Over the past century, the progress in biomedical sciences gave outstanding contributions to the understanding of human conditions, and even curing some of them. Recent advances in high-throughput technologies have enabled the profiling of Rare Diseases (RDs) trough genomics, transcriptomics, proteomics, and metabolomics. Regardless of the improvements in the efficiency of data generation, the research community struggles when stepping into bedside and translational processes. The lack of integrated networks between specialists of referral hospitals, local healthcare services and researchers represents a major challenge impacting outcomes of patients with RDs and overall family well-being.

The "Agostino Gemelli" University Hospital (GUH) is a leader in the national and international scenario on the clinical management of individuals affected by RDs with more than10.000 patients followed per year, 19 Units certifying different RDs and 16 of them belonging to the European reference Network. Nevertheless, even though the Units by themselves have a well-organized level of competencies, the network of specialists inside and outside hospital, the transition models from pediatric to adult age and the connection between referral centers and territorial services need to be optimized.

The current project aims to build the foundation of a multidimensional model helping clinicians overcoming the gaps mentioned above. Starting from the strength of a comprehensive and diversified clinical experience of specialists from GUH (HUB) and Metabolic and Genetic Unit at the Giovanni XXIII Children's Hospital in Bari (Spoke) a standardization of diagnostic and therapeutic coding according to MONDO Disease Ontology coding, Human Phenotype Ontology (HPO), and Anatomical Therapeutic Chemical (ATC) coding, will be performed. IT services will develop an electronic Case Report Form (eCRF) to collect and match data and experienced physicians will perform a functional diagnosis using the InterRAI multidimensional tools. Multiomics profiling will be performed by using the Gemelli-Science and TEchnology Park (G-STEP) facility (HUB) and the Institute for Genetic and Biomedical Research, National Research Council (Spoke) will generate induced pluripotent stem cell lines and isogenic cell lines by genome-editing technologies from selected patients with RDs. A Multidisciplinary Board for Rare Diseases (MBRD) with members of all Units will assess criteria for patient enrollment, will provide integration of clinical and molecular data and based on results, it will plan a personalized medical care strategy. To ensure the cross-sectional application of the model proposed to the broadest number of RDs, the participating Units will select four specific populations (from pediatric to adult age) affected by RDs. Measurable outcomes will arise from analysis of the mean time between first contact with the specialist and establishment of the etiologic diagnosis as well as reduction in the number of hospital visits, missed visits and loss of follow-up. The above data from patients enrolled in the present study will be compared to those collected in the two years prior Sars-Cov-2 pandemic (2017-2019) by querying data from the Regional Registry for RDs. This study performed on four major selected groups of RDs represents a model potentially applying to larger groups of RDs

Background and Rationale Over the past century, the progress in biomedical sciences gave outstanding contributions to understanding the human conditions, and even curing some of them. Recent advances in high-throughput technologies have enabled the profiling of RDs trough genomics, transcriptomics, proteomics, and metabolomics. However, integrative analysis of such datasets is complicated by the high dimensionality and heterogeneity of the data and the lack of analysis protocols. Regardless of the improvements in the efficiency of data generation, the research community struggles when stepping into bedside and translational processes. The lack of integrated networks between clinical specialists, local healthcare services, and researchers represents a major challenge impacting outcomes of patients with Rare Diseases (RDs) and overall family wellbeing.

Integrative analysis of such systems represents a major unresolved problem in managing RDs, strongly emphasize by the Sars-Cov-2 pandemic.

The "Agostino Gemelli" University Hospital (GUH) is a leader in the national and international scenario on the clinical management of individuals affected by RDs. More than 10.000 patients per year routinely receive a comprehensive healthcare plan to manage disease sequelae and comorbidities and appropriate therapeutic options when available, according to national/international recommendations. All medical records are stored in the intranet system but this is not yet customized for RDs. The interconnection with territorial services is mediated by families delivering medical documentation to local family doctors or other territorial services.

To date, 19 Units can certify RDs and 16 Units belong to the European Reference Network (ERN). Even though each Unit has a relatively well-organized level of competencies, the patient odyssey is still time and economic resource consuming. Delay in diagnosis increases the redundancy of clinical/instrumental examinations increasing costs for the National healthcare system. Therefore, the network of specialists inside/outside hospitals needs to be optimized. Moreover, the lack of standardized transition models from pediatric to adult age and connections between referral centers and territorial services lead to a high number of dropouts.

