Kristiina Heikinheimo

Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B

Although targeted gene disruption of GDF-9, an oocyte derived growth factor, leads to an arrest of folliculogenesis and causes infertility in female mice, little is known on the expression of GDF-9 protein in the ovary. We show that GDF-9 protein is expressed in rat oocytes during folliculogenesis from the early primary follicle stage onwards but the most intensive immunostaining was seen in primary and preantral follicles. Northern blot analyses of the ontogeny of GDF-9 gene expression in postnatal rat ovaries showed that the GDF-9 transcript levels are clearly increased on the second postnatal day concomitant with the appearance of primary follicles. Interestingly, Northern blot and in situ hybridization analyses indicate a similar expression pattern for GDF-9B, the rat ortholog of a mouse GDF-9 like factor for which we recently reported the partial amino acid sequence. The polypeptide sequences deduced from isolated ovarian cDNAs indicate that the rat GDF-9 prepropeptide is 440 amino acids (aa) in length and the putative mature peptide is 135 aa whereas rat GDF-9B is 391 aa long and the mature region is 125 aa. We conclude that (1) the GDF-9 protein is highly expressed in the oocytes of primary follicles of rat ovaries suggesting that it plays a role mainly in early folliculogenesis and that (2) the full-length polypeptide sequence of GDF-9B suggests that this novel TGF-beta family member is likely to be a secreted growth factor that may regulate folliculogenesis at similar developmental stages as GDF-9.

Gene Expression Profiling of Ameloblastoma and Human Tooth Germ by Means of a cDNA Microarray

The molecular and genetic characteristics of ameloblastoma are still poorly understood. We analyzed gene expression in fresh-frozen ameloblastomas and human fetal tooth germs, using a cDNA microarray. Thirty-four genes exhibited significant changes in expression levels in the ameloblastoma. Eleven genes were overexpressed more than three-fold, and 23 genes were underexpressed to below 0.4 of the control level. The oncogene FOS was the most overexpressed gene (from eight- to 14-fold), followed by tumor-necrosis-factor-receptor 1 (TNFRSF1A). Genes for sonic hedgehog (SHH), TNF-receptor-associated-factor 3 (TRAF3), rhoGTP-ase-activating protein 4 (ARHGAP4), deleted in colorectal carcinoma (DCC), cadherins 12 and 13 (CDH12 and 13), teratocarcinoma-derived growth-factor-1 (TDGF1), and transforming growth-factor-ß1 (TGFB1) were underexpressed in all tumors. In selected genes, a comparison between cDNA microarray and real-time RT-PCR confirmed similar relative gene expression changes. The gene expression profile identifies candidate genes that may be involved in the origination of ameloblastoma and several genes previously unidentified in relation to human tooth development.

Stage-specific Expression of Decapentaplegic-Vg-related Genes 2, 4, and 6 (Bone Morphogenetic Proteins 2, 4, and 6) During Human Tooth Morphogenesis

Members of the decapentaplegic-Vg-related (DVR) gene family are diffusible signaling molecules regulating inductive tissue interactions during vertebrate development. Expression of DVR/bone morphogenetic protein (BMP) 2, 4, and 6 was studied in human fetal teeth. Sequential morphogenetic stage-specific studies of DVR/BMP 2 and 4 mRNA expression by in situ hybridization revealed transcripts for DVR/BMP 4 during compaction of the dental mesenchyme. In contrast, DVR/BMP 2 mRNA appeared later during tooth development and was located in differentiated cells (odontoblasts). These results were confirmed by reverse-transcription polymerase chain reaction (RT-PCR), which detected DVR/BMP 2 and 4 mRNA in human tooth-germ samples. DVR/BMP 6 protein was distributed in the early dental epithelium and, later, in pre-odontoblasts and odontoblasts, where it remained during dentin formation. These results suggest that DVR/BMP 4 is involved in the early tooth morphogenesis. DVR/BMP 6 may, in particular, be implicated inepithelial-mesenchymalinteractionscontrolling cytodifferentiation. DVR/BMP 2 and 6 may also be involved in odontoblast secretory function. The results suggest that members of the DVR gene family may play regulatory roles during human tooth development.

High frequency of BRAF V600E mutations in ameloblastoma

Ameloblastoma is a benign but locally infiltrative odontogenic neoplasm. Although ameloblastomas rarely metastasise, recurrences together with radical surgery often result in facial deformity and significant morbidity. Development of non‐invasive therapies has been precluded by a lack of understanding of the molecular background of ameloblastoma pathogenesis. When addressing the role of ERBB receptors as potential new targets for ameloblastoma, we discovered significant EGFR over‐expression in clinical samples using real‐time RT–PCR, but observed variable sensitivity of novel primary ameloblastoma cells to EGFR‐targeted drugs in vitro. In the quest for mutations downstream of EGFR that could explain this apparent discrepancy, Sanger sequencing revealed an oncogenic BRAF V600E mutation in the cell line resistant to EGFR inhibition. Further analysis of the clinical samples by Sanger sequencing and BRAF V600E‐specific immunohistochemistry demonstrated a high frequency of BRAF V600E mutations (15 of 24 samples, 63%). These data provide novel insight into the poorly understood molecular pathogenesis of ameloblastoma and offer a rationale to test drugs targeting EGFR or mutant BRAF as novel therapies for ameloblastoma. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

