xml: <clinical_study>
    <!-- This xml conforms to an XML Schema at:
      https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
    <required_header>
      <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
      <link_text>Link to the current ClinicalTrials.gov record.</link_text>
      <url>https://clinicaltrials.gov/ct2/show/NCT05996731</url>
    </required_header>
    <id_info>
      <org_study_id>ANTHEM-RNA-Seq</org_study_id>
      <nct_id>NCT05996731</nct_id>
    </id_info>
    <brief_title>Developing a Pipeline to Employ RNA-Seq as a Complementary Diagnostic Tool in Rare Diseases</brief_title>
    <acronym>ANTHEM-RNA-Seq</acronym>
    <official_title>Developing a Pipeline to Employ RNA-Seq as a Complementary Diagnostic Tool in Rare Diseases</official_title>
    <sponsors>
      <lead_sponsor>
        <agency>Mario Negri Institute for Pharmacological Research</agency>
        <agency_class>Other</agency_class>
      </lead_sponsor>
    </sponsors>
    <source>Mario Negri Institute for Pharmacological Research</source>
    <oversight_info>
      <has_dmc>No</has_dmc>
      <is_fda_regulated_drug>No</is_fda_regulated_drug>
      <is_fda_regulated_device>No</is_fda_regulated_device>
    </oversight_info>
    <brief_summary>
      <textblock>
        This project aims to identify, through RNA-Seq technology, the genetic alterations underlying&#xD;
        undiagnosed rare diseases in pediatric and adult patients with early onset and with negative&#xD;
        WES.&#xD;
  &#xD;
          -  Objective 1: Set up and validate techniques. Set-up and validation of the transcriptome&#xD;
             analysis protocol in healthy subjects and in patients with known splicing alterations&#xD;
             and/or altered RNA expression.&#xD;
  &#xD;
          -  Objective 2: Diagnostic phase. Study of splicing alterations and RNA levels in cultured&#xD;
             fibroblasts obtained from skin biopsies of patients with rare genetic diseases and&#xD;
             negative exome.&#xD;
  &#xD;
        Exploratory goals&#xD;
  &#xD;
          -  Compare the RNA expression profile obtained from skin biopsy-derived fibroblasts with&#xD;
             the RNA expression profile from blood. The most relevant results will be validated in&#xD;
             qRT-PCR.&#xD;
  &#xD;
          -  To analyze the transcriptional and protein profile heterogeneity in skin-derived&#xD;
             fibroblasts in enrolled subjects.&#xD;
  &#xD;
        To explore the effects of genetic (from WES) and transcriptional (from RNA-seq) alterations&#xD;
        in participants' plasma and serum.&#xD;
  &#xD;
        Healthy controls Five healthy subjects will be recruited from the staff of the Mario Negri&#xD;
        Institute for Pharmacological Research. The coded samples will be used to set up the method&#xD;
        of isolation and culture of skin fibroblasts and RNA-Seq.&#xD;
  &#xD;
        Validation group For the set-up and validation of the skin fibroblast isolation and RNA-Seq&#xD;
        procedure, ten adult patients with known diagnosis and with alterations in RNA levels and/or&#xD;
        splicing will be recruited as positive controls.&#xD;
  &#xD;
        Patients who meet the requirements described above will be contacted by the doctors of the&#xD;
        Daccò Center for an interview explaining the project. Those who agree to participate in the&#xD;
        study will be asked to sign the informed consent before proceeding with the experimental&#xD;
        part.&#xD;
  &#xD;
        &quot;Discovery/Exploration&quot; group The exploration cohort will be composed of 30 symptomatic&#xD;
        undiagnosed patients with suspected genetic disease (children and adults with infantile&#xD;
        onset) belonging to the Clinical Center of the Mario Negri Institute for Pharmacological&#xD;
        Research and for whom WES investigations did not reveal causative genetic alterations.&#xD;
      </textblock>
    </brief_summary>
    <detailed_description>
      <textblock>
        Rare genetic diseases are a very heterogeneous group of diseases, often undiagnosed, for&#xD;
        which patients receive only symptomatic treatment. The development of next-generation massive&#xD;
        sequencing technology (Next-Generation-Sequencing; NGS) has made it possible to identify many&#xD;
        of the genes responsible for these pathologies, allowing the pathogenic mechanisms to be&#xD;
        outlined. The most commonly used methods, such as exome sequencing (Whole-Exome Sequencing;&#xD;
        WES) or genome sequencing (Whole-Genome Sequencing; WGS), are based on DNA sequencing and&#xD;
        allow to identify pathogenic mutations that cause loss of protein functionality, such as stop&#xD;
        mutations, large insertions and deletions and the loss of canonical splicing sites.&#xD;
        Conversely, the attribution of pathogenicity to an amino acid substitution variant (missense)&#xD;
        or a synonymous variant is often questionable and, consequently, such variants are often&#xD;
