Caracterización molecular de CNVs, mutaciones sin sentido y de splicing en enfermedades metabólicas hereditarias: investigación en terapias personalizadas

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-05-201

Hereditary primary lateral sclerosis and progressive nonfluent aphasia

Objective: to report a kindred with an association between hereditary primary lateral sclerosis (PLS) and progressive nonfluent aphasia. Patients and methods: six members from a kindred with 15 affected individuals spanning three generations, suffered from spasticity without muscle atrophy or fasciculation, starting in the lower limbs and spreading to the upper limbs and bulbar musculature, followed by effortful speech, nonfluent language and dementia, in 5 deceased members. Disease onset was during the sixth decade of life, or later. Cerebellar ataxia was the inaugural manifestation in two patients, and parkinsonism, in another. Results: neuropathological examination in two patients demonstrated degeneration of lateral corticospinal tracts in the spinal cord, without loss of spinal, brainstem, or cerebral motor neurons. Greater loss of corticospinal fibers at sacral and lumbar, rather than at cervical or medullary levels was demonstrated, supporting a central axonal dying-back pathogenic mechanism. Marked reduction of myelin and nerve fibers in the frontal lobes was also present. Argyrophilic grain disease and primary age-related tauopathy were found in one case each, and considered incidental findings. Genetic testing, including exome sequencing aimed at PLS, ataxia, hereditary spastic paraplegia, and frontotemporal lobe dementia, triplet-repeated primed polymerase chain reaction aimed at dominant spinocerebellar ataxias, and massive sequencing of the human genome, yielded negative results. Conclusion: a central distal axonopathy affecting the corticospinal tract, exerted a pathogenic role in the dominantly inherited PLS-progressive nonfluent aphasia association, described herein. Further molecular studies are needed to identify the causative mutation in this disease

Combining targeted panel-based resequencing and copy-number variation analysis for the diagnosis of inherited syndromic retinopathies and associated ciliopathies

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 retinopathiesFEDER (Fondo Europeo de Desarrollo Regional) | Ref. PI016/00425Instituto de Salud Carlos III | Ref. PT13/0010/001

Whole-exome sequencing reveals ZNF408 as a new gene associated with autosomal recessive retinitis pigmentosa with vitreal alterations

Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies that cause visual impairment as a result of photoreceptor cell death. RP is heterogeneous, both clinically and genetically making difficult to establish precise genotype–phenotype correlations. In a Spanish family with autosomal recessive RP (arRP), homozygosity mapping and whole-exome sequencing led to the identification of a homozygous mutation (c.358_359delGT; p.Ala122Leufs*2) in the ZNF408 gene. A screening performed in 217 additional unrelated families revealed another homozygous mutation (c.1621C>T; p.Arg541Cys) in an isolated RP case. ZNF408 encodes a transcription factor that harbors 10 predicted C2H2-type fingers thought to be implicated in DNA binding. To elucidate the ZNF408 role in the retina and the pathogenesis of these mutations we have performed different functional studies. By immunohistochemical analysis in healthy human retina, we identified that ZNF408 is expressed in both cone and rod photoreceptors, in a specific type of amacrine and ganglion cells, and in retinal blood vessels. ZNF408 revealed a cytoplasmic localization and a nuclear distribution in areas corresponding with the euchromatin fraction. Immunolocalization studies showed a partial mislocalization of the p.Arg541Cys mutant protein retaining part of the WT protein in the cytoplasm. Our study demonstrates that ZNF408, previously associated with Familial Exudative Vitreoretinopathy (FEVR), is a new gene causing arRP with vitreous condensations supporting the evidence that this protein plays additional functions into the human retina.This work is supported by CIBERER (06/07/0036), FIS (PI013/00226), Ministry of Economy and Competitiveness-FEDER (BFU2012-36845), RETICS (RD12/0034/0010), Fundación ONCE, Fundaluce and grants BIO2011-27069 from the Spanish Ministry of Economy and Competitiveness, and PROMETEOII/2014/025 from the Conselleria de Educacio of the Valencia Community. PC is supported by Fundación Conchita Rábago (FCR), MC by Miguel Servet ISCIII (CP/03256) and dS by CAPES Foundation, Ministry of Education of Brazil

Feasibility of nonsense mutation readthrough as a novel therapeutical approach in propionic acidemia

