A new experimental approach is required in the molecular analysis of intestinal neuronal dysplasia type B patients

5 páginas, 3 tablas.Intestinal neuronal dysplasia type B (INDB) is characterized by the malformation of the parasympathetic submucous plexus of the gut. It is generally accepted that INDB has a genetic basis, and several genes produce an INDB-like phenotype in mice when disrupted, such as EDNRB. However, no mutations associated with this disease have been identified in several series analysed. In the present studu, we sought to determine whether the EDNRB/EDN3 signalling pathway plays a role in the pathogenesis of INDB in humans. Denaturing high performance liquid chromatography (dHPLC) techniques were employed to screen the EDNRB and EDN3 coding regions in 23 INDB patients. In addition, association studies were performed on these genes with single nucleotide polymorphisms strategically selected and genotyped by TaqMan technology. Although several novel variants were detected in both genes, none of these variants appeared to play a functional role in protein function or expression. Our results indicate that additional screening of other candidate genes in larger patient series is required to elucidate the molecular basis of INDB. Additionally, the systematic lack of positive results in the screening of candidate genes for INDB reported in the literature, together with our results, leads us to propose that INDB may alternatively arise as a consequence of gain of function mutations in genes related to enteric nervous system development. Therefore, the use of different molecular approaches, such as screening for genetic duplication or enhancer mutations, is recommended for future studies on the genetic basis of INDB.This study was funded by the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (ISCIII), Spain (PI070080, and PI071315 for the E-Rare project), Consejería de Salud (PI0249-2008) de la Junta de Andalucía, Spain; and Consejería de Innovación Ciencia y Empresa (CTS-2590) de la Junta de Andalucía, Spain. The CIBER de Enfermedades Raras is an initiative of ISCIII. A.S.M. is a pre-doctoral fellow funded by ISCIII, Spain.Peer reviewe

Novel MLPA procedure using self-designed probes allows comprehensive analysis for CNVs of the genes involved in Hirschsprung disease

7 páginas, 1 figura, 1 tabla.[Background]: Hirschsprung disease is characterized by the absence of intramural ganglion cells in the enteric plexuses, due to a fail during enteric nervous system formation. Hirschsprung has a complex genetic aetiology and mutations in several genes have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. In this study, we have looked for CNVs in some of the genes related to Hirschsprung (EDNRB, GFRA1, NRTN and PHOX2B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. [Methods]: CNVs screening was performed in 208 HSCR patients using a self-designed set of MLPA probes, covering the coding region of those genes. [Results]: A deletion comprising the first 4 exons in GFRA1 gene was detected in 2 sporadic HSCR patients and in silico approaches have shown that the critical translation initiation signal in the mutant gene was abolished. In this study, we have been able to validate the reliability of this technique for CNVs screening in HSCR. [Conclusions]: The implemented MLPA based technique presented here allows CNV analysis of genes involved in HSCR that have not been not previously evaluated. Our results indicate that CNVs could be implicated in the pathogenesis of HSCR, although they seem to be an uncommon molecular cause of HSCR.This study was funded by Fondo de Investigación Sanitaria, Spain (PI070070 and PI071315 for the E-Rare project), Consejería de Innovación Ciencia y Empresa de la Junta de Andalucía (CTS-2590) and Consejería de Salud de la Junta de Andalucia (PI-0249/2008). ASM is a predoctoral fellow founded by Instituto de Salud Carlos III, Spain.Peer reviewe

Involvement of SOX10 in the pathogenesis of Hirschsprung disease: report of a truncating mutation in an isolated patient.

International audienceSOX10 protein is a key transcription factor during neural crest development. Mutations in SOX10 are associated with several neurocristopathies such as Waardenburg syndrome type IV (WS4), a congenital disorder characterized by the association of hearing loss, pigmentary abnormalities, and absence of ganglion cells in the myenteric and submucosal plexus of the gastrointestinal tract, also known as aganglionic megacolon or Hirschsprung disease (HSCR). Several mutations at this locus are known to cause a high percentage of WS4 cases, but no SOX10 mutations had been ever reported associated to isolated HSCR patient. Therefore, nonsyndromic HSCR was initially thought not to be associated to mutations at this particular locus. In the present study, we describe the evaluation of the SOX10 gene in a series of 196 isolated HSCR cases, the largest patient series evaluated so far, and report a truncating c.153-155del mutation. This is the first time that a SOX10 mutation is detected in an isolated HSCR patient, which completely changes the scenario for the implications of SOX10 mutations in human disease, giving us a new tool for genetic counseling

Is the RET proto-oncogene involved in the pathogenesis of intestinal neuronal dysplasia type B?

