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Clonal hematopoiesis, especially that of indeterminate potential (CHIP), has been associated with age-related diseases, such as those contributing to a more severe COVID-19. Four studies have attempted to associate CHIP with COVID-19 severity without conclusive findings. In the present work, we explore the association between CHIP and COVID-19 mortality. Genomic DNA extracted from peripheral blood of COVID-19 patients (n = 241 deceased, n = 239 survivors) was sequenced with the Myeloid Solutions™ panel of SOPHiA Genetics. The association between clonality and age and clonality and mortality was studied using logistic regression models adjusted for sex, ethnicity, and comorbidities. The association with mortality was performed with patients stratified into four groups of age according to the quartiles of the distribution: 60–74 years, 75–84 years, 85–91 years, and 92–101 years. Clonality was found in 38% of the cohort. The presence of CHIP variants, but not the number, significantly increased with age in the entire cohort of COVID-19 patients, as well as in the group of survivors (p p = 0.020). We confirmed the well-established linear relationship between age and clonality in the cohort of COVID-19 patients and found a significant association between pathogenic/likely pathogenic CHIP and mortality in patients from 75 to 84 years that needs to be further validated.

BackgroundThe paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-function PAX6 variants.MethodsBy locus-specific analysis of PAX6 using Sanger and/or targeted next-generation sequencing, we screened a Spanish cohort of 106 patients with PAX6-related diseases. Functional splicing assays were performed by in vitro minigene approaches or directly in RNA from patient-derived lymphocytes cell line, when available.ResultsFive out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation.ConclusionWe describe new spliceogenic mechanisms for PAX6 variants mediated by creating or strengthening five different cryptic donor sites at exon 6. Our work revealed that the activation of cryptic PAX6 splicing sites seems to be a recurrent and underestimated cause of aniridia. Our findings pointed out the importance of functional assessment of apparently silent PAX6 variants to uncover hidden genetic alterations and to improve variant interpretation for genetic counselling in aniridia.

Con esta edición se busca crear una plataforma analítica y de discusión sobre la importancia de la interdisciplina en el estudio de la forma de la ciudad. Por ello, en esta publicación se aborda el tema “Forma urbana e interdisciplina” a partir de cuatro ejes temáticos: Análisis y diagnósticos de la forma urbana; procesos históricos de la forma urbana; planes y proyectos urbanos e impactos en la forma urbana. En suma, esperamos que el material presentado cumpla con sus objetivos y que su lectura contribuya a ampliar el panorama de reflexión y discusión sobre el tema de la interdisciplina en el estudio de la forma urbana.

Laxedo, located at the foot of the Sierra del Cando, is not simply a geographical space with morphological and functional characteristic -typical of this area at the Galician rural mid-mountain range- but we are also facing a settlement made up of small nuclei that form a single and large polynuclear “village”. To speak of Laxedo is to talk about the figure of Mr. Manuel Barreiro Cabanelas, an illustrious neighbor who emigrated to Brazil. His privileged social status and his large real estate businesses will allow him to be in direct contact with the urban reality of Rio de Janeiro for decades, nourishing himself from the most modern intellectual and technical thinking of the time. Cabanelas, by means of his knowledge, applied between 1900 and 1936, a series of urban planning principles of European and American influence used in the city planning of the Rio de Janeiro -Beaurepaire, Comissão de Melhoramentos, Pereira Passos and Agache. This will change forever his native Laxedo. The study carried out combines an understanding of the urban metabolism of the nucleus through its history and its social dynamics as the effect of emigration. What really gives meaning to the place itself is the balance between traditional rural dynamics, emigration, vernacular architecture, urban influence, and the Masonic symbolism of sculpture linked to the urban layout.

