Five years' experience of the clinical exome sequencing in a Spanish single center.

Nowadays, exome sequencing is a robust and cost-efficient genetic diagnostic tool already implemented in many clinical laboratories. Despite it has undoubtedly improved our diagnostic capacity and has allowed the discovery of many new Mendelian-disease genes, it only provides a molecular diagnosis in up to 25-30% of cases. Here, we comprehensively evaluate the results of a large sample set of 4974 clinical exomes performed in our laboratory over a period of 5 years, showing a global diagnostic rate of 24.62% (1391/4974). For the evaluation we establish different groups of diseases and demonstrate how the diagnostic rate is not only dependent on the analyzed group of diseases (43.12% in ophthalmological cases vs 16.61% in neurological cases) but on the specific disorder (47.49% in retinal dystrophies vs 24.02% in optic atrophy; 18.88% in neuropathies/paraparesias vs 11.43% in dementias). We also detail the most frequent mutated genes within each group of disorders and discuss, on our experience, further investigations and directions needed for the benefit of patients.

Genetics and epidemiology of aniridia: Updated guidelines for genetic study.

Aniridia is a panocular disease characterized by iris hypoplasia, accompanied by other ocular manifestations, with a high clinical variability and overlapping with different abnormalities of the anterior and posterior segment. This review focuses on the genetic features of this autosomal dominant pathology, which is caused by the haploinsufficiency of the PAX6 gene. Mutations causing premature stop codons are the most frequent among the wider mutational spectrum of PAX6, with more than 600 different mutations identified so far. Recent advances in next-generation sequencing (NGS) have increased the diagnostic yield in aniridia and contributed to elucidate new etiopathogenic mechanisms leading to PAX6 haploinsufficiency. Here, we also update good practices and recommendations to improve genetic testing and clinical management of aniridia using more cost-effective NGS analysis. Those new approaches also allow studying simultaneously both structural variants and point-mutations in PAX6 as well as other genes for differential diagnosis, simultaneously. Some patients with atypical phenotypes might present mutations in FOXC1 and PITX2, both genes causing a wide spectrum of anterior segment dysgenesis, or in ITPR1, which is responsible for a distinctive form of circumpupillary iris aplasia present in Gillespie syndrome, or other mutations in minor genes. Since aniridia can also associate extraocular anomalies, as it occurs in carriers of PAX6 and WT1 microdeletions leading to WAGR syndrome, genetic studies are crucial to assure a correct diagnosis and clinical management, besides allowing prenatal and preimplantational genetic testing in families.

NGS and phenotypic ontology-based approaches increase the diagnostic yield in syndromic retinal diseases.

Syndromic retinal diseases (SRDs) are a group of complex inherited systemic disorders, with challenging molecular underpinnings and clinical management. Our main goal is to improve clinical and molecular SRDs diagnosis, by applying a structured phenotypic ontology and next-generation sequencing (NGS)-based pipelines. A prospective and retrospective cohort study was performed on 100 probands with an a priori diagnosis of non-Usher SRDs, using available clinical data, including Human Phenotype Ontology annotation, and further classification into seven clinical categories (ciliopathies, specific syndromes and five others). Retrospective molecular diagnosis was assessed using different molecular and bioinformatic methods depending on availability. Subsequently, uncharacterized probands were prospectively screened using other NGS approaches to extend the number of analyzed genes. After phenotypic classification, ciliopathies were the most common SRD (35%). A global characterization rate of 52% was obtained, with six cases incompletely characterized for a gene that partially explained the phenotype. An improved characterization rate was achieved addressing prospective cases (83%) and well-recognizable syndrome (62%) subgroups. The 27% of the fully characterized cases were reclassified into a different clinical category after identification of the disease-causing gene. Clinical-exome sequencing is the most appropriate first-tier approach for prospective cases, whereas whole-exome sequencing and bioinformatic reanalysis increases the diagnosis of uncharacterized retrospective cases to 45%, mostly those with unspecific symptoms. Our study describes a comprehensive approach to SRDs in daily clinical practice and the importance of thorough clinical assessment and selection of the most appropriate molecular test to be used to solve these complex cases and elucidate novel associations.

Genetics and epidemiology of aniridia: Updated guidelines for genetic study. / Genética y epidemiología de la aniridia congénita: actualización de buenas prácticas para el diagnóstico genético.