        categorized as Variants of Unknown Significance (VUS). Furthermore, exome sequencing is&#xD;
        unable to identify possible pathogenic variants located in non-coding regions, such as&#xD;
        non-canonical splice sites or deep intronic variants.&#xD;
  &#xD;
        WGS sequencing, while allowing the identification of variants present in the non-coding&#xD;
        regions of a gene, is very expensive and produces an enormous amount of data with low&#xD;
        pathogenic information. Algorithms to analyze the possible pathogenicity of intronic variants&#xD;
        have little predictive power and the pathogenicity of the identified variants must be&#xD;
        validated by lengthy and complex functional analyses. Due to these limitations, approximately&#xD;
        50% of patients with rare genetic diseases remain undiagnosed and cannot benefit from the&#xD;
        benefits of personalized medicine. Transcriptome sequencing (RNA-Seq) allows to determine&#xD;
        variations in the sequence and levels of RNA, providing complementary information to that&#xD;
        obtained with WES and WGS, thus improving the efficiency of genetic diagnosis. Splicing&#xD;
        alterations, which are among the main causes of genetic diseases and can reside both in deep&#xD;
        intronic regions and in coding regions, can be identified through RNA sequencing. Variants in&#xD;
        the regulatory regions of the gene, which are able to influence RNA expression levels, can&#xD;
        also be highlighted by RNA-Seq. Since RNA expression, in terms of levels and isoforms&#xD;
        produced by alternative splicing, is tissue-specific, the most transcriptionally informative&#xD;
        biological material is certainly the affected tissue. However, this is not always easy to&#xD;
        achieve and the collection can be too invasive for the patient. In these cases it is possible&#xD;
        to resort to the use of alternative tissues, such as whole blood or skin biopsies, which have&#xD;
        low invasiveness and a broad RNA expression profile. Fibroblasts obtained from skin biopsies,&#xD;
        in particular, have proven to be the most reliable, homogeneous and informative alternative&#xD;
        matrix for RNA-Seq analysis, in a wide range of pathologies. Yépez and collaborators&#xD;
        demonstrated that the information obtained from RNA-Seq from skin fibroblasts was superior to&#xD;
        that obtained from blood. Furthermore, the analysis of the transcriptome of skin fibroblasts&#xD;
        allowed to identify altered levels of transcripts and splicing abnormalities in neuromuscular&#xD;
        and neurodevelopmental diseases. This confirmed the efficacy of the use of skin fibroblasts&#xD;
        in the diagnosis of genetic pathologies in WES and WGS negative patients. Last but not least,&#xD;
        skin fibroblasts can be reprogrammed into other cell types for possible future analyzes.&#xD;
  &#xD;
        This project aims to identify, through RNA-Seq technology, the genetic alterations underlying&#xD;
        undiagnosed rare diseases in pediatric and adult patients with early onset and with negative&#xD;
        WES.&#xD;
  &#xD;
        Although NGS is essential for the diagnosis of genetic diseases, its success rates range from&#xD;
        30% to 50%. The advent of RNA-Seq increased the chance of diagnosing genetic diseases from 8%&#xD;
        to 36%. Through this project we intend to apply the RNA-Seq method, as an integration of the&#xD;
        WES analysis, for the diagnosis of rare genetic diseases:&#xD;
  &#xD;
          -  Objective 1: Set up and validate techniques. Set-up and validation of the transcriptome&#xD;
             analysis protocol in healthy subjects and in patients with known splicing alterations&#xD;
             and/or altered RNA expression.&#xD;
  &#xD;
          -  Objective 2: Diagnostic phase. Study of splicing alterations and RNA levels in cultured&#xD;
             fibroblasts obtained from skin biopsies of patients with rare genetic diseases and&#xD;
             negative exome.&#xD;
  &#xD;
        Exploratory goals.&#xD;
  &#xD;
          -  Compare the RNA expression profile obtained from skin biopsy-derived fibroblasts with&#xD;
             the RNA expression profile from blood. The most relevant results will be validated in&#xD;
             qRT-PCR.&#xD;
  &#xD;
          -  To analyze the transcriptional and protein profile heterogeneity in skin-derived&#xD;
             fibroblasts in enrolled subjects.&#xD;
  &#xD;
        To explore the effects of genetic (from WES) and transcriptional (from RNA-seq) alterations&#xD;
        in participants' plasma and serum.&#xD;
  &#xD;
        Healthy controls. Five healthy subjects will be recruited from the staff of the Mario Negri&#xD;
        Institute for Pharmacological Research. To ensure the protection of the category of healthy&#xD;
