Candidate gene studies in hypodontia suggest role for FGF3.

INTRODUCTION: The majority of tooth agenesis cases are mild (hypodontia) and typically not associated with the gene mutations linked to oligodontia. From this, we hypothesise that most cases of tooth agenesis fit a polygenic mode of inheritance, where several genes with small effects cause a variety of varying phenotypes. MATERIALS AND METHODS: In this study, we looked at 18 not typically studied genes in this condition, to ascertain their contribution to hypodontia. Our study subjects consisted of 167 patients with hypodontia and their parents from two cohorts (one from Brazil and one from Turkey). An additional 465 DNA samples (93 cases with hypodontia and 372 controls without family history for tooth agenesis or oral clefts) from Brazil were also available for this study. Ninety-three single nucleotide polymorphisms that maximally represent the linkage disequilibrium structure of the genes for the 18 genes were selected and genotyped using Taqman chemistry. Chi square was used to test if genotype distributions were in Hardy-Weinberg equilibrium, and 24 markers that were in Hardy-Weinberg equilibrium and had allele frequencies higher than 5 % in a panel of 50 CEPH samples were further tested. Association between hypodontia and genetic variants was tested with the transmission disequilibrium test within the programme Family-Based Association Test (FBAT) and by using Chi square and Fisher's exact tests. Alpha at a level of 0.05 was used to report results. RESULTS: Results suggest possible associations between several genes and hypodontia in the three populations. In the Turkish cohort (n = 51 parent-affected child trios) the most significant results were as follows: FGF3 rs1893047, p = 0.08; GLI3 rs929387, p = 0.03; GLI3 haplotype rs929387-rs846266, p = 0.002; and PAX9 rs2073242, p = 0.03. In the Brazilian cohort (n = 116 parent-affected child trios), the results were as follows: DLX1 rs788173, p = 0.07; FGF3 rs12574452, p = 0.03; GLI2 rs1992901, p = 0.03; and PITX2 rs2595110, p = 0.01. The second Brazilian cohort also suggested that FGF3 (rs12574452, p = 0.01) is associated with hypodontia and added EDAR (rs17269487, p = 0.04), LHX6 (rs989798, p = 0.02), and MSX1 (rs12532, p = 0.003). CONCLUSION: Our results suggest that several genes are potentially associated with hypodontia and their individual contributions may be modest. Hence, these cases may not be explained by inactivating mutations such as many oligodontia cases segregating in a Mendelian fashion but rather are influenced by one or more susceptibility alleles in multiple small effect genes.

Msx1 mutations: how do they cause tooth agenesis?

Mutations in the transcription factors PAX9 and MSX1 cause selective tooth agenesis in humans. In tooth bud mesenchyme of mice, both proteins are required for the expression of Bmp4, which is the key signaling factor for progression to the next step of tooth development. We have previously shown that Pax9 can transactivate a 2.4-kb Bmp4 promoter construct, and that most tooth-agenesis-causing PAX9 mutations impair DNA binding and Bmp4 promoter activation. We also found that Msx1 by itself represses transcription from this proximal Bmp4 promoter, and that, in combination with Pax9, it acts as a potentiator of Pax9-induced Bmp4 transactivation. This synergism of Msx1 with Pax9 is significant, because it is currently the only documented mechanism for Msx1-mediated activation of Bmp4. In this study, we investigated whether the 5 known tooth-agenesis-causing MSX1 missense mutations disrupt this Pax9-potentiation effect, or if they lead to deficiencies in protein stability, protein-protein interactions, nuclear translocation, and DNA-binding. We found that none of the studied molecular mechanisms yielded a satisfactory explanation for the pathogenic effects of the Msx1 mutations, calling for an entirely different approach to the investigation of this step of odontogenesis on the molecular level.

Genes affecting tooth morphogenesis.

