Analysis of DUX4 Expression in Bone Marrow and Re-Discussion of DUX4 Function in the Health and Disease.

OBJECTIVE: DUX4 is an embryonic transcription factor (TF) later silenced in somatic tissues, while active in germline testis cells. Re-expression in somatic cells has been revealed to be present in pathologic conditions such as dystrophy, leukemia, and other cancer types. Embryonic cells, cancer cells and testis cells that show DUX4 expression are pluri-multipotent cells. This lead us to question "Could DUX4 be a TF that is active in certain types of potent somatic cells?" As a perfect reflection of the potent cell pool, we aimed to reveal DUX4 expression in the bone marrow. MATERIAL AND METHOD: Bone marrow aspiration materials of seven healthy donors aged between 3 and 32 (2 males/5 females) were investigated with qPCR analysis after RNA isolation for the presence of DUX4 full length mRNA expression. Samples have been investigated for protein existence of DUX4 via immunohistochemistry in two donors that had sufficient aspiration material. RESULTS: DUX4 mRNA expression was present in all donors, with higher expression compared to B-actin. DUX4 positive stained cells were also detected by immunohistochemistry. CONCLUSION: With these results, novel expression for DUX4 in hematopoietic tissue is described. Further studies on the function of DUX4 in hematopoietic cells can shed light on DUX4-related pathways, and contribute to the treatment of DUX4-related diseases such as B-ALL, other cancers, and facioscapulohumeral muscular dystrophy.

Promising Perspective to Facioscapulohumeral Muscular Dystrophy Treatment: Nutraceuticals and Phytochemicals.

Facioscapulohumeral Muscular Dystrophy (FSHD) is in the top three list of all dystrophies with an approximate 1:8000 incidence. It is not a life-threatening disease; however, the progression of the disease extends over being wheelchair bound. Despite some drug trials continuing, including DUX4 inhibition, TGF-ß inhibition and resokine which promote healthier muscle, there is not an applicable treatment option for FSHD today. Still, there is a need for new agents to heal, stop or at least slow down muscle wasting. Current FSHD studies involving nutraceuticals as vitamin C, vitamin E, coenzyme Q10, zinc, selenium, and phytochemicals as curcumin or genistein, daidzein flavonoids provide promising treatment strategies. In this review, we present the clinical and molecular nature of FSHD and focus on nutraceuticals and phytochemicals that may alleviate FSHD. In the light of the association of impaired pathophysiological FSHD pathways with nutraceuticals and phytochemicals according to the literature, we present both studied and novel approaches that can contribute to FSHD treatment.

Evaluation of ETV6/RUNX1 Fusion and Additional Abnormalities Involving ETV6 and/or RUNX1 Genes Using FISH Technique in Patients with Childhood Acute Lymphoblastic Leukemia.

Childhood acute lymphoblastic leukemia (ALL) is the most common type of childhood leukemia. Specifically, ALL is a malignant disorder of the lymphoid progenitor cells, with a peak incidence among children aged 2-5 years. The t(12;21)(p13;q22) translocation occurs in 25 % of childhood B cell precursor ALL. In this study, bone marrow samples were obtained from 165 patients with childhood ALL. We analyzed the t(12;21) translocation and other related abnormalities using the fluorescent in situ hybridization (FISH) technique with the ETV6(TEL)/RUNX1(AML1) ES dual color translocation probe. Conventional cytogenetic analyses were also performed. ETV6 and RUNX1 related chromosomal abnormalities were found in 42 (25.5 %) of the 165 patients with childhood ALL. Among these 42 patients, structural changes were detected in 33 (78.6 %) and numerical abnormalities in 9 (21.4 %). The frequency of FISH abnormalities in pediatric ALL cases were as follows: 8.5 % for t(12;21)(p13;q22) ETV6/RUNX1 fusion, 6.0 % for RUNX1 amplification, 3.0 % for tetrasomy/trisomy 21, 1.8 % for ETV6 deletion, 1.21 % for ETV6 deletion with RUNX1 amplification, 1.21 % for ETV6 amplification with RUNX1 amplification, 0.6 % for polyploidy, 0.6 % for RUNX1 deletion, and 0.6 % for diminished ETV6 signal. The most common structural abnormality was the t(12;21) translocation, followed by RUNX1 amplification and ETV6 deletion, while the most commonly observed numerical abnormality was trisomy 21.

inv (4)(p13q13) in patient with essential thrombocythemia: A case report.