        volunteers, it has been envisaged that the enrollment is entrusted to the health personnel of&#xD;
        the Daccò Center, who will inform, via email, all the staff of the Institute of the&#xD;
        possibility of spontaneously joining the study. No manager or tutor will be involved in the&#xD;
        recruitment of volunteers. As a further guarantee of confidentiality, for the purposes of the&#xD;
        study, the samples of healthy volunteers will be treated in coded form The coded samples will&#xD;
        be used to set up the method of isolation and culture of skin fibroblasts and RNA-Seq.&#xD;
  &#xD;
        Validation group. For the set-up and validation of the skin fibroblast isolation and RNA-Seq&#xD;
        procedure, ten adult patients with known diagnosis and with alterations in RNA levels and/or&#xD;
        splicing will be recruited as positive controls.&#xD;
  &#xD;
        Among these, patients affected by atypical Hemolytic-Uremic Syndrome (aHUS),&#xD;
        Membranoproliferative Glomerulonephritis (MPGN), Autosomal Dominant Polycystic Kidney (ADPKD)&#xD;
        or other rare pathologies previously diagnosed at the Daccò Center will be included, who have&#xD;
        given their consent the collection and conservation (UNI EN ISO 9001 since 2016;&#xD;
        certification n° 6121) of the samples in the certified biobank (Mario Negri Biological&#xD;
        Resources Center - Rare and Kidney Diseases Biobank). Patients who meet the requirements&#xD;
        described above will be contacted by the doctors of the Daccò Center for an interview&#xD;
        explaining the project. Those who agree to participate in the study will be asked to sign the&#xD;
        informed consent before proceeding with the experimental part.&#xD;
  &#xD;
        &quot;Discovery/Exploration&quot; group. The exploration cohort will be composed of 30 symptomatic&#xD;
        undiagnosed patients with suspected genetic disease (children and adults with infantile&#xD;
        onset) belonging to the Clinical Center of the Mario Negri Institute for Pharmacological&#xD;
        Research and for whom WES investigations did not reveal causative genetic alterations. To&#xD;
        this end we plan to recruit about 60 patients, their parents and more informative family&#xD;
        members, if available, who will be analyzed, if not done previously, by WES. Based on the&#xD;
        literature and our experience, we expect that WES analysis will be curative in 40-50% of&#xD;
        cases (Lunke et al., 2023). Therefore we assume to identify 30 patients with negative WES and&#xD;
        who will therefore be able to enter the &quot;Discovery/Exploration&quot; cohort.&#xD;
  &#xD;
        After signing the informed consent to the collection and storage of samples (UNI EN ISO 9001&#xD;
        since 2016; certification n° 6121) in the certified biobank (Mario Negri Biological Resources&#xD;
        Center - Rare and Renal Diseases Biobank) and after the collection of clinical information,&#xD;
        patients will be enrolled in the project.&#xD;
  &#xD;
        Blood and skin biopsy samples for DNA and fibroblast isolation will be collected as described&#xD;
        below.&#xD;
  &#xD;
        For the WES analyses, 3 tubes of 3 ml of whole blood in EDTA will be taken from adults and 2&#xD;
        tubes of 3 ml of whole blood in EDTA from children and stored at -20°C until use. Blood&#xD;
        collection will be done at the Mario Negri Institute-Daccò Center.&#xD;
  &#xD;
        The WES analyzes will be performed at the Clinical Research Center for Rare Diseases &quot;Aldo e&#xD;
        Cele Daccò&quot; or in a specialized external laboratory, selected under our responsibility.&#xD;
  &#xD;
        An additional 3 mL aliquot of whole blood will be drawn into TempusTM Blood RNA tubes, for&#xD;
        exploratory analysis.&#xD;
  &#xD;
        A research biobank containing biological material in the form of blood, serum and plasma will&#xD;
        be set up at the &quot;Mario Negri Biological Resource Center - Rare Diseases and Kidney Diseases&#xD;
        Biobank&quot;.&#xD;
  &#xD;
        At the first visit, patients will sign a study-specific consent form, containing specific&#xD;
        information and choices regarding participation in the research biobank, including the&#xD;
        ability to use the samples in the future for additional research studies.&#xD;
  &#xD;
        If subjects give their consent, aliquots of serum and plasma (1 x 2 ml of plasma, 1 x 2 ml of&#xD;
        serum) will be collected and stored in coded form in the biobank.&#xD;
      </textblock>
    </detailed_description>
    <overall_status>Not yet recruiting</overall_status>
    <start_date type="Anticipated">September 2023</start_date>
    <completion_date type="Anticipated">September 2025</completion_date>
    <primary_completion_date type="Anticipated">September 2025</primary_completion_date>
    <phase>N/A</phase>
    <study_type>Interventional</study_type>
