Revista de la Asociación Latinoamericana de Diabetes
Presentación en modalidad oral
  Básicos y Fisiopatología
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TL 17


Dussaillant C, Serrano V, Eyheramendy S, Smalley SV, Maiz A, Chaves M, Martínez JA, Cataldo LR, Santos JL.

Lugar de Trabajo: Departamento de Nutrición, Diabetes y Metabolismo. Facultad de Medicina. Facultad de Matemáticas. Pontificia Universidad Católica de Chile. Departamento de Ciencias de la Alimentación, Fisiología y Toxicología. Universidad de Navarra.

Introduction: Severe hypertriglyceridemia (HTG) has been linked with genetic defects in LPL, APOC2, APOA5, LMF1 and GBIHBP1 genes. However, some HTG cases are polygenic or probably caused by unidentified mutations. Additionally, other candidate genes for multifactorial dyslipidaemias have emerged through genome-wide association studies. Very high triglyceride levels found in consanguineous families are strongly suggestive of a highly penetrant mutation in recessive state.

Aim: to pinpoint the genetic locus responsible of severe HTG in a three-generation consanguineous Chilean family.

Design: Genome-wide linkage study using the strategy of homozigosity mapping.

Subjects: A three-generation Chilean family of nineteen subjects was studied. The index case and her sister (daughters of a consanguineous marriage) both had plasma TG values higher than 5000 mg/dL at diagnosis. The third sister did not display dyslipidaemia and the brother and mother presented an intermediate phenotype. The normal plasma activity of LPL enzyme and APOC2 in the index case ruled out a mutation in these genes. All family members show a remarkable aggregation of features related to the metabolic syndrome (dyslipidaemia, obesity and diabetes).

Methods: DNA from saliva samples of nineteen family members was used for a genome screen of 279,241 biallelic markers (SNPs) with the Illumina HumanCytoSNP-12 panel. Through homozigosity mapping strategy, we searched for chromosome locations with excess of homozygous genotypes in the affected cases. The identification of regions with a homozigosity excess would indicate the chromosomal possible location of the mutation responsible for the disease.

Results: A large homozygous segment was found in the long arm of chromosome 11, with more than 2500 consecutive homozygous SNPs covering a region of 31 Kb in the index case and her affected sister. In the unaffected sister, the same genome region showed substantial heterozygosity (at least 25% of the genetic markers). The region found by homozigosity mapping contains the APOA5/A4/C3/A1 cluster.

Conclusion: The wholegenome screen using homozigosity mapping strategy indicates that the APOA5 and APOC3 genes are the most suggestive candidates for searching mutations responsible of HTG in this family.


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Cataldo LR, Olmos P, Díez A, Smalley SV, Fadic R, Santos JL.

Lugar de Trabajo: Departamento de Nutrición, Diabetes y Metabolismo. Departamento de Neurología. Facultad de Medicina. Pontificia Universidad Católica de Chile

Introduction: Maternally Inherited Diabetes and Deafness (MIDD) is a rare type of diabetes caused by an A-to-G transition at position 3243 of mitochondrial DNA (m.3243A>G). Severity, onset and clinical phenotype of MIDD patients are mainly determined by the proportion of mutant mitochondrial DNA copies in each cell and tissue (heteroplasmy).

Aim: to identify the m.3243A>G mutation in a Chilean case of MIDD patient and to quantitatively evaluate the heteroplasmy in different tissues (blood, saliva, hair, muscle)

Patient and Methods: DNA from blood, saliva, hair root and a muscle biopsy of a Chilean adult woman diagnosed with MIDD were studied. Conventional PCR followed by restriction fragment analysis (PCRRFLP) were used to identify the m.3243A>G mutation. PCR products were inserted in the pGEM-T Easy vector creating plasmids with 3243A (wildtype) and 3243G sequence. Mutant and wild-type vectors were mixed in different proportions to create a calibration curve used to intrapolate heteroplasmy percentages of DNA samples. Relative quantification was carried out using quantitative qPCR by the calculation of threshold cycles for 3243A or 3243G allelic discrimination assays.

Results: The m.3243A>G mutation was identified in the MIDD patient after discarding m.1310C>T and 1438A>G mutations. Quantitative PCR analysis showed that the proportion of mutant genomes in the proband was 66% (muscle), 14% (saliva), 6% (lymphocytes from blood) and 3% in hair root. The observation of grey hair in the proband since the age of ten years-old, led us to evaluate the heteroplasmy in grey hair (21%) versus dark hair (6%), suggesting a possible role of m.3243A>G in developing hair graying in the childhood of MIDD patients. However, we were not able to ascertain whether such difference in heteroplasmy in grey versus dark hair is overcome by normal variability in hair roots. The percentage of heteroplasmy calculated through qPCR and PCR-RFLP showed high concordance, although qPCR techniques are more rapid, sensitive and accurate since are based in the early-exponential amplification phase of PCR rather than in end-point measurements.

Conclusion: Quantitative PCR analysis of the percentage of heteroplasmy shows marked differences in different tissues in the MIDD patient (highest in muscle and lowest in blood). Given the relatively high heteroplasmy found in saliva, this type of sample represents an adequate non-invasive way for assessing the presence of m.3243A>G mutations.