The development of dentition is a fascinating process that encompasses a complex series of epithelial-mesenchymal interactions involving growth factors, transcription factors, signal receptors and other soluble morphogens. It is not surprising that such a complex process is prone to disturbances and may result in tooth agenesis. Initial discoveries indicating that the homeo-domain protein MSX1 and the paired-domain transcription factor PAX9 are causative genes in tooth morphogenesis were made in mice. Both genes are co-expressed in dental mesenchyme and either one, when homozygously deleted, results in an arrest at an early developmental stage. Previous studies have shown a down regulation of Bmp4 gene expression in Pax9 and Msx1 single mutant mice. Therefore, we chose to explore the molecular relationship between Pax9, Msx1 and Bmp4. In humans, unlike in mice, a heterozygous mutation in either PAX9 or MSX1 suffices to cause tooth agenesis of a predominantly molar or more premolar pattern, respectively. Our laboratory and others have identified several PAX9 and MSX1 mutations in families with non-syndromic forms of autosomal dominant posterior tooth agenesis. We have also identified families with tooth agenesis in whom PAX9 and MSX1 mutations have been excluded opening up the possibilities for the discovery of other genes that contribute to human tooth agenesis.

Genes affecting tooth morphogenesis.

The development of dentition is a fascinating process that encompasses a complex series of epithelial-mesenchymal interactions involving growth factors, transcription factors, signal receptors and other soluble morphogens. It is not surprising that such a complex process is prone to disturbances and may result in tooth agenesis. Initial discoveries indicating that the homeo-domain protein MSX1 and the paired-domain transcription factor PAX9 are causative genes in tooth morphogenesis were made in mice. Both genes are co-expressed in dental mesenchyme and either one, when homozygously deleted, results in an arrest at an early developmental stage. Heterozygous Pax9 or Msx1 mice have normal teeth, however, double heterozygous Pax9/Msx1 mice show a phenotype of arrested tooth development which can be rescued by transgenic expression of Bmp4, a very influential signaling factor in many developmental processes. We have obtained mounting evidence for a partnership between PAX9 and MSX1 within the tooth-specific Bmp4 signaling pathway. In humans, unlike in mice, a heterozygous mutation in either PAX9 or MSX1 suffices to cause tooth agenesis of a predominantly molar or more premolar pattern, respectively. Our laboratory and others have identified several PAX9 and MSX1 mutations in families with non-syndromic forms of autosomal dominant posterior tooth agenesis. We have also identified families with tooth agenesis in whom PAX9 and MSX1 mutations have been excluded opening up the possibilities for the discovery of other genes that contribute to human tooth agenesis.

Sequence diversity in genes of lipid metabolism.

Elevated plasma lipoprotein levels play a crucial role in the development of coronary artery disease. Genetic factors strongly influence the levels of plasma lipoproteins, but the genes and sequence variations contributing to the most common forms of dyslipidemias are not known. We used GeneChip probe arrays to resequence the coding regions of 10 key genes of lipid metabolism. The sequences of these genes were analyzed in 80 dyslipidemic individuals. Fourteen nonsynonymous and twenty-two synonymous single nucleotide changes were identified that could be confirmed by conventional sequencing. Seven of the fourteen nonsynonymous sequence variants were polymorphisms with allele frequency >1% in the general population. The remaining seven were not found in normolipidemic controls (25 Caucasians and 25 African-Americans). The relationship between nonsynonymous sequence variations and various dyslipidemias was explored in association and family studies. No evidence was found for coding sequence variations in any of the 10 genes contributing to dyslipidemia. Only a single sequence variation, a missense mutation in the low density lipoprotein receptor gene, co-segregated with hyperlipidemia in the proband's family. This study illustrates some of the difficulties associated with identifying sequence variations contributing to complex traits.

Hemorrhagic problems in obstetrics, exclusive of disseminated intravascular coagulation.

During pregnancy many physiologic changes occur that result in an increase in coagulation factors and a decrease in fibrinolytic activity. Because hemorrhage during pregnancy is a major cause of maternal morbidity, it is important to recognize and understand the pathophysiology of hereditary and acquired bleeding disorders. This article reviews von Willebrand's disease types 1, 2, and 3 and acquired hemophilia.

Prognostic value of K-ras mutations, ras oncoprotein, and c-erb B-2 oncoprotein expression in adenocarcinoma of the lung.

