Irene Morales-Bozo

Defining a New Candidate Gene for Amelogenesis Imperfecta: From Molecular Genetics to Biochemistry

Amelogenesis imperfecta is a group of genetic conditions that affect the structure and clinical appearance of tooth enamel. The types (hypoplastic, hypocalcified, and hypomature) are correlated with defects in different stages of the process of enamel synthesis. Autosomal dominant, recessive, and X-linked types have been previously described. These disorders are considered clinically and genetically heterogeneous in etiology, involving a variety of genes, such as AMELX, ENAM, DLX3, FAM83H, MMP-20, KLK4, and WDR72. The mutations identified within these causal genes explain less than half of all cases of amelogenesis imperfecta. Most of the candidate and causal genes currently identified encode proteins involved in enamel synthesis. We think it is necessary to refocus the search for candidate genes using biochemical processes. This review provides theoretical evidence that the human SLC4A4 gene (sodium bicarbonate cotransporter) may be a new candidate gene.

Association between the occurrence of matrix metalloproteinases 2 and 9 in parotid saliva with the degree of parotid gland damage in juvenile recurrent parotitis.

OBJECTIVE The study was aimed to investigate whether the occurrence of matrix metalloproteinases (MMP) 2 and 9 in parotid saliva of juvenile recurrent parotitis (JRP) patients is associated with the degree of glandular involvement. STUDY DESIGN Thirty-three JRP patients were included. Involvement of parotid gland was assessed by sialography. Parotid saliva was assayed for MMP-2 and MMP-9 by zymography. Medical charts were examined for number of recurrences, disease laterality, and time of follow-up. Logistic regression analysis between occurrence of either MMP, the clinical parameters, and sialographic staging was conducted. RESULTS None of the clinical parameters under analysis were found to be associated with degree of sialographic involvement. Statistical associations were found between presence of MMP-9 and MMP-2 in parotid saliva and sialographic stage (P = .017; odds ratio [OR] 6.5, 95% confidence interval [CI] 1.4-30.4; and P = .009; OR 6.1; 95% CI 1.6-23.7; respectively). CONCLUSIONS Occurrence frequency of MMP-2 and MMP-9 in parotid saliva from affected glands of JRP patients was associated with degree of gland damage.

Molecular alterations of parotid saliva in infantile chronic recurrent parotitis

Infantile chronic recurrent parotitis (ICRP) is an insidious disease whose etiopathogenesis remains an enigma. Alterations in the physical appearance of parotid saliva from ICRP patients have been frequently reported. However, sialochemical studies in regard to ICRP are very rare. The aim of this study was to determine whether saliva of ICRP patients presents major physicochemical and biochemical alterations compared with saliva from paired healthy controls. Parotid, whole, and submandibular/sublingual saliva was collected at an asymptomatic stage from 33 ICRP patients (5–16 y old, both sexes) and from 33 sex- and age-matched healthy controls. Saliva was analyzed for protein concentration, mode of protein diffusion on cellulose membranes, unidimensional sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis protein profiles and zymographic profiles of metalloproteinase 2 (MMP-2) and metalloproteinase 9 (MMP-9). Parotid saliva of ICRP patients showed an increased protein concentration, altered mode of protein diffusion, a higher frequency of polypeptide bands of 43, 37, 33, 29, 26, 16, and 10 kD, higher asymmetry in the polypeptide profiles of both contralateral parotid saliva, and an increase in the frequency of MMP-2 and MMP-9. Parotid saliva of patients with ICRP is molecularly altered with respect to normal saliva. Some of the molecular differences could be related to the etiopathogenesis of the disease.

Patterns and variability in electrophoretic polypeptide profiles of human saliva in a healthy population.

