Variantes genómicas asociadas a errores congénitos en el metabolismo de los carbohidratos en el suroccidente colombiano

Introducción: Los Errores Innatos del Metabolismo (EIM) de carbohidratos son el resultado de la interrupción de las vías catabólicas o anabólicas de diferentes carbohidratos, siendo la fructosa, la galactosa y el glucógeno los más comunes y pertenecen a un grupo heterogéneo de trastornos que pueden ser hereditarios o pueden ocurrir como resultado de variantes genéticas espontáneas. En los EIM, están las vías de los carbohidratos alteradas, tanto las vías catabólicas como las anabólicas, incluyendo el metabolismo de la fructosa, galactosa y glucógeno, dando lugar a un grupo heterogéneo de trastornos. Sin embargo, los datos sobre estas patologías en Colombia son escasos. Nuestro estudio tuvo como objetivo identificar variantes genómicas asociadas a EIM por carbohidratos en población del suroccidente colombiano carente de diagnóstico clínico. Resultados: Realizamos un estudio transversal, secuenciando 320 exomas y clasificando las variantes de acuerdo a guías estándar. Identificamos 286 variantes, incluyendo 206 nuevas y 73 variantes benignas o probablemente benignas. En particular, seis variantes en genes como GALT, GAK1, ALDOB, GAA y SLC2A1 tenían un significado incierto, y una variante patogénica en el gen GALT se asoció con la galactosemia clásica. Curiosamente, el 18% eran intrónicas, el 5% con sentido erroneo, el 10% sinónimas y el 67% variantes no notificadas. Conclusiones: Estos hallazgos ponen de relieve la necesidad de programas de diagnóstico precoz para aplicar tratamientos específicos, incluido el manejo transdisciplinar para minimizar la morbilidad y la mortalidad. El consejo genético y la educación sobre el riesgo son cruciales, facilitando enfoques de medicina anticipatoria y preventiva, avanzando así en la medicina de precisión.   (provisto por Infomedic International)

Introduction: Carbohydrate Inborn Errors of Metabolism (IEMs) result from disruption of the catabolic or anabolic pathways of different carbohydrates, fructose, galactose and glycogen being the most common and belong to a heterogeneous group of disorders that may be inherited or may occur as a result of spontaneous genetic variants. In IEM, there are altered carbohydrate pathways, both catabolic and anabolic pathways, including fructose, galactose and glycogen metabolism, resulting in a heterogeneous group of disorders. However, data on these pathologies in Colombia are scarce. The aim of our study was to identify genomic variants associated with carbohydrate IEM in the southwestern Colombian population lacking clinical diagnosis. Results: We performed a cross-sectional study, sequencing 320 exomes and classifying variants according to standard guidelines. We identified 286 variants, including 206 new variants and 73 benign or probably benign variants. Notably, six variants in genes such as GALT, GAK1, ALDOB, GAA and SLC2A1 were of uncertain significance, and one pathogenic variant in the GALT gene was associated with classic galactosemia. Interestingly, 18% were intronic, 5% missense, 10% synonymous, and 67% unreported variants. Conclusions: These findings highlight the need for early diagnostic programs to implement targeted treatments, including transdisciplinary management to minimize morbidity and mortality. Genetic counseling and risk education are crucial, facilitating anticipatory and preventive medicine approaches, thus advancing precision medicine. (provided by Infomedic International)

Importancia de la genómica en el diagnóstico de nuevas variantes asociadas a trastornos del tejido conectivo con solapamiento fenotípico

La Aracnodactilia Contractural Congénita  (ACC) es una enfermedad del tejido conectivo de herencia autosómica dominante, causada por variantes en el gen FBN2 que codifica la fibrilina-2. Tiene características específicas como contracturas congénitas, oreja con hélice superior arrugada, camptodactilia, pectus carinatum y complicaciones como escoliosis y la cifoescoliosis. Publicamos el caso de una paciente femenina de 19 años con historia de delgadez, velocidad de crecimiento acelerada, talla alta, pérdida de peso, contracturas articulares, hipotonía congénita, pubertad precoz, hábito marfanoide, pectus carinatum y leve aracnodactilia. Se sospecha de enfermedad del colágeno y se solicita secuenciación del exoma completo mediante NGS  (del inglés Next Generation Sequencing) + CNVs  (del inglés Copy Number Variations) genes relacionados con colagenopatías; se identificó una variante en el gen FBN2  (NM_001999.4): c.4394G>A; p.Cys1465Tyr; estado heterocigoto de significancia clínica probablemente patogénica. La ACC es fenotípicamente similar al síndrome de Marfán y se caracteriza por aracnodactilia, dolicostenomelia, escoliosis, contracturas congénitas múltiples y anomalías de los oídos externos. A diferencia del síndrome de Marfán; no tiene compromiso ocular ni afecta la raíz aórtica. Cuenta con variabilidad fenotípica que le dan la heterogeneidad que pueden interferir y retrasar el proceso diagnóstico y terapéutico específico al solaparse con otras condiciones médicas. Los avances en la medicina y la genómica con la utilización de nuevos métodos diagnósticos han permitido que cada día nos acerquemos más a la medicina 6P  (precisión, predicción, prevención, personalizada, participativa con enfoque poblacional) que impacta en el diagnóstico, tratamiento específico, seguimiento, pronóstico y adecuado asesoramiento genético de las enfermedades. (provisto por Infomedic International)