The inv (4)(p13q13) cytogenetic abnormality is uncommon in hematologic malignancies. So far, it has not been previously reported in patients with essential thrombocythemia (ET). We report a first case of ET with inv (4)(p13q13) karyotype in a 69-year-old female patient who developed myelofibrosis at follow up. Conventional cytogenetic analysis from a bone marrow sample showed 46, XX, inv (4)(p13q13) [3]/46, XX [4] at diagnosis and subsequent analysis revealed the same abnormal karyotype during the myelofibrosis phase (46, XX, inv (4)(p13q13) [13]/46, XX [26]). The prognostic significance of this chromosomal abnormality is unknown.

A familial interstitial 4q35 deletion with no discernible clinical effects.

Small deletions on the long arm of distal chromosome 4 do not appear to result in gross congenital malformations, with the most frequently reported clinical findings including mild to moderate intellectual disability, learning disabilities and minor dysmorphic features. Here we report on a cytogenetically detectable familial interstitial chromosome 4 long arm deletion with no discernible phenotypic effects in a mother and her two daughters. The karyotypes of the mother and her two daughters were: 46,XX,del(4)(q35.1q35.2). Based on the results of FISH analyses using whole chromosome specific and subtelomeric probes, the karyotype was designated as: 46,XX,del(4)(q35.1q35.2). ish del(4)(q35-qter)(WCP4+, 36P21+, dJ963K6-). Array-CGH analysis showed an interstitial deletion encompassing 5.75 Mb in the 4q35.1-q35.2 genomic region (chr4:184,717,878-190,469,337; hg19). This is the first report on a cytogenetically detectable familial interstitial chromosome 4 long arm deletion in which there are no discernible phenotypic effects. Both our findings and a review of the literature suggest that more detailed molecular analyses are needed in cases with distal chromosome 4 long arm deletions especially those with breakpoints in the 4q35 region to establish a more precise genotype-phenotype correlation.

Clinical expression of familial Williams-Beuren syndrome in a Turkish family.

Williams-Beuren syndrome (WBS) is a rare genetic disorder characterized by distinctive facial features, intellectual disability with a typical neurobehavioral profile, cardiovascular anomalies, and occasional infantile hypercalcemia. Majority of cases occur sporadically, and only a few cases of familial WBS have been reported. Although pre- and post-natal growth retardation is a common clinical feature of the syndrome, growth hormone deficiency is detected only in a few patients. To our knowledge, there has only been one report about familial Williams-Beuren syndrome in the Turkish population. Here, we report on the three molecular cytogenetically confirmed familial Williams-Beuren syndromes detected in a family with familial short stature. The father, daughter, and son analyzed with clinical and laboratory findings, and reasons of the short stature in Williams-Beuren syndrome are discussed through the literature.

The value of donor lymphocyte infusions in thalassemia patients at imminent risk of graft rejection following stem cell transplantation.

BACKGROUND: The aim was to evaluate the feasibility of donor lymphocyte infusion (DLI) in transplanted patients with thalassemia who were at imminent risk of graft rejection (GR). PROCEDURE: We retrospectively evaluated outcomes in a cohort of 19 patients with thalassemia who received DLI following 21 transplantations. Patients were divided into three groups depending on indication and time of DLI: group I, mixed chimerism-level-3 (MC-level-3) within 2 months and subsequently receiving DLI; group II, MC-level-3 within 2 months and receiving deferred DLI beyond post-transplant 2.5 months; group III, receiving DLI because of a gradual decrease in both donor cells and hemoglobin levels without MC-level-3 within 2 months. RESULTS: Three patients evolved to compete chimerism (16%), 9 patients had MC with transfusion independency (47%) and 7 had GR (37%). Three of 7 patients in group I, 1 of 4 patients in group II and 8 of 10 patients in group III preserved the graft. Although significant increases in the percentage of donor cells were not detected in group III, hemoglobin levels improved (median, 6.8-8.8 g/dl, P = 0.002). CONCLUSION: The risk of GR is high in patients with thalassemia who have MC-level-3 within 2 months after transplantation. DLI is a feasible method for converting unstable MC towards stable MC or full donor chimerism, but its efficacy is partially related to the percentage of residual host cells at the time of infusion. Serial chimerism studies can identify unstable MC earlier and may guide the proper timing of intervention.

Pure and complete 12p trisomy due to a maternal centric fission of chromosome 12.

