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Dora Janeth Fonseca,1,* Adrien Morel,1,* Kevin Llinás-Caballero,1 David Bolívar-Salazar,1 Paul Laissue1,2 1Center for Research in Genetics and Genomics-CIGGUR, GENIUROS Research Group, School of Medicine and Health Sciences, Universidad Del Rosario, Bogotá, Colombia; 2BIOPAS Laboratoires, Orphan Diseases Unit, BIOPAS GROUP, Bogotá, Colombia*These authors contributed equally to this workCorrespondence: Paul LaissueBIOPAS Laboratoires, BIOPAS Group, Calle 127A #53ª-45, Bogotá, CP, 11001, ColombiaTel +57 3212010179Email paullaissue@yahoo.comBackground: Adverse drug reactions (ADRs) are frequent occurring events that can essentially be defined as harmful or unpleasant symptoms secondary to the use of a medicinal product. ADRs involve a wide spectrum of clinical manifestations ranging from minor itching and rash to life-threatening reactions. Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare ADRs. SJS-TEN may be considered a polygenic pathology due to additive/epistatic effects caused by sequence variants in numerous genes. Next-generation sequencing (NGS) represents a potentially interesting exploration tool in such scenario as it facilitates the simultaneous analysis of large genomic regions and genes at affordable cost.Methods: The present study has involved using whole-exome sequencing (WES) for the first time on SJS-TEN patients. It involved robust and innovative multistep bioinformatics analysis focusing on 313 candidate genes potentially participating in the disease’s aetiology, specific drugs’ metabolism and gene regulation.Results: We identified combinations of frequently occurring and rare variants that may contribute to the disease’s pathogenesis. Depending on the specific drug being taken, different variants (and alleles) in NAT2, CYP2D8, CYP2B6, ABCC2, UGT2B7 and TCF3 were identified as coherent candidates representing potential future mar

Carlos Alberto Calderon-Ospina,1,* Mario Hernández-Sómerson,2,* Ana María García,1 Adriana Mejia,1 Caroll Tamayo-Agudelo,1 Paul Laissue,1 Dora Janeth Fonseca Mendoza1 1Center for Research in Genetics and Genomics-CIGGUR, GENIUROS Research Group, School of Medicine and Health Sciences. Universidad Del Rosario, Bogotá, Colombia; 2Medical Clinic Service, Hospital Universitario Mayor Méderi-Universidad Del Rosario, Bogotá, Colombia*These authors contributed equally to this workCorrespondence: Dora Janeth Fonseca MendozaUniversidad Del Rosario, Cra 24 N° 63c-69, Bogotá, ColombiaTel +5712920200Fax +571 3499390Email dora.fonseca@urosario.edu.coAbstract: Rosuvastatin, is a widely-used statin for the treatment of hypercholesterolemia and the prevention of cardiovascular diseases. Although rosuvastatin is well tolerated, about 3/10.000 patients can suffer severe myopathy. Rhabdomyolysis is a severe medical condition that causes injury to the skeletal muscle, electrolyte imbalances, acute renal failure and extreme creatine kinase (CK) elevation. Little is known regarding the molecular involvement of rosuvastatin-induced rhabdomyolysis (RIR). It has been demonstrated that genomic variants associated with decreased enzymatic activity of proteins are important determinants in plasmatic and skeletal muscle distribution of rosuvastatin and its toxicity. Until now, no interactions of ticagrelor, ezetimibe and rosuvastatin have been described with the consideration of pharmacogenomics predisposition. The present report involves a whole-exome sequencing (WES), in a patient affected by rosuvastatin-induced rhabdomyolysis. A pharmacogenomic dissection was performed by analyzing a comprehensive subset of candidate genes (n=160) potentially related to RIR. The genes were selected according to their implication in drug metabolism or inherited myopathies. Using an innovative approach of bioinform

A pesar de que la preeclampsia (PE) es una de las principales causas de morbimortalidad materna y fetal, su etiología es desconocida. Con el objetivo de determinar nuevos genes y mutaciones potencialmente etiológicos de la enfermedad, en el presente trabajo efectuamos la secuenciación simultánea de 386 genes, a partir de ADN placentario, en 60 mujeres afectadas por PE y restricción de crecimiento intrauterino (RCIU). Computacionalmente, utilizamos rigurosos filtros de selección de variantes. Identificamos 21 variantes (21 genes), entre las cuales 14 fueron confirmadas por medio de secuenciación de Sanger (AGT, ERAP1, F5, FOS, HTRA3, KDR, LIPC, MAN1C1, PDGFRA, SIAE, TET2, TGFB2, TGFB3, VCAN). Aquellos genes con variantes que participan en procesos biológicos como coagulación, modulación de la función inmunológica y angiogénesis llamaron especialmente la atención por su íntima relación con el desarrollo y función placentaria. Los resultados obtenidos son innovadores y sugieren una etiología poligénica de la PE. En el futuro, para una mejor comprensión de los resultados en el marco de la función placentaria, son imperativos estudios adicionales in vitro e in vivo.

