Detección de mutaciones del gen de HNF1B en niños con malformaciones congénitas renales y del tracto urinario / Detection of mutations of the HNF1B gene in children with congenital anomalies of the kidney and urinary tract

Resumen: Introducción: Las anomalías congénitas del riñón y del tracto urinario se originan de alteraciones genéticas, en su mayoría desconocidas. Las mutaciones en el gen que codifica para el factor hepatocitario nuclear 1B (HNF1B), son la causa monogénica más frecuentemente descrita. Se desconocen datos en Chile y Latinoamérica. Objetivo: Determinar la presencia de variantes del gen HNF1B en niños chilenos con anomalías congénitas del riñón y/o tracto urinario y sus características clínicas. Pacientes y Mé todo: Estudio descriptivo con pacientes entre 10 meses y 17 años, consultantes en Unidad de Nefrología Hospital Luis Calvo Mackenna, período abril - diciembre 2016, portadores de displasia renal quística, displasia/hipoplasia renal no quística y/o riñón en herradura. Se determinaron variantes de HNF1B mediante secuenciación de exones 1, 2, 3 y 4; previa extracción y amplificación de DNA. Se utilizaron enzimas de restricción para definir si variantes eran homo o heterocigotas. Familiares di rectos de casos índices se estudiaron con secuenciación del exón afectado. Resultados: Se incluyeron 32 pacientes, 43,75% varones, mediana edad 11 años. El 65,6% displasia/hipoplasia renal no quística, 31,25% displasia renal quística y 3,15% riñón en herradura. En 2 pacientes (6,25%) se detectó una misma variante genética heterocigota en exón 4, posición 1027 (C1027T), no descrita anteriormente. El estudio de familiares determinó la variante en 3 de 5 individuos, todos sin anomalías nefrouro- lógicas congénitas. Conclusiones: Confirmamos la presencia de una variante genética heterocigota del gen HNF1B, no descrita previamente, dando inicio a la búsqueda de este tipo de mutaciones en nuestro medio, lo cual nos permite aproximarnos al conocimiento de causalidad, determinación de compromiso extrarrenal y consejo genético.

Abstract Introduction: Congenital anomalies of the kidney and urinary tract are caused by genetic alterations mostly unknown. Mutations in the gene that codes for hepatocyte nuclear factor 1B (HNF1B) are the most frequently described monogenic causes. Data are unknown in Chile and Latin America. Objective: To determine the presence of variants of the HNF1B gene in Chilean children with conge nital anomalies of the kidney and/or the urinary tract and their clinical characteristics. Patients and Method: Descriptive study with children aged 10 months to 17 years, patients of the Calvo Mackenna Hospital Nephrology Unit, with cystic renal dysplasia, non cystic renal dysplasia/hypoplasia, horses hoe kidney between April and December 2016. HNF1B variants were determined by sequencing of exons 1, 2, 3 and 4 after DNA extraction and amplification. Restriction enzymes were used to define if the variants were homo or heterozygous. Direct family members of index cases were studied with sequencing of the affected exon. Results: 32 patients were included, 43.75% males, median age 11 years. 65.6% of them had non-cystic renal dysplasia, 31.25% cystic renal dysplasia, and 3.15% hor seshoe kidney. In two patients (6.25%) the same heterozygous genetic variant was detected in exon 4, position 1027 (C1027T), not previously described. The study of relatives found the same variant in three out of five individuals, all without congenital nephro-urological anomalies. Conclusions: We confirmed the presence of a not previously described heterozygous genetic variant of the HNF1B gene. This work initiates the search for this type of mutations in our region which allows us to ap proach the knowledge of causality, determination of extrarenal involvement, and genetic counseling.

[Detection of mutations of the HNF1B gene in children with congenital anomalies of the kidney and urinary tract]. / Detección de mutaciones del gen de HNF1B en niños con malformaciones congénitas renales y del tracto urinario.