    <has_expanded_access>No</has_expanded_access>
    <study_design_info>
      <allocation>Non-Randomized</allocation>
      <intervention_model>Parallel Assignment</intervention_model>
      <primary_purpose>Diagnostic</primary_purpose>
      <masking>None (Open Label)</masking>
    </study_design_info>
    <primary_outcome>
      <measure>Set up and validate the transcriptome analysis procedure in healthy controls and in patients diagnosed with known genetic diseases and with known splice/expression altering variants</measure>
      <time_frame>At day 0</time_frame>
      <description>WES analyses for DNA isolation</description>
    </primary_outcome>
    <primary_outcome>
      <measure>Analyse alterations of mRNA levels and splicing in cultured fibroblasts derived from patients with rare diseases and an inconclusive WES.</measure>
      <time_frame>At day 0</time_frame>
      <description>To investigate the heterogeneity of the transcriptomic and proteomic profile, in skin-derived fibroblasts</description>
    </primary_outcome>
    <number_of_arms>3</number_of_arms>
    <enrollment type="Anticipated">105</enrollment>
    <condition>Atypical Hemolytic Uremic Syndrome</condition>
    <condition>Membranoproliferative Glomerulonephritis</condition>
    <condition>Autosomal Dominant Polycystic Kidney</condition>
    <condition>Healthy</condition>
    <arm_group>
      <arm_group_label>Healthy subjects</arm_group_label>
      <arm_group_type>Active Comparator</arm_group_type>
      <description>Five healthy donors will be asked to participate in the study to set up the condition for isolation and culture of skin-derived fibroblasts and to establish the RNA-Seq conditions and profile.</description>
    </arm_group>
    <arm_group>
      <arm_group_label>Validation cohort</arm_group_label>
      <arm_group_type>Experimental</arm_group_type>
      <description>To develop a diagnostic pipeline for isolation and sequencing of mRNA from cultured skin fibroblasts, 10 adult patients with known genetic defects affecting RNA levels and/or splicing will be enrolled, as positive controls.</description>
    </arm_group>
    <arm_group>
      <arm_group_label>Discovery cohort</arm_group_label>
      <arm_group_type>Experimental</arm_group_type>
      <description>The second group, the discovery cohort, will be composed of 30 undiagnosed symptomatic patients with clinical suspicion of a genetic disease (both children and adults with onset in infancy or early adulthood) referred to the Clinical Research Center for Rare Diseases &quot;Aldo e Cele Daccò&quot;, and for which WES analyses did not reveal any causative genetic alteration. To this end, we plan to recruit around 60 patients, their available parents and/or their available informative relatives who will undergo WES, if not previously done. On the basis of literature and our experience we expect that WES will be resolutive in 40-50% of cases (Lunke et al., 2023). Consequently, we hypothesize to identify 30 patients with a negative WES who will enter the discovery RNA-Seq cohort.</description>
    </arm_group>
    <intervention>
      <intervention_type>Procedure</intervention_type>
      <intervention_name>Skin biopsy</intervention_name>
      <description>A punch biopsy is a 15-minute low-risk procedure performed through a sterile circular blade (usually 3-4 mm) under local anesthesia. Briefly, the instrument is rotated down through the forearm epidermis and dermis perpendicular to its physiological lines of relaxation and into the subcutaneous fat, by producing a cylindrical core. Punch biopsy site can be closed with a single suture and generally produce only a minimal scar.</description>
      <arm_group_label>Discovery cohort</arm_group_label>
      <arm_group_label>Healthy subjects</arm_group_label>
      <arm_group_label>Validation cohort</arm_group_label>
    </intervention>
    <eligibility>
      <criteria>
        <textblock>
          Healthy subjects.&#xD;
  &#xD;
          Inclusion Criteria:&#xD;
  &#xD;
            -  Male and female &gt; 18 years&#xD;
  &#xD;
            -  Written informed consent&#xD;
  &#xD;
          Exclusion Criteria:&#xD;
  &#xD;
            -  Inability to understand the potential risk and benefits of the study&#xD;
  &#xD;
            -  Legal incapacity&#xD;
  &#xD;
          Validation cohort.&#xD;
  &#xD;
          Inclusion criteria:&#xD;
  &#xD;
            -  Male and female &gt; 18 years&#xD;
  &#xD;
            -  Genetic diseases affecting RNA levels (frameshifts, stop, large deletions, alteration&#xD;
               of canonical splicing sites)&#xD;
  &#xD;
            -  Written informed consent&#xD;
  &#xD;
          Exclusion criteria:&#xD;
  &#xD;
            -  Underage patients&#xD;
  &#xD;
            -  Inability to understand the potential risk and benefits of the study&#xD;
  &#xD;
            -  Legal incapacity&#xD;
  &#xD;
          Discovery cohort.&#xD;
  &#xD;
          Inclusion criteria:&#xD;
  &#xD;
