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Cystic fibrosis (CF), an autosomal recessive disease, is caused by variants in both alleles of the CF transmembrane conductance regulator (CFTR) gene. A new assay based on allele-specific polymerase chain reaction and high-resolution melting analysis was developed for the detection of 18 CF-causing CFTR variants previously identified in Cuba and Latin America. The assay is also useful for zygosity determination of mutated alleles and includes internal controls. The reaction mixtures were normalized and evaluated using blood samples collected on filter paper. The evaluation of analytical parameters demonstrated the specificity and sensitivity of the method to detect the included CFTR variants. Internal and external validations yielded a 100% agreement between the new assay and the used reference tests. This assay can complement CF newborn screening not only in Cuba but also in Latin America.

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La uveítis anterior no infecciosa es una enfermedad inflamatoria del ojo que afecta al tracto uveal y que puede causar ceguera total y otras discapacidades visuales. Esta enfermedad se ubica en el espectro de enfermedades autoinmunes y autoinflamatorias. Se han descrito respuestas no adecuadas a la vacunación en enfermedades mediadas por el sistema inmune, por lo que se evaluaron los niveles de antitoxina tetánica y diftérica en pacientes cubanos con uveítis anterior no infecciosa, relacionada con el alelo HLA-B27. Se determinaron los niveles de antitoxina tetánica y diftérica mediante ELISA en 190 pacientes con uveítis anterior no infecciosa y controles supuestamente sanos. El 97,37 por ciento de los pacientes con uveítis mostraron niveles de protección de antitoxina tetánica mayor o igual a 0,1 UI/mL, similar a lo observado en los controles sanos (98,95 por ciento) (p=0,4385). Las proporciones de pacientes con uveítis anterior no infecciosa y sus controles en los diferentes niveles de protección de antitoxina tetánica fueron similares (p>0,05), al igual que los títulos medios geométricos (p=0,2907). En los pacientes con uveítis, de 65 años o más, se detectó una mayor proporción de individuos con títulos protectores de larga duración (>1,0 UI/mL) de antitoxina diftérica (p=0,0065). En los pacientes con uveítis no se observó asociación entre la presencia del alelo HLA-B27 y la respuesta de anticuerpos frente al toxoide tetánico (p=0,6196) y diftérico (p=0,1917). El 37,9 por ciento de los pacientes con uveítis y el 42 por ciento de los controles, presentaron títulos no protectores (<0,1 UI/mL) de antitoxina diftérica (0,1148). La mayoría de los pacientes con uveítis anterior no infecciosa y los controles supuestamente sanos presentaron protección frente al toxoide tetánico; mientras que, en los pacientes con uveítis, así como en los controles supuestamente sanos, con edad igual o más de 18 años, se debe reevaluar incluir refuerzos con toxoide diftérico para alcanzar mayores niveles de protección frente a la difteria(AU)

Non-infectious anterior uveitis is an inflammatory disease of the eye that affects the uveal tract and can cause total blindness and other visual disabilities. Autoimmune and inflammatory diseases are associated with qualitative and quantitative alterations in the immune response; therefore, the levels of tetanus and diphtheria antitoxin related to the HLA-B27 allele were evaluated in Cuban patients with non-infectious anterior uveitis. Tetanus and diphtheria antitoxin levels were determined by ELISA in 190 patients with non-infectious anterior uveitis and healthy control individuals. 97.37 percent of patients with uveitis showed protective tetanus antitoxin levels greater than and equal to 0.1 IU/mL as well as healthy controls (98.95 percent) (p=0.4385). The proportions of patients with non-infectious anterior uveitis and presumably healthy controls in the different levels of protective tetanus antitoxin were similar (p>0.05) at all levels of protection, as were the geometric mean titers for this antitoxin (p=0.2907). Patients with uveitis aged 65 years or older had a higher proportion of individuals with long-term reliable protective titers (>1.0 IU/mL) of diphtheria antitoxin (p=0.0065). In uveitis patients, no association was observed between the presence of the HLA-B27 allele and the antibody response against tetanus toxoid (p=0.6196) and diphtheria (p=0.1917). Similarly, 37.9 percent of patients with uveitis and 42 percent of their controls had non-protective titers (<0.1 IU/mL) of diphtheria antitoxin (0.1148). Most patients with anterior uveitis and control subjects were protected against tetanus (p>0.05), while in patients with uveitis and supposedly healthy controls, aged 18 years or older, the administration of booster doses with diphtheria toxoid should be reevaluated to achieve higher levels of protection against diphtheria(AU)