La insuficiencia ovárica primaria (IOP) es una patología frecuente que afecta del 1% al 3% de las mujeres de la población general menores de 40 años. No existen datos específicos en la población colombiana, lo cual sería un punto importante a determinar teniendo en cuenta que se ha reportado que esta prevalencia puede variar según el origen étnico de las pacientes. La IOP se caracteriza clínicamente por la ausencia (amenorrea primaria) o el cese de la ovulación durante al menos cuatro meses (amenorrea secundaria), con un aumento de los valores plasmáticos de FSH (hormona folículo estimulante). Aunque se han relacionado causas autoinmunes, metabólicas, infecciosas e iatrogénicas, en la mayoría de los casos su etiología es desconocida, lo que sugiere posibles causas genéticas. A pesar de numerosos intentos por determinar estas causas, solo algunos genes han sido relacionados funcionalmente con la etiología de esta enfermedad. En esta tesis de Doctorado se realizó la identificación y la validación de nuevas variantes y/o nuevos genes asociados a la etiología de la IOP. Este trabajo de tesis se divide en dos capítulos, en el primero se describe la identificación de variantes en el gen BMP15 y la evaluación de cómo las mutaciones en este gen contribuyen a la disfunción ovárica. Para esto se evaluó el efecto de 10 mutaciones en BMP15 respecto a la producción del péptido maduro, la activación de la señalización de la vía SMAD y la sinergia con GDF9. La genotipificación de BMP15 en 35 pacientes Colombianas permitió la identificación de una variante nueva (c.986C>G-p.Arg329His) y una variante previamente reportada (c.581T>C, p.Phe194Ser). La evaluación funcional de estas variantes, junto con otras 8 identificadas previamente, indicó que las mutaciones p.Leu148Phe, p.Phe194Ser y p.Tyr235Cys, están relacionadas con la reducción en la producción del péptido maduro. Las mutaciones p.Arg138His, p.Ala180Thr y p.Arg329His redujeron la actividad de BMP15 aproximadamente cuatro veces, Primary ovarian insufficiency (POI) is a frequent pathology affecting 1% - 3% of women from the general population under 40 years old. There are not specific data related Colombian population, however, this could be an interesting point to be determinate taking account the variation of POI prevalence according ethnical origin. POI is characterized by the absence (primary amenorrhea) or the cessation of ovulation (secondary amenorrhea) for at least four months, as well as high plasmatic levels of FSH (Follicle Stimulating Hormone). Autoimmune, metabolic, infectious and iatrogenic causes have been related to the disease pathogenesis. However, in the majority of cases, the aetiology remains undiscovered. Despite numerous attempts to identify these genetic causes, only a few genes have been functionally related to POI’s etiology. This Ph.D. thesis focuses on the identification and functional validation of new variants and/or new genes related to POI’s etiology. This manuscript is divided in two chapters. The first one describes the identification of new BMP15 variants and the evaluation of how these variants may contribute to ovarian dysfunction. The effect of ten BMP15 mutations in mature peptide production, activation of SMAD signaling and GDF9 synergy were assessed. The BMP15 screening on 35 Colombian patients led to the identification of a new variant (c.986C>G-p.Arg329His) and a previously reported one (c.581T>C, p.Phe194Ser) associated with POI. Assessing the expression/activity of these and other 8 BMP15 mutants allowed to describe that: (1) multiple variants, including p.Leu148Phe, p.Phe194Ser and p.Tyr235Cys reduced mature protein production; (2) other variants (p.Arg138His, p.Ala180Thr and p.Arg329His) displayed lower the activity than wild-type BMP15; and (3) some variants (p.Arg68Trp, p.Phe194Ser and p.Asn196Lys) reduced GDF9 synergy. These results showed that BMP15 mutations can disrupt ovarian function through different mechanisms. The second chapter focus