Aminoglycosides and other compounds can promote premature termination codon (PTC) readthrough constituting a potential therapy for patients with nonsense mutations. In a cohort of 190 propionic acidemia (PA) patients, we have identified 12 different nonsense mutations, six of them novel, accounting for 10% of the mutant alleles. Using an in vitro system, we establish the proof-ofprinciple that nonsense mutations in the PCCA and PCCB genes encoding both subunits of the propionyl-CoA carboxylase (PCC) enzyme can be partially suppressed by aminoglycosides, with different efficiencies depending on the sequence context. To correct the metabolic defect, the amino acid incorporated at the PTC should support protein function, and this has been evaluated in silico and by in vitro expression analysis of the predicted missense changes, most of which retain partial activity, confirming the feasibility of the approach. In patients' fibroblasts cultured with readthrough drugs, we observe a fourfold to 50-fold increase in the PCC activity, reaching up to 10-15% level of treated control cells. The ability to partially correct nonsense PCCA and PCCB alleles represents a potential therapy or supplementary treatment for a number of propionic acidemia (PA) patients, encouraging further clinical trials with readthrough drugs without toxic effects such as PTC124 or other newly developed compounds. © 2012 Wiley Periodicals, Inc.Ministerio de Ciencia e Innovación ; Centro de Investigación Biomédica en Red de Enfermedades Raras ; Fundación Ramón ArecesPeer Reviewe

Developmental and epileptic encephalopathies after negative or inconclusive genetic testing: what is next?

The redefinition of classical electroclinical syndromes and the emergence of neurogenetics has led to a revolution in the field of developmental and epileptic encephalopathies (DEEs). In this context, advances in genetic techniques are leading to the final diagnosis of a large proportion of patients with DEE. However, up to 50% of patients with DEE remain undiagnosed. For patients with uncertain genetic etiology, there is a pressing need for the implementation of new targeted treatments and precision medicine. In some undiagnosed patients, genetic reanalysis with further in-depth or reverse phenotyping are valuable diagnostic tools to clarify new variants of uncertain significance. In other cases, the implementation of new bioinformatic algorithms is required for the update and reassessment of previously generated genetic data. Moreover, many other clinical tools have been developed for the management of patients of DEEs after a negative or inconclusive genetic testing. In this review, we highlight advances and limitations of new diagnostic strategies used in DEE patients without a known genetic etiology. Finally, we provide a wide perspective on aspects that will need further research, especially in non-Mendelian inheritance DEEs, such as those related to somatic mosaicism of the central nervous system or epigenetic and oligogenic mechanisms

Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man

The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the "spf/ash" mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations

Hereditary primary lateral sclerosis and progressive nonfluent aphasia

Objective: to report a kindred with an association between hereditary primary lateral sclerosis (PLS) and progressive nonfluent aphasia. Patients and methods: six members from a kindred with 15 affected individuals spanning three generations, suffered from spasticity without muscle atrophy or fasciculation, starting in the lower limbs and spreading to the upper limbs and bulbar musculature, followed by effortful speech, nonfluent language and dementia, in 5 deceased members. Disease onset was during the sixth decade of life, or later. Cerebellar ataxia was the inaugural manifestation in two patients, and parkinsonism, in another. Results: neuropathological examination in two patients demonstrated degeneration of lateral corticospinal tracts in the spinal cord, without loss of spinal, brainstem, or cerebral motor neurons. Greater loss of corticospinal fibers at sacral and lumbar, rather than at cervical or medullary levels was demonstrated, supporting a central axonal dying-back pathogenic mechanism. Marked reduction of myelin and nerve fibers in the frontal lobes was also present. Argyrophilic grain disease and primary age-related tauopathy were found in one case each, and considered incidental findings. Genetic testing, including exome sequencing aimed at PLS, ataxia, hereditary spastic paraplegia, and frontotemporal lobe dementia, triplet-repeated primed polymerase chain reaction aimed at dominant spinocerebellar ataxias, and massive sequencing of the human genome, yielded negative results. Conclusion: a central distal axonopathy affecting the corticospinal tract, exerted a pathogenic role in the dominantly inherited PLS-progressive nonfluent aphasia association, described herein. Further molecular studies are needed to identify the causative mutation in this disease

OTC deficient patients with the c.386G>A mutation.

<p>n.d.a. no data available</p><p><b>*</b> patients referred to and genotyped at University Children’s Hospital, Zürich.</p><p>^# Liver tissue from this patient obtained after liver transplantation was used for transcript and enzyme analysis in the present study.</p><p>OTC deficient patients with the c.386G>A mutation.</p