Gema García-García

• PostDoc Position at Instituto de Investigación Sanitaria La Fe

Advances in whole-genome sequencing (WGS) have significantly enhanced our ability to detect genomic variants underlying inherited diseases. In this study, we performed long-read WGS on 24 patients with inherited retinal dystrophies (IRDs) to validate the utility of nanopore sequencing in detecting genomic variations. We confirmed the presence of al...

Background : Inherited retinal dystrophies (IRDs) are genetically heterogeneous group of conditions, with approximately 40% of cases remaining unresolved after initial genetic testing. This study aimed to assess the impact of a personalised genomic approach integrating whole-exome sequencing (WES) reanalysis, whole-genome sequencing (WGS), customis...

Spinal muscular atrophy (SMA) is a degenerative neuromuscular condition resulting from a homozygous deletion of the survival motor neuron 1 (SMN1) gene in 95% of patients. A timely diagnosis via newborn screening (NBS) and initiating treatment before the onset of symptoms are critical for improving health outcomes in affected individuals. We carrie...

Lipid metabolism plays a critical role in maintaining cellular integrity, especially within the nervous system, where lipids support neuronal structure, function, and synaptic plasticity. However, this essential metabolic pathway is highly susceptible to oxidative stress, which can lead to lipid peroxidation, a damaging process induced by reactive...

The AGBL5 gene encodes for the Cytoplasmic Carboxypeptidase 5 (CCP5), an α-tubulin deglutamylase that cleaves the γ-carboxyl-linked branching point of glutamylated tubulin. To date, pathogenic variants in AGBL5 have been associated only with isolated retinitis pigmentosa (RP). Hearing loss has not been reported in AGBL5 -caused retinal disease. In...

Spinal muscular atrophy (SMA) is a degenerative neuromuscular condition resulting from a homozygous deletion of survival motor neuron 1 (SMN1) gene in 95% of patients. A timely diagnosis via newborn screening (NBS) and initiating treatment before the onset of symptoms are critical for improving health outcomes in affected individuals. We carried ou...

Background Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45–55 DMD exons (del45–55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mu...

Introduction: TULP1 exemplifies the remarkable clinical and genetic heterogeneity observed in inherited retinal dystrophies. Our research describes the clinical and molecular characteristics of a patient manifesting an atypical retinal dystrophy pattern, marked by the identification of both a previously unreported and a rarely encountered TULP1 var...

Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive loss of vision. We report here four patients who presented with RP from three unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putat...

Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45 – 55 DMD exons (del45 – 55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation...

Simple Summary We used PCR-generated small CRISPR constructs to edit two genes (IDH2 and MYBL2) in hard-to-transfect hemopoietic cells, which are central to the progression of the devastating disease known as acute myeloid leukemia LMA (AML). MYBL2 is a transcription factor; when AML patients show an altered expression of this factor, an adverse pr...

Usher syndrome (USH) is a rare, autosomal recessively inherited disorder resulting in a combination of sensorineural hearing loss and a progressive loss of vision resulting from retinitis pigmentosa (RP), occasionally accompanied by an altered vestibular function. More and more evidence is building up indicating that also sleep deprivation, olfacto...

Inherited retinal dystrophies are a group of disorders characterized by the progressive degeneration of photoreceptors leading to loss of the visual function and eventually to legal blindness. Although next generation sequencing (NGS) has revolutionized the molecular diagnosis of these diseases, the pathogenicity of some mutations casts doubts. Aft...

Acute Myeloid Leukaemia is a complex heterogenous disease caused by clonal expansion of undifferentiated myeloid precursors. Recently, several haematological models have been developed with CRISPR/Cas9, using viral vectors, because blood cells are hard to transfect. To avoid virus disadvantages, we have developed a strategy to generate CRISPR const...

Inherited retinal dystrophies (IRD) are a group of diseases characterized by the loss or dysfunction of photoreceptors and a high genetic and clinical heterogeneity. Currently, over 270 genes have been associated with IRD which makes genetic diagnosis very difficult. The recent advent of next generation sequencing has greatly facilitated the diagno...