Aniridia is a panocular disease characterized by iris hypoplasia, accompanied by other ocular manifestations, with a high clinical variability and overlapping with different abnormalities of the anterior and posterior segment. This review focuses on the genetic features of this autosomal dominant pathology, which is caused by the haploinsufficiency of the PAX6 gene. Mutations causing premature stop codons are the most frequent among the wider mutational spectrum of PAX6, with more than 600 different mutations identified so far. Recent advances in next-generation sequencing (NGS) have increased the diagnostic yield in aniridia and contributed to elucidate new etiopathogenic mechanisms leading to PAX6 haploinsufficiency. Here, we also update good practices and recommendations to improve genetic testing and clinical management of aniridia using more cost-effective NGS analysis. Those new approaches also allow studying simultaneously both structural variants and point-mutations in PAX6 as well as other genes for differential diagnosis, simultaneously. Some patients with atypical phenotypes might present mutations in FOXC1 and PITX2, both genes causing a wide spectrum of anterior segment dysgenesis, or in ITPR1, which is responsible for a distinctive form of circumpupillary iris aplasia present in Gillespie syndrome, or other mutations in minor genes. Since aniridia can also associate extraocular anomalies, as it occurs in carriers of PAX6 and WT1 microdeletions leading to WAGR syndrome, genetic studies are crucial to assure a correct diagnosis and clinical management, besides allowing prenatal and preimplantational genetic testing in families.

Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes.

Despite the improved accuracy of next-generation sequencing (NGS), it is widely accepted that variants need to be validated using Sanger sequencing before reporting. Validation of all NGS variants considerably increases the turnaround time and costs of clinical diagnosis. We comprehensively assessed this need in 1109 variants from 825 clinical exomes, the largest sample set to date assessed using Illumina chemistry reported. With a concordance of 100%, we conclude that Sanger sequencing can be very useful as an internal quality control, but not so much as a verification method for high-quality single-nucleotide and small insertion/deletions variants. Laboratories might validate and establish their own thresholds before discontinuing Sanger confirmation studies. We also expand and validate 23 copy number variations detected by exome sequencing in 20 samples, observing a concordance of 95.65% (22/23).

Implication of non-coding PAX6 mutations in aniridia.

There is an increasing implication of non-coding regions in pathological processes of genetic origin. This is partly due to the emergence of sophisticated techniques that have transformed research into gene expression by allowing a more global understanding of the genome, both at the genomic, epigenomic and chromatin levels. Here, we implemented the analysis of PAX6, whose coding loss-of-function variants are mainly implied in aniridia, by studying its non-coding regions (untranslated regions, introns and cis-regulatory sequences). In particular, we have taken advantage of the development of high-throughput approaches to screen the upstream and downstream regulatory regions of PAX6 in 47 aniridia patients without identified mutation in the coding sequence. This was made possible through the use of custom targeted resequencing and/or CGH array to analyze the entire PAX6 locus on 11p13. We found candidate variants in 30 of the 47 patients. 9/30 correspond to the well-known described 3' deletions encompassing SIMO and other enhancer elements. In addition, we identified numerous different variants in various non-coding regions, in particular untranslated regions. Among these latter, most of them demonstrated an in vitro functional effect using a minigene strategy, and 12/21 are thus considered as causative mutations or very likely to explain the phenotypes. This new analysis strategy brings molecular diagnosis to more than 90% of our aniridia patients. This study revealed an outstanding mutation pattern in non-coding PAX6 regions confirming that PAX6 remains the major gene for aniridia.

Los defectos en las extremidades, una facies peculiar y el fallo de medro no siempre constituyen un síndrome de Cornelia de Lange / Limbs defects, peculiar face and failure to thrive is not always Cornelia de Lange Syndrome

Las malformaciones de las extremidades son raras y su etiología, variable. Existen síndromes genéticos que combinan estas malformaciones con discapacidad intelectual y otras malformaciones graves, como el síndrome de Cornelia de Lange. Presentamos el caso de una paciente con malformaciones severas en las 4 extremidades, discapacidad intelectual y características faciales peculiares que llevaron al diagnóstico presuntivo de síndrome de Cornelia de Lange. Presentamos el caso de una niña de 4 años de edad, padres consanguíneos y etnia gitana, con antecedentes de retraso del crecimiento intrauterino, bajo peso al nacer, microcefalia y múltiples malformaciones en ambas manos y pies, incluida la mano derecha hendida, con diagnóstico presuntivo de síndrome de Cornelia de Lange. Durante el primer año de vida se realizaron varios estudios con los siguientes resultados: cariotipo 46, XX; estudio de deleciones subteloméricas (técnica MLPA) normal; ecocardiograma y electrocardiograma sin hallazgos; evaluación oftalmológica y auditiva normales; ultrasonido abdominal y transfontanelar normales. A los 4 años se le aplicó una técnica de array de hibridación genómica comparada (comparative genomic hybridization) (array-CGH) con resolución de 180 kb, que detectó una deleción causal de 8,4 Mb en la citobanda 2q31.1q31.2. La deleción de 2q31 está asociada a la malformación de mano/pie hendido, y la correlación genotipo-fenotipo indica que las deleciones intersticiales de la región 2q31.1 muestran malformaciones en los miembros si incluyen una región crítica de 2,5 Mb, que incluye el cluster de HOXD y las regiones adyacentes en sentido 5’ y 3’. Concluimos que ante un paciente con malformaciones graves y signos y síntomas superpuestos de varios síndromes, es aconsejable comenzar el plan de trabajo con array-CGH, y si esta técnica no está disponible, realizar un cariotipo de alta resolución con la intención de descartar reordenamientos cromosómicos (AU)