            -  Male and female patients (children and adults with onset in infancy or early&#xD;
               adulthood) with rare genetic undiagnosed diseases&#xD;
  &#xD;
            -  Patients with no strong candidates based on previous genetic analysis such as WES, but&#xD;
               with clinically suspicion of a genetic rare disease&#xD;
  &#xD;
            -  Written informed consent&#xD;
  &#xD;
          Exclusion criteria:&#xD;
  &#xD;
            -  Inability to understand the potential risk and benefits of the study&#xD;
  &#xD;
            -  Legal incapacity&#xD;
        </textblock>
      </criteria>
      <gender>All</gender>
      <minimum_age>N/A</minimum_age>
      <maximum_age>N/A</maximum_age>
      <healthy_volunteers>Accepts Healthy Volunteers</healthy_volunteers>
    </eligibility>
    <overall_official>
      <last_name>Marina Noris, PhD</last_name>
      <role>Principal Investigator</role>
      <affiliation>Istituto di Ricerche Farmacologiche Mario Negri IRCCS</affiliation>
    </overall_official>
    <overall_contact>
      <last_name>Marina Noris, PhD</last_name>
      <phone>+3903545351</phone>
      <email>marina.noris@marionegri.it</email>
    </overall_contact>
    <overall_contact_backup>
      <last_name>Elena Bresin</last_name>
      <phone>+3903545351</phone>
      <email>elena.bresin@marionegri.it</email>
    </overall_contact_backup>
    <location>
      <facility>
        <name>Centro di Ricerche Cliniche per le Malattie Rare &quot;Aldo e Cele Daccò&quot;</name>
        <address>
          <city>Ranica</city>
          <state>BG</state>
          <zip>24020</zip>
          <country>Italy</country>
        </address>
      </facility>
      <contact>
        <last_name>Elena Bresin</last_name>
        <phone>0039 035 45351</phone>
        <email>elena.bresin@marionegri.it</email>
      </contact>
    </location>
    <location_countries>
      <country>Italy</country>
    </location_countries>
    <reference>
      <citation>Kremer LS, Bader DM, Mertes C, Kopajtich R, Pichler G, Iuso A, Haack TB, Graf E, Schwarzmayr T, Terrile C, Konarikova E, Repp B, Kastenmuller G, Adamski J, Lichtner P, Leonhardt C, Funalot B, Donati A, Tiranti V, Lombes A, Jardel C, Glaser D, Taylor RW, Ghezzi D, Mayr JA, Rotig A, Freisinger P, Distelmaier F, Strom TM, Meitinger T, Gagneur J, Prokisch H. Genetic diagnosis of Mendelian disorders via RNA sequencing. Nat Commun. 2017 Jun 12;8:15824. doi: 10.1038/ncomms15824.</citation>
      <PMID>28604674</PMID>
    </reference>
    <reference>
      <citation>Lee H, Huang AY, Wang LK, Yoon AJ, Renteria G, Eskin A, Signer RH, Dorrani N, Nieves-Rodriguez S, Wan J, Douine ED, Woods JD, Dell'Angelica EC, Fogel BL, Martin MG, Butte MJ, Parker NH, Wang RT, Shieh PB, Wong DA, Gallant N, Singh KE, Tavyev Asher YJ, Sinsheimer JS, Krakow D, Loo SK, Allard P, Papp JC; Undiagnosed Diseases Network; Palmer CGS, Martinez-Agosto JA, Nelson SF. Diagnostic utility of transcriptome sequencing for rare Mendelian diseases. Genet Med. 2020 Mar;22(3):490-499. doi: 10.1038/s41436-019-0672-1. Epub 2019 Oct 14.</citation>
      <PMID>31607746</PMID>
    </reference>
    <reference>
      <citation>Yepez VA, Mertes C, Muller MF, Klaproth-Andrade D, Wachutka L, Fresard L, Gusic M, Scheller IF, Goldberg PF, Prokisch H, Gagneur J. Detection of aberrant gene expression events in RNA sequencing data. Nat Protoc. 2021 Feb;16(2):1276-1296. doi: 10.1038/s41596-020-00462-5. Epub 2021 Jan 18.</citation>
      <PMID>33462443</PMID>
    </reference>
    <verification_date>August 2023</verification_date>
    <study_first_submitted>August 2, 2023</study_first_submitted>
    <study_first_submitted_qc>August 9, 2023</study_first_submitted_qc>
    <study_first_posted type="Actual">August 18, 2023</study_first_posted>
    <last_update_submitted>August 9, 2023</last_update_submitted>
    <last_update_submitted_qc>August 9, 2023</last_update_submitted_qc>
    <last_update_posted type="Actual">August 18, 2023</last_update_posted>
    <responsible_party>
      <responsible_party_type>Sponsor</responsible_party_type>
    </responsible_party>
    <keyword>Undiagnosed genetic rare diseases</keyword>
    <keyword>Whole-exome sequencing</keyword>
    <keyword>RNA-Sequencing</keyword>
    <keyword>Molecular diagnosis</keyword>
    <keyword>Skin-derived fibroblasts</keyword>
    <condition_browse>
      <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
      <mesh_term>Polycystic Kidney Diseases</mesh_term>
      <mesh_term>Glomerulonephritis</mesh_term>
      <mesh_term>Polycystic Kidney, Autosomal Dominant</mesh_term>
      <mesh_term>Hemolytic-Uremic Syndrome</mesh_term>
      <mesh_term>Atypical Hemolytic Uremic Syndrome</mesh_term>
      <mesh_term>Glomerulonephritis, Membranoproliferative</mesh_term>
      <mesh_term>Rare Diseases</mesh_term>
    </condition_browse>
    <patient_data>
      <sharing_ipd>No</sharing_ipd>
    </patient_data>
    <!-- Results have not yet been posted for this study                                          -->
  </clinical_study>
  