Usher syndrome (USH) is an autosomal recessive syndromic ciliopathy characterized by sensorineural hearing loss, retinitis pigmentosa and, sometimes, vestibular dysfunction. There are three clinical types depending on the severity and age of onset of the symptoms; in addition, ten genes are reported to be causative of USH, and six more related to t...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Purpose: We aimed to establish correlations between the clinical features of a cohort of Usher syndrome (USH) patients with pathogenic variants in MYO7A, type of pathogenic variant, and location on the protein domain. Methods: Sixty-two USH patients from 46 families with biallelic variants in MYO7A were examined for visual and audiological featu...

Purpose: To highlight the challenge of correct reproductive and therapeutic counselling in complex pedigrees with different inherited retinal dystrophies. Methods: 208 patients diagnosed with non-syndromic inherited retinal dystrophies underwent full ophthalmologic examination and molecular analysis using targeted next-generation sequencing. Re...

Fingertip mechanoreceptors comprise sensory neuron endings together with specialized skin cells that form the end-organ. Exquisitely sensitive, vibration-sensing neurons are associated with Meissner’s corpuscles in the skin. In the present study, we found that USH2A, a transmembrane protein with a very large extracellular domain, was found in termi...

A cohort of 128 patients from 118 families diagnosed with non-syndromic or syndromic hearing loss (HL) underwent an exhaustive clinical evaluation. Molecular analysis was performed using targeted next-generation sequencing (NGS) with a custom panel that included 59 genes associated with non-syndromic HL or syndromic HL. Variants were prioritized ac...

Usher type 1 syndrome is a rare autosomal recessive disorder involving congenital severe-to-profound hearing loss, development of vision impairment in the first decade, and severe balance difficulties. The PCDH15 gene, one of the five genes implicated in this disease, is involved in 8–20% of cases. In this study, we aimed to identify and characteri...

Inherited retinal dystrophies are an assorted group of rare diseases that collectively account for the major cause of visual impairment of genetic origin worldwide. Besides clinically, these vision loss disorders present a high genetic and allelic heterogeneity. To date, over 250 genes have been associated to retinal dystrophies with reported causa...

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 clinic...

A cohort of 172 patients diagnosed clinically with non-syndromic retinal dystrophies, from 110 families underwent full ophthalmologic examination, including retinal imaging, electrophysiology and optical coherence tomography, if feasible. Molecular analysis was performed using targeted next-generation sequencing (NGS). Variants were filtered and pr...

Purpose: Usher syndrome (USH) is a rare disorder characterized by retinitis pigmentosa (RP) and sensorineural hearing loss. Several genes are responsible for the disease, but not all cases are explained by mutations in any of these, supporting the fact that there remain other unknown genes that have a role in the syndrome. We aimed to find the gen...

Non‐syndromic hearing loss (NSHL), a common sensory disorder, is characterized by high clinical and genetic heterogeneity (i.e. approximately 115 genes and 170 loci so far identified). Nevertheless, almost half of patients submitted for genetic testing fail to receive a conclusive molecular diagnosis. We used next‐generation sequencing to identify...

Background Usher syndrome (USH) is a recessive inherited disease characterized by sensorineural hearing loss, retinitis pigmentosa, and sometimes, vestibular dysfunction. Although the molecular epidemiology of Usher syndrome has been well studied in Europe and United States, there is a lack of studies in other regions like Africa or Central and Sou...

Usher syndrome is a rare disorder causing retinitis pigmentosa, together with sensorineural hearing loss. Due to the phenotypic and genetic heterogeneity of this disease, the best method to screen the causative mutations is by high-throughput sequencing. In this study, we tested a semiconductor chip based sequencing approach with 77 unrelated patie...

Choroideremia is a monogenic X‐linked recessive chorioretinal disease linked to pathogenic variants in the CHM gene. These variants are commonly base‐pair changes, frameshifts or large deletions. However, a few rare or unusual events comprising large duplications, a retrotransposon insertion, a pseudo‐exon activation and two c‐98 promoter substitut...