Contractural arachnodactyly congenita  (CCA) is an autosomal dominantly inherited connective tissue disease caused by variants in the FBN2 gene encoding fibrillin-2. It has specific features such as congenital contractures, wrinkled upper helix ear, camptodactyly, pectus carinatum and complications such as scoliosis and kyphoscoliosis. We publish the case of a 19-year-old female patient with a history of thinness, accelerated growth velocity, tall stature, weight loss, joint contractures, congenital hypotonia, precocious puberty, marfanoid habitus, pectus carinatum and mild arachnodactyly. Collagen disease was suspected and whole exome sequencing by NGS  (Next Generation Sequencing) + CNVs  (Copy Number Variations) genes related to collagenopathies was requested; a variant was identified in the FBN2 gene  (NM_001999.4): c.4394G>A; p.Cys1465Tyr; heterozygous state of probably pathogenic clinical significance. CCA is phenotypically similar to Marfan syndrome and is characterized by arachnodactyly, dolichostenomelia, scoliosis, multiple congenital contractures, and external ear anomalies. Unlike Marfan syndrome, it has no ocular involvement and does not affect the aortic root. It has phenotypic variability that gives it heterogeneity that can interfere and delay the specific diagnostic and therapeutic process by overlapping with other medical conditions. Advances in medicine and genomics with the use of new diagnostic methods have allowed us to get closer to 6P medicine  (precision, prediction, prevention, personalized, participatory with a population approach) that impacts on the diagnosis, specific treatment, follow-up, prognosis and adequate genetic counseling of diseases. (provided by Infomedic International)

Actuales retos diagnósticos genómicos en los Síndromes Hemofagocíticos en pediatría: reporte de caso

Introducción: La linfohistiocitosis hemofagocítica familiar (FHL) es una enfermedad del sistema autoinmune que se presenta con un síndrome inflamatorio excesivo causado por linfocitos T activados e histiocitosis. Cursa con herencia autosómica recesiva ligada al cromosoma X. Aproximadamente el 90% de los niños diagnosticados son menores de 2 años y la incidencia es de aproximadamente 0.12 por 100.000. Se puede dividir en cinco subtipos según la variante genética causante. Las variantes patogénicas más involucradas son en los genes de la perforina 1 (PRF1) y homólogo D de la proteína UNC-13 (UNC13D). Caso clínico: Se presenta el caso de un preadolescente de 11 años, con antecedente de infecciones recurrentes, quien cursa con síndrome convulsivo asociado a fiebre, peso y talla bajas para la edad, hepatomegalia y discapacidad cognitiva. En el abordaje inicial se descartan enfermedades infecciosas, inmunológicas, hematológicas, metabólicas y oncológicas. El exoma clínico para inmunodeficiencias primarias muestra una variante patogénica p.A91V homocigota en el gen de la PRF1 de herencia autosómica recesiva, resultado relacionado con linfohistiocitosis hemofagocítica familiar tipo 2 (FHL2). Discusión y conclusión: El cambio conformacional del PRF1 alterado reduce la actividad citotóxica de la proteína y provoca la enfermedad. Los pacientes portadores de defectos en el gen PRF1 son vulnerables a infecciones, enfermedades autoinmunes y tumores malignos. Con un diagnóstico definido y preciso es posible orientar las acciones en salud, pautas de seguimiento, evaluación de riesgo de heredabilidad a través de un caso índice para así encontrar otros posibles portadores, realizar un asesoramiento genético completo, implementar e iniciar tratamientos dirigidos que aminoren la morbilidad y mortalidad asociada a esta patología. Actualmente se cuenta con varios estudios en diferentes fases de investigación sobre moléculas que pueden intervenir en la historia natural de la enfermedad. (provisto por Infomedic International)

Introduction: Familial hemophagocytic lymphohistiocytosis (FHL) is a disease of the autoimmune system that presents with an excessive inflammatory syndrome caused by activated T lymphocytes and histiocytosis. It occurs with autosomal recessive inheritance linked to the chromosome X. Approximately 90% of diagnosed children are under 2 years of age and the incidence is approximately 0.12 per 100,000. It can be divided into five subtypes depending on the causative genetic variant. The most involved pathogenic variants are in the perforin 1 (PRF1) and UNC-13 protein homolog D (UNC13D) genes. Clinical case: The case of an 11-year-old preadolescent is presented, with a history of recurrent infections, who presents with convulsive syndrome associated with fever, low weight and height for age, hepatomegaly and cognitive disability. In the initial approach, infectious, immunological, hematological, metabolic and oncological diseases are ruled out. The clinical exome for primary immunodeficiencies shows a homozygous pathogenic variant p.A91V in the PRF1 gene of autosomal recessive inheritance, a result related to familial hemophagocytic lymphohistiocytosis type 2 (FHL2). Discussion and conclusion: The altered PRF1 conformational change reduces the cytotoxic activity of the protein and causes disease. Patients carrying defects in the PRF1 gene are vulnerable to infections, autoimmune diseases and malignant tumors. With a defined and precise diagnosis, it is possible to guide health actions, follow-up guidelines, evaluation of heritability risk through an index case in order to find other possible carriers, carry out complete genetic counseling, implement and initiate targeted treatments that reduce the morbidity and mortality associated with this pathology. Currently, there are several studies in different phases of research on molecules that may intervene in the natural history of the disease. (provided by Infomedic International)

Diagnostic yield of genetic testing in adults with sensorineural hearing loss.