Since the start of the Living Atlases community in 2013, participants have given us feedback on how to enhance our community. One of the recommendations was to simplify the deployment of a new Living Atlases data portal. As a result, the technical coordinator, Vicente J. Ruiz Jurado, of the Living Atlases community, designed a procedure that uses remote sessions to deploy a new Living Atlas instance at an institution. Two options are available. The first one is dedicated to newcomers to help them understand how the Living Atlases modules work by installing a demo in several Virtual Machines, which are provided. The demo installation remains available for some weeks after the session to aid learning. The second option focuses on helping participants during their first deployment on their own production machines. We will explain both solutions during the presentation. During this talk, we will also explore other tools that simplify the installation like the Living Atlases Ansible Generator. We will detail how this technical tool helps developers install chosen modules from the Atlas of Living Australia suite. The feedback from those who have participated in these sessions will be shared.

The paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-functionBy locus-specific analysis ofFive out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation.We describe new spliceogenic mechanisms for

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients’ features. Our data support the notion that CPAMD8 loss-offunction underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.

Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K–AKT–mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.

Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the NHS gene cause Nance-Horan syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical NHS isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced NHS expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting NHS, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the NHS defects.

Purpose The aim of the present work is the molecular diagnosis of three patients with deafness and retinal degeneration. Methods Three patients from two unrelated families were initially analyzed with custom gene panels for Usher genes, non-syndromic hearing loss, or inherited syndromic retinopathies and further investigated by means of clinical or whole exome sequencing. Results The study allowed us to detect likely pathogenic variants in PEX6, a gene typically involved in peroxisomal biogenesis disorders (PBDs). Beside deaf–blindness, both families showed additional features: Siblings from Family 1 showed enamel alteration and abnormal peroxisome. In addition, the brother had mild neurodevelopmental delay and nephrolithiasis. The case II:1 from Family 2 showed intellectual disability, enamel alteration, and dysmorphism. Conclusions We have reported three new cases with pathogenic variants in PEX6 presenting with milder forms of the Zellweger spectrum disorders (ZSD). The three cases showed distinct clinical features. Thus, expanding the phenotypic spectrum of PBDs and ascertaining exome sequencing is an effective strategy for an accurate diagnosis of clinically overlapping and genetically heterogeneous disorders such as deafness–blindness association.

International audience; GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identified four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identified in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical significance were also identified. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identified heterozygous 1q21 microdeletions in five families with coloboma and other ocular and/or extraocular findings. However, the exact genotype-phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confirming the importance of this gene in early eye development.

Human iPSC line, IISHDOi006-A, was obtained from fibroblasts of a patient with Dominant Optic Atrophy (DOA) carrying a heterozygous mutation in the gene ACO2: c.1999G>A; p.Glu667Lys. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using a non-integrative methodology that involves the use of Sendai virus.

The power and configurability of the the Atlas of Living Australia tools have enabled more and more institutions and participants of the Global Biodiversity Information Facility adapt and install biodiversity platforms. For six years, we have demonstrated that the community around this platform was needed and ready for its adoption. During the symposium organized for the SPNHC+TDWG 2018, we started a discussion that has led us to the creation of a more structured and sustainable community of practice. We want to create a community that follows the structure of open-source technical projects such as Linux or Apache foundation. After the GBIF Governing Board (GB25), the Kilkenny accord was agreed among 8 country or institution partners and early adopters of ALA platform to outline the scope of the new Living Atlases community. Thanks to this accord, we have begun to set up a new structure based on the Community of Practice (CoP) model. In summary, the governance will be held by a Management committee and a Technical advisory committee. Adding to these, the Living Atlases community will have two coordinators with technical and administrative duties. This presentation will briefly summarise the community history leading up to the agreement of the Kilkenny accord and provide information and context of the key points contained. Then, we will present and launch the new Living Atlases Community of Practice . Through this presentation, we aim to collect lessons learned and good practices from other CoP in topics like governance, communications, sustainability, among others to incorporate them in the consolidation process of the Living Atlases community.