Introduction Mutations in USH2A cause both isolated Retinitis Pigmentosa (RP) and Usher syndrome (that implies RP and hearing impairment). One of the most frequent variants identified in this gene and among these patients is the p.(Cys759Phe) change. However, the pathogenic role of this allele has been questioned since it was found in homozygosity...

Gene Ontology terms (biological process) enriched in USH2A interactome. USH2A interacting proteins (with a STRING combined score > 0.400) compared to the whole genome. Abbreviations: GO, gene ontology; FDR, false discovery rate. (DOC)

Cox proportional hazards models. The table shows the results of the Cox models for the risk of presenting blindness and hearing loss, comparing p. (Cys759Phe) heterozygous patients (missense + truncating) with p. (Cys759Phe) homozygous patients. The models are summarized by the hazard ratio, its 95% confidence interval and the p value. Abbreviation...

Genes related to IRD included in the different NGS approaches used during this study. (A) In house IRD_NGS panel with 68 genes. (B) In house IRD_NGS panel with 75 genes. (C) Virtual panel with 205 genes selected for clinical exome analysis. (D) Virtual panel with 83 genes associated with deafness. (DOC)

Pedigree of homozygous and heterozygous families for USH2A p.(Cys759Phe) variant. (A) Pedigrees of homozygous p.(Cys759Phe) families. (B) Pedigrees of compound heterozygous families. (C) Pedigrees of families with causative mutations in other RP genes. Co-segregation analyses in family members are displayed when available. Abbreviations: m1, p.(Cys...

Number of patients/families that underwent different analysis during this study. *Three patients (RP-1574, RP-0391 and RP-1016/982) initially characterized using classical techniques were not re-analyzed by NGS due to lack of sample with enough quantity and/or quality. ** RP-2424 was excluded from phenotype studies, since no clinical information wa...

Audiograms from two Usher type II patients (families RP-0061 and RP-1031). Audiograms show a typical Usher type II down-sloping pattern with bilateral hypoacusis from moderate to severe degree at high frequencies. (DOC)

Clinical features of 63 patients from 56 families with at least one p.(Cys759Phe) allele. Proband from family RP-2424 was excluded from this table, since only molecular information was available. Families are organized in categories: category A, homozygous patients for p.(Cys759Phe) allele; category B, compound heterozygous patients p.(Cys759Phe) +...

Hearing loss is the most common sensory disorder and because of its high genetic heterogeneity, implementation of Massively Parallel Sequencing (MPS) in diagnostic laboratories is greatly improving the possibilities of offering optimal care to patients. We present the results of a two-year period of molecular diagnosis that included 207 French fami...

Usher syndrome (USH) is a rare autosomal recessive disease and the most common inherited form of combined visual and hearing impairment. Up to 13 genes are associated with this disorder, with USH2A being the most prevalent, due partially to the recurrence rate of the c.2299delG mutation. Excluding hearing aids or cochlear implants for hearing impai...

Background Norrie disease (ND) is a rare X-linked disorder characterized by bilateral congenital blindness. ND is caused by a mutation in the Norrie disease pseudoglioma ( Methods Biochemical and genetic analyses were performed to understand the atypical phenotype and radiological findings. Biogenic amines in cerebrospinal fluid (CSF) were measure...

Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients wor...

Enrichment capture methods for NGS are widely used, however, they evolve rapidly and it is necessary to periodically measure their strengths and weaknesses before transfer to diagnostic services. We assessed two recently released custom DNA solution-capture enrichment methods for NGS, namely Illumina NRCCE and Agilent SureSelectQXT, against a refer...

Background Usher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and tim...

Purpose The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa. Methods The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analy...

We show that massively parallel targeted sequencing of 19 genes provides a new and reliable strategy for molecular diagnosis of Usher syndrome (USH) and nonsyndromic deafness, particularly appropriate for these disorders characterized by a high clinical and genetic heterogeneity and a complex structure of several of the genes involved. A series of...