INTRODUCTION: The contribution of genetic causes to sensorineural hearing loss (SNHL) in adults is less clear than in children, and genetic diagnosis is still not standardized in adults. In this study we present the genetic results obtained in a cohort of adult patients with SNHL. MATERIALS AND METHODS: We included 63 adults with SNHL that received genetic testing between 2019 and 2022. Whole exome sequencing was performed and variants in genes related to hearing loss (virtual panel with 244 genes) were prioritised and analysed. RESULTS: 24% (15/63) of patients were genetically diagnosed: 87% (13/15) of patients had non-syndromic hearing loss and 13% (2/15) had syndromic hearing loss. We identified pathogenic and likely pathogenic variants in 11 different genes. CONCLUSIONS: Our results show that a significant proportion of adults with SNHL have a genetic origin, and that implementation of genetic testing improves diagnostic accuracy and allows personalized management of these patients.

Diagnóstico prenatal, síndrome Freeman-Sheldon mediante ultrasonido y estudio genético. Reporte de caso / Prenatal diagnosis of Freeman-Sheldon syndrome using ultrasound and genetic testing. Case report

Objetivos: Describir un caso de diagnóstico prenatal de síndrome de Freeman-Sheldon mediante hallazgos ecográficos y secuenciación completa del exoma fetal. Materiales y métodos: Mujer de 33 años, con antecedentes de hipotiroidismo en tratamiento, a quien en semana 19 se realizó ecografía de detalle anatómico, en la cual se observaron deformidades en el feto en más de dos áreas corporales (extremidades superiores e inferiores), sugiriendo el diagnóstico de artrogriposis. Posteriormente, se brindó asesoría genética y se realizó amniocentesis en semana 20 de gestación, con análisis de la hibridación in situ por fluorescencia, seguido de secuenciación completa del exoma fetal. Este último examen permitió identificar una variante patogénica heterocigota en el gen MYH3, la cual se asocia con la artrogriposis distal tipo 2A. Conclusiones: La realización de la secuenciación completa de exoma fetal es un factor clave para identificar la mutación del gen MYH3, y confirma que las deformidades evidenciadas por ultrasonido estaban relacionadas con la artrogriposis distal tipo 2A. Es importante hacer la secuenciación de exoma fetal en fetos que muestren hallazgos de malformaciones articulares en el ultrasonido prenatal.

Objectives: To describe a case of prenatal diagnosis of Freeman-Sheldon syndrome based on ultrasound findings and complete fetal exome sequencing. Materials and methods: A 33-year-old woman currently on treatment for hypothyroidism in whom a 19-week detailed anatomical ultrasound scan showed fetal deformities in more than two body areas (upper and lower limbs), suggesting a diagnosis of arthrogryposis. Genetic counseling was provided and amniocentesis was performed at 20 weeks for fluorescence in situ hybridization (FISH) analysis and complete fetal exome sequencing, with the latter allowing the identification of a heterozygous pathogenic variant of the MYH3 gene which is associated with type 2A distal arthrogryposis. Conclusions: Complete fetal exome sequencing was a key factor in identifying the MYH3 gene mutation and confirmed that the deformities seen on ultrasound were associated with type 2A distal arthrogryposis. It is important to perform complete fetal exome sequencing in cases of joint malformations seen on prenatal ultrasound.

Introducción al filtrado, análisis y curación de variantes genéticas en pacientes con Discapacidad Intelectual / Introduction to filtering, analysis and curation of genetic variants in patients with Intellectual Disability

Resumen Actualmente la secuenciación del exoma completo (WES; Whole-exome sequencing) mediante la técnica NGS (Next-generation sequencing) es uno de los estudios genéticos más solicitados dentro del abordaje de pacientes con Discapacidad Intelectual con o sin otras anomalías. Al igual que con otros proce dimientos y estudios clínicos, es conveniente que los médicos prescriptores tengan una comprensión clara de los alcances y limitaciones del uso de WES, del proceso de análisis de las variantes genéticas identificadas, así como de aspectos a evaluar acerca de la calidad y estructura de los informes de los estudios de NGS, con el objetivo de que puedan interpretar mejor los resultados de un estudio y plantear de la mejor manera la correlación de los mismos con la clínica observada.

Abstract Currently, Whole exome sequencing (WES) using NGS (Next-generation sequencing) technology is one of the most requested genetic studies within the approach of patients with intellectual disability with or without other anomalies. As with other procedures and clinical studies, it is convenient for prescribing physicians to have a clear understanding of the scope and limitations of the use of WES, the analysis process of the genetic variants identified, as well as aspects to be evaluated regarding quality and structure of the reports of the NGS studies, with the aim that they can better interpret the results of a study, evaluate its quality, and propose in the best way the correlation of the same with the observed phenotype.

Intervention of next-generation sequencing in diagnosis of Alzheimer's disease: challenges and future prospects / Intervenção do sequenciamento de nova geração no diagnóstico da doença de Alzheimer: desafios e perspectivas futuras

ABSTRACT Clinical diagnosis of several neurodegenerative disorders based on clinical phenotype is challenging due to its heterogeneous nature and overlapping disease manifestations. Therefore, the identification of underlying genetic mechanisms is of paramount importance for better diagnosis and therapeutic regimens. With the emergence of next-generation sequencing, it becomes easier to identify all gene variants in the genome simultaneously, with a system-wide and unbiased approach. Presently various bioinformatics databases are maintained on discovered gene variants and phenotypic indications are available online. Since individuals are unique in their genome, evaluation based on their genetic makeup helps evolve the diagnosis, counselling, and treatment process at the personal level. This article aims to briefly summarize the utilization of next-generation sequencing in deciphering the genetic causes of Alzheimer's disease and address the limitations of whole genome and exome sequencing.