This trial was undertaken to determine the prognostic role of K-ras (p21), c-erb B-2 (p185) protein expression, and the presence or nonpresence of a K-ras gene mutation in patients with adenocarcinoma of the lung. This was a retrospective study of 103 patients with adeno- or large-cell carcinoma of the lung who had available paraffin-stored tumor material. The relation of several clinical variables to survival was analyzed. Immunohistochemical techniques were used to determine expression of p21 and p185. Polymerase chain reaction (PCR) and sequencing were used to determine K-ras mutation status. Tumor stage was the only nonmolecular clinical variable predictive of survival (p=0.0001). A combination of K-ras mutation and p 185 expression (p=0.0144), ras mutation and strong p21 expression (p=0.0137), and K-ras mutation and the combined expression of p21 and p185 were predictive of poor survival (p=0.0415) in univariate analysis of all patients. The sole presence of K-ras mutation was predictive of survival. Additionally, when combined with elevated p21 or p185 expression in a subset of patients with 4 or more years of follow-up, negative correlation with survival was observed.

Irinotecan hydrochloride (CPT-11) resistance identified by K-ras mutation in patients with progressive colon cancer after treatment with 5-fluorouracil (5-FU).

OBJECTIVE: To determine the prognostic role of a K-ras mutation in tumor tissue of patients with refractory colon cancer who received irinotecan hydrochloride (CPT-11). METHODS: DNA was extracted from paraffin-stored tumor tissue of 35 patients with progressive colon cancer failing treatment with 5-fluorouracil who subsequently received CPT-11 (100 mg/m2 i.v. per week x 4 weeks with 2 weeks off per course). The first exon of the K-ras gene was amplified by polymerase chain reaction by using K-ras-specific primers followed by mutant enrichment sequencing. Survival differences of patients with a K-ras mutation were compared with those of patients with a normal K-ras status. RESULTS: A total of 21 patients had a normal K-ras sequence and 14 patients had a K-ras mutation [GAT, n = 7; TGT, n = 3; and GCT, AGT, GTT, GAC (codon 13), n = 1 each]. Median survival of patients with a normal ras sequence from time of treatment with CPT-11 was 332 days compared with 169 days for patients with a K-ras mutation (p = 0.0036). No differences in age, sex, cancer stage, surgical treatment, or chemotherapy treatment were observed. CONCLUSION: Determination of the presence of a K-ras mutation may predict survival in patients with progressive colon cancer after treatment with 5-fluorouracil who receive CPT-11.

Human hair follicles as a peripheral source of tyrosine hydroxylase and aromatic L-amino acid decarboxylase mRNA.

Total RNA from human hair follicles was reverse transcribed and amplified using primers specific for aromatic L-amino acid decarboxylase (AADC) and tyrosine hydroxylase (TH). Agarose electrophoresis of the amplified products showed reverse transcription polymerase chain reaction (RT-PCR) products of the expected size for both TH and AADC. Sequencing showed that the amplified products matched the known sequences of TH and AADC. This study identifies hair follicles as a convenient, uninvasive, source of the mRNA for TH and AADC. Analysis of these mRNA's may be useful in the diagnosis and investigation of conditions resulting from qualitative changes in the genes that code for these enzymes.

Chemosensitization of human hepatocellular carcinoma cells with cyclosporin A in post-liver transplant patient plasma.