INTRODUCTION: Congenital anomalies of the kidney and urinary tract are caused by genetic alterations mostly unknown. Mutations in the gene that codes for hepatocyte nuclear factor 1B (HNF1B) are the most frequently described monogenic causes. Data are unknown in Chile and Latin America. OBJECTIVE: To determine the presence of variants of the HNF1B gene in Chilean children with conge nital anomalies of the kidney and/or the urinary tract and their clinical characteristics. PATIENTS AND METHOD: Descriptive study with children aged 10 months to 17 years, patients of the Calvo Mackenna Hospital Nephrology Unit, with cystic renal dysplasia, non cystic renal dysplasia/hypoplasia, horses hoe kidney between April and December 2016. HNF1B variants were determined by sequencing of exons 1, 2, 3 and 4 after DNA extraction and amplification. Restriction enzymes were used to define if the variants were homo or heterozygous. Direct family members of index cases were studied with sequencing of the affected exon. RESULTS: 32 patients were included, 43.75% males, median age 11 years. 65.6% of them had non-cystic renal dysplasia, 31.25% cystic renal dysplasia, and 3.15% hor seshoe kidney. In two patients (6.25%) the same heterozygous genetic variant was detected in exon 4, position 1027 (C1027T), not previously described. The study of relatives found the same variant in three out of five individuals, all without congenital nephro-urological anomalies. CONCLUSIONS: We confirmed the presence of a not previously described heterozygous genetic variant of the HNF1B gene. This work initiates the search for this type of mutations in our region which allows us to ap proach the knowledge of causality, determination of extrarenal involvement, and genetic counseling.

[Comparison of real-time polymerase chain reaction and antigenemia assay to detect cytomegalovirus in pediatric transplants]. / Comparación de las técnicas de reacción de polimerasa en cadena en tiempo real y antigenemia para la detección de citomegalovirus en sangre de niños sometidos a trasplantes.

BACKGROUND: Cytomegalovirus (CMV) infections are an important cause of morbidity and mortality in transplant recipient. To date, the antigenemia assay is the most used technique for diagnostic and management of CM V infections. However, quantification of CMV viral load by real time polymerase chain reaction (RT-PCR) has becoming the method of choice to detect CMV in a rapid, sensitive and specific manner. OBJECTIVE: To compare antigenemia and RT-PCR assays in the detection of CMV in blood sample from solid organ and bone marrow transplant (BMT) in children attended at the Dr. Luis Calvo Mackenna Hospital. METHODS: In a prospective study, we detect the presence of CMV in blood sample by RT-PCR and antigenemia assays. RESULTS: We analyzed 219 blood samples from 68 children subjected to kidney, liver and BMT. Out of 219 samples analyzed, 147 were negative and 33 were positive for CMV by both techniques. Thirty-seven samples were positive only by RT-PCR and 2 by antigenemia. Considering the antigenemia as a reference, RT-PCR shows 94%, 80%, 34% and 99% sensitivity, specificity, positive and negative predictive values, respectively. The kappa coefficient between both techniques was 0.528. CONCLUSION: Quantitative determination of CMV viral load by RT-PCR is a sensitive technique with excellent negative predictive valué compared to antigenemia. Our results support the use of RT-PCR as a technique that might facilítate the diagnostic and treatment of active CMV infection in pediatric transplants.

Comparación de las técnicas de reacción de polimerasa en cadena en tiempo real y antigenemia para la detección de citomegalovirus en sangre de niños sometidos a trasplantes / Comparison of real-time polymerase chain reaction and antigenemia assay to detect cytomegalovirus in pediatric transplants