RESUMO O diagnóstico clínico de vários distúrbios neurodegenerativos com base no fenótipo clínico é difícil devido à sua natureza heterogênea e às manifestações da doença que se sobrepõem. Portanto, a identificação dos mecanismos genéticos subjacentes é de suma importância para um melhor diagnóstico e regimes terapêuticos. Com o surgimento do sequenciamento de próxima geração, o diagnóstico se tornou mais acessível com uma abordagem imparcial em todo o sistema para identificar simultaneamente todas as variantes de genes no genoma. Atualmente, vários bancos de dados de bioinformática sobre variantes genéticas descobertas e indicações fenotípicas estão disponíveis online. Uma vez que os indivíduos são únicos em seu genoma, a avaliação com base em sua composição genética ajudou na evolução do processo de diagnóstico, aconselhamento e tratamento em nível pessoal. Este artigo teve como objetivo resumir brevemente a utilização do sequenciamento de próxima geração para decifrar as causas genéticas da doença de Alzheimer (DA) e abordar as limitações do sequenciamento completo do genoma e do exoma.

Alterações sistêmicas na assinatura do ciclo celular de pacientes com COVID-19 / Systemic changes in the cell cycle signature of patients with COVID-19

Com base nas perturbações fosfoproteômicas de moléculas associadas ao ciclo celular em células infectadas pelo coronavírus causador da síndrome respiratória aguda grave (SARSCoV)-2, a hipótese de inibidores do ciclo celular como uma terapia potencial para a doença de coronavírus 2019 (COVID-19) foi proposta. No entanto, o cenário das alterações do ciclo celular em COVID-19 permanece inexplorado. Aqui, realizamos uma análise integrativa de sistemas imunológicos de proteoma publicamente disponível (espectrometria de massa) e dados de transcriptoma (sequenciamento de RNA em massa e de célula única [scRNAseq]), com o objetivo de caracterizar mudanças globais na assinatura do ciclo celular de pacientes com COVID-19. Além de módulos de co-expressão de genes significativos enriquecidos associados ao ciclo celular, encontramos uma rede interconectada de proteínas diferencialmente expressas associadas ao ciclo celular (DEPs) e genes (DEGs) integrando dados moleculares de 1.480 indivíduos (974 pacientes infectados por SARS-CoV-2 e 506 controles [controles saudáveis ou indivíduos com outras doenças respiratórias]). Entre esses DEPs e DEGs estão várias ciclinas (CCNs), ciclo de divisão celular (CDCs), quinases dependentes de ciclinas (CDKs) e proteínas de manutenção de minicromossomos (MCMs). Embora os pacientes com COVID-19 compartilhem parcialmente o padrão de expressão de algumas moléculas associadas ao ciclo celular com outras doenças respiratórias, eles exibiram uma expressão significativamente maior de moléculas associadas ao ciclo celular relacionadas à gravidade da doença. Notavelmente, a assinatura do ciclo celular predominou nos leucócitos do sangue dos pacientes, mas não nas vias aéreas superiores. Os dados de scRNAseq de 229 indivíduos (159 pacientes com COVID- 19 e 70 controles) revelaram que as alterações das assinaturas do ciclo celular predominam nas células B, T e NK. Esses resultados fornecem uma compreensão global única das alterações nas moléculas associadas ao ciclo celular em pacientes com COVID-19, sugerindo novas vias putativas para intervenção terapêutica

Based on phosphoproteomics perturbations of cell cycle-associated molecules in severe acute respiratory syndrome coronavirus (SARS-CoV)-2-infected cells, the hypothesis of cell cycle inhibitors as a potential therapy for Coronavirus disease 2019 (COVID-19) has been proposed. However, the landscape of cell cycle alterations in COVID-19 remains mostly unexplored. Here, we performed an integrative systems immunology analysis of publicly available proteome (mass spectrometry) and transcriptome data (bulk and single-cell RNA sequencing [scRNAseq]), aiming to characterize global changes in the cell cycle signature of COVID-19 patients. Beyond significant enriched cell cycle-associated gene co-expression modules, we found an interconnected network of cell cycle-associated differentially expressed proteins (DEPs) and genes (DEGs) by integrating molecular data of 1,480 individuals (974 SARS-CoV- 2 infected patients and 506 controls [either healthy controls or individuals with other respiratory illness]). Among these DEPs and DEGs are several cyclins (CCNs), cell division cycle (CDCs), cyclin-dependent kinases (CDKs), and mini-chromosome maintenance proteins (MCMs). Although COVID-19 patients partially shared the expression pattern of some cell cycleassociated molecules with other respiratory illnesses, they exhibited a significantly higher expression of cell cycle-associated molecules associated with disease severity. Notably, the cell cycle signature predominated in the patients blood leukocytes but not in the upper airways. The scRNAseq data from 229 individuals (159 COVID-19 patients and 70 controls) revealed that the alterations of cell cycle signatures predominate in B, T, and NK cells. These results provide a unique global comprehension of the alterations in cell cycle-associated molecules in COVID-19 patients, suggesting new putative pathways for therapeutic intervention

Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly / Novo mapeamento de mutações patogênicas do gene ASPM em famílias consanguíneas com microcefalia primária do Paquistão

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing", with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser "causador de doenças", com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly

Abstract Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be disease causing, with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Resumo Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser causador de doenças, com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly / Novo mapeamento de mutações patogênicas do gene ASPM em famílias consanguíneas com microcefalia primária do Paquistão

Abstract Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing," with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Resumo Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser "causador de doenças", com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Exonic variants of the P2RX7 gene in familial multiple sclerosis.