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BACKGROUND: There are several studies that show a good genotype-phenotype correlation in congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD). However, there is well-documented evidence of inconsistency in some cases. OBJECTIVES: To determine if there is a correlation between the identified mutations and the clinical manifestations of 21OHD in the Cuban population. METHODS: A cross-sectional descriptive study of all patients referred for a molecular diagnosis of 21OHD in Cuba from January 2000 to December 2018. The clinical manifestations of each patient were identified and classified according to the phenotype. The CYP21A2 gene was analyzed for the presence of 5 point mutations involved in the pathogenesis of 21OHD (intron 2, deletion of 8bp, I172N, P30L, and Q318X); correlation was sought between the phenotypic characteristics and the frequencies of point mutations in the patients using the Spearman test. RESULTS: A total of 55 patients underwent direct analysis of the CYP21A2 gene in order to determine the presence of the 5 point mutations. Point mutations were identified in 31 patients, which corresponded to 56%. A statistically significant genotype-phenotype correlation was found. CONCLUSIONS: The correlation between the detected molecular defect and the clinical expression of 21OHD was reasonable in the Cuban population, which could allow phenotypic predictions to be made from the genotype.

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BACKGROUND: Congenital adrenal hyperplasia (CAH) is an autosomal recessive group of diseases. 21-Hydroxylase deficiency (21OHD) accounts for between 95 and 99% of all CAH cases. OBJECTIVES: To characterize the genotype of patients clinically diagnosed with 21OHD and to identify the most frequent mutations in the Cuban population. METHODS: Cross-sectional descriptive study that included all patients diagnosed with 21OHD from January 2000 to December 2018. For the molecular analysis of the CYP21A2 gene, a protocol was used that used the polymerase chain reaction in 2 stages; in the first stage genomic DNA was amplified and 5 point mutations were detected in the second stage (Intron 2, Deletion of 8 bp, G318X, I172N and P30L). RESULTS: The 5 point mutations were identified in 31 of the 55 (56%) studied patients, 16/21 (76%) in the salt-wasting, 12/18 (67%) in the simple virilizing and 3/16 (19%) in the nonclassical form. The Intron 2 mutation was the most frequent, followed by G318X and 8 bp deletion. Compound heterozygotes were found in 10 patients, all corresponded to classic forms of the disease. CONCLUSIONS: The causal CYP21A2 gene mutation was detected in 56% (72% in classic CAH), which makes the method encouraging. The most frequent mutations observed were Intron 2 and G318X. The detection of mutations offers confirmation of diagnosis, prediction of phenotype and genetic counseling.

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Resumen Fundamento: la hiperplasia suprarrenal congénita constituye la causa más frecuente de ambigüedad sexual en la infancia y aproximadamente el 95 % de los casos son producto de mutaciones que ocurren en el gen que codifica la enzima 21α-hidroxilasa. El diagnóstico molecular constituye un elemento a considerar para el manejo y asesoramiento genético a pacientes y familiares en riesgo. Objetivo: identificar la mutación I172N, determinar su frecuencia en la población estudiada y su posible relación con los fenotipos clínicos encontrados. Métodos: se realizó un estudio descriptivo, de corte transversal, durante el período 2014-2016 a pacientes cubanos con diagnóstico de hiperplasia suprarrenal congénita por insuficiencia de 21-OH atendidos en el Instituto de Endocrinología de La Habana. El universo de estudio quedó constituido por 32 pacientes. Los resultados se presentaron en tablas o gráficos según fue más factible mostrar la información. Resultados: la mutación I172N fue identificada en individuos de ambos sexos y se estableció su relación con las formas clásicas de la enfermedad. En la población estudiada, se ubicó dentro de las tres mutaciones más frecuentes de las pesquisadas hasta el momento en el Centro Nacional de Genética Médica de la Habana a pacientes con hiperplasia suprarrenal congénita por insuficiencia de 21 OH. Conclusiones: el método estandarizado demostró ser reproducible y confiable para el diagnóstico molecular de los individuos con hiperplasia suprarrenal congénita por insuficiencia de 21-OH. La mutación I172N se ubica dentro de las tres más frecuentes en la población cubana estudiada y se relaciona con las formas clásicas de la enfermedad.