  
text: download date: ClinicalTrials.gov processed this data on September 20, 2023
  link text: Link to the current ClinicalTrials.gov record.
  url: https://clinicaltrials.gov/ct2/show/NCT05996731
  org study id: ANTHEM-RNA-Seq
  nct id: NCT05996731
  lead sponsor: 
  has dmc: No
  is fda regulated drug: No
  is fda regulated device: No
  This project aims to identify, through RNA-Seq technology, the genetic alterations underlying
        undiagnosed rare diseases in pediatric and adult patients with early onset and with negative
        WES.
  
          -  Objective 1: Set up and validate techniques. Set-up and validation of the transcriptome
             analysis protocol in healthy subjects and in patients with known splicing alterations
             and/or altered RNA expression.
  
          -  Objective 2: Diagnostic phase. Study of splicing alterations and RNA levels in cultured
             fibroblasts obtained from skin biopsies of patients with rare genetic diseases and
             negative exome.
  
        Exploratory goals
  
          -  Compare the RNA expression profile obtained from skin biopsy-derived fibroblasts with
             the RNA expression profile from blood. The most relevant results will be validated in
             qRT-PCR.
  
          -  To analyze the transcriptional and protein profile heterogeneity in skin-derived
             fibroblasts in enrolled subjects.
  
        To explore the effects of genetic (from WES) and transcriptional (from RNA-seq) alterations
        in participants' plasma and serum.
  
        Healthy controls Five healthy subjects will be recruited from the staff of the Mario Negri
        Institute for Pharmacological Research. The coded samples will be used to set up the method
        of isolation and culture of skin fibroblasts and RNA-Seq.
  
        Validation group For the set-up and validation of the skin fibroblast isolation and RNA-Seq
        procedure, ten adult patients with known diagnosis and with alterations in RNA levels and/or
        splicing will be recruited as positive controls.
  
        Patients who meet the requirements described above will be contacted by the doctors of the
        Daccò Center for an interview explaining the project. Those who agree to participate in the
        study will be asked to sign the informed consent before proceeding with the experimental
        part.
  
        "Discovery/Exploration" group The exploration cohort will be composed of 30 symptomatic
        undiagnosed patients with suspected genetic disease (children and adults with infantile
        onset) belonging to the Clinical Center of the Mario Negri Institute for Pharmacological
        Research and for whom WES investigations did not reveal causative genetic alterations.
  Rare genetic diseases are a very heterogeneous group of diseases, often undiagnosed, for
        which patients receive only symptomatic treatment. The development of next-generation massive
        sequencing technology (Next-Generation-Sequencing; NGS) has made it possible to identify many
        of the genes responsible for these pathologies, allowing the pathogenic mechanisms to be
        outlined. The most commonly used methods, such as exome sequencing (Whole-Exome Sequencing;
        WES) or genome sequencing (Whole-Genome Sequencing; WGS), are based on DNA sequencing and
        allow to identify pathogenic mutations that cause loss of protein functionality, such as stop
        mutations, large insertions and deletions and the loss of canonical splicing sites.
        Conversely, the attribution of pathogenicity to an amino acid substitution variant (missense)
        or a synonymous variant is often questionable and, consequently, such variants are often
        categorized as Variants of Unknown Significance (VUS). Furthermore, exome sequencing is
        unable to identify possible pathogenic variants located in non-coding regions, such as
        non-canonical splice sites or deep intronic variants.
  
        WGS sequencing, while allowing the identification of variants present in the non-coding
        regions of a gene, is very expensive and produces an enormous amount of data with low
        pathogenic information. Algorithms to analyze the possible pathogenicity of intronic variants
        have little predictive power and the pathogenicity of the identified variants must be
        validated by lengthy and complex functional analyses. Due to these limitations, approximately
        50% of patients with rare genetic diseases remain undiagnosed and cannot benefit from the
        benefits of personalized medicine. Transcriptome sequencing (RNA-Seq) allows to determine
        variations in the sequence and levels of RNA, providing complementary information to that
        obtained with WES and WGS, thus improving the efficiency of genetic diagnosis. Splicing
        alterations, which are among the main causes of genetic diseases and can reside both in deep
        intronic regions and in coding regions, can be identified through RNA sequencing. Variants in
        the regulatory regions of the gene, which are able to influence RNA expression levels, can
        also be highlighted by RNA-Seq. Since RNA expression, in terms of levels and isoforms
        produced by alternative splicing, is tissue-specific, the most transcriptionally informative
        biological material is certainly the affected tissue. However, this is not always easy to
        achieve and the collection can be too invasive for the patient. In these cases it is possible
        to resort to the use of alternative tissues, such as whole blood or skin biopsies, which have
        low invasiveness and a broad RNA expression profile. Fibroblasts obtained from skin biopsies,
        in particular, have proven to be the most reliable, homogeneous and informative alternative
        matrix for RNA-Seq analysis, in a wide range of pathologies. Yépez and collaborators
        demonstrated that the information obtained from RNA-Seq from skin fibroblasts was superior to
        that obtained from blood. Furthermore, the analysis of the transcriptome of skin fibroblasts
        allowed to identify altered levels of transcripts and splicing abnormalities in neuromuscular
        and neurodevelopmental diseases. This confirmed the efficacy of the use of skin fibroblasts
        in the diagnosis of genetic pathologies in WES and WGS negative patients. Last but not least,
        skin fibroblasts can be reprogrammed into other cell types for possible future analyzes.
  
        This project aims to identify, through RNA-Seq technology, the genetic alterations underlying
        undiagnosed rare diseases in pediatric and adult patients with early onset and with negative
        WES.
  
