One year in review 2020: economic and organisational aspects in rare and complex connective tissue diseases.

Rare and complex connective tissue diseases (rCTDs) encompass a considerable number of diseases and syndromes and their variability highly impacts on the clinical management, resulting in variable economic and organisational burden that might represent a challenge for healthcare systems. This paper is aimed at providing an overview of the most recent evidence regarding the economic and organisational impact of rCTDs. In particular, this work discusses the most relevant data on specific aspects related to health economics in rCTDs published in 2019.

Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy.

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes approximately 60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non-HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome.

Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome-associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development.

A restricted spectrum of mutations in the SMAD4 tumor-suppressor gene underlies Myhre syndrome.

Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor ß and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth.

Complete sequence of the IncT-type plasmid pT-OXA-181 carrying the blaOXA-181 carbapenemase gene from Citrobacter freundii.

The gene encoding the carbapenemase OXA-181 (an OXA-48 variant) was identified from a Citrobacter freundii isolate coproducing NDM-1. The whole sequence of plasmid pT-OXA-181 bearing the blaOXA-181 gene was determined and revealed a 84-kb mobilizable but non-self-conjugative IncT-type plasmid. It totally differs from the 7.6-kb ColE-type and blaOXA-181-bearing plasmid recently identified in a Klebsiella pneumoniae isolate. However, in both plasmids, insertion sequence ISEcp1 might have played a role in acquisition of the blaOXA-181 gene.

A boy with Burkitt lymphoma associated with Noonan syndrome due to a mutation in RAF1.

This article reports on an association between Burkitt lymphoma and Noonan syndrome (NS) due to a RAF1 gene mutation. The patient was a 7-year-old boy with NS, who was included in the first series reporting the association between Noonan and RAF1, and who later presented with a 2-week history of asymptomatic unilateral tonsillar swelling and ipsilateral cervical lymphadenopathy. Histological and biological examinations of the tonsillar biopsy led to the diagnosis of Burkitt lymphoma. While there is a well-established association between NS and solid cell tumors, this is the first case described in the literature of Burkitt lymphoma in a patient with NS, and adds to the growing list of data supporting neoplasia's association with NS.

Resources and tools for rare disease variant interpretation.

Collectively, rare genetic disorders affect a substantial portion of the world's population. In most cases, those affected face difficulties in receiving a clinical diagnosis and genetic characterization. The understanding of the molecular mechanisms of these diseases and the development of therapeutic treatments for patients are also challenging. However, the application of recent advancements in genome sequencing/analysis technologies and computer-aided tools for predicting phenotype-genotype associations can bring significant benefits to this field. In this review, we highlight the most relevant online resources and computational tools for genome interpretation that can enhance the diagnosis, clinical management, and development of treatments for rare disorders. Our focus is on resources for interpreting single nucleotide variants. Additionally, we present use cases for interpreting genetic variants in clinical settings and review the limitations of these results and prediction tools. Finally, we have compiled a curated set of core resources and tools for analyzing rare disease genomes. Such resources and tools can be utilized to develop standardized protocols that will enhance the accuracy and effectiveness of rare disease diagnosis.

Unmet needs in countries participating in the undiagnosed diseases network international: an international survey considering national health care and economic indicators.

Background: Patients, families, the healthcare system, and society as a whole are all significantly impacted by rare diseases (RDs). According to various classifications, there are currently up to 9,000 different rare diseases that have been recognized, and new diseases are discovered every month. Although very few people are affected by each uncommon disease individually, millions of people are thought to be impacted globally when all these conditions are considered. Therefore, RDs represent an important public health concern. Although crucial for clinical care, early and correct diagnosis is still difficult to achieve in many nations, especially those with low and middle incomes. Consequently, a sizeable amount of the overall burden of RD is attributable to undiagnosed RD (URD). Existing barriers and policy aspects impacting the care of patients with RD and URD remain to be investigated. Methods: To identify unmet needs and opportunities for patients with URD, the Developing Nations Working Group of the Undiagnosed Diseases Network International (DNWG-UDNI) conducted a survey among its members, who were from 20 different nations. The survey used a mix of multiple choice and dedicated open questions covering a variety of topics. To explore reported needs and analyze them in relation to national healthcare economical aspects, publicly available data on (a) World Bank ranking; (b) Current health expenditure per capita; (c) GDP per capita; (d) Domestic general government health expenditure (% of GDP); and (e) Life expectancy at birth, total (years) were incorporated in our study. Results: This study provides an in-depth evaluation of the unmet needs for 20 countries: low-income (3), middle-income (10), and high-income (7). When analyzing reported unmet needs, almost all countries (N = 19) indicated that major barriers still exist when attempting to improve the care of patients with UR and/or URD; most countries report unmet needs related to the availability of specialized care and dedicated facilities. However, while the countries ranked as low income by the World Bank showed the highest prevalence of referred unmet needs across the different domains, no specific trend appeared when comparing the high, upper, and low-middle income nations. No overt trend was observed when separating countries by current health expenditure per capita, GDP per capita, domestic general government health expenditure (% of GDP) and life expectancy at birth, total (years). Conversely, both the GDP and domestic general government health expenditure for each country impacted the presence of ongoing research. Conclusion: We found that policy characteristics varied greatly with the type of health system and country. No overall pattern in terms of referral for unmet needs when separating countries by main economic or health indicators were observed. Our findings highlight the importance of identifying actionable points (e.g., implemented orphan drug acts or registries where not available) in order to improve the care and diagnosis of RDs and URDs on a global scale.