ABSTRACT Background: congenital adrenal hyperplasia is the most frequent cause of sexual ambiguity in childhood. Molecular diagnosis is an element to be considered for the management and genetic counseling of patients and relatives at risk. Objective: to identify the I172N mutation, to determine its frequency in the studied population and its possible relationship with the clinical phenotypes found. Methods: a descriptive, cross-sectional study was conducted during the 2014-2016 period for Cuban patients diagnosed with congenital adrenal hyperplasia due to 21-OH insufficiency treated at the Institute of Endocrinology of Havana. The universe consisted of 32 patients. The variables analyzed were: age, social sex, age at diagnosis, clinical form of hyperplasia, diagnosis by screening program, family history, consanguinity, nonspecific neonatal death, genital crisis of the newborn, previous molecular diagnosis, mutations studied previously, mutation I172N gene CYP21A. The results were presented in tables or graphs as it was more feasible to show the information. Results: the I172N mutation was identified in individuals of both sexes and its relation with the classic forms of the disease was established. In the studied population the three most frequent mutations of the researched ones, so far in the National Center of Medical Genetics of Havana to patients with congenital adrenal hyperplasia due to 21 OH insufficiency. Conclusions: the standardized method proved to be reproducible and reliable for the molecular diagnosis of individuals with congenital adrenal hyperplasia due to 21-OH insufficiency. The I172N mutation is among the three most frequent in the studied Cuban population and is related to the classic forms of the disease.

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Introducción: La Enfermedad de Wilson es una enfermedad con patrón de herencia autosómico recesivo. Es causada por las mutaciones en el gen atp7b. El exón 3 del gen atp7b es polimórfico y se informan más de 120 polimorfismos en el gen atp7b. Objetivo: Identificar los cambios conformacionales en el exón 3 del gen atp7b y detectar polimorfismos en pacientes cubanos con diagnóstico clínico presuntivo de la enfermedad de Wilson. Materiales y Métodos: Se realizó un estudio descriptivo, en el Centro Nacional de Genética Médica y en el Instituto Nacional de Gastroenterología, durante el período 2007-2013, que incluyó 105 pacientes con diagnóstico clínico presuntivo de la enfermedad de Wilson. La extracción del ADN fue por la técnica de precipitación salina. Se utilizó la técnica de Reacción en Cadena de la Polimerasa para la amplificación del fragmento de interés, y para detectar los cambios conformacionales y la presencia del polimorfismo p.L456V, se usó la técnica de Polimorfismo Conformacional de Simple Cadena, en el exón 3 del gen atp7b. Resultados: En el exón 3 se detectan los cambios conformacionales denominados b y c que correspondieron al polimorfismo p.L456V en estado heterocigótico y homocigótico respectivamente. La frecuencia alélica del polimorfismo p.L456V es de 41 por ciento. Las manifestaciones más frecuentes en los pacientes que presentaron este polimorfismo son las hepáticas. Conclusiones: Se identificó el polimorfismo p.L456V en 64 pacientes cubanos con diagnóstico clínico de la enfermedad de Wilson, lo cual posibilitará hacer estudios moleculares por métodos indirectos(AU)

