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IMPLEMENTATION OF MOLECULAR DIAGNOSTIC PATHWAYS

2017-03-20 21:23:22 | BioPortfolio

Summary

Most neurological and neurodegenerative diseases have a multifactorial nature. For some of them it is documented a genetic component (sometimes described as Mendelian, other times as a multifactorial), while others have not been reported genes or family segregation evidence.

Therefore, the presence of these scenarios sometimes makes it impossible or unlikely to arrive at a molecular result following a clinical evidence.

To date there are new technologies such as Next Generation Sequencing (NGS), to help you analyze hundreds of genes with content and in very short time costs. The implementation of these technologies, already used synergistically to molecular pathways, will identify new genes and new variants associated with neurological diseases. All this will allow to have a greater number of molecular diagnosis with reproducible results and significant, for use in clinical practice.

Description

1. INTRODUCTION Most neurological and neurodegenerative diseases have a multifactorial nature. For some of them it is documented a genetic component (sometimes described as Mendelian, other times as a multifactorial), while others have not been reported genes or family segregation evidence.

For some monogenic diseases, such as Huntington's, there are validated molecular protocols and guidelines that must be met to ensure a quality diagnostic results. The application of these protocols allows you to use the molecular result in diagnostic clinical and pre-symptomatic pathways.

Unfortunately, this scenario is not reproducible for most of the neurological and neurodegenerative disorders in which a strong genetic component is documented. This is due to:

- Polygenicity, where different genes can contribute to the same phenotype (eg Spastic Paraplegia, associated with over 50 genes)

- Multifactorial, which the genetic explains only a part of the etiology of the disease (such as Parkinson's disease in which the identified genes are responsible for only 15% of patients with a clinical diagnosis)

- Disorders with clear genetic component, but which were not identified genes responsible.

Therefore, the presence of these scenarios sometimes makes it impossible or unlikely to arrive at a molecular result following a clinical evidence.

To date there are new technologies such as Next Generation Sequencing (NGS), to help you analyze hundreds of genes with content and in very short time costs. The implementation of these technologies, already used synergistically to molecular pathways, will identify new genes and new variants associated with neurological diseases. All this will allow to have a greater number of molecular diagnosis with reproducible results and significant, for use in clinical practice.

2. DESIGN STUDY The target of this study is to enrich the diagnostic pathways in molecular genetics to characterize more precisely genomic variations and new genes responsible for neurological and neurodegenerative diseases.

They will seek to identify:

1. New genes associated with diseases characterized by genetic heterogeneity, Mendelian and polygenic heredity.

2. New variations responsible for disease or that can increase susceptibility.

3. EXPERIMENTAL PHASE (Attachment 1)

1. After Neurology consulting, is required a Genetic Counseling.

2. In Genetic Counseling the molecular testing to be performed (level I). Here you collect a blood sample after getting a signed informed consent form (informed consent Neuromed version 2.12.2015) for the Diagnostic Study. About 6 milliliters of blood will be taken and then a fractional part will be in serum and lymphocytes what will be stored at -80 ° C.

3. They are applied classical diagnostic test based on specific national guidelines for each disease (SIGU: http://www.sigu.net/show/attivita/5/1/LINEE%20GUIDA%20SIGU) Molecular analyzes are carried out at the Institute of Molecular Genetics Center IRCCS INM Neuromed using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA) and microsatellites.

4. When the first level test is positive we will proceed to reporting the variant identified associated with suspected pathology. Instead, if the test is negative it will proceed with an analysis of molecular level II (NGS on a large scale of Patients with complexes phenotypes). This can provide a positive result, and then a report of the variant for the condition in question, or a negative result to which then the test sample will be preserved for future inclusion in research protocols adapted to define the pathology methods with which to today are not yet available.

4. MATERIALS AND METHODS

- DNA-RNA extracting The extraction of DNA and RNA will be performed using kits designed to maximize its efficiency and cleanliness.

- MLPA (Multiplex ligation-dependent probe amplification) MLPA (Multiplex Ligation-dependent Probe Amplification) is a multiplex PCR method detecting abnormal copy numbers of up to 50 different genomic DNA or RNA sequences, which is able to distinguish sequences differing in only one nucleotide.

Typical for MLPA is that it is not target sequences that are amplified, but MLPA probes that hybridise to the target sequence. In contrast to a standard multiplex PCR, a single pair PCR primers is used for MLPA amplification. The resulting amplification products of a SALSA MLPA kits range between 130 and 480 nt in length and can be analysed by capillary electrophoresis. Comparing the peak pattern obtained to that of reference samples indicates which sequences show aberrant copy numbers.