Pure and complete 12p trisomy are rare. Here, we report on a unique patient with trisomy 12p syndrome due to centric fission of maternal chromosome 12. Conventional cytogenetic and fluorescence in situ hybridization (FISH) techniques revealed the proposita's karyotype to be 47,XX,+fis(12)(p10)mat whereas the maternal one was 47,XX,-12,+fis(12)(p10),+fis(12) (q10). This is the first report on centric fission of chromosome 12 leading to stable telocentrics, each with a fully functional centromere. Our observation shows that the centric fission of chromosome 12 can be a new mechanism for generation of a partial centromere and trisomy 12p syndrome.

Complete gonadal dysgenesis 46,XY (Swyer syndrome) in two sisters and their mother's maternal aunt with a female phenotype.

OBJECTIVE: To present a familial case of Swyer syndrome. DESIGN: Case report. SETTING: Academic medical center. PATIENT(S): Two sisters with a main complaint of primary amenorrhea and another case, their mother's maternal aunt with the same history of primary amenorrhea but married with no consanguinity and no children. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The patients were studied from clinical, endocrinologic, and genetic perspectives. RESULT(S): Chromosome analyses revealed a 46,XY male karyotype with no detectable mosaicism in both sisters and their mother's maternal aunt. Molecular studies of sex-determining region Y and molecular investigation undertaken for the two sisters revealed SRY negativity. CONCLUSION(S): Gonadal dysgenesis can also be inherited as an X-linked disorder, and evidence exists from familial studies of perhaps autosomal inheritance.

Evaluation of PTEN and Mcl-1 expressions in NSCLC expressing wild-type or mutated EGFR.

Signaling pathways activated by epidermal growth factor receptors (EGFRs) are important in lung carcinogenesis. New treatment strategies with EGFR-targeting drugs provided improvements in management of lung cancer. However, molecular mechanisms underlying resistance to these drugs need to be evaluated. Surgically resected samples were obtained from 50 patients with non-small-cell-lung cancer. PTEN, Mcl-1 and EGFR protein expression levels were evaluated by Western-blot. Direct sequencing was performed to investigate EGFR tyrosine kinase domain mutations. We detected c.2235-2249 (pGlu746-Ala750del) mutation in exon 19 in two patients with adenocarcinoma histology. Elevated expression levels of both Mcl-1 isoforms (Mcl-1S and Mcl-1XL) and EGFR proteins were found in 15 (30%) and 23 (46%) of the cases, respectively. Reduced PTEN protein expression levels were observed in 17 (34%) of the cases. PTEN expression level was reduced in 26% of cases that showed increased EGFR expression. Also, increased expression of Mcl-1 protein was observed in 26% of cases with EGFR overexpression. One of the cases harboring pGlu746-Ala750del mutation had increased levels of Mcl-1 and decreased PTEN expression levels. Our results indicate that, in addition to lack of PTEN expression, elevated levels of the Mcl-1 protein might be one of the important intrinsic mechanisms protecting non-small-cell-lung cancer cells from apoptosis induced by several compounds. Therefore, EGFR mutations in conjunction with evaluation of Mcl-1 and PTEN expression levels in large cohorts might provide important clues for improvements of new treatment strategies in non-small-cell-lung cancer management.

Evaluation of eNOS gene polymorphisms in relation to BMD in postmenopausal women.

OBJECTIVE: The aim of the present study was to evaluate the relations between T(-786)C and Glu298Asp polymorphisms of the endothelial nitric oxide synthase (eNOS) gene and BMD in postmenopausal Turkish women. METHODS: The T(-786)C and Glu298Asp polymorphisms were genotyped by PCR-RFLP method in 311 postmenopausal osteoporotic women (OP) and in 305 age-matched postmenopausal females (CG) with normal BMD. RESULTS: None of the SNPs of the eNOS gene was significantly associated with BMD at the lumbar spine, femoral neck, Ward's triangle and femoral trochanter in the combined group. Mean BMD values were therefore found to be similar across the genotypes in postmenopausal Turkish women. However, there was a significant association between the T(-786)C polymorphism and BMD values at the lumbar spine in the normal control group (P=0.005), and at the femoral trochanter in the osteoporotic patients (P=0.046). The mean value of the lumbar spine BMD in the normal controls was significantly higher in women with the TC genotype of the T(-786)C polymorphism than in women with the TT genotype (P=0.0012). Women with the CC genotype of the T(-786)C polymorphism in the osteoporotic patients had significantly higher BMD value at the femoral trochanter than those with the TC (P=0.018) and TT genotypes (P=0.024). Frequencies of the TC heterozygotes for T(-786)C polymorphism were significantly higher among osteoporotic subjects than normal controls. Also, the CC and TT genotype frequencies of control group were significantly higher than those of the osteoporotic group at the femoral neck. CONCLUSIONS: We conclude that, although the biological role of the nitric oxide synthases is well established, our study does not suggest that eNOS gene polymorphisms, T(-786)C and Glu298Asp, are major contributors to adult bone mineral density in the postmenopausal Turkish women.