Introduction: Wilson's disease is a rare inherited autosomal recessive disorder caused by mutations in the ATP7B gene. The exon 3 of the ATP7B gene is polymorphic, and more than 120 polymorphisms of this type have been reported in the literature. Objective: To identify conformational band shifts in exon 3 and detect polymorphisms of the ATP7B gene in Cuban patients, clinically diagnosed with Wilson's disease. Materials and Methods: A descriptive study including 105 patients with the clinical diagnosis of Wilson's disease was conducted at the National Center for Medical Genetics and the National Institute of Gastroenterology from 2007 to 2013. Salting-out protocol was used for DNA extraction. The Polymerase Chain Reaction was used to amplify the fragment of interest and the Single-Strand Conformational Polymorphism was applied in the region of exon 3 of the ATP7B gene to identify conformational changes and the presence of the polymorphism p.L456V. Results: The conformational change called B and C corresponded to the p.L456V polymorphism in the heterozygous and homozygous states, respectively. The allelic frequency of the p.L456V polymorphism in 105 Cuban patients clinically diagnosed with Wilson's disease was 41 percent. The most common manifestations in patients with this polymorphism were related to the liver. Conclusion: The p.L456V polymorphism was identified in 64 Cuban patients with Wilson disease, which will enable us to conduct molecular studies by indirect methods(AU)

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Introducción: La Lepra es una enfermedad infecciosa causada por el Mycobacterium leprae. Los patrones dermatoglíficos de pacientes cubanos con lepra lepromatosa mostraron indicios probatorios de que existe predisposición genética para el desarrollo de esta enfermedad, que sugiere la búsqueda de la asociación con polimorfismos moleculares, de mayor precisión. Entre estos, unos de los más estudiados son el T352C del gen del receptor de la vitamina D y el A16974C del gen IL12p40, cuya utilidad relativa depende de la población. Objetivo: Determinar si existe asociación entre la presencia de los polimorfismos T352C y A16974C con la lepra lepromatosa en pacientes cubanos. Material y Métodos: Se realizó un estudio observacional, analítico, de tipo caso-control de asociación genética donde se estudiaron pacientes con lepra lepromatosa y controles. Fueron identificados los genotipos relacionados con los polimorfismos T352C y A16974C en cada grupo. La prueba Chi-cuadrado de Pearson fue utilizada para determinar si los controles se hallaban en equilibrio de Hardy Weinberg, así como si existía relación entre los polimorfismos y la presencia de la enfermedad. Resultados: Los pacientes estudiados fueron 32 para el polimorfismo T352C y 44 para el A16974C. Los controles fueron 64 y 44, respectivamente; estos se hallaron en equilibrio Hardy-Weinberg. No se detectó asociaciónentre los polimorfismos A16974C y T352C con la lepra lepromatosa. Conclusiones: Los polimorfismos T352C y A16974C no son útiles como factor de riesgo predisponente en el grupo de pacientes cubanos con lepra lepromatosa estudiados(AU)

Introduction: Leprosy is an infectious disease caused by Mycobacterium leprae. Dermatoglyphic patterns of Cuban patients with lepromatose leprosy showed evidential signs of the existence of genetic predisposition to the development of this disease, which suggests a search for the association with molecular polymorphisms of higher degree of accuracy. Among them, some of the most studied are the T352C vitamin D receptor gene and the A16974Cof the IL12p40 gene, which relative usefulness depends on the population. Objective: To determine whether there is an association between the T352Cand A16974C polymorphisms with lepromatose leprosy in Cuban patients. Material and methods: An observational analytical case-control type genetic association study was conducted where patients with lepromatose leprosy and controls were studied. Genotypes related to T352Cand A16974C polymorphisms were identified in each group. Pearson´s chi square test was used to determine whether the controls were in Hardy-Weinberg equilibrium, and also whether there was a relation between polymorphisms and the presence of diseases. Results: There were 32 patients under study for T352C polymorphism, and 42 for A16974C. The controls were 64 and 44, respectively; and these were in Hardy-Weinberg equilibrium. No association between T352Cand A16974C polymorphisms with lepromatose leprosy was detected. Conclusions: T352Cand A16974C polymorphisms are not useful as a predisposing risk factor in the group of Cuban patients with lepromatose leprosy studied(AU)