Electrophoretic polypeptide profiles of normal human saliva differ markedly between different reports. Since both methodological variations and polymorphism may explain these differences, in this study we aimed to establish whether or not the salivary electrophoretic polypeptide profiles of subjects from a healthy population share discrete molecular features. To this end, parotid, submandibular/sublingual and whole salivas were collected separately from each of 40 young and 34 elderly clinically healthy adults and processed for SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Each type of glandular saliva displayed a different group of invariant (i.e. present in every subject) electrophoretic polypeptide bands while whole saliva showed a profile that reflected mostly the combined contribution of the major salivary glands. Some minor variant (i.e. absent in some subjects) bands were identified in each type of saliva. Regarding those interindividual variations, no ageor sex-dependence was appreciated. Altogether, these results demonstrate the occurrence of distinctive electrophoretic polypeptide patterns, in addition to some minor variations, for each type of normal saliva, thus providing a background for further populational studies on salivary polypeptide profiles.ResumenLos perfiles electroforéticos de la saliva humana normal publicados presentan marcadas diferencias. Puesto que estas diferencias podrían deberse a polimorfismo poblacional o a variaciones metodológicas, en el presente estudio pretendemos determinar si los perfiles polipeptídicos electroforéticos salivales de una población sana comparten rasgos moleculares. Con este propósito, se colectó saliva parotídea, saliva submandibular/sublingual y saliva total de 40 sujetos adultos jóvenes y 34 sujetos adultos mayores, clínicamente sanos. Las muestras de saliva fueron sometidas a fraccionamiento electroforético en geles de poliacrilamida-SDS y reveladas por tinción con azul de Coomassie. Cada tipo de saliva glandular presentó un grupo característico de bandas polipeptídicas invariables (presentes en todos los sujetos). En la saliva total se pudo observar un perfil que reflejaba principalmente la contribución proteica de las glándulas salivales mayores. En todos los tipos salivales analizados, se pudo observar algunos polipéptidos variables (ausentes en algunos sujetos). Estas variaciones interindividuales no se relacionaron con el sexo o la edad de los individuos. El conjunto de los resultados demuestra la existencia de patrones polipeptídicos distintivos para cada tipo salival junto con la presencia de algunos pocos rasgos variantes en condiciones de normalidad, lo que podría servir de base en futuros estudios poblacionales sobre perfiles polipeptídicos salivales.

Novel missense mutation of the FAM83H gene causes retention of amelogenin and a mild clinical phenotype of hypocalcified enamel

OBJECTIVE Amelogenesis imperfecta (AI) is a group of clinically and genetically heterogeneous inherited conditions, causing alterations in the structure of enamel and chemical composition of enamel matrix during development. The objective of this study was to compare the clinical, radiographic, histological and immunohistochemical phenotypes of subjects affected with hypocalcified AI from three Chilean families and identify causal mutations in the FAM83H gene. DESIGN The diagnosis was made using clinical, radiographic, histological and genealogical data from the patients, who were evaluated according to the classification criteria by Witkop. PCR and Sanger sequencing of the complete coding sequence and surrounding intron regions of the FAM83H gene were conducted. The structural study of the affected teeth was performed with light microscopy, scanning electron microscopy and immunohistochemistry. RESULTS The probands of the three families were diagnosed with hypocalcified AI, but in only one of them the missense variant p.Gly557Cys was identified. This variant was not present in the SNP database or in 100 healthy controls and segregated with the disease in the affected family. Using light microscopy, a normal prismatic structure was observed in all three cases. However, the ultrastructure was found to be affected in two of the cases, showing persistence of organic matter including amelogenins. CONCLUSIONS These results suggest that FAM83H missense mutation reported in one of the families analyzed in this study might cause a phenotype of hypocalcified enamel more attenuated with retention of amelogenin.

Determination of susceptibility to sensitization to dental materials in atopic and non-atopic patients

Introduction: Some studies report that atopic patients have a greater frequency of delayed-type sensitization than non-atopic patients. Objective: To determine the influence of the atopic condition on delayed sensitization to dental materials. Design: cross-sectional study. Methods: Forty (40) atopic subjects and forty (40) non-atopic subjects, of both sexes, between 20 and 65 years of age were included. The determination of delayed sensitization to dental materials was performed using patch test. An oral exam was also carried out to check for lesions of the oral mucosa. Results: 61.25% of the patients were positive for delayed-type sensitization to one or more allergens, being palladium chloride (21.25%), ammoniated mercury (20%), benzoyl peroxide (12.5%) and amalgam (10%) the most frequent. The frequency of sensitization was 67.5% in the group of atopic patients, compared to 55% in the non atopic group (p>0.05). The materials with the greatest difference of sensitization in atopic compared to non-atopic patients were ammoniated mercury, benzoyl peroxide, amalgam and Bisphenol A Dimethacrylate (BIS-GMA). Conclusion: The atopic condition is not related to a higher frequency of delayed sensitization to a battery of dental materials. Key words: Patch test, delayed-type sensitization, allergy contact, atopia, dental materials.