E-learning is a training method increasingly used as it enhances opportunities for participation, allows active tracking of courses and enables wider flexibility for timetables and organization. The implementation of an e-learning platform managed by GBIF Spain was aimed at exploring other ways of creating capacity, reaching a broader audience and multiplying the impact of a well-consolidated training facility. The first platform, used by the Spanish node in 2012, was the open source tool ATutor, which was migrated to Chamilo in 2017 and is available at http://elearning.gbif.es. Since then, these platforms have hosted courses focused on biodiversity data mobilization and data use, addressing both users and data publishers. GBIF.es offers the platform to other organizations in the community so the entire GBIF network can benefit from this service, taking advantage of the already invested resources. Additionally, the GBIF Secretariat uses the platform for developing training activities in the Biodiversity Information for Development (BID) and Biodiversity Information for Asia (BIFA) programmes reaching participants in sub-Saharan Africa, the Caribbean and the Pacific, and has implemented the Biodiversity Data Mobilization course for use in other programmes such as Biodiversity Data Management Skills for Students (BioDATA) and Biodiversity Information Management and Reporting (BIMR), reaching communities in Eurasia and South-East Europe respectively. GBIF Argentina has also used it to offer a course on biodiversity data quality and publication. In this session, we will overview the GBIF.es e-learning platform, highlight some successful use cases of the virtual training tool and show how it helped GBIF Spain reach a broader community. We will also present plans for future expansion.

A Living Atlas is an informatics infrastructure that aggregates biodiversity data from multiple sources and makes it freely available and usable online. It is based on the Atlas of Living Australia platform and has been adopted since 2014 by numerous GBIF Nodes and other organizations to support integrated access and use of their national biological and environmental information. Its implementation at national level has been justified by the need to provide data services tailored to the specific needs of national users and communities at a level of detail not offered in international portals. As all these countries and organizations share the same technology to make biodiversity data available, it makes perfect sense to synchronize efforts in producing documentation that can be useful for all of them. The Living Atlases Community has been working on improving the national atlases documentation for end-users during the last year. Efforts were focused in producing two video tutorials Fig. 1 about different components of the Atlas tools and practical online exercises about how to use these data portals. Resources are available in different languages so they can meet the needs of all users. During this presentation, we will show the videos and make a demonstration of the documentation available. We will also present a draft roadmap for enhancing this important part of the community and collect inputs from participants in the symposium.

International audience; 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.

Mutations in PAX6 are involved in several developmental eye disorders. These disorders have considerable phenotypic variability, ranging from panocular forms of congenital aniridia and microphthalmia to isolated anomalies of the anterior or posterior segment. Here, we describe 3 families with variable inter-generational ocular expression of aniridia, iris coloboma, or microphthalmia, and an unusual transmission of PAX6 mutations from an unaffected or mildly affected parent; all of which raised suspicion of gonosomal mosaicism. We first identified two previously known nonsense mutations and one novel likely pathogenic missense variant in PAX6 in probands by means of targeted NGS. The subsequent segregation analysis by Sanger sequencing evidenced the presence of highly probable mosaic events in paternal blood samples. Mosaicism was further confirmed by droplet digital PCR analysis in several somatic tissues of mosaic fathers. Quantification of the mutant allele fraction in parental samples showed a marked deviation from 50%, with a range between 12 and 29% depending on cell type. Gonosomal mosaicsm was definitively confirmed in one of the families thanks to the availability of a sperm sample from the mosaic father. Thus, the recurrence risk in this family was estimated to be about one-third. This is the first report confirming parental PAX6 mosaicism as a cause of disease recurrence in aniridia and other related phenotypes. In addition, we demonstrated that post-zygotic mosaicism is a frequent and underestimated pathogenic mechanism in aniridia, explaining intra-familial phenotypic variability in many cases. Our findings may have substantial implications for genetic counseling in congenital aniridia. Thus, we also highlight the importance of comprehensive genetic screening of parents for new sporadic cases with aniridia or related developmental eye disease to more accurately assess recurrence risk. In conclusion, somatic and/or gonosomal mosaicism should be taken into consideration as a genetic factor to explain not only families with unaffected parents despite multiple affected children but also variable expressivity, apparent de novo cases, and even uncharacterized cases of aniridia and related developmental eye disorders, apparently lacking PAX6 mutations.

Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). Here, Rodriguez-Laguna et al. report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of GLA., Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K–AKT–mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.

Inherited syndromic retinopathies are a highly heterogeneous group of diseases that involve retinal anomalies and systemic manifestations. They include retinal ciliopathies, other well-defined clinical syndromes presenting with retinal alterations and cases of non-specific multisystemic diseases. The heterogeneity of these conditions makes molecular and clinical characterization of patients challenging in daily clinical practice. We explored the capacity of targeted resequencing and copy-number variation analysis to improve diagnosis of a heterogeneous cohort of 47 patients mainly comprising atypical cases that did not clearly fit a specific clinical diagnosis. Thirty-three likely pathogenic variants were identified in 18 genes (ABCC6, ALMS1, BBS1, BBS2, BBS12, CEP41, CEP290, IFT172, IFT27, MKKS, MYO7A, OTX2, PDZD7, PEX1, RPGRIP1, USH2A, VPS13B, and WDPCP). Molecular findings and additional clinical reassessments made it possible to accurately characterize 14 probands (30% of the total). Notably, clinical refinement of complex phenotypes was achieved in 4 cases, including 2 de novo OTX2-related syndromes, a novel phenotypic association for the ciliary CEP41 gene, and the co-existence of biallelic USH2A variants and a Koolen-de-Vries syndrome-related 17q21.31 microdeletion. We demonstrate that combining next-generation sequencing and CNV analysis is a comprehensive and useful approach to unravel the extensive phenotypic and genotypic complexity of inherited syndromic retinopathies, This study was funded by several grants from the Instituto de Salud Carlos III (ISCIII) from the Spanish Ministry of Health, including CIBERER (06/07/0036), FIS - FEDER (European Regional Development Fund) (PI016/00425), IIS-FJD Biobank PT13/0010/0012. In addition, sponsored chair HU-FJD-UAM “Cátedra de Patrocinio Medicina Genómica”, the Spanish National Organization of the Blind (ONCE) and the Spanish Fighting Blindness Foundation (FUNDALUCE) also supported our work. ISN and RSA are sponsored by Sara Borrell Postdoctoral Program (CD13-00085 and CD12-00676) from ISCIII/FEDER. MC is supported by the Miguel Servet Program (CP12/03256) from ISCIII/FEDER. RPC is supported by Fundación Conchita Rábago and LRJS is supported by CAPES Foundation, Ministry of Education of Brazil

There is no sign of saturation in accumulation of alien species (AS) introductions worldwide, additionally the rate of spread for some species has also been shown to be increasing. However, the challenges of gathering information on AS are recognized. Recent developments in citizen science (CS) provide an opportunity to improve data flow and knowledge on AS while ensuring effective and high quality societal engagement with the issue of IAS (Invasive Alien Species). Advances in technology, particularly on-line recording and smartphone apps, along with the development of social media, have revolutionized CS and increased connectivity while new and innovative analysis techniques are emerging to ensure appropriate management, visualization, interpretation and use and sharing of the data. In early July 2018 we launched a European CO-operation in Science and Technology (COST) Action to address multidisciplinary research questions in relation to developing and implementing CS, advancing scientific understanding of AS dynamics while informing decision-making specifically implementation of technical requirements of relevant legislation such as the EU Regulation 1143/2014 on IAS. It will also support the EU biodiversity goals and embedding science within society. The Action will explore and document approaches to establishing a European-wide CS AS network. It will embrace relevant innovations for data gathering and reporting to support the implementation of monitoring and surveillance measures, while ensuring benefits for society and citizens, through an AS CS European network. The Action will, therefore, increase levels of participation and quality of engagement with current CS initiatives, ensuring and evaluating educational value, and improve the value outcomes for potential users including citizens, scientists, alien species managers, policy-makers, local authorities, industry and other stakeholders., peer-reviewed