La Preeclampsia (PE) es una enfermedad exclusiva del embarazo caracterizada por hipertensión arterial y compromiso multisistémico. Afecta entre el 2% y el 8% de las mujeres gestantes en el mundo y es la principal causa de morbilidad y mortalidad materna y neonatal. El principal evento relacionado con la fisiopatología de la PE es la disfunción endotelial, la cual depende del control poligénico de múltiples cascadas moleculares. Diversas proteínas han sido estudiadas con el objetivo de esclarecer la fisiopatología de la enfermedad, entre ellas el factor de transcripción STOX1. Esta proteína regula diferentes procesos biológicos implicados en la fisiopatología de la PE como la angiogénesis, el estrés oxidativo, la inflamación y la apoptosis. Sin embargo, se desconoce qué factores de transcripción se fijan al promotor de STOX1 participando en su regulación. Durante el presente trabajo de tesis se propuso identificar los factores que interactúan potencialmente con la región promotora de STOX1 e identificar, por medio de secuenciación del promotor, variantes que puedan afectar sitios de unión a factores de transcripción en una muestra de pacientes francesas afectadas con PE. Para cumplir este objetivo fueron utilizadas técnicas experimentales in vivo como el ensayo de monohíbrido en levaduras y bioinformáticas in silico. Se identificó al factor de transcripción HEY-L como un potencial regulador de STOX1 y fue encontrada la variante c.-735T>G en la región donde potencialmente interactúa este factor de transcripción. Sin embargo, deben ser realizados estudios funcionales para validar la interacción definitiva y si la mutación genera una modificación en la transactivación del promotor.

Jubby Marcela Galvez,1 Carlos Martin Restrepo,1 Nora Constanza Contreras,1 Clara Alvarado,1 Carlos-Alberto Calderón-Ospina,1 Nidia Peña,1 Ricardo A Cifuentes,2 Daniela Duarte,1 Paul Laissue,1 Dora Janeth Fonseca1 1GENIUROS Research Group, Center For Research in Genetics and Genomics – CIGGUR, School of Medicine and Health Sciences, Universidad del Rosario, Bogotá, Colombia; 2Area of Basic Sciences, College of Medicine, Universidad Militar Nueva Granada, Bogotá, Colombia Purpose: Warfarin is an oral anticoagulant associated with adverse reaction to drugs due to wide inter- and intra-individual dosage variability. Warfarin dosage has been related to non-genetic and genetic factors. CYP2C9 and VKORC1 gene polymorphisms affect warfarin metabolism and dosage. Due to the central role of populations’ ethnical and genetic origin on warfarin dosage variability, novel algorithms for Latin American subgroups are necessary to establish safe anticoagulation therapy.Patients and methods: We genotyped CYP2C9*2 (c.430C > T), CYP2C9*3 (c.1075A > C), CYP4F2 (c.1297G > A), and VKORC1 (-1639 G > A) polymorphisms in 152 Colombian patients who received warfarin. We evaluated the impact on the variability of patients’ warfarin dose requirements. Multiple linear regression analysis, using genetic and non-genetic variables, was used for creating an algorithm for optimal warfarin maintenance dose.Results: Median weekly prescribed warfarin dosage was significantly lower in patients having the VKORC1-1639 AA genotype and poor CYP2C9*2/*2,*2/*3 metabolizers than their wild-type counterparts. We found a 2.3-fold increase in mean dose for normal sensitivity patients (wild-type VKORC1/CYP2C9 genotypes) compared to the other groups (moderate and high sensitivity); 31.5% of the patients in our study group had warfarin sensitivity-related genotypes. The estimated regression equation account

Los síndromes de enanismo esencial microcefálico son un grupo de enfermedades monogénicas infrecuentes que se caracterizan principalmente por talla baja extrema proporcionada de inicio prenatal y microcefalia severa. En los pacientes que formaron parte del presente estudio se investigaron variantes en el gen PCNT debido a que presentaban hallazgos clínicos compatibles con el síndrome MOPD II (enanismo esencial osteodisplásico microcefálico tipo II). Posteriormente, se amplió el estudio con una secuenciación de exoma en una paciente con variantes heterocigotas en el gen PCNT que no explicaban el fenotipo, encontrando una variante nueva en el gen DDX11 y realizando el diagnóstico de Síndrome de Warsaw Breakage en esta paciente., Microcephalic primordial dwarfism syndromes are a group of rare monogenic diseases that are characterized primarily by extreme low stature of prenatal onset and severe microcephaly. In patients who participated in the study, variants in the gene PCNT was investigated because they had clinical findings consistent with the MOPD II (Microcephalic osteodisplastic primordial dwarfism type II) syndrome. The study was expanded with exome sequencing in a patient who presented heterozygous variants in the PCNT gene, which could not explain the phenotype. This find gave room for the discovery of a new variant in the DDX11 gene that allowed for the diagnosis of the Warsaw Breakage syndrome in the patient.