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BACKGROUND: Mutation scanning methods in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene may not distinguish between a Cystic Fibrosis (CF) causing mutation and a benign variant. We have developed a simple and fast method for scanning 14 selected CF-causing mutations which have high frequency in Latin America. METHODS: In a group of 35 samples coming from CF patients previously characterized and using two allele-specific real-time multiplex PCRs targeting wild-type and mutant alleles respectively, we detect the presence of mutations by analyzing the Ct variation. Twenty-five samples without mutations considered non-carrier samples, were also included in this study. High Resolution Melting Analysis (HRMA) was performed to confirm the result of the scanning method and in most cases allowed the genotype determination. RESULTS: The results validate this method for CF diagnosis. A least one CFTR gene mutation was detected in the samples of CF patients, as predicted by their ΔCt values. The ΔCt value also indicated the zygosity of the sample according to the distribution of CFTR gene mutations. In most cases, HRMA allowed the identification of the mutation(s), thereby confirming the efficiency of this scanning strategy. CONCLUSIONS: This strategy simplifies the detection of CF, reducing the analysis of 14 CF-causing mutations to two parallel reactions and making the procedure compatible with the analysis of a large number of samples. As the method is fast, inexpensive and highly reliable, it is advisable for scanning CFTR gene mutations in newborns, patients with a clinical suspicion of CF as well as in the preconception carrier screening.

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Fundamento: la enfermedad de Wilson se caracteriza por la acumulación de cobre, fundamentalmente en el hígado y cerebro. Se transmite con un patrón de herencia autosómico recesivo. La causa molecular que la provoca son las mutaciones en el gen atp7b, que se encuentra en el cromosoma 13. Se han informado en la literatura más de 139 polimorfismos en el gen atp7b, tanto en los exones como en los intrones.Objetivo: identificar el polimorfismo T991T en pacientes cubanos con diagnóstico clínico de enfermedad de Wilson.Métodos: se realizó un estudio descriptivo, en el Centro Nacional de Genética Médica, durante el período 2011-2013, que incluyó 35 pacientes con diagnóstico clínico de enfermedad de Wilson. Para la amplificación del fragmento de interés, se utilizó la técnica de Reacción en Cadena de la Polimerasa y para identificar los cambios conformacionales, la técnica de Polimorfismo Conformacional de Simple Cadena, en el exón 13 del gen atp7b. El polimorfismo T991T fue identificado por secuenciación del fragmento analizado y la mutación L708P por digestión enzimática.Resultados: la frecuencia alélica del polimorfismo T991T en los 35 pacientes cubanos estudiados fue de 10 %. Las manifestaciones hepáticas fueron las más frecuentes (57,1 %). En el 71,4 % de los pacientes que presentaron el polimorfismo T991T se identificó además el polimorfismo K832R. En un paciente se identificó la mutación L708P.Conclusión: la identificación del polimorfismo T991T permitirá en un futuro inmediato el diagnóstico molecular por métodos indirectos y su correlación con las manifestaciones clínicas presentes en los pacientes con enfermedad de Wilson.

Foundation: Wilson’s disease is characterized by the accumulation of copper, mainly in the liver and the brain. It is transmitted with an autosomal recessive inheritance pattern. The mutations of atp7b gene are the cause which produces it, which is found in chromosome 13.  In literature it has been informed more than 139 polymorphisms in atp7b gene, both in exons and in introns.Objective: To identify T991T polymorphism in Cuban patients with clinical diagnosis of Wilson’s disease.Methods: A descriptive study was done, in the Center of Medical Genetic, during the period of 2011-2013, which included 35 patients with clinical diagnosis of Wilson’s disease. For the amplification of fragment of interest, it was used the Polymerase Chain Reaction technique and to identify conformational changes, the Simple Chain Conformational Polymorphism technique, in exon 13 of the atp7bgene. T991T polymorphism was identified by the sequence of the fragment analyzed and the L708P mutation by enzymatic digestion.Results: T991T polymorphism allele frequency in the 35 Cuban studied patients was of 10 %. Hepatic manifestations were the most frequent (57.1 %). In the 71.4 % of the patients who presented T991T polymorphism it was identified K832R polymorphism as well. In one patient it was identified mutation L708P.Conclusion: the identification of T991T polymorphism will allow in the immediate future the molecular diagnosis by indirect methods and its correlations with clinical manifestations   present in patients with Wilson’s disease.