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Ho-Urriola JA, Smalley SV, Cataldo LR, Hodgson MI, González A, González D, Valladares M, Weisstaub G, Santos JL.

Lugar de Trabajo: Department of Nutrition, Diabetes and Metabolism. School of Medicine. Pontificia Universidad Católica de Chile. Santiago, Chile.

Introduction: MC4R gene is involved in the leptin-melanocortin pathway that controls energy homeostasis. Genetic variations of MC4R are important, as it has been demonstrated in animal models and in rare MC4R mutations to cause hyperphagia and severe human obesity. A common genetic variant located 188 kb downstream of the MC4R gene (rs17782313; g.5641961T>C) on chromosome 18 has been consistently associated with increased body weight and fat mass in multifactorial obesity through genome-wide association studies.

Methods: A case-control study for childhood obesity (6 to 12 years old; both genders) was carried out in Santiago (Chile). We recruited a total of 285 obese children (BMI above 95th percentile according to NCHS/CDC 2000) and 97 normal-weight children (BMI between 10th -85th percentile according to NCHS/CDC 2000). The rs17782313 variant was genotyped in all subjects through quantitative PCR using an allelic discrimination Taqman assays. Eating behavior was assessed using the Child Eating Behavior Questionnaire (CEBQ; 35 items and 8 subscales) and the Eating in the Absence of Hunger Questionnaire (EAHQ; 14 items and 3 subscales). Non-parametric tests were used to compare eating behavior scores across genotypes.

Results: The rs17782313 genotype distribution TT, CT and CC was 76.7%, 20.9% and 2.36% respectively. The C allele at rs17782313 was significantly associated with childhood obesity (p-value=0.008). When we tested the association between MC4R rs17782313-C allele and eating behavior, we found a significant statistical association with the score sof food responsiveness subscale (p-value=0.0034). Near-significant associations were found with enjoyment of food (p-value=0.09), desire for drinks (p-value=0.10), satiety responsiveness (p-value=0.10) subscales. We have not found significant associations between rs17782313 genotypes and EAH questionnaire subscales such as negative affect, external eating and fatigue/boredom.

Conclusion: The MC4R rs17782313 polymorphism appears to contribute to childhood obesity in Chile and is possibly involved in childhood eating behavior dimensions such as food responsiveness.


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Paola Jara, Tiare de la Barra, Arturo Osorio, Paolo Giraudo*, Claudio Aguayo, Liliana Lamperti y Carlos Grant&

Lugar de Trabajo: Facultad de Farmacia y Facultad de Medicina&, Universidad de Concepción, Laboratorio Diagnomed*, Concepción.

Contenido: La Diabetes Mellitus (DM) constituye un serio problema de salud pública que incrementa la morbi-mortalidad por complicaciones crónicas micro y macrovasculares. La hiperglicemia como factor patogénico primario, inicia estas complicaciones provocando un efecto negativo sobre la función del endotelio vascular, promoviendo aumento de la producción de especies reactivas de oxígeno (ROS) y por lo tanto estrés oxidativo.

Objetivo: Determinar la producción de ROS en células endoteliales cultivadas en presencia de plasma de pacientes sometidos a prueba de tolerancia oral a la glucosa estandarizada (TTGO).

Metodología: Células endoteliales de línea celular ECV-304 se incubaron por 12h. con plasmas de 39 sujetos a tiempo basal y 120 min del TTGO. Con sonda fluorescente se midió la producción de ROS durante 10 min. Se calcularon parámetros cinéticos de velocidad inicial (Vi), fluorescencia máxima (Fmáx.) y tiempo medio (t1/2) de producción de ROS. En relación a la razón de las pendientes de ROS en (Vi) los pacientes fueron divididos en 2 grupos: G1 (Vi<1; 24 pacientes) y G2 (Vi>1; 15 pacientes). Como índice de producción de ROS se calculó ROS como la diferencia entre las razones de (Fmáx)/(t1/2) a tiempo basal - (Fmáx)/(t1/2) a los 120 min. Se utilizó pruebas de chi-cuadrado y test t-student.

Resultados: Grupo G1 mostró una mayor producción de ROS (Fmáx) con el plasma a 120 min en comparación al G2 (p<0,017). En G1, ROS fue significativamente menor en comparación al G2 (p< 0,0001). Las alteraciones del metabolismo hidrocarbonado: glicemia ayuna alterada (GAA), intolerancia a la glucosa (ITG), diabetes mellitus (DM) según grupos se muestran en Tabla 1.  

Se observó aumento de ROS en un 33,3% de los sujetos normales, en un 62,5% de los pacientes con GAA y en 14,3% en categoría ITG más DM.

Conclusión: En sujetos con aumento de ROS, se observa mayor porcentaje de alteración del metabolismo hidrocarbonado, la glicemia de ayuna alterada es la alteración predominante. En este grupo, el aumento de ROS constituye un indicador de disfunción endotelial, y por tanto, una etapa crucial de intervención para evitar complicaciones micro y macrovasculares.

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