Las proteínas ADAMTS (desintegrinas y metaloproteinasas con motivos trombospondina) han sido asociadas a diversos procesos biológicos entre ellos el desarrollo folicular y la ovulación. Algunos miembros son considerados proteínas huérfanas debido a que no se han descrito sus sustratos específicos (e.g. ADAMTS19). Específicamente ADAMTS19 ha sido sugerido recientemente como un gen candidato de FOP. En el presente trabajo se realizó un tamizaje de las potenciales proteínas de interacción (partners) de ADAMTS19 mediante el sistema de doble híbrido en levaduras. Se realizó coinmunoprecipitación en células transfectadas y en líquido folicular humano para verificar la interacción directa entre ADAMTS19 y COL6A2. Ensayos de RT-PCR efectuados para evaluar la coexpresión de Adamts19 y Col6a2 en ovarios de ratón. Adicionalmente, la técnica de inmunoflurescencia indirecta usó para evaluar la colocalización de las proteínas. Se encontró que ADAMTS19 se une directamente a COL6A2 en un contexto ovárico lo que se correlaciona con los hallazgos de coexpresión y de colocalización en líquido folicular humano. Proponemos que ADAMTS19 actúa sobre las microfibras de COL6A2 durante el crecimiento folicular y la expansión de la lámina basal. Encontramos que la mutación p.Thr943Ile ADAMTS19 identificada previamente en una paciente con FOP no alteraba la interacción ADAMTS19/COL6A2. Sin embargo, no descartamos que la mutación contribuya al fenotipo de FOP. Los resultados de este trabajo, que describen la primera proteína de interacción directa de ADAMTS19, arrojan nuevos datos sobre la función de las metaloproteinasas en la biología del ovario y la fertilidad., ADAMTS proteins (disintegrins and metalloproteinases with thrombospondin motifs) have been associated with various biological processes including follicular development and ovulation. Some members are considered orphan proteins because their specific substrates have not been described (e.g. ADAMTS19). Specifically ADAMTS19 has recently been suggested as a candidate gene for FOP. In the present study a screening of the potential interaction proteins (partners) of ADAMTS19 was carried out using yeast two hybrid assay. Co-immunoprecipitation was performed on transfected cells and human follicular fluid to verify the direct interaction between ADAMTS19 and COL6A2. RT-PCR assays werw performed to evaluate co-expression of Adamts19 and Col6a2 in mouse ovaries. In addition, the indirect immunofluorescence technique used to evaluate the colocalization of proteins. It was found that ADAMTS19 binds directly to COL6A2 in an ovarian context which correlates with the findings of coexpression and colocalization in human follicular fluid. We propose that ADAMTS19 acts on the microfibers of COL6A2 during follicular growth and expansion of the basal lamina. We found that the p.Thr943Ile ADAMTS19 mutation previously identified in a patient with FOP did not alter the ADAMTS19 / COL6A2 interaction. However, we do not rule out that the mutation contributes to the FOP phenotype. The results of this work, which describe the first direct interaction protein of ADAMTS19, yield new data on the role of metalloproteinases in ovarian biology and fertility., Universidad del Rosario

Se describe la variante homocigota c.320-2A>G de TGM1 en dos hermanas con ictiosis congénita autosómica recesiva. El clonaje de los transcritos generados por esta variante permitió identificar tres mecanismos moleculares de splicing alternativos., The variant c.320-2A> G of TGM1 is described in two sisters with autosomal recessive congenital ichthyosis. The cloning of the transcripts generated by this variant allowed the identification of three alternative molecular splicing mechanisms., Unidad de Genética, Facultad de Medicina, Colegio Mayor de Nuestra Señora del Rosario, Grupo CIGGUR, centro de Investigación en Genética y Genómica Universidad del Rosario, Unidad de Dermatología, Clínica Carlos Ardila Lule, Bucaramanga.

