Anna Papadopoulou

8q24 Copy number gains and expression of the c‐myc mRNA stabilizing protein CRD‐BP in primary breast carcinomas

The coding region determinant binding protein (CRD‐BP) was isolated by virtue of its high affinity to the c‐myc mRNA coding region stability determinant and shown to shield this message from nucleolytic attack, prolonging its half‐life. CRD‐BP is normally expressed during fetal life but is also activated de novo in tumors. Considering that aberrant CRD‐BP expression may represent an additional mechanism interfering with c‐myc regulation, we screened 118 primary breast carcinomas for CRD‐BP expression, 60 of which had also been analyzed by comparative genomic hybridization (CGH). Copy number gains encompassing 8q24, the chromosome band that contains the c‐myc locus, were detected in 48.3% (29/60) of tumors, whereas gains involving band 17q21, which contains the CRD‐BP locus, were observed in 18.3% (11/60) of tumors. CRD‐BP expression was detected in 58.5% (69/118) of tumors, implying mechanisms of activation alternative to gene amplification. Altogether, some 75% of the tumors had alterations pertaining to c‐myc since they either harbored 8q24 gains and/or expressed CRD‐BP. Significant associations were detected between CRD‐BP expression and the absence of estrogen receptors (p = 0.005) and between the presence of 8q24 gains and an increased number of genomic changes as measured by CGH (p = 0.0017). Tumors were divided into 4 groups according to CRD‐BP expression and 8q24 gains. The odds for tumors having both characteristics to be classified as poorly differentiated (grade III vs. grade I and II) were 19.6 times the corresponding odds for tumors neither expressing CRD‐BP nor harboring 8q24 gains. For tumors either harboring 8q24 gains only or expressing CRD‐BP alone, the corresponding odds were 6.4 and 3, respectively.

Effects of anticonvulsant therapy on vitamin D status in children : Prospective monitoring study

Reports of hypovitaminosis D associated with anticonvulsant drugs in pediatric patients are conflicting. The effects of carbamazepine or sodium valproate on vitamin D status were evaluated prospectively in 51 ambulatory epileptic children who were followed during the first year of the study and in 25 and 6 children during the second and third year, respectively. Serum 25-hydroxyvitamin D, parathyroid hormone, calcium, and phosphorus levels were determined before and every 3 months during anticonvulsant therapy. Our subjects were grouped into four classes (0, 1, 2, and 3 consisted of the patients before and during the first, second, and third years of the treatment, respectively). The control group consisted of 80 healthy children. Comparisons between controls and patients of class 0 for the means for each season of all variables showed no significant differences. A decreasing trend in serum 25-hydroxyvitamin D (P < .03) and an increasing trend in serum parathyroid hormone (P < .04) levels were noticed in all seasons from class 0 to class 3. Twenty-five patients (49%) acquired hypovitaminosis D during the study period. The effects of seasonality on serum 25-hydroxyvitamin D, parathyroid hormone, and calcium were noticed in our patients grouped in classes 0, 2 and 3, as well as in controls. Evidence is provided that carbamazepine or sodium valproate can cause hypovitaminosis D in children. (J Child Neurol 2006; 21:205—209; DOI 10.2310/7010.2006.00050)

Assessment of bone mineral density and markers of bone turnover in children under long-term oral anticoagulant therapy.

Oral anticoagulants antagonize vitamin K action and potentially impair the carboxylation of osteocalcin, a protein essential for normal bone matrix formation. In the present study, bone mineral density (BMD) and bone turnover markers were evaluated in 23 children under long-term oral anticoagulant therapy. BMD of the lumbar spine was assessed (Dual Energy x-ray Absorptiometry) and reported as z score. Osteoblast [bone alkaline phosphatase, osteocalcin (Gla-Oc), amino-terminal procollagen 1 extension peptide] and osteoclast (urinary calcium and deoxypyridinoline, serum cross-linked C telopeptide) activity markers were measured. Vitamin D {[25(OH) D], parathormone, calcium, phosphorus, magnesium} and vitamin K status [factors II, VII, IX, X, protein C, protein S, undercarboxylated osteocalcin (Glu-Oc)] were determined. The above parameters were also evaluated in 25 healthy controls. Patients presented with higher levels in Glu-Oc, parathormone, and bone resorption markers, lower levels in bone formation markers and 25(OH) D, whereas 52% of them showed signs of osteopenia (−1> BMD z score >−2.5). Statistical analysis demonstrated that anticoagulant therapy was an independent predictor of alterations in Glu-Oc, Gla-Oc, bone alkaline phosphatase, amino-terminal procollagen 1 extension peptide, and serum cross-linked C telopeptide levels. It seems that long-term use of coumarin derivatives may cause osteopenia in children with the risk of developing osteoporosis later in life.

Cytogenetic profile of unknown primary tumors: clues for their pathogenesis and clinical management.