We previously showed that combined neoadjuvant doxorubicin (DOX) treatment and orthotopic liver transplantation produced a 3-year tumor-free survival rate of 54% in stage II-IVa nonresectable hepatocellular carcinomas (HCCs). These patients received posttransplant immunosuppressive doses of cyclosporin A (CsA). CsA has been shown to modify the function of a membrane P-glycoprotein (Pgp) whose overexpression is associated with a multidrug-resistant (MDR1) phenotype. This study utilized HCC cell lines to characterize the in vitro chemomodulatory properties of CsA as found in posttransplant patient plasma to consider the hypothesis that CsA may prolong posttransplant survival by enhancing the therapeutic efficacy of DOX against multidrug-resistant hepatoma cells. We characterized Pgp expression in the HCC lines Hep3B, Hep G2, and SK-HEP-1 by immunohistochemistry and the reverse transcription-polymerase chain reaction. The combined cytotoxicity of DOX + CsA was examined by [3H]thymidine uptake and flow cytometric drug-retention assays. Pgp expression was assessed further after prolonged (10-day) treatment with CsA. Hep3B and Hep G2 cells expressed low to moderate levels of Pgp. The effective DOX dose required for inhibiting MDR1(+) Hep3B and Hep G2 cell proliferation by 50% (DOX IC50) was 44.5 ng/ml and 43.5 microgram/ml, as compared with 10.7 ng/ml for Pgp-negative SK-HEP-1 cells. Optimal concentrations of CsA (0.8 micrometer) lowered DOX IC50 for Hep3B cells and Hep G2 cells by 6-fold and 4-fold, respectively. Similarly, plasma from patients containing immunosuppressive levels of CsA lowered DOX IC50 of the MDR1(+) Hep G2 cells by up to 4-fold. Prolonged exposure to CsA did not affect its chemosensitizing capacity or Pgp expression of HCC cells. PSC-833, a nonimmunosuppressive analogue of CsA, was equally effective in reducing the DOX IC50 of MDR1(+) HCC cells. CsA and PSC-833 increased drug retention by approximately 75%, but did not significantly affect hepatoma cell viability or Pgp expression. Pharmacological concentrations of cyclosporin analogues, including one nonimmunosuppressive form, enhance DOX cytotoxicity of MDR1(+) HCC cells by modulating drug retention. CsA as found in posttransplant patient plasma enhanced DOX cytotoxicity to human MDR1(+) hepatoma cells in vitro, albeit at less than optimal chemosensitizing concentrations. Prolonged exposure to CsA did not affect its chemosensitizing properties or block Pgp expression of HCC cells. These findings support our hypothesis that in vivo immunosuppressive levels of CsA may enhance DOX chemotherapeutic efficacy on MDR1(+) HCC cells.

Alternative splicing in the coding region of human aromatic L-amino acid decarboxylase mRNA.

Total RNA from human neuroblastoma cells (SK-N-SH) was reverse transcribed and amplified using primers specific for aromatic L-amino acid decarboxylase (AADC). Two polymerase chain reaction (PCR) products were observed following agarose electrophoresis. Cycle sequencing of the PCR products revealed the larger fragment (414 bp) to be identical to the published human cDNA sequence (Type I). Sequencing of the smaller band (300 bp) demonstrated a form missing exon three (Type II). Both types of the mRNA were colocalized in human brain regions (gray matter and white matter) and other human tissues (liver, kidney, adipose, heart, adrenal gland and keratinocytes). The relative concentrations varied in each tissue studied but specific neuronal or non-neuronal patterns were not apparent. The study demonstrates alternative splicing within the coding region of the human AADC mRNA and the results suggest the possibility that two proteins are derived from the AADC gene in human tissues.

Anti-CD40 antibody binding modulates human multiple myeloma clonogenicity in vitro.

Ligand binding of the B-cell lineage antigen CD40 enhances growth and interleukin-6 (IL-6) secretion in human B cells (the CD40/IL-6 loop). IL-6 has an autocrine and paracrine role in human multiple myeloma (MM) cell growth. With the use of the CD40 monoclonal antibody (MoAb) G28-5, we examined CD40 expression and the effect of CD40 binding on MM clonogenic colony (MCC) formation to characterize the IL-6/CD40 loop activity in MM. CD40 was expressed on plasmacytoid cells in 21 of 28 plasma cell dyscrasia (PCD) bone marrow (BM) biopsies tested (10 of 14 MM, 2 of 2 Waldenstrom's macroglobulinemia [WM], 2 of 2 plasma cell leukemia [PCL], 6 of 8 monoclonal gammopathy of undetermined significance [MGUS], and 1 of 2 primary amyloidosis [AL]). G28-5 binding increased MCCs by 35% to 150% in 11 of 17 CD40+ PCD BM cultures, but did not affect MCC formation in CD40- specimens or normal BM colony forming units (CFU-GEMM, CFU-GM, BFU-E). Responsive cultures originated from BM of patients with MM (2 of 5 cases tested), WM (2 of 2), PCL (2 of 2), and MGUS (5 of 6). CD40-responsiveness was not significantly inhibited by the presence of an anti-IL-6 MoAb (2 of 2 MGUS cultures tested), and did not correlate with the capacity to respond to IL-6 stimulation (n = 17, P > .05) or a detectable level of endogenous IL-6 (n = 15, P > .05). Additional studies were performed with PCD cell lines to characterize the interrelationship of CD40 activation and IL-6 production. Fifty percent to greater than 95% of cells from the RPMI 8226 and ARH77 lines expressed CD40, whereas 6% of U266 cells were CD40+. For RPMI 8226, ARH-77, and U266 cells, the increased MCC formation after anti-CD40 stimulation was not affected by the presence of an anti-IL-6 neutralizing MoAb and was not accompanied by detectable IL-6 secretion. There was no apparent increase in IL-6 mRNA transcription following G28-5 treatment of U266 or RPMI 8226 cells. Our observations indicate that CD40 is expressed in a subset of human myeloma cells present in various PCDs. Cell-line studies suggest that the CD40+ myeloma cell may regulate MM clonogenic colony formation without activating the IL-6 pathway.