General Objectve

The current project aims to build the foundation of a multidimensional model helping clinicians overcoming the gaps mentioned above. In order to reach this main goal, the project will pursue the following specific objectives trough the implementation of different tasks:

Specific objectives

AIM 1 Standardization of the clinical characterization of people with RDs given there is currently no unified model nationwide.

• Task 1. To standardize diagnostic and therapeutic coding. To develop an eCRF according to MONDO Disease Ontology coding, Human Phenotype Ontology (HPO), and Anatomical Therapeutic Chemical (ATC) coding.
• Task 2. To standardize the evaluation of a person's function, needs, and burden of care using the InterRAI multidimensional tools suite. For patients with severe complex diseases, a telemedical consult allows for a first clinical evaluation, the possible enrollment in the study, and follow-up planning.
• Task 3. To standardize the coding of the other clinically significant parameters derived from biological specimens and instrumental examinations to be included in the eCRF.

AIM 2 Multi-omics-based profiling and IPSCs-based modelling for RDs. - Task 1. Multi-omics profiling (clinical exome sequencing, transcriptomics, proteomics, and metabolomics) will be performed at Unit 1 using the G-STEP research facility on various biological specimens collected by RDs patients from Units 1 and 3.

The profiling will be completed for undiagnosed genetic patients, while for those already diagnosed, the profiling will be limited to transcriptomics, proteomics, and metabolomics.

• Task 2. Generation of induced pluripotent stem cell lines (iPSCs) from selected patients with RDs and the creation of isogenic cell lines by genome editing technologies will be performed at Unit 2. These cell lines will be used for biobanking and will be instrumental in studying pathogenic disease processes and drug discovery.
• Task 3. Dedicating biobanking for biological samples of recruited patients will be set up at Units 1 and 2.

AIM 3 Integration of data collected to guide patients trough targeted drug therapies, personalized clinical management, and transition.

• Task 1. Based on data collected from clinical and multi-omics characterization, the MBRD will be able to direct the patient into two main therapeutic pathways: a targeted therapy track in cases where a clear genetic cause underlying the RD emerged and for which targeted drug therapy is available; a personalized clinical management track with specific attention to care transition in all other cases.
• Task 2. Regardless of the therapeutic pathway, the MBRD will establish the clinical follow-up plans shared with patients, family, and territorial services through a telemedicine platform. According to the specific medical condition of the patient, a modular model for remote care with increasing levels of complexity will be used.
• Task 3. Once patient care priorities are clearly defined, in those situations where study subjects are patients above 14 years, MRDB will coordinate and plan the transition to adult care through the participation of adult specialists in medical care assessment decisions (at least 3 MRDB meetings).
• Task 4. The large volume of clinical and multi-omics assessment data will be made available in a dedicated data warehouse following participating Units' request for future specific collaborative research programs.

Methods

Study Design This is a multicenter, experimental study that will be performed at the "Agostino Gemelli" University Hospital (Rome), Institute of Genetic and Biomedical Research - National Research Council (CNR) Monserrato (CA) and Giovanni XXIII Children's Hospital Bari.

Endpoints Primary endpoint

- Mean time from first clinical evaluation performed by an experienced physician to etiologic diagnosis Secondary endopoints Number (n°) of days lost at work for caregivers, n° of visits performed in the hospital, n° of visits lost, n° of patients lost at follow up, n° of patients enrolled in targeted therapies programs, n° telemedicine visits, n° of patients transitioned from pediatric to adult services, n° of patients transitioned to adult care services that come back to pediatric ones, patient/caregiver satisfaction trough Patient-Related Outcome Measures and Patient Assessment of Chronic Illness and satisfactory questionnaires

Population Patient population A total number of 100 patients is enrolled by the MBRD at Unit 1 and 3 (1/3 affected by syndromic conditions and 2/3 with muscular disorders including mitochondrial diseases and muscular dystrophies.

The 100 patients enrolled in the present project (POST group) will be compared with a 100 PRE group patients admitted and managed from 2017 to 2019 in Unit 1 and 3, before the implementation of the multidimensional model.

Clinical and instrumental evaluation All enrolled subjects will undergo a complete clinical, laboratory and instrumental evaluation, according to routine clinical practice.