INTRODUCTION: Several studies have analysed the presence of P2RX7 variants in patients with MS, reporting diverging results. METHODS: Our study analyses P2RX7 variants detected through whole-exome sequencing (WES). RESULTS: We analysed P2RX7, P2RX4, and CAMKK2 gene variants detected by whole-exome sequencing in all living members (n = 127) of 21 families including at least 2 individuals with multiple sclerosis. P2RX7 gene polymorphisms previously associated with autoimmune disease. Although no differences were observed between individuals with and without multiple sclerosis, we found greater polymorphism of gain-of-function variants of P2RX7 in families with individuals with multiple sclerosis than in the general population. Copresence of gain-of-function and loss-of-function variants was not observed to reduce the risk of presenting the disease. Three families displayed heterozygous gain-of-function SNPs in patients with multiple sclerosis but not in healthy individuals. We were unable to determine the impact of copresence of P2RX4 and CAMKK2 variants with P2RX7 variants, or the potential effect of the different haplotypes described in the gene. No clinical correlations with other autoimmune diseases were observed in our cohort. CONCLUSIONS: Our results support the hypothesis that the disease is polygenic and point to a previously unknown mechanism of genetic predisposition to familial forms of multiple sclerosis. P2RX7 gene activity can be modified, which suggests the possibility of preventive pharmacological treatments for families including patients with familial multiple sclerosis.

Análisis del rendimiento de la secuenciación del exoma clínico en hipogonadismo hipogonadotropo congénito teniendo en cuenta el grado de alteración del olfato / Diagnostic yield of clinical exome sequencing in congenital hypogonadotropic hypogonadism considering the degree of olfactory impairment

Introducción: El hipogonadismo hipogonadotropo congénito (HHC) puede presentarse de manera aislada o acompañado de anosmia o de malformaciones congénitas. Más de 30 genes han sido implicados en la patogénesis de HHC; además, se han descrito varios patrones de herencia asociados a esta entidad. La creciente disponibilidad de técnicas de secuenciación masiva (NGS) ha permitido que aumente el rendimiento diagnóstico del estudio de esta patología. Pacientes y métodos: Evaluamos el rendimiento diagnóstico del estudio mediante NGS de pacientes con HHC, usando la secuenciación del exoma clínico filtrado por paneles virtuales. Además, se analizó si el diseño de estos paneles, basándose en la presencia/ausencia de microsmia/anosmia aumentaban este rendimiento diagnóstico. Resultados: Usando un panel virtual compuesto de 34 genes pudimos confirmar el diagnóstico de HHC en cinco de nueve pacientes (55%). En dos de nueve individuos (22%) estudiados se obtuvieron resultados no concluyentes. La ausencia/presencia de microsmia para la elección de genes a estudiar no mejora el rendimiento diagnóstico. Conclusiones: El abordaje del estudio genético de pacientes con HHC puede variar en función de las técnicas disponibles en cada centro, por lo que la sensibilidad del test utilizado variará, dependiendo si se utiliza secuenciación de paneles, exoma clínico o exoma completo. El análisis de todos los genes relacionados con HHC independientemente de la presencia/ausencia de microsmia pareciera el abordaje con mejor rendimiento. (AU)

Introduction: Congenital hypogonadotropic hypogonadism (CHH) can present alone or in association with anosmia or other congenital malformations. More than 30 genes have been identified as being involved in the pathogenesis of CHH with different patterns of inheritance, and the increasing availability of next generation sequencing (NGS) has increased the diagnostic yield. Methods: We analysed the diagnostic yield of NGS in patients with CHH using the clinical exome filtered with virtual panels. We also assessed whether designing panels based on the presence/absence of microsmia increased the diagnostic yield. Results: The use of a 34-gene virtual panel confirmed the diagnosis of CHH in 5 out of 9 patients (55%). In 2 out of 9 (22%), the findings were inconclusive. Applying the presence/absence of microsmia criterion to choose genes for analysis did not improve the diagnostic yield. Conclusions: The approach to the genetic study of patients with CHH varies depending on the resources of each healthcare facility, so the sensitivity of testing may vary substantially depending on whether panels, clinical exome sequencing or whole exome sequencing (WES) are used. The analysis of every genes related to CHH regardless of the presence/absence of microsmia seems to be the best approach. (AU)

Variant rs4149584 (R92Q) of the TNFRSF1A gene in patients with familial multiple sclerosis.

INTRODUCTION: Genomic studies have identified numerous genetic variants associated with susceptibility to multiple sclerosis (MS); however, each one explains only a small percentage of the risk of developing the disease. These variants are located in genes involved in specific pathways, which supports the hypothesis that the risk of developing MS may be linked to alterations in these pathways, rather than in specific genes. We analyzed the role of the TNFRSF1A gene, which encodes one of the TNF-α receptors involved in a signaling pathway previously linked to autoimmune disease. METHODS: We included 138 individuals from 23 families including at least 2 members with MS, and analyzed the presence of exonic variants of TNFRSF1A through whole-exome sequencing. We also conducted a functional study to analyze the pathogenic mechanism of variant rs4149584 (-g.6442643C > G, NM_001065.4:c.362 G > A, R92Q) by plasmid transfection into human oligodendroglioma (HOG) cells, which behave like oligodendrocyte lineage cells; protein labeling was used to locate the protein within cells. We also analyzed the ability of transfected HOG cells to proliferate and differentiate into oligodendrocytes. RESULTS: Variant rs4149584 was found in 2 patients with MS (3.85%), one patient with another autoimmune disease (7.6%), and in 5 unaffected individuals (7.46%). The 2 patients with MS and variant rs4149584 were homozygous carriers and belonged to the same family, whereas the remaining individuals presented the variant in heterozygosis. The study of HOG cells transfected with the mutation showed that the protein does not reach the cell membrane, but rather accumulates in the cytoplasm, particularly in the endoplasmic reticulum and near the nucleus; this suggests that, in the cells presenting the mutation, TNFRSF1 does not act as a transmembrane protein, which may alter its signaling pathway. The study of cell proliferation and differentiation found that transfected cells continue to be able to differentiate into oligodendrocytes and are probably still capable of producing myelin, although they present a lower rate of proliferation than wild-type cells. CONCLUSIONS: Variant rs4149584 is associated with risk of developing MS. We analyzed its functional role in oligodendrocyte lineage cells and found an association with MS in homozygous carriers. However, the associated molecular alterations do not influence the differentiation into oligodendrocytes; we were therefore unable to confirm whether this variant alone is pathogenic in MS, at least in heterozygosis.

