Identification of somatic and germ-line DICER1 mutations in pleuropulmonary blastoma, cystic nephroma and rhabdomyosarcoma tumors within a DICER1 syndrome pedigree.

BACKGROUND: DICER1 syndrome is a pediatric cancer predisposition condition causing a variety of tumor types in children and young adults. In this report we studied a family with two relatives presenting a variety of neoplastic conditions at childhood. METHODS: Germ-line mutation screening of the complete coding region of the DICER1 gene in genomic DNA from the proband was performed. The presence of somatic DICER1 mutation and further alterations in driver genes was investigated in genomic DNA obtained from available tumor samples. RESULTS: A nonsense germ-line mutation in DICER1 causing a truncated protein at the IIIb domain level was identified segregating within a family including two affected relatives who developed in one case cystic nephroma and pleuropulmonary blastoma, and rhabdomyosarcoma and multinodular goiter in the other. Additional in trans DICER1 missense somatic mutations in the IIIb DICER1 domain were found both in the cystic nephroma and in the rhabdomyosarcoma, suggesting that neoplasms in this family might arise from the unusual two-hit mechanism for DICER-derived tumorigenesis in which after the presence of a truncated constitutive protein, a neomorphic DICER1 activity is somatically adquired. Additional genetic alterations, such as TP53 mutations, were identified in the rhabdomyosarcoma. CONCLUSIONS: Besides DICER1 loss of standard activity, oncogenic cooperation of other genes, as mutated TP53, may involve developing higher grade tumors within this syndrome. Given the broad clinical spectrum that may arise, genetic counseling and close surveillance must be offered to all family members at risk of DICER1 syndrome.

Mutational analysis of BRCA1 and BRCA2 in hereditary breast and ovarian cancer families from Asturias (Northern Spain).

BACKGROUND: The prevalence of BRCA1 and BRCA2 mutations in Spain is heterogeneous and varies according to geographical origin of studied families. The contribution of these mutations to hereditary breast and ovarian cancer has not been previously investigated in Asturian populations (Northern Spain). METHODS: In the present work, 256 unrelated high-risk probands with breast and/or ovarian cancer from families living in Asturias were analyzed for the presence of a BRCA1 or BRCA2 gene mutation from October 2007 to May 2012. The entire coding sequences and each intron/exon boundaries of BRCA1/2 genes were screened both by direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: A total of 59 families (23%) were found to carry a pathogenic germ line mutation, 39 in BRCA1 and 20 in BRCA2. Twenty nine additional families (12%) carried an unknown significance variant. We detected 28 distinct pathogenic mutations (16 in BRCA1 and 12 in BRCA2), of which 3 mutations in BRCA1 (c.1674delA, c.1965C>A and c.2900_2901dupCT) and 5 in BRCA2 (c.262_263delCT, c.2095C>T, c.3263dupC, c.4030_4035delinsC, c.8042_8043delCA) had not been previously described.The novel mutations c.2900_2901dupCT in BRCA1 and c.4030_4035delinsC in BRCA2 occurred in 8 and 6 families respectively and clustered in two separated small geographically isolated areas suggesting a founder effect. These 2 mutations, together with the Galician BRCA1 mutation c.211A>G (9 families), and the common BRCA1 mutation c.3331_3334delCAAG (6 families), account for approximately 50% of all affected families. By contrast, very frequent mutations in other Spanish series such as the BRCA1 Ashkenazi founder mutation c.68_69delAG, was found in only one family. CONCLUSIONS: In this study we report the BRCA1 and BRCA2 spectrum of mutations and their geographical distribution in Asturias, which largely differ from other areas of Spain. Our findings may help design a first step recurrent mutation panel for screening high-risk breast and/or ovarian cancer families from this specific area.

Hemophilic Arthropathy of the Knee and Its Association with Reduced Muscle Strength and Activation and the Pressure Pain Threshold: A Case-Control Study.