Methods for specific AIMs

Aim 1

• Task 1: A customized eCRF is set up at Unit 1 by IT services for each group of individuals recruited. The MONDO Disease Ontology, HPO and ATC coding is used to standardize description of phenotypic features, clinical and instrumental findings.
• Task 2: The Intellectual Disability (ID) for individuals older than 18 years and Child and Youth Mental Health Instrument for Developmental Disabilities (ChYMH-DD) tools for patients younger than 18 years from the InterRAI is performed at baseline and after 12 months from recruitment

The individual status is checked by using:

• Status and Outcome Scales (standardized);
• Collaborative Action Points: to support continuity of care planning;
• Personal Health Profile: a summarized patient sheet including medical record and functional level.
• Task 3: Pathogenic variants and key diagnostic findings (instrumental/radiological examinations) coded trough MONDO, HPO systems are included in the eCRF

Aim 2

• Task 1: We plan to perform:
• Genomic DNA and total RNA sequencing on the Illumina Novaseq6000 platform
• Peptides and proteins identification by Nano-high performance liquid chromatography (HPLC)/high-resolution Nanoelectrospray ionization (ESI)-mass spectrometry (MS)/MS (top-down and bottom up analysis)
• Targeted metabolomic panels: a 44 amino acids; a 14 long chain fatty acids; a methylated arginine metabolites; a thiols panel;
• Fourier Transform infrared (FTIR) spectroscopy for lipid and protein content (19);
• NFLs, human TNF-a, circulating biomarkers such as DAMPs and ccf-mtDNA, and a comprehensive miRNA profiling.
• Task 2: PBMCs or NECs from at least 20 patients carrying pathogenic variants are shipped to Unit 2 for iPSCs generation. Sendai virus vectors encoding Yamanaka factors are used to reprogram PBMCs/NECs. The CRISPR/Cas9 genome-editing approach is used to correct the specific mutation.
• Task 3: Biological samples handling and processing is performed at Unit 1. Samples collected are: 1 blood sample (plasma, serum, EDTA), 1 nasal brush, 1 buccal swab in all participants; tissue samples (skin biopsy for syndromes due to somatic mutations; muscular biopsy in patients with muscular dystrophies/mitochondrial disorders).

Aim 3

• Task 1: If pharmacological therapies are available, specific therapeutic protocols are activated at Unit 1 otherwise a personalized clinical management at Unit 1,3 or through telemedicine is guaranteed. The care transition process is set up by the MBRD for subjects above 14 years
• Task 2: The televisit system is integrated to the Hospital information system (Trakcare, at Unit 1). The platform has to be implemented for Unit 3. Mobile and Web Application (MFA/SPID authentication) are used to connect doctors with patients. Different type of telemedicine services are available:
• Basic level: video call consultation with 1 specialist;
• Intermediate level: teleconsultation with a multidisciplinary team supported by questionnaires or video tutorials for caregivers;
• Intermediate level "Plus": if interventions are required (e.g. physical therapy, gastrostomy and tracheostomy care)
• Advanced level: if a tele-monitoring routinely assessed in hospitals (e.g., pulse oximetry) is needed (digital devices sent at home);
• Task 3: The transition program is provided by the MBRD. The effective transition to adults is measured by the following Key Performance Indicators (KPI):
• No. of patients transitioned to adult care;
• No. of patients who leave adult care to come back to the previous care setting (indicator assessed at 12 months after the 1st visit evaluation in the adult medicine setting);
• Patient-Related Outcome Measures and Patient Assessment of Chronic Illness Care tool.
• Task 4: Implementation of the virtual memory actually available at Unit 1 with a dedicated data warehouse

Statistic plan All patients with clinical criteria for inclusion (complex syndromic RDs without a genetic diagnosis, ultra-rare diseases with or without a genetic confirmation, muscular dystrophies, mitochondrial disorders), managed at Unit 1 and 3 will be enrolled.

One hundred patients will be followed using the multidimensional model proposed with the aim of testing whether this model is able to reduce the time between the medical consultation and the etiological diagnosis and improve both decision making and treatment process of patients.

The study is designed as a multi-centers, quasi-experimental, pre-post study comparing the impact of the multidimensional model before and after its implementation on two cohort of patients. The 100 patients enrolled in the present project (POST group) will be compared with a 100 PRE group patients admitted and managed from 2017 to 2019 in Unit 1 and 3, before the implementation of the multidimensional model.

Data (demographic data, clinical data and omics data) are presented as mean and standard deviation (±SD) or median and interquartile range (IQR) in case of continuous variables and as a percentage and absolute values in case of discrete variables. The normality of the sample distribution will be assessed using the Kolmogorov Smirnov test.

Outcomes are measured and reported using the KPIs below summarized.