La falla ovárica prematura es una enfermedad común que conduce a la infertilidad femenina y cuya etiología no es identificable en más del 50% de los casos, lo cual sugiere un origen genético en la patogénesis de esta enfermedad. La función crucial del gen BMP15 en la biología de la reproducción fue propuesta cuando modelos KO de ratón y mutaciones naturales en ovejas revelaron fenotipos ováricos específicos. Aunque la secuenciación de la región codificante de BMP15 en grandes paneles de pacientes afectadas con Falla Ovárica Prematura (FOP) ha identificado algunas mutaciones, estas variaciones explican una baja proporción de casos. Nosotros hipotetizamos que una variante en la secuencia reguladora (promotor BMP15) podrían estar asociada a la etiología de la FOP no-sindrómica. Con la evidencia de los estudios previos que sugirieron la potencial implicación de la variante de secuencia c.-9C>G del promotor de BMP15 en los fenotipos reproductivos incluyendo FOP, evaluamos si este polimorfismo podría modificar las propiedades de transactivación de un factor de transcripción específico. Empleamos aproximaciones in-silico para predecir potenciales sitios de unión a factores de transcripción (TFBS: Transcription Factor Binding Sites) en la región 5´ de BMP15. El ensayo reportero de luciferasa se usó para determinar la modificación de la transacativación del promotor de BMP15 causada por la variante de c-9C>G. Se demostró que aunque los dos constructos del promotor de BMP15 (BMP15- prom-G and BMP15-prom-C) fueron transactivados por el factor de transcripción PITX1, el constructo BMP15- prom-G aumentó 1.6 veces la actividad transcripcional del factor de transcripción de una manera estadísticamente significativa. Por otro lado, se demostró por primera vez que BMP15 y PITX1 son co-expresados en tejido ovárico de humano y de ratón., BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15 c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF.

Blepharophimosis syndrome (BPES) is caused by loss-of-function mutations in the single-exon forkhead transcription factor gene FOXL2 and by genomic rearrangements of the FOXL2 locus. Here, we focus on 92 new intragenic FOXL2 mutations, 34 of which are novel. Specifically, we found 10 nonsense mutations (11%), 13 missense mutations (14%), 40 deletions or insertions leading to a frameshift (43%), and 29 in-frame changes (32%), of which 28 (30%) lead to a polyalanine expansion. This study confirms the existence of two previously described mutational hotspots. Moreover, we gained novel insights in genotype-phenotype correlations, emphasizing the need to interpret genotype-phenotype correlations individually and always in the context of further clinical observations.

Blepharophimosis syndrome (BPES) is caused by loss-of-function mutations in the single-exon forkhead transcription factor gene FOXL2 and by genomic rearrangements of the FOXL2 locus. Here, we focus on 92 new intragenic FOXL2 mutations, 34 of which are novel. Specifically, we found 10 nonsense mutations (11%), 13 missense mutations (14%), 40 deletions or insertions leading to a frameshift (43%), and 29 in-frame changes (32%), of which 28 (30%) lead to a polyalanine expansion. This study confirms the existence of two previously described mutational hotspots. Moreover, we gained novel insights in genotype-phenotype correlations, emphasizing the need to interpret genotype-phenotype correlations individually and always in the context of further clinical observations., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

Blepharophimosis syndrome (BPES) is caused by loss-of-function mutations in the single-exon forkhead transcription factor gene FOXL2 and by genomic rearrangements of the FOXL2 locus. Here, we focus on 92 new intragenic FOXL2 mutations, 34 of which are novel. Specifically, we found 10 nonsense mutations (11%), 13 missense mutations (14%), 40 deletions or insertions leading to a frameshift (43%), and 29 in-frame changes (32%), of which 28 (30%) lead to a polyalanine expansion. This study confirms the existence of two previously described mutational hotspots. Moreover, we gained novel insights in genotype-phenotype correlations, emphasizing the need to interpret genotype-phenotype correlations individually and always in the context of further clinical observations.

Blepharophimosis syndrome (BPES) is caused by loss-of-function mutations in the single-exon forkhead transcription factor gene FOXL2 and by genomic rearrangements of the FOXL2 locus. Here, we focus on 92 new intragenic FOXL2 mutations, 34 of which are novel. Specifically, we found 10 nonsense mutations (11%), 13 missense mutations (14%), 40 deletions or insertions leading to a frameshift (43%), and 29 in-frame changes (32%), of which 28 (30%) lead to a polyalanine expansion. This study confirms the existence of two previously described mutational hotspots. Moreover, we gained novel insights in genotype-phenotype correlations, emphasizing the need to interpret genotype-phenotype correlations individually and always in the context of further clinical observations., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published