Journal Article;Hirschsprung disease (HSCR) is defined by the absence of intramural ganglia of Meissner and Auerbach along variable lengths of the gastrointestinal tract. Intestinal neuronal dysplasia (IND) type B is characterized by the malformation of the parasympathetic submucous plexus of the gut. A connection appears to exist between these two enteric nervous system abnormalities. Due to the major role played by the RET proto-oncogene in HSCR, we sought to determine whether this gene was also related to INDB. dHPLC techniques were employed to screen the RET coding region in 23 patients presenting with INDB and 30 patients with a combined HSCR+INDB phenotype. In addition, eight RET single nucleotide polymorphisms (SNPs) were strategically selected and genotyped by TaqMan technology. The distribution of SNPs and haplotypes was compared among the different groups of patients (INDB, HSCR+INDB, HSCR) and the controls. We found several RET mutations in our patients and some differences in the distribution of the RET SNPs among the groups of study. Our results suggest an involvement of RET in the pathogenesis of intestinal INDB, although by different molecular mechanisms than those leading to HSCR. Further investigation is warranted to elucidate these precise mechanisms and to clarify the genetic nature of INDB.The study was funded by Fondo de Investigación Sanitaria, Spain (PI070070 and PI071315 for the E-Rare project) and Consejería de Innovación Ciencia y Empresa de la Junta de Andalucía (CTS2590).Ye

INSERT-seq enables high-resolution mapping of genomically integrated DNA using Nanopore sequencing

Comprehensive characterisation of genome engineering technologies is relevant for their development and safe use in human gene therapy. Short-read based methods can overlook insertion events in repetitive regions. We develop INSERT-seq, a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration. The experimental pipeline improves the number of mappable insertions at repetitive regions by 4.8-7.3% and larger repeats are processed with a computational peak calling pipeline. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex vivo and in vivo samples.We thank funding received from UPGRADE (European Union Horizon 2020, grant agreement No 825825), Fundación Ramón Areces (“Advanced gene editing technologies to restore LAMA2 on merosin-deficient congenital muscular dystrophy type 1A”), MdM projecte de recerca “Unidad de Excelencia María de Maeztu”, funded by the AEI (CEX2018-000792-M)

CRISPR-Analytics (CRISPR-A): A platform for precise analytics and simulations for gene editing

Gene editing characterization with currently available tools does not always give precise relative proportions among the different types of gene edits present in an edited bulk of cells. We have developed CRISPR-Analytics, CRISPR-A, which is a comprehensive and versatile genome editing web application tool and a nextflow pipeline to give support to gene editing experimental design and analysis. CRISPR-A provides a robust gene editing analysis pipeline composed of data analysis tools and simulation. It achieves higher accuracy than current tools and expands the functionality. The analysis includes mock-based noise correction, spike-in calibrated amplification bias reduction, and advanced interactive graphics. This expanded robustness makes this tool ideal for analyzing highly sensitive cases such as clinical samples or experiments with low editing efficiencies. It also provides an assessment of experimental design through the simulation of gene editing results. Therefore, CRISPR-A is ideal to support multiple kinds of experiments such as double-stranded DNA break-based engineering, base editing (BE), primer editing (PE), and homology-directed repair (HDR), without the need of specifying the used experimental approach.This work was supported by the European Commission (European Union Horizon 2020 grant 825825 to MG), Ramón y Cajal program (grant RYC-2015-17734 to MG), Fundación Ramón Areces (grant “Advanced gene editing technologies to restore LAMA2 on merosin-deficient congenital muscular dystrophy type 1A” to MG) and Ministerio de Ciencia e Innovación de España (Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020 «Advanced methodologies for precise and efficient gene delivery» grant PID2020-118597RB-I00 to MG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Find and cut-and-transfer (FiCAT) mammalian genome engineering

While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Here we develop a gene delivery tool (FiCAT) combining the precision of a CRISPR-Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut-and-transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. We demonstrate efficient delivery and programmable insertion of small and large payloads in cellulo (human (Hek293T, K-562) and mouse (C2C12)) and in vivo in mouse liver. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes.The authors received funding from UPGRADE (European Union Horizon 2020, grant agreement No 825825); Fundació La Caixa: Caixa Impulse 2018 (grant agreement CI 1800036), Caixa Impulse Consolidate 2019 (grant agreement cF91-00010) and Programa Captació de Talent; Fundación Ramón Areces (“Advanced gene editing technologies to restore LAMA2 on merosin-deficient congenital muscular dystrophy type 1A”); Ministerio de Economia, Industria y Competitividad de España (Plan Estatal 2013-2016 (Grant agreement SAF2017-88784-R, Ramón y Cajal program (Grant agreement RYC-2015-17734)). All grants were received by M.G