• mean time between contact with the specialist and establishment of the etiologic diagnosis
• n° of days lost at work for caregivers
• n° of hospital visits
• n° of missed visits and lost at follow-up;
• n° of patients transitioned to a target therapy treatments during the study time;
• n° of telemedicine visits performed
• n° of patients transitioned to adult care
• n° of patients who leave adult care to come back to the previous care setting (indicator assessed at 12 months after the 1st visit evaluation in the adult medicine setting)
• Patient-Related Outcome Measures and Patient Assessment of Chronic Illness Care tool
• patient/caregiver satisfaction. To compare the groups, as appropriate, the following tests will be used: t-test, Wilcoxon test, chi-square test, or Fisher's exact test. We will report between-group differences with their associated 95% confidence interval (CI). Exploratory subgroup analysis will be carried out considering the different classes of conditions.

Sample size As this study involves all eligible patients during the pre-specified period, a prior calculation of sample size has not been performed. However, according preliminary data of the PRE group, the mean time-laps between first outpatient evaluation and etiological diagnosis is 18.7 months (standard deviation 5). Therefore, the sample sizes of 100 (PRE group) and 100 (POST group) achieve 80.7% power to reject the null hypothesis of equal mean time when the population mean difference is 2 months with a standard deviation for both groups of 5 and with a significance level (alpha) of 0,05 (two-sided twosample equal-variance z-test).

Expected outcomes For patients enrolled without a genetic diagnosis (POST group) we expect a reduction of the mean time between contact with the specialist and establishment of the etiologic diagnosis (molecular profiling). In patients with a genetic diagnosis fulfilling criteria for enrolment, we expect to improve overall clinical management as measured using the above mentioned secondary outcomes. Moreover, we expect to identify some reliable biomarkers to be confirmed by future research projects. Finally, we expect to improve the time-laps between patient evaluation at the baseline and access to targeted therapies, QoL and Patient/caregiver satisfaction.

Experimental procedures A subgroup of patients will undergo a skin biopsy, nasal brush and buccal swab. These procedures determine a minimum risk for the patients, as they are widely used in the clinical practice of the management of subjects suffering from rare diseases.

Significance and innovation Major goal of this proposal is to present a new model of integrated approach for RDs with the aim of reducing time-laps between physician consult and etiological diagnosis, sharing decision making process on clinical follow-up and access to personalized therapies, building transition programs from pediatric to adult care and finally helping patients with complex chronic conditions to receive the standards of care through telemedicine.

This pilot project benefits of a well-established network of physicians inside Unit 1 and 3, and integrated facilities for multiomics profiling at Unit 1.

Moreover, the connection with Unit 3 for creation of patient-specific iPSC-cells represents an important step for translational research and future studies.

Data collection and management

• we have standardizes protocols for data collection, including training of study personnel, in order to minimize inter-observer variability; whenever possible, data will be collected blindly;
• we will use a standardized way to interview patients;
• for subjects lost to follow-up we will schedule personalized patient contact via phone or email and home visits;
• we will use, as much as possible, objective diagnostic studies and validated measures as outcomes;
• the personal data of each patient will be cripted by using a unique aphanumeric code (anonymous data)

Dissemination of Results The results obtained will be published in international peer-reviewed journals. An annual meeting will be performed at the "Agostino Gemelli" University Hospital in order to provide a clinical update to colleagues and other healthcare professionals or researchers interested in these genetic disorders.

Protocol's deviations and amendments Any deviation of the Protocol not approved by the Ethics Committee will involve study discontinuation for that patient. Protocol's amendaments must be submitted to the Ethics Committee.

Study documents and biological samples The clinical documentation of the study will be kept for at least 15 years after its conclusion at the "Agostino Gemelli" University Hospital. The biological material will be kept in the XBiogem Biobank located at the "Agostino Gemelli" University Hospital.

Inclusion Criteria:

• complex syndromic RDs without a genetic diagnosis, ultra-rare diseases with or without a genetic confirmation, muscular dystrophies, mitochondrial disorders
• Evidence of a personally signed and dated informed consent document indicating that the subject has been informed of all pertinent aspects of the study

Exclusion Criteria:

• inability to understand or unwilling to follow the study requirements including attendance at the study center, completion of questionnaires and participation in laboratory testing as called for by the protocol
• inability to sign an informed consent;
• severe psychiatric diseases.

Patients with complex syndromic RDs without a genetic diagnosis, ultra-rare diseases with or without a genetic confirmation, muscular dystrophies, mitochondrial disorders.