Extracellular matrix and growth factors in the pathogenesis of some craniofacial malformations.

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.

Lung regions differently modulate bronchial branching development and extracellular matrix plays a role in regulating the development of chick embryo whole lung.

Normal branching development is dependent on the correlation between cells and extracellular matrix. In this interaction glycosaminoglycans, cytokines and growth factors play a fundamental role. In order to verify the distribution and influence of extracellular matrix and related enzymes on chick embryo lung development, 6 day-old whole lungs were maintained in vitro with testicular hyaluronidase, beta-N-acetyl-D-glucosaminidase and chondrotinase ABC or in linkage with apical, medial and caudal lung regions of 6-day development before and after enzyme treatment. In a separate lung region beta-N-acetyl-D-glucosaminidase and hyaluronidase were determined. Our data show that the whole lung cultures increase bronchial branching development when the medial region is admixed separately, while the separate apical or caudal regions or apical combined with caudal region do not affect bronchial branching development. The enzyme treatment of medial region prevents the branching development in associated whole lung. The bronchial branching development of whole lung cultured in medium containing the enzymes related to glycosaminoglycans turnover is significantly altered. In conclusion, these data show that the different influence of separate apical, medial, caudal lung regions on bronchial branching development is related to the extracellular matrix composition.

FHIT oncosuppressor gene expression profile in human anal cancers.

The FHIT gene, a member of the histidine triad gene family, is a tumor suppressor gene exhibiting deletions in the majority of human cancers. Aberrant transcripts of this gene have been found in about 50% of esophageal, stomach and colon carcinomas. Little is known about the molecular mechanisms involved in malignant transformation of the lining cells of the anus. In this study FHIT gene expression was investigated in this particular kind of human cancer. FHIT expression was comparatively analyzed at the mRNA level, by RT-PCR, in squamous anal cancers, normal anal tissue and peripheral blood samples. cDNA analyses showed variability in FHIT transcripts, without apparent effects on the predicted amino acid sequence. These different FHIT mRNAs could represent transcripts from an alternative splicing event. Our data indicate that the FHIT mRNA detected in anal cancers and in normal samples is heterogeneous. Immunohistochemical data suggest that the Fhit protein is expressed only in a fraction of the tumor cells, while it is strongly expressed in the epithelial cells of glands of the normal anal mucosa. The absence or poor expression of the Fhit protein in anal cancers suggests a role for this tumor suppressor gene product, as a risk factor, in the onset of this human cancer, as reported before for other human gastrointestinal tumors.

Study of the PVRL1 gene in Italian nonsyndromic cleft lip patients with or without cleft palate.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a complex genetic trait and little is known about its aetiology. Recent investigations on rare clefting syndromes provided interesting clues about genes involved in face development. The PVRL1 gene encodes nectin1, a cell-to-cell adhesion molecule. Mutations in its sequence have been shown to cause the rare autosomal recessive syndrome CL/P-ectodermal dysplasia syndrome (CLPED1), while heterozygosity for the mutation W185X seemed to increase the risk of non syndromic CL/P in a population from northern Venezuela. In the present study, we screened 143 Italian CL/P patients for mutations in PVRL1. Three rare sequence variants in exon 3 that create amino-acid changes were detected in a total of 7 patients. Two of these mutations were not found in a panel of 292 unaffected controls, while the third was found in two controls. This study describes new mutations that may represent genetic risk factors for CL/P. Even though a study to look at the effects of the mutations on nectin1 function was not feasible, supporting evidence was reported, thus confirming the involvement of PVRL1 in the aetiology of non-syndromic CL/P malformation.

Gene expression profile analysis in human T lymphocytes from patients with Down Syndrome.

Down Syndrome (DS) is caused by the presence of three copies of the whole human chromosome 21 (HC21) or of a HC21 restricted region; the phenotype is likely to have originated from the altered expression of genes in the HC21. We apply the cDNA microarray method to the study of gene expression in human T lymphocytes with trisomy 21 in comparison to normal cells. Two patients with DS were investigated, along with two normal subjects as a control, all being tested in independent, duplicated cell culture experiments. The most consistent finding was the overexpression of the superoxide dismutase gene (SOD1), located on 21q, and of MHC DR beta 3 (HLA-DRB3), GABA receptor A gamma 2 (GABRG2), acetyltransferase Coenzyme, A 2 (ACAT2) and ras suppressor protein 1 (RSU1) genes. When the data were clustered according to chromosome localization, the HC21 gene set showed, on average, the highest expression in DS cells in all the experiments. Moreover, separate clustering of patients and controls was obtained when analysis was restricted to HC21 gene expression values. These findings reinforce the specific gene dosage theory for the pathogenesis of the DS phenotype, and show a consistent overexpression of the SOD1 gene on 21q.