[Purpose]: CLAPO syndrome is a rare vascular disorder characterized by capillary malformation of the lower lip, lymphatic malformation predominant on the face and neck, asymmetry, and partial/generalized overgrowth. Here we tested the hypothesis that, although the genetic cause is not known, the tissue distribution of the clinical manifestations in CLAPO seems to follow a pattern of somatic mosaicism., [Methods]: We clinically evaluated a cohort of 13 patients with CLAPO and screened 20 DNA blood/tissue samples from 9 patients using high-throughput, deep sequencing., [Results]: We identified five activating mutations in the PIK3CA gene in affected tissues from 6 of the 9 patients studied; one of the variants (NM_006218.2:c.248T>C; p.Phe83Ser) has not been previously described in developmental disorders., [Conclusion]: We describe for the first time the presence of somatic activating PIK3CA mutations in patients with CLAPO. We also report an update of the phenotype and natural history of the syndrome., This research was supported by the project “Genetics of vascular and lymphatic malformations” financed with funds donated by Asociación Ultrafondo and Villareal FC, cofinanced by project IP-17 from the funding call “Todos Somos Raros” (Telemaraton TVE promoted by Fundación Isabel Gemio, Federación ASEM, and Federación Española de Enfermedades Raras), cofinanced by the Instituto de Salud Carlos III, FEDER FUNDS FIS PI15/01481, and IIS-Fundación Jiménez Díaz UAM Genome Medicine Chair.

Choroideremia (CHM) is a rare X-linked disease leading to progressive retinal degeneration resulting in blindness. The disorder is caused by mutations in the CHM gene encoding REP-1 protein, an essential component of the Rab geranylgeranyltransferase (GGTase) complex. In the present study, we evaluated a multi-technique analysis algorithm to describe the mutational spectrum identified in a large cohort of cases and further correlate CHM variants with phenotypic characteristics and biochemical defects of choroideremia patients. Molecular genetic testing led to the characterization of 36 out of 45 unrelated CHM families (80%), allowing the clinical reclassification of four CHM families. Haplotype reconstruction showed independent origins for the recurrent p.Arg293* and p.Lys178Argfs*5 mutations, suggesting the presence of hotspots in CHM, as well as the identification of two different unrelated events involving exon 9 deletion. No certain genotype-phenotype correlation could be established. Furthermore, all the patients´ fibroblasts analyzed presented significantly increased levels of unprenylated Rabs proteins compared to control cells; however, this was not related to the genotype. This research demonstrates the major potential of the algorithm proposed for diagnosis. Our data enhance the importance of establish a differential diagnosis with other retinal dystrophies, supporting the idea of an underestimated prevalence of choroideremia. Moreover, they suggested that the severity of the disorder cannot be exclusively explained by the genotype.

Chromosomal deletions at 11p13 are a frequent cause of congenital Aniridia, a rare pan-ocular genetic disease, and of WAGR syndrome, accounting up to 30% of cases. First-tier genetic testing for newborn with aniridia, to detect 11p13 rearrangements, includes Multiplex Ligation-dependent Probe Amplification (MLPA) and karyotyping. However, neither of these approaches allow obtaining a complete picture of the high complexity of chromosomal deletions and breakpoints in aniridia. Here, we report the development and validation of a customized targeted array-based comparative genomic hybridization, so called WAGR-array, for comprehensive high-resolution analysis of CNV in the WAGR locus. Our approach increased the detection rate in a Spanish cohort of 38 patients with aniridia, WAGR syndrome and other related ocular malformations, allowing to characterize four undiagnosed aniridia cases, and to confirm MLPA findings in four additional patients. For all patients, breakpoints were accurately established and a contiguous deletion syndrome, involving a large number of genes, was identified in three patients. Moreover, we identified novel microdeletions affecting 3' PAX6 regulatory regions in three families with isolated aniridia. This tool represents a good strategy for the genetic diagnosis of aniridia and associated syndromes, allowing for a more accurate CNVs detection, as well as a better delineation of breakpoints. Our results underline the clinical importance of performing exhaustive and accurate analysis of chromosomal rearrangements for patients with aniridia, especially newborns and those without defects in PAX6 after diagnostic screening.