Severe limbs deformities are rare and its etiology is variable. There are known genetic syndromes that combine limbs deformities, mental disability and other mayor malformations, such as Cornelia de Lange syndrome. We present the case of a patient with severe deformities in 4 limbs, mental disability and minor facial features that lead to the presumptive diagnosis of Cornelia de Lange syndrome. Four years old female her parents are consanguineous and from gipsy ethnicity. History of intrauterine growth retardation, low birth weight, microcephaly and multiple deformities in both hands and both feet including split-hand deformity of the right hand. Presumptive diagnosis of Cornelia de Lange syndrome, during the first year of life these studies were performed: kariotype 46, XX, subtelomeric deletion study (MLPA technique): normal, echocardiogram and EKG without abnormalities, oftalmologic and audition evaluation: normal and cranial and abdominal ultrasound also normal. At four years old array comparative genomic hybridization 180 kb was performed and it showed a causal deletion of 8.4 Mb at cytoband 2q31.1q31.2. 2q31 deletion is associated with the split hand/foot malformation, the genotype-phenotype correlation of interstitial deletions of the 2q31.1 region shows that limbs malformation are associated to a critical 2.5 Mb deletion containing the HOXD cluster and surrounding 5’ and 3’ regions. We conclude that when a patient presents major malformations and overlapping signs and symptoms of various syndromes it is wise to begin the workup with high resolution karyotype and/or, where available, array-CGH in order to rule out cytogenetic rearrangements (AU)

Guidelines for genetic study of aniridia.

INTRODUCTION: Aniridia is a panocular disorder which occurs in 1/50,000 to 1/100,000 live births and can appear either in isolated form or in the context of a syndrome. Isolated aniridia is inherited as an autosomal dominant condition and is caused by mutations of the PAX6 gene. A variety of techniques and methodologies within molecular genetics and cytogenetics are used to study these mutations. OBJECTIVE: To identify the different aspects of this disease and to provide a guide for proper genetic diagnosis leading to improved clinical management of the disease. DEVELOPMENT: Aniridia is an autosomal dominant disease that primarily affects the iris, though it can impact most of the ocular structures. The disease is mainly caused by mutations in the PAX6 gene located on chromosome 11p13 which encodes a transcription factor that is involved in the development of the eye. Genetic analysis of aniridia is complex and requires the use of both molecular genetics and cytogenetics techniques. These procedures are indicated in all cases of aniridia. It is important bear certain clinical and technical aspects in mind prior to starting analysis or providing genetic counseling for patients and their families. CONCLUSIONS: The use of molecular genetic techniques in the genetic diagnosis of aniridia enables patients and their families to receive better clinical management.

Guía para el estudio genético de la aniridia / Guidelines for genetic study of aniridia

Introducción: La aniridia es una enfermedad panocular con una incidencia de entre 1/50.000 a 1/100.000 nacidos vivos, que puede presentarse de forma aislada o en el contexto de un síndrome. Presenta una herencia autosómica dominante y en la mayoría de los casos está causada por mutaciones en el gen PAX6, para cuyo estudio de mutaciones se emplea una gran variedad de técnicas y metodologías de genética molecular y citogenéticas. Objetivo: Recoger los distintos aspectos de esta enfermedad y ofrecer una guía para el adecuado diagnóstico genético que ayude a un mejor manejo clínico de la misma. Desarrollo: La aniridia es una enfermedad autosómica dominante que afecta fundamentalmente al iris, pero también puede afectar a la mayoría de las estructuras oculares. Está causada principalmente por mutaciones en el gen PAX6, ubicado en la región cromosómica 11p13, que codifica para una proteína reguladora de la transcripción imprescindible en el desarrollo del ojo. El análisis genético de la aniridia es complejo y requiere tanto de técnicas de genética molecular (secuenciación, CGH-array o MLPA) como citogenéticas (cariotipo y FISH). Este estudio está indicado en todos los casos de aniridia y es importante tener en cuenta ciertas consideraciones tanto clínicas como técnicas antes de abordar su análisis y el asesoramiento genético de los pacientes y familias afectados por esta enfermedad. Conclusiones: La aplicación de técnicas de genética molecular al diagnóstico genético de la aniridia permite un mejor manejo clínico tanto de los afectados como de sus familiares(AU)