Maternal MTHFR variant forms increase the risk in offspring of isolated nonsyndromic cleft lip with or without cleft palate.

The pathogenesis of cleft lip with or without cleft palate (CL/P) is complex; its onset could be due to the interaction of various genetic and environmental factors. Recently MTHFR functional polymorphisms were found to increase the risk of this common malformation; however, this finding is still debated. We investigated 110 sporadic CL/P patients, their parents and 289 unrelated controls for c.665C>T (commonly known as 677C>T; p.Ala222Val) and c.1286A>C (known as 1298A>C; p.Glu429Ala) polymorphism in the MTHFR gene. Transmission disequilibrium test (TDT) showed no distortion in allele transmission. Nevertheless, association studies revealed significant differences in allele frequencies between mothers of CL/P patients and controls. This work supports the hypothesis that a lower MTHFR enzyme activity in pregnant women, mostly related to the c.665C>T variant form, is responsible for a higher risk of having CL/P affected offspring.

GeneRecords: a relational database for GenBank flat file parsing and data manipulation in personal computers.

UNLABELLED: Extracting the desired data from a database entry for later analysis is a constant need in the biological sequence analysis community; GeneRecords 1.0 is a solution for GenBank biological flat file parsing, as it implements a structured representation of each feature and feature qualifier in GenBank following import in a common database managing system usable in a personal computer (Macintosh and Windows environments). This collection of related databases enables the local management of GenBank records, allowing indexing, retrieval and analysis of both information and sequences on a personal computer. AVAILABILITY: The current release, including the FileMaker Pro runtime application (built for Windows and Macintosh environments), is freely available at http://apollo11.isto.unibo.it/software/

Bronchial branching correlates with specific glycosidase activity, extracellular glycosaminoglycan accumulation, TGF beta(2), and IL-1 localization during chick embryo lung development.

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), beta-N-acetyl-D-glucosaminidase activity, the presence of transforming growth factor beta(2) (TGF beta(2)), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of beta-N-acetyl-D-glucosaminidase with biochemical methods. Our data show that beta-N-acetyl-D-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that beta-N-acetyl-D-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.

Interactions of fibroblasts with soldered and laser-welded joints.

Relatively little is known about the biocompatibility of the soldered or laser-welded joints of dental appliances. We investigated the reaction of human gingival fibroblasts cultured in vitro in direct contact with samples of soldered and laser-welded joints from orthodontic lingual arches. Contrast phase light microscopy was used to evaluate cell adhesion, morphology and proliferation after 6 and 24h and after 7 and 16 days. Scanning electron microscopy (SEM) was performed at 16 days. Our in vitro findings provide evidence that laser-welded orthodontic appliances have superior fibroblast biocompatibility.

Genetic expression profiling of six odontogenic tumors.

Odontogenic tumors are rare neoplasms arising from the odontogenic apparatus. We aimed to identify molecular characteristics associated with odontogenic tumorigenesis and malignancy. To this end, we investigated the expression level of human genes by using, for the first time in odontogenic tumors, the technique of expression profiling. Gene expression alterations common to all six odontogenic tumors were identified by the use of cDNA microarrays containing 19,000 human cDNAs. Statistical analysis on a subset of 4974 cDNAs present in the biopsies identified 506 distinct genes associated with the tumors (p-value < 0.01). Gene ontology analysis of the cellular processes which were differentially regulated in odontogenic tumors was accomplished by the use of a subset of 1409 annotated genes. Finally, 43 cDNAs differentiated the three malignant odontogenic tumors (ameloblastic carcinoma, clear cell odontogenic tumor, granular cell odontogenic tumor) from the three benign ameloblastoma biopsies (p < 0.01). The identified genes might help us better classify borderline odontogenic tumors.

Classification of oral clefts by affection site and laterality: a genotype-phenotype correlation study.