The MLPA reaction can be divided in five major steps: 1) DNA denaturation and hybridisation of MLPA probes; 2) ligation reaction; 3) PCR reaction; 4) separation of amplification products by electrophoresis; and 5) data analysis

- Sequencing The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides.

The mixture is first heated to denature the template DNA (separate the strands), then cooled so that the primer can bind to the single-stranded template. Once the primer has bound, the temperature is raised again, allowing DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide.

This process is repeated in a number of cycles. By the time the cycling is complete, it's virtually guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the target DNA in at least one reaction. That is, the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA (see figure below). The ends of the fragments will be labeled with dyes that indicate their final nucleotide. After the reaction is done, the fragments are run through a long, thin tube containing a gel matrix in a process called capillary gel electrophoresis. Short fragments move quickly through the pores of the gel, while long fragments move more slowly. As each fragment crosses the "finish line" at the end of the tube, it's illuminated by a laser, allowing the attached dye to be detected.

The smallest fragment (ending just one nucleotide after the primer) crosses the finish line first, followed by the next-smallest fragment (ending two nucleotides after the primer), and so forth. Thus, from the colors of dyes registered one after another on the detector, the sequence of the original piece of DNA can be built up one nucleotide at a time. The data recorded by the detector consist of a series of peaks in fluorescence intensity, as shown in the chromatogram above. The DNA sequence is read from the peaks in the chromatogram.

- Microsatellite Microsatellite analysis includes PCR amplification of the microsatellite loci using fluorescently labeled primers (6-FAM, TET, HEX, NED); labeled PCR products are then analyzed by capillary electrophoresis (ABI PRISM 310 and 3130 XL Applied Biosystem) (CE) or electrophoresis to separate the alleles by size.

The results were processed using the GENESCAN and GENOTYPER5 programs. Once established the values of individual alleles, they were assigned to each individual.

- Next Generation Sequencing (NGS)

In principle, the concept behind NGS technology is similar to CE sequencing—DNA polymerase catalyzes the incorporation of fluorescently labeled deoxyribonucleotide triphosphates (dNTPs) into a DNA template strand during sequential cycles of DNA synthesis. During each cycle, at the point of incorporation, the nucleotides are identified by fluorophore excitation. The critical difference is that, instead of sequencing a single DNA fragment, NGS extends this process across millions of fragments in a massively parallel fashion. Illumina sequencing by synthesis (SBS) chemistry is the most widely adopted chemistry in the industry and delivers the highest accuracy, the highest yield of error-free reads, and the highest percentage of base calls above Q30.6-8 The Illumina NGS workflows include 4 basic steps (Figure 3):

1. Library Preparation—The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5' and 3' adapter ligation. Alternatively, "tagmentation" combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process.9 Adapter-ligated fragments are then PCR amplified and gel purified.

2. Cluster Generation—For cluster generation, the library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. Each fragment is then amplified into distinct, clonal clusters through bridge amplification. When cluster generation is complete, the templates are ready for sequencing.

3. Sequencing—Illumina technology utilizes a proprietary reversible terminator-based method that detects single bases as they are incorporated into DNA template strands. As all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias and greatly reduces raw error rates compared to other technologies.6, 7 The result is highly accurate base-by-base sequencing that virtually eliminates sequence-context-specific errors, even within repetitive sequence regions and homopolymers.

4. Data Analysis—During data analysis and alignment, the newly identified sequence reads are then aligned to a reference genome. Following alignment, many variations of analysis are possible such as single nucleotide polymorphism (SNP) or insertion-deletion (indel) identification, read counting for RNA methods, phylogenetic or metagenomic analysis, and more.

5. STATISTICS

In order to determine the pathogenicity of the identified variants, will be performed:

- Molecular test in the proband's family.

- In-silico analyzes using bioinformatics software (Sift: http://sift.jcvi.org/; PolyPhen: http://genetics.bwh.harvard.edu/pph2/).

- Frequency analysis in the general population (dbSNP: https: //www.ncbi.nlm.nih.gov/projects/SNP/; EXAC: http://exac.broadinstitute.org/)

6. ETHICAL ASPECTS This study follow the ethical standards set out in the Helsinki Declaration and its revisions. The study will be conducted taking into account the regulatory requirements and compliance with the law. The informed consent already approved previously by the ethics committee.

Study Design

Conditions

Genetic Disease

Intervention

neurological and neurodegenerative diseases

Location

Stefano Gambardella
Pozzilli
Isernia
Italy
86077

Status

Not yet recruiting

Source

Neuromed IRCCS

Results (where available)

View Results

Links

Published on BioPortfolio: 2017-03-20T21:23:22-0400

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