(1) Background: Hemophilia is characterized by recurrent hemarthrosis leading to degenerative arthropathy. The aim was to evaluate the differences in muscle strength and activity and the pressure pain threshold between patients with knee arthropathy and their healthy peers; (2) Methods: A case-control study in which 23 adult patients with knee arthropathy and 24 healthy peers matched in terms of characteristics were recruited. The study variables were quadriceps muscle strength, muscle activation and the pressure pain threshold; (3) Results: There were significant differences between the two groups in quadriceps strength on the dominant (CI95%: 64.69, 129.2) and non-dominant (CI95%: 29.95, 93.55) sides and in the pressure pain threshold on the dominant (CI95%: 3.30, 43.54) and non-dominant (CI95%: 3.09, 45.25) sides. There were differences in neuromuscular fatigue on the non-dominant side in the vastus medialis (CI95%: 8.72, 21.51), vastus lateralis (CI95%: 4.84, 21.66) and rectus femoris (CI95%: 6.48, 24.95) muscles; (4) Conclusions: Muscle strength and the pressure pain threshold are lower in patients with hemophilia. Quadriceps muscle activation in patients with hemophilic knee arthropathy does not in any way differ from activation in healthy subjects. However, muscle fatigue is greater in patients with knee arthropathy. Strength training in patients with hemophilia should focus on the activation of the vastus medialis and lateralis muscles.

Germ-line mutations in epidermal growth factor receptor (EGFR) are rare but may contribute to oncogenesis: a novel germ-line mutation in EGFR detected in a patient with lung adenocarcinoma.

BACKGROUND: A subset of lung cancer patients harbour EGFR somatic mutations in their tumours and are candidates for treatment with EGFR tyrosine kinase inhibitors. In a few cases EGFR mutations have also been found in the germ line, suggesting a role in lung carcinogenesis. Objetives of this study were: 1) To analyze the EGFR gene mutations in a population diagnosed with lung adenocarcinoma from Northern Spain. 2) To determine the frequency of a new germ-line mutation found in our laboratory as well as the frequency in our population of three other EGFR germ-line mutations detected by other authors. 3) To determine whether the novel mutation detected may have a functional effect on the EGFR protein. METHODS: Tumour DNA samples were obtained from frozen or paraffin embedded tumour tissues. Samples of DNA from peripheral blood cells were obtained from 912 individuals with lung cancer recruited from the CAPUA study 12, 477 unrelated healthy donor individuals and 32 individuals with other types of cancer. EGFR gene exons 18 to 21 were studied by direct standard dideoxy sequencing. Specific mutations were determined either by direct sequencing or by specific RFLP analysis. Cell lines were transfected with EGFR-mutant plasmids and analysed by western blot with antibodies specific for total or phosphorylated-EGFR. RESULTS: We found EGFR mutation in 12 of the 71 tumour samples (17%). One tumour contained two mutations. One mutation (p.R776G) was present as a germ line. Using an RFLP analysis, this mutation was not found in 954 alleles from healthy individuals studied, concluding that it is not a polymorphism. The mutation was not found either in genomic DNA from 912 lung cancer patients. Three additional EGFR germ-line mutations that were already described were not found in any of the studied samples. These observations show that EGFR mutated alleles are rare in the population. In vitro studies revealed that tyrosine autophosphorylation is enhanced in p.R776G-mutant EGFR when compared with wild-type EGFR. This enhanced autophosphorylation in the absence of ligand may be associated with a proliferative advantage. CONCLUSIONS: Germ-line mutations in EGFR are rare but may contribute to oncogenesis.

Residue 826 in the calcium-sensing receptor is implicated in the response to calcium and to R-568 calcimimetic compound.