Introduction: Aniridia is a panocular disorder which occurs in 1/50,000 to 1/100,000 live births and can appear either in isolated form or in the context of a syndrome. Isolated aniridia is inherited as an autosomal dominant condition and is caused by mutations of the PAX6 gene. A variety of techniques and methodologies within molecular genetics and cytogenetics are used to study these mutations. Objective: To identify the different aspects of this disease and to provide a guide for proper genetic diagnosis leading to improved clinical management of the disease. Development: Aniridia is an autosomal dominant disease that primarily affects the iris, though it can impact most of the ocular structures. The disease is mainly caused by mutations in the PAX6 gene located on chromosome 11p13 which encodes a transcription factor that is involved in the development of the eye. Genetic analysis of aniridia is complex and requires the use of both molecular genetics and cytogenetics techniques. These procedures are indicated in all cases of aniridia. It is important bear certain clinical and technical aspects in mind prior to starting analysis or providing genetic counseling for patients and their families. Conclusions: The use of molecular genetic techniques in the genetic diagnosis of aniridia enables patients and their families to receive better clinical management(AU)

Novel p.M96T variant of NRL and shRNA-based suppression and replacement of NRL mutants associated with autosomal dominant retinitis pigmentosa.

Mutations in the gene encoding the transcription factor neural retina leucine zipper (NRL) are known to cause autosomal dominant (adRP) or recessive (arRP) retinitis pigmentosa (RP). In an adRP Spanish family, we detected a novel sequence variation (c.287T>C) in the NRL gene that results in the p.M96T protein change. A functional test of the ability of NRL, in conjunction with cone-rod homeobox (CRX), to transactivate a human rhodopsin (RHO) promoter was used to evaluate the pathogenic mechanisms of NRL. We found upregulation of the RHO promoter by p.M96T protein similar to that shown by other missense NRL mutations that cause adRP. Affected RP patients of the family carry the nucleotide change, although two other family members that also carry the c.287T>C variation remain asymptomatic. This result complicates the genetic counselling of the family. The pathogenic mechanisms associated with adRP NRL mutations appear to be caused by a gain of function. To suppress the negative effect of an NRL mutant, the suppression and replacement strategy seems to be the most suitable therapeutic approach capable of overcoming the mutational heterogeneity associated with NRL-linked adRP. Thus, we evaluated this methodology in the NRL gene for the first time.

Members 6B and 14 of the TNF receptor superfamily in multiple sclerosis predisposition.

TNFRSF6B and TNFRSF14 genes were recently associated with Crohn's disease and rheumatoid arthritis. TNFRSF14 is known as herpes virus entry mediator (HVEM), and herpes viruses have been involved in the aetiology of multiple sclerosis (MS). MS patients present human herpes virus 6 (HHV6) in active plaques and increased antibody responses to HHV6. We aimed to ascertain the role of these genes in MS susceptibility and to investigate the relationship of the gene encoding the widely expressed HVEM receptor with the active replication of HHV6 found in some MS patients. Genotyping of 1370 Spanish MS patients and 1715 ethnically matched controls was performed. HHV6A DNA levels (surrogate of active viral replication) were analysed in serum of MS patients during a 2-year follow-up. Both polymorphisms were associated with MS predisposition, with stronger effect in patients with HHV6 active replication-TNFRSF6B-rs4809330(*)A: P=0.028, OR=1.13; TNFRSF14-rs6684865(*)A: overall P=0.0008, OR=1.2; and HHV6-positive patients vs controls: P=0.017, OR=1.69.

Glypican 5 is an interferon-beta response gene: a replication study.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. Interferon-beta is the most usual therapy in relapsing-remiting MS. However, approximately 50% of the treated patients do not respond adequately. Very recently, a genome-wide association study on interferon-beta pharmacogenetics has described polymorphisms at several genes that are associated with response to this treatment. Our aim is to replicate the results obtained at the two loci most strongly implicated in the response to interferon-beta treatment, HAPLN1 and GPC5. PATIENTS AND METHODS: We performed a case-control study, analyzing 199 patients with MS treated with interferon-beta for at least 2 years and at least two documented relapses over the 2 years, previous to treatment onset. Responders had neither relapses nor increase in expanded disability status scale (EDSS) over the 2-year follow-up period, whereas nonresponders had at least two relapses or an increase in EDSS of at least 1 point. We studied three single-nucleotide polymorphisms (SNPs) in the GPC5 locus and three SNPs in the HAPLN1 locus by TaqMan technology. Allelic frequencies between responders and nonresponders were compared by a chi-square test. RESULTS: An association was found between GPC5 polymorphisms and the response to interferon-beta therapy in patients with MS, in agreement with earlier data (responder vs nonresponder patients: rs10492503, P = 0.0005). The other locus studied (HAPLN1) did not show association with treatment response to interferon-beta (all SNPs P > 0.05). CONCLUSIONS: We confirm the association of polymorphisms within GPC5 with response to interferon-beta therapy in patients with MS.