymphoma

ZDHHC11 and ZDHHC11B are critical novel components of the oncogenic MYC-miR-150-MYB network in Burkitt lymphoma

Overexpression of miR-652-5p in new onset type 1 diabetes

Introduction. MicroRNAs (miRNAs) are small noncoding RNA regulating gene expression at the posttranscriptional level. miRNAs have emerged as an important regulators of central and peripheral immune tolerance, therefore study the RNA molecules in the context of type 1 diabetes (T1D) pathogenesis is an important issue. The aim of this study was to investigate miR-652-5p expression level in the new onset T1D and an impact on ADAR and MARCH5, potential target genes. Material and methods. The miR-652-5p expression was investigated in the peripheral blood mononuclear cell of newly diagnosed T1D pediatric patients (n = 28) and age-matched controls (n = 28) by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). miRNA targets were analyzed by luciferase reporter assays. Results. Expression analysis revealed upregulation of miR-652-5p in T1D group compared to non-diabetic controls (p Conclusion. Our study revealed miR-652-5p as potential marker of new onset type 1 diabetes.