Within the extracellular loops of the seven-transmembrane domain of the calcium-sensing receptor (CaR) there is a region (I819-E837) relevant for calcimimetic activity. As the naturally occurring variant Ala826Thr is within this important region, it may be postulated that this change may influence the CaR response to calcium and R-568. Human embryonic kidney (HEK-293) cells transiently transfected with three different human CaRs (wild-type [A826], variant allele [T826], and artificial mutant [W826]) were used to test the ability of calcium alone or in combination with the calcimimetic R-568 to modulate CaR activity. CaR activation was detected by flow cytometry using a fluorescent probe. Intracellular calcium changes were measured in response to changes in extracellular calcium alone or with different R-568 concentrations. The change of the alanine in the 826 position (A826) for threonine (T826) worsened calcium sensitivity, increasing the EC(50) value from 2.34 +/- 0.48 mM (A826, wild-type) to 2.96 +/- 0.75 mM (T826) (P < 0.05). The T826 receptor reached a similar response with 1 muM R-568 compared with the wild-type receptor. On the contrary, the artificial introduction of a tryptophan in the same position (W826) did not affect calcium sensitivity (EC(50) = 2.64 +/- 0.81 mM) but reduced the ability of the receptor to respond to R-568. The results demonstrate the importance of the 826 residue in the CaR response to calcium and calcimimetics. Since the A826T change was described as a natural variant, the differences in the calcium and calcimimetic responses observed between the alleles could have potential clinical impact.

Carbohydrate- and conformation-dependent cargo capture for ER-exit.

Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture mechanism, but the signals required for the cargo-receptor interaction are largely unknown. Here, we describe a novel targeting motif that is composed of a high-mannose type oligosaccharide intimately associated with a surface-exposed peptide beta-hairpin loop. The motif accounts for lectin ERGIC-53-assisted ER-export of the lyososomal enzyme procathepsin Z. The second oligosaccharide chain of procathepsin Z exhibits no binding activity for ERGIC-53, illustrating the selective lectin properties of ERGIC-53. Our data suggest that the conformation-based motif is only present in fully folded procathepsin Z and that its recognition by ERGIC-53 reflects a quality control mechanism that acts complementary to the primary folding machinery in the ER. A similar oligosaccharide/beta-hairpin loop structure is present in cathepsin C, another cargo of ERGIC-53, suggesting the general nature of this ER-exit signal. To our knowledge this is the first documentation of an ER-exit signal in soluble cargo in conjunction with its decoding by a transport receptor.

TP53 p.R72P genotype is a marker of poor prognosis in lung cancer.

BACKGROUND: Lung cancer is a leading cause of death worldwide, with poor survival rates despite diagnostic and therapeutic advances. Markers are needed in order to improve clinical patient management and survival. TP53 is frequently involved in lung cancer development with polymorphic sites potentially having a role in it. This study aims to determine the value of codon 72 missense polymorphic variant genotyping, TP53 R72P, as a prognostic factor in NSCLC patients. METHODS: One hundred and fifteen NSCLC samples from patients exposed to tobacco smoke and silica dust from Asturias (Northern Spain) were genotyped by direct sequencing. RESULTS: Seventy-five percent tumour samples alleles coded for Arg. The R72P genotype was an independent predictor of lymph node status (HR = 3.6). The heterozygous genotype was associated to a reduced 5-year survival rate (28% vs 51% for homozygotes). Importantly, this result was specifically observed in these subsets of patients: those over 67 years, patients with silicosis, current smokers, patients with squamous cell carcinomas and, notably, with tumour free lymph nodes. CONCLUSION: Our results indicate a remarkable application of R72P genotyping in the clinical setting: refine patient subclassification to identify those with an adverse clinical course despite tumour free lymph node status.

Impact of PI3K/AKT/mTOR pathway activation on the prognosis of patients with head and neck squamous cell carcinomas.

The PI3K/AKT/mTOR signaling pathway has emerged as one of the most frequently deregulated in head and neck squamous cell carcinomas (HNSCC). Numerous alterations of various upstream and downstream components have been described; however, their prognostic significance and impact on HNSCC patient survival remains to be established. This was addressed using an unbiased cohort of 93 consecutive and homogeneous surgically treated HNSCC patients and results confirmed in 432 HNSCC patients. Our findings reveal the high prevalence of S6 phosphorylation, a surrogate marker of mTORC1 activation, in HNSCC specimens (>70%) and, more importantly, demonstrate its relevance on clinical outcome. Phosphorylation of ribosomal protein S6 on either Ser235/236 or Ser240/244 was consistently and significantly correlated with favorable prognosis, although with differences depending on the tumor site. Thus, p-S6 expression was significantly correlated with better disease-specific survival specifically in the subgroup of laryngeal carcinoma patients (P< 0.001). In addition, multivariate regression models revealed p-S6 to be an inverse and independent predictor of lymph-node metastasis (P= 0.004) and distant metastasis (P= 0.006). Taken together, this study unveils an unprecedented correlation of mTOR activation with improved clinical outcome in patients with laryngeal carcinomas and uncovers the potential of p-S6 expression as a good prognostic biomarker and an inverse predictor of lymph node and distant metastases. These results should be of broad interest as immunohistochemical detection of p-S6 may help to stratify patients and guide treatment decisions.