OBJECTIVES: The aim of this study was to classify the phenotypes found in a series of patients with non-syndromic cleft lip (CL) with or without cleft palate (CP) and isolated cleft palate. Additionally, the frequency distribution of cases belonging to families linked to markers on chromosomes 6 and 2 within these phenotypic patterns were estimated. DESIGN: A retrospective examination of all the available affected cases collected in Italy. SETTING AND SAMPLE POPULATION: Ninety-seven affected subjects aged 5-18 years belonging to 38 families were considered. Patterns were identified by variance of the cleft (lip, primary palate, secondary palate) and stratified according to the side of occurrence (right, left, or bilateral). Latent class analysis was used as main statistical tool for carrying out the results. RESULTS: Three homogenous classes were identified (P < 0.0001) by means of latent class analysis. Individuals were assigned to the most suited class. All three variables (lip, primary and secondary cleft palate) generated a specific class. Optimal findings were reported in cases having 'any isolated cleft lip' (class 1); 'secondary CP with or without bilateral/right primary cleft palate + bilateral/right cleft lip' (class 2); and 'left primary cleft palate + left/bilateral cleft lip with or without secondary CP' (class 3). Correspondence to the evidence of linkage to chromosome 6 showed that 9 of 10 cases presenting with 'right primary CP + right CL with secondary cleft palate' (class 2) belonged to a linked family. The same combination, but occurring on the left side (class 3), revealed that only three of nine cases belong to families linked to chromosome 6 (P-value = 0.02). The two patterns (right and left) never occurred in the same family. Three reliable groups were identified based on laterality and the presence of a cleft. A single right sided pattern displayed a statistically different distribution of linkage to chromosome 6 when compared with the homologous left side. CONCLUSION: Non-syndromic CL with/without CP can be classified according to laterality that can be under genetic control.

Glycosidases during chick embryo lung development and their colocalization with proteoglycans and growth factors.

During development, the epithelial component of the lung goes through a complex orderly process of branching, following strict patterns of space and time. Proteoglycans, glycosaminoglycans and growth factors are fundamental components of the extracellular matrix and perform a key role in differentiative processes. The embryonic chick lung shows a specific glycosaminoglycan composition at different levels of branching and at different embryonic stages. Proteoglycan and glycosaminoglycan accumulation is the result of secretion, absorption and degradation processes. In this pathway, enzymes, such as glycosidases, growth factors and cytokines are involved. We examined the behaviour of glycosidases, such as beta-hexosaminidases (beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-D-galactosaminidase), beta-glucuronidase and beta-galactosidase, during the development of the lung bud. Our data show that the activity of the enzymes is closely linked to the processes of epithelial proliferation, bronchial tubule lengthening and infiltration of the surrounding mesenchyme. The glycosaminoglycans colocalize with transforming growth factor beta2 and interleukin-1 in the basement membrane and in the mesenchymal areas where the epithelium grows, and are complementary to the presence of the glycosidases. In conclusion, the activity of these glycosidases is spatially and temporally programmed and favors the release of the factors and the events which they influence.

Expression profile of epidermal differentiation complex genes in normal and anal cancer cells.

Anal cancer originates from a peculiar histological region and provides a useful model for investigating alterations in proliferation and/or differentiation of neoplastic keratinocytes. Epidermal differentiation complex (EDC) genes, which form one of the major gene clusters in the human genome, are involved in the terminal differentiation of epithelial cells and in many instances have been implicated in epithelial tumours. We constructed a DNA macroarray capable of characterising the expression profiles of the entire EDC gene complex in normal mucosa and anal cancer biopsies of seven unrelated patients. Brain tissue and cultured keratinocytes were used as controls. All anal cancer samples showed expression profiles in which none of the EDC genes was silent, as evaluated by phosphor-imager analysis. Variance analysis showed significantly lower expression of SPRR2 with respect to SPRR1 or SPRR3, and significantly higher expression of S100A8 than of other S100A subfamily members. At hierarchical clustering analysis, the four basaloid anal cancer cases conglomerated in the top five positions. The macroarray method used by us provides the first demonstration of the expression profile of the EDC gene family in anal cancer, and is capable of producing significant information on the subgrouping of epithelial tumours such as anal cancer.

Epithelial-mesenchymal interactions and lung branching morphogenesis. Role of polyamines and transforming growth factor beta1.