Unknown primary tumors (UPTs) represent an entity of great clinical and biological interest, whose origin cannot be determined even after medical workup. To better understand their pathogenesis by outlining their genetic composition, 20 UPTs were investigated by G-banding, supplemented with Fluorescence In Situ Hybridization and Comparative Genomic Hybridization analyses. The data obtained were sufficient to reach a diagnosis in five cases-four lymphomas and one Ewing sarcoma-demonstrating that in a subset of UPTs, cytogenetics can be an adjunct for differential diagnosis. In the remaining 15 UPTs, an aggressive cytogenetic pattern was revealed. The most frequently rearranged chromosome regions were 1q21, 3p13, 6q15-23, 7q22, 11p12-5, and 11q14-24, pinpointing gene loci probably associated with the peculiar pathogenesis of UPTs. The preferential involvement of 4q31, 6q15, 10q25, and 13q22 in adenocarcinomas (whereas 11q22 is involved in the rest of the carcinomas)-in addition to the marked divergence in the mean average of chromosomal changes, 16 and 3, respectively-demonstrates genotypic differences between the two histologic subgroups. Furthermore, the significantly shorter survival in cases displaying massive chromosome changes compared with those having a few changes indicates that the cytogenetic pattern might be used as a tool to assess prognosis in UPTs, even without the detection of their primary site.

Low TSH levels are not associated with osteoporosis in childhood

INTRODUCTION A recent study on TSH receptor (TSHR) null mice suggested that skeletal loss occurring in hyperthyroidism is caused by the low TSH rather than high thyroid hormone levels. The aim of this study was to examine whether low TSH results in osteoporosis in the human. SUBJECTS AND METHODS We determined bone mineral density (BMD) and markers of bone metabolism in two male siblings aged 9.8 and 6.8 years with isolated TSH deficiency, due to a mutation of the TSH beta-subunit gene. BMD was measured in the lumbar spine (L1-L4) by dual-energy X-ray absorptiometry. Laboratory investigation included the determination of serum calcium, phosphate, 25-hydroxy-vitamin D, parathyroid hormone concentrations, and urine calcium (Ca)/creatinine (Cr) ratio. Osteoblast activity was measured by serum bone alkaline phosphatase and osteocalcin levels, and osteoclast activity by urine cross-linked amino-terminal, carboxy-terminal telopeptides of type I collagen and deoxypyridinoline concentrations. RESULTS BMD of both patients was within the normal range for age and sex; z-scores were -0.55 and -0.23 for patients 1 and 2 respectively. Serum calcium, phosphate, urine Ca/Cr ratio, and specific markers of bone metabolism were also within normal range. CONCLUSION In childhood, chronic extremely low TSH levels, in the face of normal thyroid hormone levels, are not related to bone loss.

Genome Profiling of Breast Cancer Cells Selected Against In Vitro Shows Copy Number Changes

About 20% of breast carcinomas show no clonal chromosome abnormalities when analyzed after short‐term culturing. An interesting question is whether this subset of breast carcinomas really is karyotypically normal or if selection for normal cells occurred in vitro. To address this issue, 26 breast carcinomas that had shown no cytogenetic changes by chromosome banding analysis were examined by comparative genomic hybridization (CGH), a technique that does not require culturing or tumor metaphase cells. All but one case showed copy number changes by CGH (median, four). A comparison of these findings with those of a karyotypically abnormal series analyzed using the same CGH protocol found that the cytogenetically “normal” cases were typically genetically less complex (median, four and eight, respectively; P = 0.0058). Although largely the same alterations were found in both series, some differences with respect to the frequencies of specific imbalances were seen. Gains of 3p and 6q and losses of 10q, 14q, and 17p more often were found in the cytogenetically abnormal series than in the normal tumors. We conclude that in most instances cells found to be normal by chromosome banding analysis after short‐term culture do not belong to the tumor parenchyma. Furthermore, when we compared the distribution of the number of imbalances detected by CGH in the total data set according to the mitotic index in vivo (scored from 1 to 3), the median values were three, seven, and 18, respectively (P < 0.001). These data indicate not only that karyotypically normal breast carcinomas may represent a genetically simpler subgroup that grows poorly in vitro but also that this subset of tumors already has a slow growth rate in vivo.

Calcium and Vitamin D Metabolism in Hypocalcemic Vitamin D-Resistant Rickets Carriers

Background/Aims: Hypocalcemic vitamin D-resistant rickets (HVDRR) is a rare monogenic autosomal recessive disorder associated with mutations in the gene of the vitamin D receptor (VDR), the mediator of 1,25(OH)2D3 action. Although many investigations have discussed the clinical manifestations and molecular etiology of this disease, only a few have investigated the biochemical and hormonal status of heterozygous HVDRR. The aim of the current work was to investigate the profile of selected biochemical and hormonal parameters related to the vitamin D endocrine system in a large number of HVDRR heterozygotes. Methods: 67 relatives of 2 HVDRR patients, all members of an extended Greek kindred of five generations with a common ancestor, were included in the study. Direct sequencing was used to identify VDR gene mutations. Serum Ca, P, 25(OH)D, iPTH, and 1,25(OH)2D levels were determined in all members of the kindred. Results: DNA analysis of the participants led to the design of two study groups: the HVDRR carriers (24) and the control subjects (43). Our results showed elevated circulating serum levels of 1,25(OH)2D3 and lower levels of PTH than their age- and sex-matched controls. No hypocalcemia or hypophosphatemia were detected in HVDRR carriers. Conclusions: Our findings suggest that HVDRR carriers may have compensatory elevated serum levels of 1,25(OH)2D3 through which they restrain PTH secretion. The study of HVDRR carriers could be a useful tool for the investigation of the vitamin D endocrine system.

