On-going projects of Genetics

ACCORDANCE WITH ERA-NET E-RARE-3-HETER-OMICS-2, 88/2019

The approach of multi-OMICS of the malformation in cortical brain

The total value of the contract: 585.063,00 RON; representing the 125.550,00 €

Date: 01/06/2019-31/05/2022

Team: Aurora Arghir, PhD; Sorina Mihaela Papuc, PhD; Maria Dobre, PhD; Alina Erbescu

 

Abstract

Heterotopias is very abnormal activity of neurons in the cerebral cortex of the brain. Heterotopiile are rare disorders of the neuro-development, which lead to a severe form of epilepsy, and intellectual disability. These disorders form a spectrum that ranges from sub-group of neural abnormalities in the cerebral ventricles (periventricular) to the heterotopias located in the white matter (subcortical). In order to investigate specific pathways, converging, which are at the basis of these disorders, it is very important to an understanding of the types of cells that functions abnormally, and the trajectories of their development is abnormal, as well as the characterization of the consequences of the functional characteristics of the latter. In addition, the accumulation of clinical data and, based on the patients ' neinvestigati the previous provides a perspective of clinical importance. The goal of this project is to characterize the organisms of a population-based cohort of children with heterotopia, and to integrate the genetic data to the clinical setting. In this project, through the integration of multi-dimensional data, will allow for the identification of new variants of genes in candidate and potential sources of treatment. In addition, the families of these patients will not benefit from the possibility of a better characterization of clinical and genetic defects.

Objectiv

The characterization of the genetics of a comprehensive, a new cohort of patients with heterotopias and the correlation with clinical data and imaging

Step 1
December 2019

Malformations of the developing cortex of the brain (CDM) is an important cause of epileptic seizures, and delayed development. One of the MCD, heterotopiile are the ailments of the heterogeneous, from the genetic point of view, with a wide range of faults reported by the microdeletiii and microduplicatii at the mutation point which requires the use of panel data techniques for the investigation of the genetics, which will try to detect such anomalies, quantitative, genome-based as well as on the sequence.

The objectives of this phase 1

– Initiation of the study group, and the collection of tissue samples of primary and processed that have been obtained from patients with heterotopia

– Optimization of the protocols for the experimental screening of mutational targeted by the sequencing of the classic

– The establishment of a protocol for the investigation of genomic, by microarray found

The methods employed and the results obtained

At this stage, they have been conducted of the activities of the initiation of the basis of the data, and the collection of biological samples, as well as the set-up and optimization of the molecular tests are based on the sequencing and microarray. They were included in the study, patients with different types of heterotopia, and available blood samples from probanzi and their parents. They were made of the activities of processing the initial screening of biological samples and DNA isolation. It has been optimized a method for sequencing a direct analysis of the sequence of the gene FLNA and DCX using the system for capillary electrophoresis Applied Biosystems ABI 3500 Genetic Analyzer is set the protocol for the later stages. It has started screening the genome for abnormalities in the number of copies of DNA by hybridization, comparative genomic approach based on the microarray (array-CGH), and this is done on the platform of the Agilent SureScan. They were obtained in the first few sections genome by microarray slides the oligonucleotide to its high density, OGT 4x180K, and they have been analyzed, the imbalances in genome.

Conclusions:

At this stage, they have been met in full the objectives of the phase have been proposed.

– It has been founded based on the data of patients with heterotopias and it has been recorded, data on the patients included up to the date of the report.

They have been set forth uniform procedures for the processing and extraction of DNA from biological samples, and has initiated the collection of nucleic acids in the context of the project.

They were made of the activities of the optimization is the art of sequencing, Many of the genes FLNA and DCX using DNA in controls, without changes in the level of the genes tested, confirming the work.

The established protocol and that it has initiated the screening of genomic-based array-CGH.

They have been carried out of scientific communication in the form of a two posters and one oral communication in various scientific events in the field of genetics, and neuropsychiatry (OF the ELEVENTH Conference of the Genetics of Health, with the Participation of the International gallery and A PROJECT of the National Conference of the Romanian Association for the Fight Against Epilepsy, Usa).