Lung branching morphogenesis is a result of epithelial-mesenchymal interactions, which are in turn dependent on extracellular matrix composition and cytokine regulation. Polyamines have recently been demonstrated as able to modify chick embryo skin differentiation. In this work we have examined the effects of putrescine and spermidine during chick embryo lung morphogenesis in organotypic cultures by morphological, histochemical and biochemical examination. To verify the role of polyamines, we used specific inhibitors, such as bis-cyclohexylammonium sulphate and alfa-difluoromethylornithine, and transforming growth factor beta1, an ornithine decarboxylase and polyamine stimulator. Our data show that lung morphogenesis is significantly altered following the induced mesenchymal glycosaminoglycan changes. The increase of mesenchymal glycosaminoglycans is correlated with a stimulation of lung development in the presence of polyamines, and with its inhibition when transforming growth factor beta1 is added to the culture medium. The morphometric data show a uniform increase of both the mesenchyme and epithelial branching with spermidine and putrescine stimulus, whereas the mesenchymal substance alone is significantly increased in apical-median lung sections with transforming growth factor beta1 and transforming growth factor beta1 + spermidine lung cultures. Transforming growth factor beta1 and transforming growth factor beta1 + spermidine confirm the blocking of epithelial branching formations and fibroblast activation, and show that polyamines are unable to prevent the blocking of epithelial cells due to the inhibitory effect of transforming growth factor beta1.

Lack of mutations of type 1 11beta-hydroxysteroid dehydrogenase gene in patients with abdominal obesity.

There is increasing evidence that in human obesity, particularly the abdominal phenotype, the activity of the hypothalamic-pituitary-adrenal (HPA) axis is disregulated. At least two distinct alterations have been reported: one is characterized by several neuroendocrine abnormalities and hyperresponsiveness of the HPA axis to different neuropeptides, the other is characterized by elevated cortisol traffic and probably by supranormal cortisol production. The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes interconvert cortisol and cortisone in human. Two different isoforms have been identified. A possible modification of the activity of the enzyme 11beta-HSD1 in subjects with abdominal obesity has been described in the literature. We decided to test the hypothesis that mutated isoforms of type 11beta-HSD1 protein could be responsible for alterations of cortisol metabolism in patients with abdominal obesity. A mutational screening of the whole coding sequence and exon-flanking regions of the 11B-HSD1 gene has been performed in 8 patients. The main results of our study are the exclusion of a common association of 11beta-HSD1 mutations to obesity and the identification of two novel allelic variants for the gene 11beta-HSD1 in the Italian population, not previously described in any database.

C677T variant form at the MTHFR gene and CL/P: a risk factor for mothers?

Maternal folic acid supplementation in early pregnancy has been suggested to play a role in the prevention of nonsyndromic orofacial cleft, i.e., cleft lip with or without cleft palate (CL/P). Moreover, some authors demonstrated association of the C-->T mutation (C677T), converting an alanine to a valine residue in 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, with other congenital anomalies such as neural tube defects (NTDs). Because of MTHFR's involvement in the metabolism of folate, we investigated 64 CL/P patients and their parents for C677T MTHFR mutation. No linkage disequilibrium was found using the transmission disequilibrium test (TDT). However, a significantly higher mutation frequency was detected in mothers of CL/P patients compared to controls. The odds ratios calculated for mothers having CT or TT genotype, compared to the normal CC genotype, were 2.75 (95% confidence interval 1.30-5.57) and 2.51 (1.00-6.14), respectively. These results support the involvement of the folate pathway in the etiology of CL/P, and indicate an effect of the maternal genotype, rather than influence of the embryo's genotype.

Linkage analysis of three candidate regions of chromosome 1 in nonsyndromic familial orofacial cleft.

Linkage analysis and mouse model knockout studies indicate that loci/genes mapping in different chromosome 1 regions are good candidates for nonsyndromic orofacial cleft (OFC) malformation. On this basis, three different regions of the chromosome 1 have been analysed, by linkage analysis, in 38 families with nonsyndromic OFC. Positive scores were obtained by pairwise analysis and a non-parametric linkage approach for the 1p36 region, with markers close to the MTHFR locus. Additional results allowed us to exclude the presence of an OFC susceptibility gene in the 1q21 and 1q32-42.3 regions.

Survival analysis of transfused fetuses affected by Rh-alloimmunization.