The KDM6B mutation: Phenotype and clinical characteristics-Report of a case.

INTRODUCTION: Alterations in the genes of lysine methylation as Lysine-specific demethylase 6B (KDM6B) have been associated with multiple neurodevelopmental disorders. Until now, there are few cases in the literature attributed to KDM6B mutations. This gap may be due to the fact that the exome sequencing technique is still being implemented in routine clinical practice. MATERIAL AND METHODS: A case is presented with its clinical and phenotypic characteristics. The sequence exome analysis was done with the Nimblegen SeqCap EZ MedExome capture kit+mtDNA 47Mb. The psychopathological approach from mental health was carried out through individual and family interviews, the Conner's questionnaires, ADHD rating scale, as well as the psychometry. RESULTS: A frameshift variant in the KDM6B gene related to neurodevelopmental disorders with facial and body dysmorphia was obtained. The case was oriented as a neurodevelopmental disorder secondary to a genetic alteration and a comorbid Attention Deficit Hyperactivity Disorder (ADHD). CONCLUSIONS: The clinical peculiarities shared by patients identified with the KDM6B mutation, raises the need to recognize it as a particular entity. The possibility of applying the exome sequencing technique to patients with syndromic phenotype and developmental impairment may clarify its etiopathogenesis. It is highly probable that the complexity of these cases requires an approach by a multidisciplinary team that includes genetics, neurology and psychiatry, among other specialties. The coordinated approach is essential to have a comprehensive vision of the case.

Preliminary identification of pathogenic variants in an Afro-Colombian Raizal family with risk factors for glaucoma / Identificación preliminar de variantes patógenas en una familia raizal afrocolombiana con factores de riesgo para glaucoma

Abstract Objective: To identify pathogenic variants in an Afro-Colombian Raizal family with risk factors for glaucoma. Methods: In the present study, whole exome sequencing was performed on seven members of a Raizal family from the archipelago of San Andrés, Providencia, and Santa Catalina, in the Caribbean region of Colombia. Four of them had been diagnosed with glaucoma. In addition, two healthy volunteers from the island were included. Results: Of the 198 single nucleotide variants associated with glaucoma, previously reported by the DisGeNET database, four were identified in members of the Raizal family: rs11938093, rs7336216, rs3817672, and rs983034. Furthermore, single nucleotide variant rs983034 was identified in the Wnt ligand secretion mediator gene in all members of the family but not in healthy volunteers. Notably, WLS dysfunctions have been linked to pathology in the trabecular meshwork of the eye. Trabecular meshwork is an important regulator of the outflow of aqueous humor that maintains intraocular pressure (intraocular pressure) at normal levels. Damage to trabecular meshwork is associated with ocular hypertension, which leads to glaucoma progression. In relation to the other single nucleotide variants that were identified, their presence was confirmed in some members of the Raizal family. However, it is still unclear the pathophysiological cause that associates these single nucleotide variants with glaucoma. Conclusions: It was possible to identify four non-synonymous single nucleotide variants that predict significant damage to the structure and function of genes associated with glaucoma pathology in an Afro-Colombian.

Resumen Objetivo: Identificar las variantes patogénicas en una familia raizal afrocolombiana con factores de riesgo para el glaucoma. Métodos: En el presente estudio, se realizó una secuenciación de exoma completo en siete miembros de una familia Raizal del archipiélago de San Andrés, Providencia y Santa Catalina del Caribe colombiano. La mitad de ellos habían sido diagnosticados con glaucoma. Además, se incluyeron dos voluntarios sanos de la isla. Resultados: De las 198 variantes de un solo nucleótido (SNV) asociadas con el glaucoma, previamente informadas por la base de datos DisGeNET, se identificaron cuatro en los miembros de la familia Raizal: rs11938093, rs7336216, rs3817672 y rs983034. Ademas, en todos los miembros de la familia, pero no en voluntarios sanos, se identificó SNV rs983034 en el gen mediador de secreción de ligando Wnt (WLS). Notablemente, las disfunciones WLS se han relacionado con patologías en la red trabecular (TM) del ojo. TM es un regulador importante del flujo de salida del humor acuoso que mantiene la presión intraocular (presión intraocular) en niveles normales. El daño a la TM se asocia con hipertensión ocular que conduce a la progresión del glaucoma. En relación con los demás SNV identificados, se constató su presencia en algunos miembros de la familia Raizal. Sin embargo, aún no está clara la causa fisiopatológica que asocia estas SNV con el glaucoma. Conclusiones: Fue posible identificar cuatro SNVs no sinónimos con predicción de daño significativo en la estructura y función de genes asociados a patología de glaucoma en un afrocolombiano.