Step 2
December 2020

Malformations of the developing cortex of the brain (CDM) is an important cause of epileptic seizures, and delayed development. One of the MCD, heterotopiile are the ailments of the heterogeneous, from the genetic point of view, with a wide range of faults reported by the (micro -) deletiii, and (micro -) duplicatii the anomalies in the sequence, which in turn requires the use of panel data techniques for the investigation of the genetics of which is to detect that the imbalances in the genomic and the variation of the sequence.

 

The objectives of phase 2

– The addition to the collection of biological samples

– Screening of mutational by sequencing the ability of the gene FLNA

Screening of the genomic abnormalities of a number of copies of DNA, based on array CGH

– Optimization of the protocols for the experimental screening of mutational by next generation sequencing of the whole exom

– Dissemination of the results

The methods employed and the results obtained

At this stage, they have been conducted of the activities of the characterization of the genetics of the patients included in the study and optimization of molecular tests based on the next generation sequencing of the whole exom.

They have been carried out activities to add to the database, and the collection of biological samples, through the receipt and processing of biological samples from 10 patients with heterotopias and their parents.

There have been experiments with the microarray chromosome (blades, 4x180k, OGT Constitutional v3) for 12 patients, and identified a CNV of the pathological, and the three of HOW sites of significance to be determined. Screening of mutational gene FLNA it was performed by the sequencing classic Sanger in 3 patients, with no detection of variants of the sequence of the pathological-or by the significance of the uncertainties in the ampliconii review.

It has been adapted and optimized the protocol for sequencing of the whole exom, using state of the art for a method for the synthesis of libraries based on the PCR method, with the AmpliSeq for Illumina Exome Panel. This method allows to obtain a uniform coatings of high and specific (>90%) to obtain a large amount of data (approx. 59 MB). Prepare the libraries for sequencing were conducted for up to 8 samples from 3 of the patients and their parents.

The dissemination activities have been carried out by the participation of the European Conference of the Genetics of Human work in the form of a poster ( "Brain malformations diagnosis and clinical management in pediatric patients"), and submitted to be published in the journal of Experimental and Therapeutical Medicine, he has a dream "Clinical and genomic findings in the brain heterotopias – report of a pediatric patient cohort from Romania," which is currently in the evaluation phase, the decision of the revision is minor.

In addition, on 28 and 29 January, 2020, has been held in Paris, the consortium HETEROMICS, led by Professor Fiona, Francis. Institutions and research groups are involved in the consortium that submitted the materials for the description of the team, and the themes of the research, in general, as well as a description of the activities implemented in the first phase of the project and the achievement of the objectives of the phase.

Conclusions:

In this stage, they have been met in full the objectives of the phase have been proposed.

– Has been completed in the study group, and the collection of biological samples;

He was done for ' the mutational through it a classic of the FLNA in the patients with a given clinical and imaging features suggestive of, and it was performed the analysis of the data and the interpretation of the significance of the variations in the sequence of the detected;

He has since been screening the genome by array CGH for the patients of the group 1 of the study, and an additional 4 patients in group 2. It was performed the analysis of the profiles and the genome, and have been classified in the CNV-s, on the basis of the significance of the clinical trials;

The optimized experimental protocol for next generation sequencing of the whole exom, and has been secventiate 8 samples;

– The results of the activities carried out within the framework of the project have been disseminated to the scientific events, and submitted for publication in specialized journals.

Step 3
December 2021

Congenital, delayed development and disability, intellectual, these are the most common health problems in the population of hyderabad, and they represent a heterogeneous group of diseases with a significant impact on the quality of your life. Malformations of cerebral cortical (MCD) are often the cause is genetic, the progress of technologies in the investigation, bringing a permanent information on the architecture of the genetics of these disorders. The characterization of the genetic defects in the CDM contributes to deciphering the pathways of the pathogenesis and have the potential to identify molecular targets for personalized therapies. At the same time, the elucidation of the etiology, genetic, it can help to improve the clinical management and treatment, to assess the prognosis and evolution of the disease.