Response of parathyroid glands to calcitriol in culture: Is this response mediated by the genetic polymorphisms in vitamin D receptor?

BACKGROUND: Recently, we have developed a model of parathyroid tissue culture that allows the study of the response of the parathyroid glands to long-term effectors, such as calcitriol, and that is also useful to study the likely effect of the genetic polymorphisms in the functionality of the glands. The aim of this study was to evaluate the response to calcitriol of cultured parathyroid tissue from patients with secondary hyperparathyroidism (HPT) and the possible effect of vitamin D receptor (VDR) gene polymorphisms on this response. METHODS: Parathyroid glands (N = 37) from 34 parathyroidectomized patients (17 men, 17 women) were used. Several gland fragments were cultured for 60 hours in the presence of calcitriol 10(-9) mol/L or 10(-8) mol/L. DNA from each fragment was extracted to normalize the hormone secretion levels and to genotype the restriction sites ApaI, BsmI, TaqI, and FokI in the VDR gene. RESULTS: The percentages of secretion observed in the response to calcitriol were: 69%+/- 28% (range, 3-100) and 46%+/- 19% (range, 8-78) for calcitriol 10(-9) mol/L and 10(-8) mol/L, respectively (P = 0.004). None of the polymorphisms showed statistical differences in response to calcitriol with any of the concentrations used. CONCLUSION: Parathyroid glands cultured in vitro from patients with secondary HPT are able to respond to calcitriol decreasing PTH synthesis. These results, however, do not support the current hypothesis that VDR polymorphisms are involved in the modulation of the parathyroid gland response.

Influence of polymorphisms in VDR and COLIA1 genes on the risk of osteoporotic fractures in aged men.

BACKGROUND: Osteoporosis in chronic renal failure is a common finding caused by several factors, including age. In the last decade, the likely effect of genetic markers related with the appearance and evolution of osteoporosis has been mainly studied in women, with no categorical results. The aim of this study was to assess the influence of polymorphisms of the vitamin D receptor (VDR) and COLIA1 genes on the risk of osteoporotic fractures in men older than 50 years. METHODS: The study population comprised 156 men, aged 64 +/- 9 (50-86), randomly selected from the population list of Oviedo, Spain. Prevalent vertebral fractures and incident non-vertebral fractures were identified, as well as several genetic polymorphisms. Prevalent vertebral fractures were considered according to the Genant grade 2 classifications. The analyzed genetic polymorphisms were located on restriction sites BsmI (B,b), ApaI (A,a), and TaqI (T,t) in the VDR and on Sp1 (S,s) in COLIA1. RESULTS: Although none of the VDR gene polymorphisms separately analyzed showed any differences between fractured and non-fractured men, the utilization of haplotypes could be employed in order to find osteoporotic fractures in men. By contrast, the COLIA1 polymorphism was associated with osteoporotic fractures. The percentage of prevalent vertebral fractures was significantly higher in the "ss" genotype with respect to the other genotypes. These results show that in men, the "ss" genotype of COLIA1 polymorphism could be the best osteoporotic fracture risk genetic predictor, independent of bone mass values.

Effect of VDR gene polymorphisms on osteocalcin secretion in calcitriol-stimulated human osteoblasts.