The KDM6B mutation: Phenotype and clinical characteristics—Report of a case / La mutación KDM6B: fenotipo y características clínicas—Informe de un caso

Introduction: Alterations in the genes of lysine methylation as Lysine-specific demethylase 6B (KDM6B) have been associated with multiple neurodevelopmental disorders. Until now, there are few cases in the literature attributed to KDM6B mutations. This gap may be due to the fact that the exome sequencing technique is still being implemented in routine clinical practice.Material and methods: A case is presented with its clinical and phenotypic characteristics. The sequence exome analysis was done with the Nimblegen SeqCap EZ MedExome capture kit+mtDNA 47Mb. The psychopathological approach from mental health was carried out through individual and family interviews, the Conner's questionnaires, ADHD rating scale, as well as the psychometry.Results: A frameshift variant in the KDM6B gene related to neurodevelopmental disorders with facial and body dysmorphia was obtained. The case was oriented as a neurodevelopmental disorder secondary to a genetic alteration and a comorbid Attention Deficit Hyperactivity Disorder (ADHD).Conclusions: The clinical peculiarities shared by patients identified with the KDM6B mutation, raises the need to recognize it as a particular entity. The possibility of applying the exome sequencing technique to patients with syndromic phenotype and developmental impairment may clarify its etiopathogenesis. It is highly probable that the complexity of these cases requires an approach by a multidisciplinary team that includes genetics, neurology and psychiatry, among other specialties. The coordinated approach is essential to have a comprehensive vision of the case. (AU)

Introducción: Las alteraciones en los genes de metilación de lisina como la desmetilasa 6B de lisina (KDM6B) se han asociado con múltiples trastornos del neurodesarrollo. Hasta ahora, existen pocos casos en la literatura atribuidos a mutaciones del gen KDM6B. Esta brecha puede deberse al hecho de que la técnica de secuenciación del exoma aún está en fase de implementación en la práctica clínica habitual.Material y métodos: Se presenta un caso con características clínicas y fenotípicas. El análisis de secuenciación exómica se realizó con el kit de captura Nimblegen SeqCap EZ MedExome + mtDNA 47Mb. La aproximación psicopatológica desde salud mental se realizó a través de entrevistas individuales, familiares, los cuestionarios Conners, ADHD rating scale, así como la psicometría.Resultados: Se obtuvo una variante de cambio en el gen KDM6B que ha sido relacionado con trastornos del neurodesarrollo con dismorfias faciales y corporales. El caso fue orientado como un trastorno del neurodesarrollo secundario a una alteración genética, un trastorno por déficit de atención e hiperactividad (TDAH).Conclusiones: Las peculiaridades clínicas que comparten los pacientes identificados con la mutación KDM6B, plantea la necesidad de reconocerla como una entidad particular. La posibilidad de aplicar la técnica de secuenciación del exoma a pacientes con fenotipo sindrómico y retraso generalizado en el desarrollo puede aclarar su etiopatogenia. Es muy probable que la complejidad de estos casos requiera el abordaje de un equipo multidisciplinar que incluya genetistas, neurólogos y psiquiatras, entre otras especialidades. El enfoque coordinado es fundamental para obtener una visión integral del caso. (AU)

Cribado neonatal genómico. Perspectiva de la Comisión de ética de la Asociación Española de Genética Humana. Parte I. Las tecnologías de secuenciación masiva (NGS) y su aplicación al cribado neonatal. Desafíos y oportunidades / Genomic newborn screening. Perspective from the Ethics commission of the Spanish Society for Human Genetics. Part I. Next generation sequencing technologies applied to newborn screening. Challenges and opportunities

En 2003, cuando finalizó el Proyecto Genoma Humano, surgió la idea de secuenciar el genoma a todos los recién nacidos, archivarlo en la historia clínica y usarlo a lo largo de toda la vida para manejar riesgos de enfermedades y respuesta a medicamentos. Dieciocho años más tarde, las promesas de la medicina genómica y el extraordinario abaratamiento de las tecnologías secuenciadoras, siguen alimentando este sueño que todavía plantea grandes desafíos prácticos, éticos y sociales y la secuenciación genómica se presenta como el próximo gran cambio histórico en los programas de cribado neonatal. En el presente artículo, se analizan los retos y oportunidades de las tecnologías secuenciadoras de nueva generación, sus costos reales, la problemática inherente a la gestión, almacenamiento y protección de la enorme cantidad de datos genómicos que generan y finalmente, en base a las conclusiones de investigaciones recientes, se considera el potencial y limitaciones de su aplicación en dos escenarios, el recién nacido enfermo con finalidades diagnósticas y el recién nacido sano, asintomático, con finalidades de salud pública(programas de cribado neonatal). En una segunda parte de este artículo se tendrán en cuenta los aspectos éticos, legales y sociales (AELS). El objetivo final es contribuir al debate científico, profesional, ético y social, promoviendo que la secuenciación genómica en el recién nacido no sea usada indiscriminadamente constituyendo un riesgo, sino que bien empleada sea una aliada en la promoción de la salud y prevención de las consecuencias de las enfermedades genéticas.(AU)

In 2003 at the ending of the Human Genome Project, it aroused the idea that all newborns could be sequenced and its genome archived in the clinical record, in order to manage risks of diseases and response to medicaments along his whole life. Eighteen years later, promises of genomic medicine and tremendous decrease of costs of next generation sequencing technologies, continues feeding this dream that shows important practical, ethical and social challenges and genomic sequencing is presented as the next historical change in newborn screening programs. In this article we analyze challenges and opportunities of next generation sequencing technologies, their real costs, problems associated to management, storage and protection of the enormous amount of genomic data produced and finally, according to conclusions of recent researches, there are considered the conclusions in two contexts, sick newborn with diagnostic purposes and healthy asymptomatic newborns with public health purposes (newborn screening programs). In a second part of this article it will be considered ethical, legal and social issues (ELSI). Final objective is to contribute to scientific, professional, ethics and social debate in order to promote that genome sequencing in newborn don’t be used indiscriminately constituting a risk, but properly done, as a partner in the promotion of health and prevention of consequences of genetic diseases.(AU)

Diagnostic yield of clinical exome sequencing in congenital hypogonadotropic hypogonadism considering the degree of olfactory impairment.