Comparison of the promoter region of the human and porcine choline acetyltransferase genes: localization of an important enhancer region.

Genomic clones of human and porcine choline acetyltransferase were obtained by screening genomic libraries with synthetic oligonucleotides. The human and porcine genes exhibit significant conservation of both their intron/exon structure and the nucleotide sequence in their 5' flanking regions. However, the two genes differ in several respects, including the absence of a "TATA" box in the human gene and differences in the position of the methionine start codon. Analysis of the promoter region of the two genes has led to the localization of an enhancer element that appears necessary for efficient transcription of the gene.

A human gene family with sequence homology to Drosophila melanogaster Hsp70 heat shock genes.

A growing literature describes the structure and regulation of prokaryotic and eukaryotic heat shock genes. We here report the isolation of several members of a human heat shock protein 70 (hsp 70) multigene family which contains at least 10 different genes and/or pseudogenes exhibiting sequence homology to the hsp70 gene of Drosophila melanogaster. Eight nonoverlapping recombinant lambda phages from a lambda-Charon4A human genomic library were studied by restriction mapping. One lambda clone was sequenced and characterized as a hsp70 pseudogene inserted into a rearranged human HindIII 1.9-kilobase repeated DNA sequence. This pseudogene is probably located on the X chromosome. Its predicted amino acid sequence shows extensive homology to those of Drosophila hsp70, trout hsp70, Xenopus hsp70, yeast hsp70, and some homology to the heat-inducible dnaK gene product of Escherichia coli. Amino acid homology is clustered, suggesting evolutionary conservation of domains critical to the function of this protein in both prokaryotic and eukaryotic cells.

Blood pressure elevation in rats by peripheral administration of Tyr-Gly-Gly-Phe-Met-Arg-Phe and the invertebrate neuropeptide, Phe-Met-Arg-Phe-NH2.

Phe-Met-Arg-Phe-NH2 (FMRFamide), injected at less than 1 mumol/kg intravenously in the anesthetized rat, produces sharp elevations of blood pressure and changes in respiration. The effects were dependent on the carboxyterminal Arg-Phe (RF) configuration and were stereospecific for these two amino acids. A related peptide with RF carboxyterminus, gamma 1-melanotropic stimulating hormone, also had potent blood pressure stimulating activity. The mechanisms underlying the pressor effect of FMRFamide have not yet been established but this pressor action was not significantly attenuated by standard pharmacologic antagonists or prevented by removal of the adrenal or pituitary gland.

Normal limits of urinary excretion of eleven enzymes.

Urinary excretion of lactate dehydrogenase, hydroxybutyrate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, arylsulphatase A, alpha-glucosidase, beta-galactosidase, trehalase, N-acetyl-beta-glucosaminidase, beta-glucuronidase, and leucinearylamidase was studies in a carefully selected group of 100 healthy subjects, 50 women and 50 men. Enzyme activities were assayed in 3-h morning samples after gel filtration of the urine. Activities were related to time volume, and to urinary creatinine concentration. Several transforming functions had to be applied to enzyme output data to obtain an approximation to gaussian frequency distribution. Men showed a significantly higher excretion of gamma-glutamyltransferase, alpha-glucosidase, trehalase, N-acetyl-beta-glucosaminidase,beta-glucuronidase, and leucine arylamidase activity than did women if enzyme activity was related to urinary time volume. Women excreted more lactate dehydrogenase, hydroxybutyrate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, alpha-glucosidase, trehalase, and N-acetyl-beta-glucosaminidase activity than did men, if urinary creatinine was used as the basis of reference. Reference intervals were calculated as 2.5 and 97.5 percentiles for both sexes.