Evaluation of the efficacy of two mouthrinses formulated for the relief of xerostomia of diverse origin in adult subjects

OBJECTIVE To evaluate the efficacy of two new mouthrinses in the reduction of xerostomía-associated symptomatology. BACKGROUND Xerostomia is a common chronic health condition that affects a great number of adults and significantly deteriorates quality of life, such that treatment is necessary. MATERIALS AND METHODS Sixty-seven adult subjects of both sexes presenting xerostomia of diverse origin were selected. Mouthrinses were tested using a double-blind, randomized, cross-over clinical trial with an intervining wash out period. RESULTS The 100% of subjects presented sensation of dry mouth, and 86% stated sensation of thick saliva. Burning tongue sensation, need to drink liquids to swallow and the sensation of swallowing difficulty were recorded in more than 50% of the patients. The most frequent pathologies in the sample were depression, arthritis, and arterial hypertension. Results of the clinical tests showed that mouthrinse 1 relieves sensation of dry mouth, need to drink liquids, and swallowing difficulty. In contrast, mouthrinse 2 relieves only latter two symptoms. Both rinses were more effective in relieving xerostomía-associated symptomatology in patients taking 3 or more medicines simultaneously. CONCLUSION Both mouthrinses were effective in relieving various xerostomia symptoms, could be distributed at a low cost, thereby improving the quality of life of population affected.

Characterization of mouse salivary polypeptide secretion after oral administration of pilocarpine

Se caracterizo la secrecion salival murina producida por administraciones orales unicas o multiples del agente colinergico pilocarpina. La saliva fue colectada desde varios ratones a la vez mediante un accesorio disenado para tal fin y manipulado por un solo operador. Una simple estimulacion mediante una dosis del agonista en el rango 40-400 µg provoco una respuesta secretoria dependiente de la dosis. La respuesta secretoria duro alrededor de 40 min y en ella se pudo definir cuatro etapas, a saber: lag (5-10 min), flujo maximo (10 min), enlentecimiento (15-20 min) y reposo post-secretorio. En la respuesta, los parametros usuales fueron: velocidad de flujo maxima, 30-40 µl/min; volumen total de saliva, 250-350 µl/raton y proteina salival total, alrededor de 700 µg/raton. Se observo desensibilizacion temporal de la respuesta secretoria durante la primera hora siguiente a una estimulacion simple por pilocarpina. Sin embargo, estimulaciones sucesivas a intervalos de 24 h con este agonista produjeron una respuesta secretoria igualmente intensa, lo que sugirio resensibilizacion en tales intervalos. La composicion polipeptidica de cada una de las salivas obtenidas a partir de una serie de ratones estimulados por una vez con pilocarpina fueron indistinguibles entre si. Tal patron polipeptidico salival tambien fue observado en series de salivas obtenidas dia a dia a partir de ratones unicos estimulados con pilocarpina a intervalos de 24 h. De esta manera, tanto las caracteristicas de la respuesta secretoria como la composicion polipeptidica de la saliva murina obtenida mediante estimulaciones unicas o multiples con pilocarpina resultaron altamente consistentes. Asi, estos estudios abren la posibilidad de emprender una tipificacion molecular sistematica de la saliva de ratones individuales vivos obtenidos ya sea de poblaciones naturales o de aquellos sometidos a condiciones experimentales de laboratorio

α-2-Macroglobulin in Saliva Is Associated with Glycemic Control in Patients with Type 2 Diabetes Mellitus

Background. Subjects with type 2 diabetes mellitus (DM2) require an adequate glycemic control to avoid diabetic complications. Currently, saliva biomarkers are used as a diagnostic tool and can be indicative of the degree of progression and control of various diseases. Several studies indicate that α-2-macroglobulin levels are elevated in diabetic patients. Methods. 120 subjects with DM2 were enrolled and classified into two groups according to their glycemic control (percentage of glycated hemoglobin-A1c (HbA1c), <7% adequate glycemic control group; >7% inadequate glycemic control group). The relationship between α-2-macroglobulin levels from saliva samples and HbA1c was subsequently evaluated. Results. We found a positive correlation between α-2-macroglobulin and HbA1c (r = 0.778 and P < 0.0001). Area under the receivers operating characteristic (ROC) curve of α-2-macroglobulin indicated a positive discrimination threshold of α-2-macroglobulin (AUC = 0.903, CI 95%: 0.847–0.959, P < 0.0001) to diagnose glycemic control. Conclusions. Our data strongly suggest that the level of saliva α-2-macroglobulin is an indicator for the degree of glycemic control in diabetic patients and represents a promising alternative method to evaluate this parameter.

DNA from oral bacteria by sodium hydroxide–paper method suitable for polymerase chain reaction

In the oral cavity, we can find a complex mixture of microorganisms, commensals, and pathogens. The studies of normal oral microbiota, as well as the studies of much oral pathology (e.g., caries, periodontitis), involve the isolation and cultivation of these microorganisms and their molecular analysis. The aim of this study was to validate a quick, easy, efficient, and inexpensive DNA extraction method for the recovery of genomic DNA from gram-positive and gram-negative oral bacteria to be used in polymerase chain reaction amplification. This method worked great with all samples analyzed, providing an approach to extract DNA for different microorganisms.