The aim of the present study was to evaluate the survival rate of a group of 86 fetuses affected by Rh-alloimmunization submitted to intrauterine red blood cell transfusion. All the women had antibody titres> or = 1:32 at the time of their enrollment in the study. Crude fetal survival rate was 89.5% (77/86 cases). Data were stratified according to specific cut-off points of (1) pre-transfusion fetal haemoglobin expressed as the rate between the observed and the estimated value for each gestational age at the time of the first transfusion; (2) the difference between the haemoglobin at the beginning of the second-transfusion less that at the end of the first transfusion (delta haemoglobin); and (3) presence of ultrasound detected anomalies. Statistically significant stratification of the survival rate was observed for the level of pre-transfusion fetal haemoglobin (95% and 76.9%, respectively, p= 0.009) using a cut-off value of < 70% and > or = 70% of the expected value. Again, delta haemoglobin showed a different survival rate when a cut-off value of 6 g/dl was used to generate subgroups of fetuses: 94.6% and 80%, respectively (p= 0.0145). Among the ultrasound anomalies, the presence of hydrops showed a correlation with the survival rates. The quoted values were 97.83% (absence) and 80.0% (presence) respectively (p= 0.0058). Cox regression showed a significant association of the studied variables with the outcome (survival). The presence of hydrops was the best predictor (Odds ratio= 8.7, p= 0.0073) followed by Delta haemoglobin (Odds ratio= 2.0, p= 0.0422). The rate of pre-treatment fetal haemoglobin < 70% of the expected value did not add any significant valu and was thus removed from the final model. Weight at delivery expressed in grams showed a direct correlation with the survival rate (Odds ratio= 0.9, p= 0.1529) and was added into the model as an adjustment quantitative variable.

Role of the extracellular matrix and growth factors in skull morphogenesis and in the pathogenesis of craniosynostosis.

The complex and largely obscure regulatory processes that underlie ossification and fusion of the sutures during skull morphogenesis are dependent on the conditions of the extracellular microenvironment. The concept that growth factors are involved in the pathophysiology of craniosynostosis due to premature fusion of skull bone sutures, is supported by recent genetic data. Crouzon and Apert syndromes, for example, are characterized by point mutations in the extracellular or transmembrane domains of fibroblast growth factor-2 receptor. In primary cultures of periosteal fibroblasts and osteoblasts obtained from Apert and Crouzon patients, we observed that Crouzon and Apert cells behaved differently with respect to normal cells as regards the expression of cytokines and extracellular matrix (ECM) macromolecule accumulation. Further modulation of ECM components observed after the addition of cytokines provides support for an autocrine involvement of these cytokines in Crouzon and Apert phenotype. Changes in ECM composition could explain the altered osteogenic process and account for pathological variations in cranial development. We suggest that a correlation exists between in vitro phenotype, clinical features and genotype in the two craniosynostotic syndromes. New research into signal transduction pathways should establish further connections between the mutated genotype and the molecular biology of the cellular phenotype.

The murine DSCR1-like (Down syndrome candidate region 1) gene family: conserved synteny with the human orthologous genes.

A recently recognized gene family, conserved from yeast to humans, includes Down syndrome candidate region 1 gene (DSCR1), Adapt78 (recognized as the hamster ortholog of the DSCR1 isoform 4), ZAKI-4 (renamed DSCR1-like 1, DSCR1L1) and DSCR1L2 (a novel gene on human chromosome 1), along with yeast and C. elegans single members (Strippoli P., Lenzi L., Petrini M., Carinci P., Zannotti M., 2000. A new gene family including DSCR1 (Down Syndrome Candidate Region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member. Genomics 64, 252-263). The proposed family labels were a putative single-strand nucleic acid binding domain similar to the RNA recognition motif, and a unique, highly-conserved serine-proline motif. We have used a bioinformatics-driven molecular biology approach to characterize the murine members of DSCR1-like gene family. Systematic expressed-sequence-tags (EST) database search and reverse-transcription polymerase chain rection (RT-PCR) product sequencing allowed identification of the murine DSCR1, DSCR1L1 and DSCR1L2. The sequences of the respective protein products are of 198, 197 and 241 amino acids, respectively, and are very similar to the corresponding human proteins. The very broad expression pattern of the murine DSCR1 genes is similar to that of the human genes. Using a radiation hybrid panel, we mapped the murine DSCR1-like family members. The murine DSCR1 ortholog is located on the chromosome 16, in a region corresponding to that on human chromosome 21 just upstream of the Down syndrome candidate region. DSCR1L1 and DSCR1L2 murine genes are also located in chromosomal segments of chromosome 17 and 4, respectively, exactly corresponding to those containing the respective human homologs on chromosomes 6 and 1. Description of the mouse orthologs for DSCR1-like genes will allow knockout mice to be obtained for specific family members.