BACKGROUND: The impact of vitamin D receptor (VDR) gene polymorphisms in bone metabolism remains controversial. Some authors have found a beneficial effect of some VDR gene polymorphisms, while others found no differences, or even a lower bone mass in subjects with the same type of polymorphisms. The aim of this study was to assess if the VDR gene polymorphisms could have an effect on the calcitriol-stimulated osteocalcin in human osteoblasts. METHODS: Osteoblasts were obtained from human femoral necks replaced because of osteoarthritis. Bones were cut into pieces of 1 to 2 mm and placed in a nylon mesh. After the migration of osteoblasts, the pieces were collected and cultured with different concentrations of calcitriol (10(-8), 10(-9), and 10(-1)0 mol/L). After 48 hours of incubation with calcitriol, the osteocalcin secreted into the medium (corrected by either total proteins or total DNA content) was measured. The DNA was extracted from the osteoblasts, amplified by polymerase chain reaction (PCR), and analyzed for target sequences sites of the BsmI, ApaI, TaqI, and FokI restriction enzymes. RESULTS: The response observed in osteocalcin secretion in the bb or TT genotypes doubled the response observed in the BB or tt genotypes (calcitriol 10(-8) and 10(-9) mol/L). A slight trend was also observed with the aa genotype. Men showed higher levels of osteocalcin secretion than women. Age did not show any influence in osteocalcin secretion. CONCLUSION: VDR alleles and gender demonstrated an effect on the osteocalcin secretion. BB or tt genotypes, and also the "A" allele, showed the lowest calcitriol-stimulated osteocalcin secretion.

Chromosomal aberrations, the consequence of refractory hyperparathyroidism: its relationship with biochemical parameters.

BACKGROUND: It has been shown that refractory hyperparathyroidism (HPT) correlates biologically with a monoclonal true neoplasm, but the chromosomal changes and their relationship with biochemical variables such as high levels of phosphate, low levels of calcium (Ca), and calcitriol deficiency are still in need of a deeper analysis. METHODS: Comparative genomic hybridization was used to scan for DNA copy number changes in two groups of samples: 57 glands from refractory secondary HPT and 28 glands from refractory HPT after kidney transplantation. Biochemical HPT-related parameters from these patients were collected and analyzed. RESULTS: Sixty-one percent of the glands from dialysis patients and 53.6% of the glands from transplanted patients suffering severe secondary hyperparathyroidism had clonal chromosomal imbalances. Losses were far more common than gains. The most recurrent changes were losses of 1p (71%), monosomies of chromosomes 19 and 22 (45%), and losses of 20q (44%) and 16p (42%). The most frequent gains were 5q, 6q, and 13q. Biochemical parameters suggested that Ca excess is related to the development of these chromosomal aberrations, although it is not known if it is by playing a role in producing the alterations or merely as a reflection of HPT severity. Phosphate levels, despite their known effect in increasing the proliferation of the parathyroid glands, were not related to the chromosomal aberrations found in severe secondary HPT. CONCLUSION: Clonal recurrent chromosomal changes are present in more than half of the glands from patients with refractory HPT, which undergo extreme biochemical levels in hyperparathyroidism effectors. These changes support the idea of the monoclonal neoplastic nature of this disorder.

Progression of secondary hyperparathyroidism involves deregulation of genes related to DNA and RNA stability.

BACKGROUND: Renal secondary hyperparathyroidism in its late stages becomes autonomous, so excessive parathyroid hormone (PTH) secretion no longer responds to physiologic stimuli or to aggressive medical treatment. METHODS: To gain molecular understanding of progression of renal secondary hyperparathyroidism, normal and hyperplastic parathyroid tissue with diffuse and nodular growth were analyzed. The results were also compared to parathyroid adenomas. The analysis was performed by high-density oligonucleotide microarray and bidirectional subtraction library. RESULTS: Analysis of the DNA arrays found 16 overexpressed and 132 repressed genes in the nodules while the subtraction library produced 34 overexpressed and 40 repressed genes. The differentially expressed genes between diffuse and nodular samples included some related to DNA stability and repair (TALDO1, PRDX2, DDB1, XRCC1, and POLB), RNA stability and degradation (OASL and AUF1), protein synthesis and processing (PFDN5, HSPD1, and NACA), cell growth (CDC25C and GRPR), and tumorigenesis and cell cycle (VIL2 and TPD52). CONCLUSION: According to the function described for the deregulated genes, when secondary hyperparathyroidism becomes autonomous and refractory to treatment, RNA degradation may be increased while DNA integrity may be compromised. These two mechanisms, combined with deregulation of genes related to growth and differentiation show the complex pathway of parathyroid glands' evolution in renal hyperparathyroidism and may explain the large amount of molecular cytogenetic aberrations found in refractory hyperparathyroidism. Considering that some of the genes with altered expression in nodular hyperplasia lead to irreversible consequences in the genomic integrity of the cells, an adequate and early management of the secondary hyperparathyroidism of chronic kidney disease becomes mandatory.