Compound Heterozygous Desmoplakin Mutations Result in a Phenotype with a Combination of Myocardial, Skin, Hair, and Enamel Abnormalities

Desmoplakin (DP) anchors the intermediate filament cytoskeleton to the desmosomal cadherins and thereby confers structural stability to tissues. In this study, we present a patient with extensive mucocutaneous blisters, epidermolytic palmoplantar keratoderma, nail dystrophy, enamel dysplasia, and sparse woolly hair. The patient died at the age of 14 years from undiagnosed cardiomyopathy. The skin showed hyperplasia and acantholysis in the mid- and lower epidermal layers, whereas the heart showed extensive fibrosis and fibrofatty replacement in both ventricles. Immunofluorescence microscopy showed a reduction in the C-terminal domain of DP in the skin and oral mucosa. Sequencing of the DP gene showed undescribed mutations in the maternal and paternal alleles. Both mutations affected exon 24 encoding the C-terminal domain. The paternal mutation, c.6310delA, leads to a premature stop codon. The maternal mutation, c.7964 C to A, results in a substitution of an aspartic acid for a conserved alanine residue at amino acid 2655 (A2655D). Structural modeling indicated that this mutation changes the electrostatic potential of the mutated region of DP, possibly altering functions that depend on intermolecular interactions. To conclude, we describe a combination of DP mutation phenotypes affecting the skin, heart, hair, and teeth. This patient case emphasizes the importance of heart examination of patients with desmosomal genodermatoses.

Genomic profiles and CRTC1–MAML2 fusion distinguish different subtypes of mucoepidermoid carcinoma

Mucoepidermoid carcinoma is the most common salivary gland malignancy, and includes a spectrum of lesions ranging from non-aggressive low-grade tumors to aggressive high-grade tumors. To further characterize this heterogeneous group of tumors we have performed a comprehensive analysis of copy number alterations and CRTC1–MAML2 fusion status in a series of 28 mucoepidermoid carcinomas. The CRTC1–MAML2 fusion was detected by RT-PCR or fluorescence in situ hybridization in 18 of 28 mucoepidermoid carcinomas (64%). All 15 low-grade tumors were fusion-positive whereas only 3 of 13 high-grade tumors were fusion-positive. High-resolution array-based comparative genomic hybridization revealed that fusion-positive tumors had significantly fewer copy number alterations/tumor compared with fusion-negative tumors (1.5 vs 9.5; P=0.002). Twelve of 18 fusion-positive tumors had normal genomic profiles whereas only 1 out of 10 fusion-negative tumors lacked copy number alterations. The profiles of fusion-positive and fusion-negative tumors were very similar to those of low- and high-grade tumors. Thus, low-grade mucoepidermoid carcinomas had significantly fewer copy number alterations/tumor compared with high-grade mucoepidermoid carcinomas (0.7 vs 8.6; P<0.0001). The most frequent copy number alterations detected were losses of 18q12.2-qter (including the tumor suppressor genes DCC, SMAD4, and GALR1), 9p21.3 (including the tumor suppressor genes CDKN2A/B), 6q22.1-q23.1, and 8pter-p12.1, and gains of 8q24.3 (including the oncogene MAFA), 11q12.3-q13.2, 3q26.1-q28, 19p13.2-p13.11, and 8q11.1-q12.2 (including the oncogenes LYN, MOS, and PLAG1). On the basis of these results we propose that mucoepidermoid carcinoma may be subdivided in (i) low-grade, fusion-positive mucoepidermoid carcinomas with no or few genomic imbalances and favorable prognosis, (ii) high-grade, fusion-positive mucoepidermoid carcinomas with multiple genomic imbalances and unfavorable prognosis, and (iii) a heterogeneous group of high-grade, fusion-negative adenocarcinomas with multiple genomic imbalances and unfavorable outcome. Taken together, our studies indicate that molecular genetic analysis can be a useful adjunct to histologic scoring of mucoepidermoid carcinoma and may lead to development of new clinical guidelines for management of these patients.

Cell proliferation and chromosomal changes in human ameloblastoma

Cell proliferation and chromosomal imbalances, important parameters in relation to tumor progression, were studied in ameloblastoma (n=20), a benign odontogenic tumor of locally recurrent nature. Immunocytochemical staining with MIB-1 antibody and comparative genomic hybridization (CGH) were performed on formalin-fixed paraffin-embedded ameloblastomas. The mean follow-up time was 12.4 years. An MIB-1-index was formed by counting 5000 tumor-cell nuclei in 10-15 randomly chosen high-power fields and calculating percentages of positively stained cells. CGH involved hybridization of FITC-dUTP-labeled tumor DNA with Texas-red-labeled normal DNA. Images were digitally analyzed. The MIB-1-index (range 0-2.51) was low for all tumors. No statistically significant correlation between MIB-1 index and tendency to recurrence was found. Chromosomal aberrations were detected in 2 of 17 cases. The results suggest that formation of an MIB-1 index is not helpful in assessing future clinical behavior of an ameloblastoma and that chromosomal imbalances are uncommon.