Background: Cytomegalovirus (CMV) infections are an important cause of morbidity and mortality in transplant recipient. To date, the antigenemia assay is the most used technique for diagnostic and management of CM V infections. However, quantification of CMV viral load by real time polymerase chain reaction (RT-PCR) has becoming the method of choice to detect CMV in a rapid, sensitive and specific manner. Objective: To compare antigenemia and RT-PCR assays in the detection of CMV in blood sample from solid organ and bone marrow transplant (BMT) in children attended at the Dr. Luis Calvo Mackenna Hospital. Methods: In a prospective study, we detect the presence of CMV in blood sample by RT-PCR and antigenemia assays. Results: We analyzed 219 blood samples from 68 children subjected to kidney, liver and BMT. Out of 219 samples analyzed, 147 were negative and 33 were positive for CMV by both techniques. Thirty-seven samples were positive only by RT-PCR and 2 by antigenemia. Considering the antigenemia as a reference, RT-PCR shows 94 percent, 80 percent, 34 percent and 99 percent sensitivity, specificity, positive and negative predictive values, respectively. The kappa coefficient between both techniques was 0.528. Conclusion: Quantitative determination of CMV viral load by RT-PCR is a sensitive technique with excellent negative predictive valué compared to antigenemia. Our results support the use of RT-PCR as a technique that might facilítate the diagnostic and treatment of active CMV infection in pediatric transplants.

Antecedentes: Las infecciones por citomegalovirus (CMV) corresponden a una importante causa de morbilidad y mortalidad en pacientes sometidos a trasplantes. Hasta la fecha, la detección de CMV en células infectadas en sangre periférica mediante la técnica de inmunofluorescencia (antigenemia) es la más utilizada para el diagnóstico y monitorización de la infección por este agente. Sin embargo, en el último tiempo la cuantificación de la carga de ácido nucleico (ADN) de CMV en sangre mediante la técnica de reacción de polimerasa en cadena en tiempo real (RPC-TR) ha permitido la detección de CMV de forma más rápida, sensible y específica. Objetivos: Comparar las técnicas de antigenemia y RPC-TR para la detección de CMV en sangre en niños sometidos a trasplante de órganos sólidos y trasplante de precursores hematopoyéticos (TPH) en el Hospital Dr. Luis Calvo Mackenna. Metodología: En un estudio prospectivo de seguimiento preventivo de reactivación se detectó la presencia de CMV en muestras de sangre utilizando las técnicas de RPC-TR y antigenemia. Resultados: Se analizaron 219 muestras de sangre, correspondiente a 68 niños sometidos a trasplante de hígado, riñon y TPH. De las muestras analizadas, 147 fueron negativas y 33 positivas para CMV utilizando ambas técnicas. Treinta y siete muestras resultaron ser positivas sólo por RPC-TR y dos sólo por antigenemia. Tomando en cuenta la antigenemia como referencia, la RPC-TR mostró una sensibilidad, especificidad, valor predictor positivo y negativo de 94 por ciento, 80 por ciento, 34 por ciento y 99 por ciento, respectivamente. El índice de concordancia entre ambas técnicas tuvo un valor de kappa = 0,528. Conclusión: La determinación cuantitativa de ADN de CMV por RPC-TR es una técnica sensible, con un gran valor predictor negativo comparada con la antigenemia. Los resultados obtenidos en este trabajo apoyan el uso de RPC-TR para el diagnóstico y tratamiento oportuno de las infecciones activas por CMV en niños sometidos a trasplantes.

Caracterización molecular de mecanismos de resistencia a cloranfenicol en cepas de shigella flexneri aisladas en niños chilenos con diarrea aguda / Molecular characterization of resistance mechanisms to chloramphenicol in shigella flexneri strains isolated from chilean children with acute diarrhea

Background: Chloramphenicol is one of the therapeutic options for shigellosis, but resistance to this antimicrobial is increasing. Aim: To characterize molecular mechanisms conferring resistance to chloramphenicol (CmR) in Shigella flexneri strains isolated from Chilean children with acute diarrhea. Material and methods: Thirty one Shigella filexneri strains, including 22 with the CmR phenotype were analyzed. Strains were tested for antimicrobial susceptibility by plate dilution and for the presence of an internal fragment of the cat gene encoding for chloramphenicol o-acetyl-transferase, by polymerase chain reaction and Southern blot analysis. Results: All CmR strains had a minimal inhibitory concentration over 64 µg/ml and amplified the internal fragment of the cat gene. Southern blot analyses indicated that this gene was located in the bacterial chromosome. Conclusions: Resistance to chloramphenicol in this group of Shigella flexneri strains was mediated by a chromosomally located cat gene