        Although NGS is essential for the diagnosis of genetic diseases, its success rates range from
        30% to 50%. The advent of RNA-Seq increased the chance of diagnosing genetic diseases from 8%
        to 36%. Through this project we intend to apply the RNA-Seq method, as an integration of the
        WES analysis, for the diagnosis of rare genetic diseases:
  
          -  Objective 1: Set up and validate techniques. Set-up and validation of the transcriptome
             analysis protocol in healthy subjects and in patients with known splicing alterations
             and/or altered RNA expression.
  
          -  Objective 2: Diagnostic phase. Study of splicing alterations and RNA levels in cultured
             fibroblasts obtained from skin biopsies of patients with rare genetic diseases and
             negative exome.
  
        Exploratory goals.
  
          -  Compare the RNA expression profile obtained from skin biopsy-derived fibroblasts with
             the RNA expression profile from blood. The most relevant results will be validated in
             qRT-PCR.
  
          -  To analyze the transcriptional and protein profile heterogeneity in skin-derived
             fibroblasts in enrolled subjects.
  
        To explore the effects of genetic (from WES) and transcriptional (from RNA-seq) alterations
        in participants' plasma and serum.
  
        Healthy controls. Five healthy subjects will be recruited from the staff of the Mario Negri
        Institute for Pharmacological Research. To ensure the protection of the category of healthy
        volunteers, it has been envisaged that the enrollment is entrusted to the health personnel of
        the Daccò Center, who will inform, via email, all the staff of the Institute of the
        possibility of spontaneously joining the study. No manager or tutor will be involved in the
        recruitment of volunteers. As a further guarantee of confidentiality, for the purposes of the
        study, the samples of healthy volunteers will be treated in coded form The coded samples will
        be used to set up the method of isolation and culture of skin fibroblasts and RNA-Seq.
  
        Validation group. For the set-up and validation of the skin fibroblast isolation and RNA-Seq
        procedure, ten adult patients with known diagnosis and with alterations in RNA levels and/or
        splicing will be recruited as positive controls.
  
        Among these, patients affected by atypical Hemolytic-Uremic Syndrome (aHUS),
        Membranoproliferative Glomerulonephritis (MPGN), Autosomal Dominant Polycystic Kidney (ADPKD)
        or other rare pathologies previously diagnosed at the Daccò Center will be included, who have
        given their consent the collection and conservation (UNI EN ISO 9001 since 2016;
        certification n° 6121) of the samples in the certified biobank (Mario Negri Biological
        Resources Center - Rare and Kidney Diseases Biobank). Patients who meet the requirements
        described above will be contacted by the doctors of the Daccò Center for an interview
        explaining the project. Those who agree to participate in the study will be asked to sign the
        informed consent before proceeding with the experimental part.
  
        "Discovery/Exploration" group. The exploration cohort will be composed of 30 symptomatic
        undiagnosed patients with suspected genetic disease (children and adults with infantile
        onset) belonging to the Clinical Center of the Mario Negri Institute for Pharmacological
        Research and for whom WES investigations did not reveal causative genetic alterations. To
        this end we plan to recruit about 60 patients, their parents and more informative family
        members, if available, who will be analyzed, if not done previously, by WES. Based on the
        literature and our experience, we expect that WES analysis will be curative in 40-50% of
        cases (Lunke et al., 2023). Therefore we assume to identify 30 patients with negative WES and
        who will therefore be able to enter the "Discovery/Exploration" cohort.
  
        After signing the informed consent to the collection and storage of samples (UNI EN ISO 9001
        since 2016; certification n° 6121) in the certified biobank (Mario Negri Biological Resources
        Center - Rare and Renal Diseases Biobank) and after the collection of clinical information,
        patients will be enrolled in the project.
  
        Blood and skin biopsy samples for DNA and fibroblast isolation will be collected as described
        below.
  
        For the WES analyses, 3 tubes of 3 ml of whole blood in EDTA will be taken from adults and 2
        tubes of 3 ml of whole blood in EDTA from children and stored at -20°C until use. Blood
        collection will be done at the Mario Negri Institute-Daccò Center.
  
        The WES analyzes will be performed at the Clinical Research Center for Rare Diseases "Aldo e
        Cele Daccò" or in a specialized external laboratory, selected under our responsibility.
  
        An additional 3 mL aliquot of whole blood will be drawn into TempusTM Blood RNA tubes, for
        exploratory analysis.
  
        A research biobank containing biological material in the form of blood, serum and plasma will
        be set up at the "Mario Negri Biological Resource Center - Rare Diseases and Kidney Diseases
        Biobank".
  
        At the first visit, patients will sign a study-specific consent form, containing specific
        information and choices regarding participation in the research biobank, including the
        ability to use the samples in the future for additional research studies.
  