Regeneration of bone and periodontal ligament induced by recombinant amelogenin after periodontitis.

Regeneration of mineralized tissues affected by chronic diseases comprises a major scientific and clinical challenge. Periodontitis, one such prevalent disease, involves destruction of the tooth‐supporting tissues, alveolar bone, periodontal‐ligament and cementum, often leading to tooth loss. In 1997, it became clear that, in addition to their function in enamel formation, the hydrophobic ectodermal enamel matrix proteins (EMPs) play a role in the regeneration of these periodontal tissues. The epithelial EMPs are a heterogeneous mixture of polypeptides encoded by several genes. It was not clear, however, which of these many EMPs induces the regeneration and what mechanisms are involved. Here we show that a single recombinant human amelogenin protein (rHAM+), induced in vivo regeneration of all tooth‐supporting tissues after creation of experimental periodontitis in a dog model. To further understand the regeneration process, amelogenin expression was detected in normal and regenerating cells of the alveolar bone (osteocytes, osteoblasts and osteoclasts), periodontal ligament, cementum and in bone marrow stromal cells. Amelogenin expression was highest in areas of high bone turnover and activity. Further studies showed that during the first 2 weeks after application, rHAM+ induced, directly or indirectly, significant recruitment of mesenchymal progenitor cells, which later differentiated to form the regenerated periodontal tissues. The ability of a single protein to bring about regeneration of all periodontal tissues, in the correct spatio‐temporal order, through recruitment of mesenchymal progenitor cells, could pave the way for development of new therapeutic devices for treatment of periodontal, bone and ligament diseases based on rHAM+.

Distribution of extracellular matrix proteins in odontogenic tumours and developing teeth.

SummaryThe distribution of two cellular fibronectins (cFn), tenascin, laminin, as well as type VII collagen was studied in 14 benign odontogenic tumours of epithelial (ameloblastoma) and epithelial-ectomesenchymal (ameloblastic fibroma) origins, as well as in developing human teeth by immunocytochemical means using monoclonal antibodies (Mabs). An extradomain sequence-A-containing form of cFn (EDA-cFn) was seen in the extracellular matrix (ECM) of all tumours studied and in the mesenchyme of the developing tooth germs, indicating that cFn in these tissues are predominantly produced locally. A form of cFn containing an oncofetal domain (Onc-cFn), hitherto found only in carcinomas, was detected focally in the stroma of most ameloblastomas but was absent from ameloblastic fibromas and tooth germs. Tenascin was strongly expressed in the basement membrane (BM) zone of all odontogenic tumours and in that of the early tooth germs. Focal absence of laminin and type VII collagen from the BM of some ameloblastomas and the presence of Onc-cFn in the ECM of most ameloblastomas may correlate with their aggressive behaviour. The results also suggest that EDA-cFn and tenascin are involved in epithelial-mesenchymal interactions during tooth development and in odontogenic tumours.

Analysis of cervical ribs in a series of human fetuses

In humans, an increasing body of evidence has linked the frequency of cervical ribs to stillbirths, other malformations and early childhood cancers. However, the frequency of cervical ribs in a putatively healthy fetal population is not sufficiently known to assess the actual medical risks of these prenatal findings. We therefore analyzed the presence of skeletal anomalies in a series of 199 electively aborted fetuses, which were whole‐mount stained with alizarin red specific for skeletal tissues. Results show that approximately 40% of the fetuses had cervical ribs, even though external congenital abnormalities such as craniofacial and limb defects were absent. A literature overview indicates that the observed frequency of cervical ribs is comparable to results previously obtained for deceased fetuses with no or minor congenital anomalies, and higher than expected for healthy fetuses. This unexpected result can probably in part be explained by a higher detection rate of small cervical ribs when using alizarin red staining instead of radiographs. Additionally, studies in the literature suggest that the size of a cervical rib may indicate the severity of abnormalities, but this possibility requires further research. Anomalies of the axial skeleton are known to be caused by a disturbance of early development, which alters Hox gene expression, but in this study the origin of the stress could not be verified as maternal medical data were not available. The co‐occurrence of rudimentary or absent 12th ribs in 23.6% of the cases with cervical ribs indicates that in approximately 8% of the fetuses a homeotic shift occurred over a larger part of the vertebral column. This suggests that the expression of multiple Hox genes may have been affected in these fetuses. Together, the high incidence of cervical ribs and also their co‐occurrence with rudimentary or absent 12th ribs suggests that there may have been a disturbance of early development such that the studied fetuses are probably not informative about the general population. Future studies determining the frequency of cervical ribs in a more healthy fetal population are therefore needed to evaluate their potential as an indicator of medical risks.