The objectives of phase 3

  • The expansion of the collection of biological samples;
  • Screening of mutational by the sequencing of the classic gene FLNA and DCX in patients with similar clinical and imaging suggestive of defects in these genes;
  • Screening of the genomic abnormalities of a number of copies of DNA, based on array CGH;
  • Screening of mutational by next generation sequencing of the whole exom (Whole Exome Sequencing WES) in patients selected on the basis of the phenotype of the clinical and imaging, without any genetic changes relevant to the malformation of the brain;
  • The analysis and interpretation of the data;
  • The dissemination of the results that have been achieved.

The methods employed and the results obtained

At this stage, they have been conducted of the activities of the characterization of the genetics of the patients included in the study, including the investigation is based on the next generation sequencing of the whole exom, the patients selected. They have been carried out activities to add to the database, and the collection of biological samples. At this stage, they received blood samples from 10 patients with heterotopias and their parents.

The profile of the genome has been characterized by the study of the microarray are found for the 12 samples, using the blade to the microarray 4x180K (OGT of the Constitution of v3 with the design is specific to the diseases of the gene in development). Screening of the genome by array CGH has been completed for all the patients, the identification of this phase of the versions, with significance determined.  

Have been investigated by sequencing classic Sanger patients with phenotypes suggestive of a genetic test target: three of the patients, using the primers for the regions of the encoders of the FLNA two of the patients, using primers for exons of the gene DCX. They have not been detected variants in the sequence of the pathological-or by the significance of the uncertainties in the gene FLNA. It has been detected, a variant of the pathology in the gene The DCX file, in conjunction with the heterotopias subcorticala on the tape on a female patient and a phenotype characteristic of loss of function of the gene The DCX file.

A protocol for the preparation of libraries for sequencing were conducted for up to 8 samples you have selected. Libraries results were skewed, and secventiate on the platform of the NextSeq 550 (light up) with the kit on the NextSeq 500/550 300 cycles (the access to and the logistical support provided by the members of the team, CRGM Dolj, romania in the framework of the SCJU price and PHARMACY of Craiova). The data were analyzed and interpreted using the programs in the successive stages in the second module, the analysis of the individual (one case), and the analysis of the correlation analysis (trio). Special attention has been paid to the genes known to be involved in the occurrence of malformations of the brain, subsequently being made to step out of the interrogation of the whole exom for the potential gene candidates.

Activitatile de diseminare au fost realizate prin comunicarea cate unei lucrari  la Conferinta Europeana de Citogenomica, Conferinta Excellence in Pediatrics, Congresul European de Psihiatrie, Congresul National de Pediatrie, si Conferinta Societatii Romane Impotriva Epilepsiei. A fost publicat in revista Experimental and Therapeutic Medicine un articol stiintific original cu titlul “Clinical and genomic findings in brain heterotopia – report of a pediatric patient cohort from Romania”, (1 decembrie 2021, https://doi.org/10.3892/etm.2021.11024). In plus, a fost depus pentru evaluare la revista Revista Romana de Medicina de Laborator, indexata in Web of Science (factor de impact 1,027), un manuscris cu rezultate originale intitulat „Novel DCX pathogenic variant in a girl with subcortical band heterotopia”. In primul semestru al anului 2022, articolul a fost acceptat pentru publicare.

In the 13th and 14th of January 2021, has taken place in the virtual of the business activity HETEROMICS, led by Professor Fiona, Francis, the University of the Sorbonne, in Paris. Institutions and research groups engaged in the development, they presented the preliminary results obtained in the second phase of the project and the achievement of the objectives of the phase.