No association between bone mineral density and transforming growth factor beta gene T(861-20)-C polymorphism in Turkish postmenopausal women.

The aim of the study was to examine whether the TGF-beta1 T(861-20)-C gene polymorphism might be useful in identifying individuals with increased susceptibility to postmenopausal bone loss within the Turkish women population. T(861-20)-C polymorphism was genotyped in 616 postmenopausal women selected from the Turkish population: 311 postmenopausal osteoporotic women (OP) aged 45-65 years (mean age 58 years) and a control group (CG) of 305 postmenopausal women in the same age range (mean age 53 years) with normal bone mineral density. We have not found any significant differences in the frequency of the individual genotypes between the osteoporotic and control groups. The distribution of the T(861-20)-C genotypes was for Lumbar spine, CC, 74.0% in OP, 75.1% in CG; TC, 24.1% in OP, 23.9% in CG; TT, 1.9% in OP, 1.0% in CG; and for femoral neck, CC, 76.8% in OP, 72.8% in CG; TC, 22.1% in OP, 25.5% in CG; TT 1.1% in OP, 1.7% in CG. T(861-20)-C polymorphism was not found to be associated with bone mineral density in postmenopausal Turkish women. It was argued that this will be a pioneering study for the future research and therapies.

Lack of BCL-2 confers interferon-alpha sensitivity to B-cell lymphomas.

Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder with pathological manifestations usually including splenomegaly and pancytopenia. Interferons (IFNs), specifically of the alpha subtypes, have shown a significant anti-tumor effect in HCL patients, with improvement of hematological parameters within the first few months of treatment. However, the therapeutic effect of IFN-alpha is still rather limited. The mechanisms responsible for the beneficial action of IFN-alpha in HCL patients are unclear. A continuous line of cells (Eskol) from a patient diagnosed with HCL was established and shown to have several properties of HCL. Even though, Eskol cells are very resistant to anti-proliferative activity of IFN-alpha, Daudi cells, another human B-cell-derived cell line, are very sensitive to anti-proliferative activity of IFN-alpha and are commonly used as a model cell to test anti-proliferative effect of IFN-alpha. To understand the molecular reason(s) behind the observed obvious differences to IFN sensitivity of above cells, we have analyzed the expression levels of BCL2, caspase-1, Laminin and PARP in these cells. We found that Daudi cells do not express BCL2 at all, and probably because of that, these cells have constantly cleaved, and probably activated form of caspase-1. However, when we over-expressed BCL2 in these cells, they lost processed form of caspase-1 and became resistant to anti-proliferative activity of IFN-alpha. These results let us to suggest that IFN-alpha sensitivity of B-cell lymphomas, once again, depends on the presence or absence of BCL2.

Donor cell-derived acute myeloblastic leukemia after allogeneic peripheral blood hematopoietic stem cell transplantation for juvenile myelomonocytic leukemia.

Despite its rarity, donor cell leukemia (DCL) is a most intriguing entity. We report here the case of a 5 year-old girl with juvenile myelomonocytic leukemia and normal female karyotype who developed acute myeloblastic leukemia with a karyotype of 46, X, t(X; 7) (p21; p11.2), der(7) t(3; 7) (q13.3; q22) 5 months after peripheral blood hematopoietic stem cell transplantation from her HLA-matched sister. We performed the analysis of short tandem repeat sequence markers to DNA obtained from donor peripheral blood, patient's peripheral blood including leukemic blasts and patient's hair root. This analysis showed that the leukemic blood DNA matched the donor blood DNA and not the patient's DNA, thus confirming DCL. To our knowledge, this is the first case of DCL after peripheral blood SCT for juvenile myelomonocytic leukemia.

Existence of acute lymphoblastic leukemia and osteosarcoma in a child.

The existence of acute lymphoblastic leukemia (ALL) and osteosarcoma is described. An 8-year-old girl had osteosarcoma diagnosed on radiologic and pathologic examination during ALL maintenance treatment. Cytogenetic analyses in primary cell culture of osteosarcoma tissue from the patient showed complex chromosomal abnormalities including t(1;19), usually seen in B precursor cell ALL, and del 13, found in a great majority of primary osteosarcomas. To show the possibility of the existence of the genetic susceptibility caused by gene rearrangements, we used molecular technique. But we could not determine any association between gene and genetic susceptibility.