PURPOSE. Choroideremia (CHM) is an X-linked ophthalmic disease. The gene associated with CHM (REP-1) encodes a ubiquitously expressed protein that is indispensable for the posttranslational activation of retina-specific Rab protein. Different mutations, including large genomic rearrangements involving the REP-1 gene, are responsible for CHM, but they all cause the protein to be truncated or absent. The authors screened 20 Spanish families with clinical diagnoses of CHM to determine the molecular cause of the disease. METHODS. First, the authors performed haplotype analyses to determine whether the disease is linked to the REP-1 gene. In families in whom the disease segregated with the CHM locus (n = 14), mutational screening of the REP-1 gene was performed. RESULTS. In 13 of the 14 families in which the phenotype segregated with the CHM locus, the authors identified the mutation associated with the disease. Eight different molecular defects that led to truncation and one that led to complete absence of the REP-1 protein were found in nine families and one family, respectively. Furthermore, the authors identified a novel type of mutation in the REP-1 gene in three families. This novel type of mutation did not result in a truncated or absent protein. Rather, these patients lost different parts of the REP-1 mRNA in-frame that in all the cases encode a conserved protein domain implicated in the interaction with Rab proteins. CONCLUSIONS. Based on the different mutations found, the authors propose a four-step protocol for the molecular diagnosis of CHM.

Leber congenital amaurosis (LCA) is the most severe inherited retinopathy with the earliest age of onset. To date, eleven genes have been reported to cause the non-syndromic LCA phenotype. The CEP290 gene has been shown to account for Joubert and Senior-Loken syndromes and to represent a frequent cause of non-syndromic LCA. The aim of the present study was to establish the prevalence of CEP290 c.2991_1655AG in non-syndromic Spanish patients having LCA or early-onset retinitis pigmentosa (RP).We used automated sequencing to examine 49 non-syndromic Spanish families with LCA and 126 Spanish families with early-onset RP for the CEP290 c.2991_1655AG mutation. As a control, we recruited 50 unrelated Spanish healthy individuals.The frequencies of mutated alleles were 6% in LCA cases and 0% in early-onset RP and healthy individual controls. These results were compared to other populations.The CEP290 c.2991_1655AG mutation frequency in Spanish non-syndromic LCA families is lower than that of other countries.

Purpose The purpose of this study was to evaluate the clinical usefulness of the array comparative genomic hybridization technique for the genetic analysis of patients with congenital ocular malformations. Design Laboratory investigation. Methods This was a multicenter study. Samples were collected from 37 patients with negative results for the routine diagnostic work-up, including normal karyotype and mutation analysis of appropriate genes. Samples from both parents also were tested. High-resolution genome-wide Agilent 244K oligoarray (Agilent Technologies) was applied. Confirmation of the results was obtained with independent techniques. Results Causal deletions were identified in 5 (13%) patients, affecting OTX2, FOXC1 and VPS13B (COH1), the downstream regulatory region of PAX6, and a 1,5 Megabases de novo deletion on chromosome 16. Conclusions This high frequency of causal submicroscopic chromosomal aberrations in patients with congenital ocular malformation warrants implementation of array comparative genomic hybridization in the diagnostic work-up of these patients. Moreover, this screening technique broadens the phenotypic and mutational spectrum associated with genes known to cause congenital ocular malformation.