Structural and enzymatic characterization of Drosophila Dm2-MMP, a membrane-bound matrix metalloproteinase with tissue-specific expression.

We report the isolation and characterization of a cDNA encoding Dm2-MMP, the second matrix metalloproteinase (MMP) identified in the Drosophila melanogaster genome. The cloned cDNA codes for a polypeptide of 758 residues that displays a domain organization similar to that of other MMPs, including signal peptide, propeptide, catalytic, and hemopexin domains. However, the structure of Dm2-MMP is unique because of the presence of an insertion of 214 amino acids between the catalytic and hemopexin domains that is not present in any of the previously described MMPs. Dm2-MMP also contains a C-terminal extension predicted to form a cleavable glycosylphosphatidylinositol anchor site. Western blot and immunofluorescence analysis of S2 cells transfected with the isolated cDNA confirmed that Dm2-MMP is localized at the cell surface. Production of the catalytic domain of Dm2-MMP in Escherichia coli and analysis of its enzymatic activity revealed that this proteinase cleaves several synthetic peptides used for analysis of vertebrate MMPs. This proteolytic activity was abolished by MMP inhibitors such as BB-94, confirming that the isolated cDNA codes for an enzymatically active metalloproteinase. Reverse transcription-PCR analysis showed that Dm2-MMP is expressed at low levels in all of the developmental stages of Drosophila as well as in adult flies. However, detailed in situ hybridization at the larval stage revealed a strong tissue-specific expression in discrete regions of the brain and eye imaginal discs. According to these results, we propose that Dm2-MMP plays both general proteolytic functions during Drosophila development and in adult tissues and specific roles in eye development and neural tissues through the degradation and remodeling of the extracellular matrix.

Cloning of a new familial t(3;8) translocation associated with conventional renal cell carcinoma reveals a 5 kb microdeletion and no gene involved in the rearrangement.

This study describes the molecular cloning of a familial translocation, t(3;8)(p14.2;q24.2), that segregates with the conventional renal cell carcinoma (conventional RCC). We had previously reported the family history and, through loss of heterozygosity and comparative genomic hybridization, detected the loss of the 3p chromosome arm and somatic mutation in the retained von Hippel-Lindau gene in some members of the family. With the help of array painting and sequence tagged site-PCR on flow-sorted derivative chromosomes, we have cloned the breakpoints of the translocation. We have studied the junctions on both derivative chromosomes at the genomic and expression levels. The analysis of the sequence revealed a 5 kb microdeletion at the chromosome 3 breakpoint together with a high density of repetitive motifs (Alu, short interspersed nuclear element) and an AT-rich region. Both chromosome 3 and 8 rearranged regions were very poor in gene content. We tested an expressed sequence tag, two predicted genes, one novel gene and LRIG1, a gene located more than 200 kb apart from the breakpoint on chromosome 3. None of these genes, except LRIG1, showed expression in any of the tested tissues (including normal adult and fetal kidney, sporadic kidney tumours and tumour samples from the proband's family). Taken together, all these data suggest that, rather than deregulation of specific genes that may be rearranged by the translocation, the proposed three-step model of tumour development (translocation, loss of the 3p chromosome, and mutation in a tumour suppressor gene located within that region) could be the biological mechanism that takes place in this familial form of conventional RCC.