INTRODUCTION: Congenital hypogonadotropic hypogonadism (CHH) can present alone or in association with anosmia or other congenital malformations. More than 30 genes have been identified as being involved in the pathogenesis of CHH with different patterns of inheritance, and the increasing availability of next generation sequencing (NGS) has increased the diagnostic yield. METHODS: We analysed the diagnostic yield of NGS in patients with CHH using the clinical exome filtered with virtual panels. We also assessed whether designing panels based on the presence/absence of microsmia increased the diagnostic yield. RESULTS: The use of a 34-gene virtual panel confirmed the diagnosis of CHH in 5 out of 9 patients (55%) patients. In 2 out of 9 (22%), the findings were inconclusive. Applying the presence/absence of microsmia criterion to choose genes for analysis did not improve the diagnostic yield. CONCLUSIONS: The approach to the genetic study of patients with CHH varies depending on the resources of each healthcare facility, so the sensitivity of testing may vary substantially depending on whether panels, clinical exome sequencing or whole exome sequencing (WES) are used. The analysis of all genes related to CHH regardless of the presence/absence of microsmia seems to be the best approach.

Análisis clínico, molecular y bioinformático de paciente con enfermedad de Cori-Forbes en el Suroccidente Colombiano / Clinical, molecular and bioinformatic analysis a patient with Cori-Forbes disease in the Southwest of Colombia

Introducción: La enfermedad por almacenamiento del glucógeno tipo III (GSDIII, Glycogen storage disease type III) o Enfermedad de Cori Forbes es un trastorno del proceso de glucogenólisis ocasionado por variantes del gen AGL que codifica la enzima desramificante del glucógeno; se encuentra ubicado en el cromosoma 1p21.2 y su alteración genera una degradación incompleta del glucógeno, llevando a una acumulación de dextrina límite en órganos blanco, ocasionando organomegalia y disfunción. Objetivo: Caracterizar molecularmente un paciente lactante mayor con diagnóstico clínico y bioquímico sospechoso de GSDIII. Materiales y Métodos: Paciente lactante mayor masculino con antecedente de displasia broncopulmonar, infección respiratoria aguda, reflujo gastroesofágico, hepatomegalia e intolerancia a la lactosa. Se realizó estudio molecular mediante secuenciación de exoma completo; las variantes reportadas fueron evaluadas por Software de predicción como: Mutation Tas-ter, PROVEAN, UMD-Predictor, POLYPHEN, SIFT, Human Splicing Finder. Finalmente, se realizó una red de interacción génica mediante el programa GeneMania para determinar asociaciones génicas cercanas. Resultados: Se identifi caron 3 variantes heterocigotas ubicadas en el gen AGL: p.Arg910* que ocasiona pérdida del dominio amilo-1,6 glucosidasa y el dominio de unión al glucógeno, y las variantes p.Trp373Cys, p.Asn565Ser que generan cambios missense en la proteína. El análisis de significancia clínica por medio de métodos in-sílico determinó una clasificación patogénica para todas las variantes. La red de interacción permitió observar asociaciones entre el gen AGL y los genes FOXA2, PPP1R3B, NHLRC1 y GCK, que tienen relación con procesos metabólicos. Conclusión: una sospecha clínica inicial, a través de una buena historia clínica y la pertinencia de estudios bioquímicos-metabólicos-genómicos dirigidos, permite brindar un correcto diagnóstico, tratamiento y seguimiento, acercándonos a la medicina de precisión.

Introduction: Glycogen storage disease type III (GSDIII) or Cori Forbes disease is a disorder of the glycogeno-lysis process caused by variants of the AGL gene that encodes the glycogen debranching enzyme; It is located on chromosome 1p21.2 and its alteration generate an incomplete degradation of glycogen, leading to an accumu-lation of borderline dextrin in target organs, causing organomegaly and dysfunction. Objective: To characterize at the molecular level an elderly male lactating patient from southwestern Colombia with a clinical, biochemical diagnosis suspected of GSDIII. Materials and methods: An elderly male infant with a history of bronchopul-monary dysplasia, acute respiratory infection, gastroesophageal refl ux, hepatomegaly, and lactose intolerance. A molecular study was performed by whole exome sequencing; the reported variants were evaluated by prediction software such as Mutation Taster, PROVEAN, UMD-Predictor, POLYPHEN, SIFT, Human Splicing Finder. Fi-nally, a gene interaction network was performed using the GeneMania program to determine close gene associa-tions. Results: 3 heterozygous variants located in the AGL gene were identifi ed: p.Arg910 * that causes loss of the amyl-1,6 glucosidase domain and the glycogen-binding domain, and the variants p.Trp373Cys, p.Asn565 in the protein. The analysis of clinical signifi cance by means of in-silico methods determined a pathogenic classifi cation for all the variants. The interaction network will observe associations between the AGL gene and the FOXA2, PPP1R3B, NHLRC1 and GCK genes, which are related to metabolic processes. Conclusion: an initial clinical suspicion, through a good clinical history and the relevance of directed biochemical-metabolic-genomic studies, allows us to provide a correct diagnosis, treatment, and follow-up, bringing us closer to precision medicine