Association of vitamin D with adiposity measures and other determinants in a cross-sectional study of Cypriot adolescents

OBJECTIVE To assess vitamin D status among Cypriot adolescents and investigate potential determinants including BMI and body fat percentage (BF%). DESIGN Participants had cross-sectional assessments of serum vitamin D, physical activity, dietary vitamin D intake and sun exposure. Linear and logistic regression models were used to explore the associations of vitamin D with potential predictors. SETTING Hospitals, Cyprus, November 2007-May 2008. SUBJECTS Adolescents (n 671) aged 16-18 years. RESULTS Mean serum vitamin D was 22·90 (sd 6·41) ng/ml. Only one in ten children had sufficient levels of vitamin D (≥30 ng/ml), while the prevalence of vitamin D deficiency (12-20 ng/ml) and severe deficiency (<12 ng/ml) was 31·7 % and 4·0 %, respectively. Lower vitamin D was associated with winter and spring season, female gender, reduced sun exposure in winter and darker skin. Participants with highest BMI and BF% when compared with a middle reference group had increased adjusted odds of vitamin D insufficiency (OR = 3·00; 95 % CI 1·21, 7·45 and OR = 5·02; 95 % CI 1·80, 13·97, respectively). A similar pattern, although not as strong, was shown for vitamin D deficiency with BF% (OR = 1·81; 95 % CI 1·04, 3·16) and BMI (OR = 1·51; 95 % CI 0·85, 2·67). Participants in the lowest BMI and BF% groups also displayed compromised vitamin D status, suggesting a U-shaped association. CONCLUSIONS Vitamin D deficiency in adolescence is very prevalent in sunny Cyprus, particularly among females, those with darker skin and those with reduced sun exposure in winter. Furthermore, vitamin D status appears to have a U-shaped association with adiposity measures.

Combined 22q11.1-q11.21 deletion with 15q11.2-q13.3 duplication identified by array-CGH in a 6 years old boy

BackgroundDeletions of chromosome 22q11 are present in over 90% of cases of DiGeorge or Velo-Cardio-Facial syndrome (DGS/VCFS). 15q11-q13 duplication is another recognized syndrome due to rearrangements of several genes, belonging to the category of imprinted genes. The phenotype of this syndrome varies but has been clearly associated with developmental delay and autistic spectrum disorders. Co-existence of the two syndromes has not been reported so far.ResultsHere we report a 6-year-old boy presenting growth retardation, dysmorphic features and who exhibited learning difficulties. Fluorescence in situ hybridization (FISH) analysis of the proband revealed a deletion of DiGeorge Syndrome critical region (TUPLE). Array-CGH analysis revealed an interstitial duplication of 12 Mb in size in the area 15q11.2-q13.3, combined with a 3.2 Mb deletion at region 22q11.1-q11.21. FISH analysis in the mother showed a cryptic balanced translocation between chromosome 15 and chromosome 22 (not evident by classic karyotyping).DiscusionThe clinical manifestations could be related to both syndromes and the importance of array-CGH analysis in cases of unexplained developmental delay is emphasized. The present case further demonstrates how molecular cytogenetic techniques applied in the parents were necessary for the genetic counseling of the family.

Telomerase Activity and Genetic Alterations in Primary Breast Carcinomas

It has been proposed that the structural and numerical chromosome abnormalities recorded in breast cancer could be the result of telomere dysfunction and that telomerase is activated de novo to provide a survival mechanism curtailing further chromosomal aberrations. However, recent in vivo and in vitro data show that the ectopic expression of telomerase promotes tumorigenesis via a telomere length-independent mechanism. In this study, the relation between telomerase expression and the extent of chromosomal aberrations was investigated in 62 primary breast carcinomas. Telomerase activity was measured using a polymerase chain reaction-based telomeric repeat amplification protocol assay and 92% of the tumors were found to express telomerase with a relative activity ranging from 0 to 3839.6. Genetic alterations were determined by G-banding and comparative genomic hybridization analysis and 97% of the tumors exhibited chromosomal aberrations ranging from 0 to 44 (average: 10.98). In the overall series, the relationship between telomerase activity levels and genetic changes could be best described by a quadratic model, whereas in tumors with below-average genetic alteration numbers, a significant positive association was recorded between the two variables (coefficient=0.374, P=.017). The relationship between telomerase activity levels and the extent of genetic alteration may reflect the complex effect of telomerase activation upon tumor progression in breast carcinomas.