        If subjects give their consent, aliquots of serum and plasma (1 x 2 ml of plasma, 1 x 2 ml of
        serum) will be collected and stored in coded form in the biobank.
  allocation: Non-Randomized
  intervention model: Parallel Assignment
  primary purpose: Diagnostic
  masking: None (Open Label)
  measure: Set up and validate the transcriptome analysis procedure in healthy controls and in patients diagnosed with known genetic diseases and with known splice/expression altering variants
  time frame: At day 0
  description: WES analyses for DNA isolation
  measure: Analyse alterations of mRNA levels and splicing in cultured fibroblasts derived from patients with rare diseases and an inconclusive WES.
  time frame: At day 0
  description: To investigate the heterogeneity of the transcriptomic and proteomic profile, in skin-derived fibroblasts
  arm group label: Healthy subjects
  arm group type: Active Comparator
  description: Five healthy donors will be asked to participate in the study to set up the condition for isolation and culture of skin-derived fibroblasts and to establish the RNA-Seq conditions and profile.
  arm group label: Validation cohort
  arm group type: Experimental
  description: To develop a diagnostic pipeline for isolation and sequencing of mRNA from cultured skin fibroblasts, 10 adult patients with known genetic defects affecting RNA levels and/or splicing will be enrolled, as positive controls.
  arm group label: Discovery cohort
  arm group type: Experimental
  description: The second group, the discovery cohort, will be composed of 30 undiagnosed symptomatic patients with clinical suspicion of a genetic disease (both children and adults with onset in infancy or early adulthood) referred to the Clinical Research Center for Rare Diseases "Aldo e Cele Daccò", and for which WES analyses did not reveal any causative genetic alteration. To this end, we plan to recruit around 60 patients, their available parents and/or their available informative relatives who will undergo WES, if not previously done. On the basis of literature and our experience we expect that WES will be resolutive in 40-50% of cases (Lunke et al., 2023). Consequently, we hypothesize to identify 30 patients with a negative WES who will enter the discovery RNA-Seq cohort.
  intervention type: Procedure
  intervention name: Skin biopsy
  description: A punch biopsy is a 15-minute low-risk procedure performed through a sterile circular blade (usually 3-4 mm) under local anesthesia. Briefly, the instrument is rotated down through the forearm epidermis and dermis perpendicular to its physiological lines of relaxation and into the subcutaneous fat, by producing a cylindrical core. Punch biopsy site can be closed with a single suture and generally produce only a minimal scar.
  arm group label: Discovery cohort
  arm group label: Healthy subjects
  arm group label: Validation cohort
  criteria: 
  gender: All
  minimum age: N/A
  maximum age: N/A
  healthy volunteers: Accepts Healthy Volunteers
  last name: Marina Noris, PhD
  role: Principal Investigator
  affiliation: Istituto di Ricerche Farmacologiche Mario Negri IRCCS
  last name: Marina Noris, PhD
  phone: +3903545351
  email: marina.noris@marionegri.it
  last name: Elena Bresin
  phone: +3903545351
  email: elena.bresin@marionegri.it
  facility: 
  contact: 
  country: Italy
  citation: Kremer LS, Bader DM, Mertes C, Kopajtich R, Pichler G, Iuso A, Haack TB, Graf E, Schwarzmayr T, Terrile C, Konarikova E, Repp B, Kastenmuller G, Adamski J, Lichtner P, Leonhardt C, Funalot B, Donati A, Tiranti V, Lombes A, Jardel C, Glaser D, Taylor RW, Ghezzi D, Mayr JA, Rotig A, Freisinger P, Distelmaier F, Strom TM, Meitinger T, Gagneur J, Prokisch H. Genetic diagnosis of Mendelian disorders via RNA sequencing. Nat Commun. 2017 Jun 12;8:15824. doi: 10.1038/ncomms15824.
  PMID: 28604674
  citation: Lee H, Huang AY, Wang LK, Yoon AJ, Renteria G, Eskin A, Signer RH, Dorrani N, Nieves-Rodriguez S, Wan J, Douine ED, Woods JD, Dell'Angelica EC, Fogel BL, Martin MG, Butte MJ, Parker NH, Wang RT, Shieh PB, Wong DA, Gallant N, Singh KE, Tavyev Asher YJ, Sinsheimer JS, Krakow D, Loo SK, Allard P, Papp JC; Undiagnosed Diseases Network; Palmer CGS, Martinez-Agosto JA, Nelson SF. Diagnostic utility of transcriptome sequencing for rare Mendelian diseases. Genet Med. 2020 Mar;22(3):490-499. doi: 10.1038/s41436-019-0672-1. Epub 2019 Oct 14.
  PMID: 31607746
  citation: Yepez VA, Mertes C, Muller MF, Klaproth-Andrade D, Wachutka L, Fresard L, Gusic M, Scheller IF, Goldberg PF, Prokisch H, Gagneur J. Detection of aberrant gene expression events in RNA sequencing data. Nat Protoc. 2021 Feb;16(2):1276-1296. doi: 10.1038/s41596-020-00462-5. Epub 2021 Jan 18.
  PMID: 33462443
  responsible party type: Sponsor
  mesh term: Polycystic Kidney Diseases
  mesh term: Glomerulonephritis
  mesh term: Polycystic Kidney, Autosomal Dominant
  mesh term: Hemolytic-Uremic Syndrome
  mesh term: Atypical Hemolytic Uremic Syndrome
  mesh term: Glomerulonephritis, Membranoproliferative
  mesh term: Rare Diseases
  sharing ipd: No