So, In conclusion, in the second stage, they achieved all of the objectives for the stage have been proposed:

  • It was a complete collection of the biological sample;
  • It was done for ' the mutational through it a classic of the FLNA and DCX in all patients with a given clinical and imaging features suggestive of the investigation of the target of these genes has been performed the data analysis and the interpretation of the significance of the variations in the sequence of the detected;
  • It was completed screening of the genome by array CGH for all patients, the analysis of the profiles and the genome, and the classification of CNV-shows, in line with the contribution of the potential of the;
  • It was carried out the experimental design of the next generation sequencing of the whole exom on the selected samples, and the obtained data were analyzed and interpreted;
  • They have been communicated to the scientific papers of three of the conferences of the european, and the two conferences of the national; and he published an article in the original and it was sent to you for evaluation of a manuscript with the original results, the is a journal indexed in the Web of Science, accepted for publication in the first half of the year is 2022.

Step 4:
December 2022

The objectives of phase 4

  • The assessment of the genetic data of the preliminary as well as the clinical and imaging, in conjunction with a partner, a 5 in the framework of the consortium for the application gets divided the whole exom – WES
  • Screening of mutational by next generation sequencing of the whole exom of the patients selected, and their parents (open-exome)
  • The analysis of the data and the interpretation of the results generated by the prospect of detection of new variants in the known genes, or the genes of the candidate with the potential to be the targets of therapeutic
  • The dissemination of the results through the communication of the work at national and international conferences, publication of scientific articles

The methods employed and the results obtained

At this stage, there were carried out investigations based on next generation sequencing of the whole exom five of the patients and their parents. It has been so completed, the group proposed a 12-investigation of WES in the trio (the patient and both parents).

The blood samples were represented in the peripheral blood, which were collected from the patients and their parents. A protocol for the preparation of the library for sequencing was performed on 16 samples you have selected. Libraries results were skewed, and secventiate on the platform of the NextSeq 550 (Illumina) with the kit on the NextSeq 500/550 300 cycles. The data were analyzed and interpreted by using the software NextGene (SoftGenetics), in successive steps. It has been used in the module you created in the previous phase for the analysis of the individual (one case), and the modulus of comparative analysis (analysis of the trio). A special attention has been given to you, in the first stage of the analysis, the genes known to be involved in the development of malformations in the brain. Subsequently, they were analyzed in all the other genes that do not encode proteins. Assessment of the significance of the clinical variants of that detected it was done by querying the database population (for example, GnomAD), and the variants reported by the patient with the pathology of neurodevelopmental (ClinVar, Decipher, OMIM).

The dissemination activities have been carried out by the communication of a work in four international conferences (the Conference of the European Human Genetics Congress of the European Society of Pediatric Neurology, the International Congress of child Neurology, Pediatric care, and the Conference of the Excellence in Pediatrics), and in the Congress of the Romanian Society of Medical Genetics. It was published in the Romanian Journal of Laboratory Medicine (impact factor 1,027) of the article the science of the original “Novel DCX pathogenic variant in a girl with subcortical band heterotopias” (DOI:10.2478/rrlm-2022-0031). In addition, it has been submitted for assessment in the journal Genes, indexed to the WEB of Science (impact factor 4,141), a manuscript with the results of the original paper entitled "Autistic behavior as novel clinical finding in the OFD1 syndrome", which was published in the month of January 2023.

So, In conclusion, at this stage, they achieved the objectives of the phase have been proposed:

  • The complete collection of biological samples;
  • It was carried out the experimental design of the next generation sequencing of the whole exom, and they were obtained in a library for 5 samples, totalizandu be like for the 12 tests in a threesome proposed, and in addition to the analysis of two patients, additional;
  • They have been analyzed and interpreted the data from the sequencing of the whole exom in 5 of the patients, thus completing the group of 14 patients tested by WES by the correlation of the data with those in the literature and the results obtained by different groups of experts, including in the framework of the business activity HETEROMICS;
  • It has been rated the in silico the impact of the functional characteristics of the variants were detected by using various software programs to predict the functional classification of the contribution of the;
  • They have been communicated to the scientific papers from the 4 conferences of the european and the national conference; it has been published, an article in the original and it was sent to you for evaluation of a manuscript with the original results, the is a journal indexed in WEB of Science.
en_GBEnglish (UK)
en_GBEnglish (UK) ro_RORomână