Patients suffering from Usher syndrome (USH) exhibit sensorineural hearing loss, retinitis pigmentosa (RP) and, in some cases, vestibular dysfunction. USH is the most common genetic disorder affecting hearing and vision and is included in a group of hereditary pathologies associated with defects in ciliary function known as ciliopathies. This syndr...

Comparison between useful data generated by NGS and Sanger sequencing in Usher test sample. Numbers in the sphere correspond to the sum of variants identified in each approach.

Patients included in the study.

Box-plots of average DOC for targeted regions. Detailed coverage for the 634 regions is shown gene by gene. Boxes are showed in red when mean DOC in the region is lower than 409. Red line represents a DOC of 40 reads, the minimum limit for a proper validation. The horizontal gray line corresponds to the median. The mean value is marked with a white...

Inclusion of NGS in the decision-making diagram for molecular diagnosis of USH patients.

List of 47 Usher patients included in test sample. The genes previously studied using Sanger sequencing or aCGH for each subject are marked with a cross and the identified putative mutations are displayed (all the mutations were detected in the heterozygous state).

Table of 19 human genes targeted for the nextgeneration sequencing. Additional exon(s) refers to the number of exons differing from the main isoform.

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital profound deafness, vestibular areflexia and prepubertal retinitis pigmentosa. The first purpose of this study was to determine the pathologic nature of eighteen USH1 putative splicing variants found in our series and their effect in the splicing process by m...

Usher syndrome type 2 (USH2) is an autosomal recessive disease characterized by moderate to severe hearing loss and retinitis pigmentosa. To date, three disease-causing genes have been identified, USH2A, GPR98, and DFNB31, of which USH2A is clearly the major contributor. The aim of this work was to determine the contribution of GPR98 and DFNB31 gen...

To identify the genetic defect in Spanish families with Usher syndrome (USH) and probable involvement of the CLRN1 gene. DNA samples of the affected members of our cohort of USH families were tested using an USH genotyping array, and/or genotyped with polymorphic markers specific for the USH3A locus. Based on these previous analyses and clinical fi...

USH2A sequencing in three affected members of a large family, referred for the recessive USH2 syndrome, identified a single pathogenic alteration in one of them and a different mutation in the two affected nieces. As the patients carried a common USH2A haplotype, they likely shared a mutation not found by standard sequencing techniques. Analysis of...

Usher Syndrome type II (USH2) is an autosomal recessive disorder, characterized by moderate to severe hearing impairment and retinitis pigmentosa (RP). Among the three genes implicated, mutations in the USH2A gene account for 74-90% of the USH2 cases. To identify the genetic cause of the disease and determine the frequency of USH2A mutations in a c...

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by severe-profound sensorineural hearing loss, retinitis pigmentosa, and vestibular areflexia. To date, five USH1 genes have been identified. One of these genes is Usher syndrome 1C (USH1C), which encodes a protein, harmonin, containing PDZ domains. The aim of the present...

PCDH15, encoding protocadherin 15, is mutated in Usher syndrome type 1F (USH1F) patients. Not only point mutations, but also large deletions have been detected within this gene. However, the detection and characterization of gross deletions in the USH1F locus have been difficult. The purpose of the present work was to identify large genomic rearran...

Usher syndrome is defined by the association of sensorineural hearing loss, retinitis pigmentosa and variable vestibular dysfunction. Many disease-causative mutations have been identified in MYO7A and USH2A genes, which play a major role in Usher syndrome type I and type II, respectively. The pathogenic nature of mutations that lead to premature st...

It has been demonstrated that mutations in deafness, autosomal recessive 31 (DFNB31), the gene encoding whirlin, is responsible for nonsyndromic hearing loss (NSHL; DFNB31) and Usher syndrome type II (USH2D). We screened DFNB31 in a large cohort of patients with different clinical subtypes of Usher syndrome (USH) to determine the prevalence of DFNB...

The purpose of this study was to test the ability of the genotyping microarray for Usher syndrome (USH) to identify the mutations responsible for the disease in a cohort of 183 patients with USH. DNA from 183 patients with Usher syndrome from the Spanish population was analyzed using a genotyping microarray containing 429 previously identified dise...