Undiagnosed diseases: Needs and opportunities in 20 countries participating in the Undiagnosed Diseases Network International.

Introduction: Rare diseases (RD) are a health priority worldwide, overall affecting hundreds of millions of people globally. Early and accurate diagnosis is essential to support clinical care but remains challenging in many countries, especially the low- and medium-income ones. Hence, undiagnosed RD (URD) account for a significant portion of the overall RD burden. Methods: In October 2020, the Developing Nations Working Group of the Undiagnosed Diseases Network International (DNWG-UDNI) launched a survey among its members, belonging to 20 countries across all continents, to map unmet needs and opportunities for patients with URD. The survey was based on questions with open answers and included eight different domains. Conflicting interpretations were resolved in contact with the partners involved. Results: All members responded to the survey. The results indicated that the scientific and medical centers make substantial efforts to respond to the unmet needs of patients. In most countries, there is a high awareness of RD issues. Scarcity of resources was highlighted as a major problem, leading to reduced availability of diagnostic expertise and research. Serious equity in accessibility to services were highlighted both within and between participating countries. Regulatory problems, including securing informed consent, difficulties in sending DNA to foreign laboratories, protection of intellectual property, and conflicts of interest on the part of service providers, remain issues of concern. Finally, most respondents stressed the need to strengthen international cooperation in terms of data sharing, clinical research, and diagnostic expertise for URD patients in low and medium income countries. Discussion: The survey highlighted that many countries experienced a discrepancy between the growing expertise and scientific value, the level of awareness and commitment on the part of relevant parties, and funding bodies. Country-tailored public health actions, including general syllabus of medical schools and of the education of other health professionals, are needed to reduce such gaps.

Klebsiella pneumoniae ST258 producing KPC-3 identified in italy carries novel plasmids and OmpK36/OmpK35 porin variants.

A carbapenemase-resistant Klebsiella pneumoniae strain, clone ST258 producing KPC-3, was fully characterized. The entire plasmid content was investigated, thereby identifying plasmids of the IncFII(k) (two of them similar to pKPQIL and pKPN3, respectively), IncX, and ColE types, carrying a formidable set of resistance genes against toxic compounds, metals, and antimicrobial drugs and a novel iron(III) uptake system.

Complete sequencing of an IncH plasmid carrying the blaNDM-1, blaCTX-M-15 and qnrB1 genes.

OBJECTIVES: To characterize plasmid pNDM-MAR recovered from a Klebsiella pneumoniae isolate of sequence type (ST) 15 from Morocco, carrying the genes encoding the metallo-ß-lactamase NDM-1, the extended-spectrum ß-lactamase (ESBL) CTX-M-15 and the qnrB1 quinolone resistance determinant. METHODS: The plasmid DNA sequence was obtained by using the 454-Genome Sequencer FLX procedure on a library constructed from total plasmid DNA obtained from an Escherichia coli J53 transconjugant. Contig assembly and predicted gaps were confirmed and filled by PCR-based gap closure. RESULTS: Plasmid pNDM-MAR was 267 242 bp long and encoded 177 predicted proteins. It harboured novel replicons and transfer loci, defining a novel plasmid type within the IncH plasmid family. The bla(NDM-1) gene was flanked by genetic elements that are distinct from those observed in other bla(NDM-1)-positive plasmids, including the groES and groEL chaperonin genes. This plasmid harboured the ESBL gene bla(CTX-M-15) together with the quinolone resistance gene qnrB1. In addition, it harboured genes encoding resistance to tellurite, mercury, chloramphenicol and aminoglycosides. Interestingly, pNDM-MAR did not carry any 16S rRNA methylase gene, in contrast to other bla(NDM-1)-positive plasmids. CONCLUSIONS: This study underlines the diversity of genetic vehicles involved in the spread of the bla(NDM-1) gene. Plasmid pNDM-MAR differed significantly from all known bla(NDM-1)-bearing plasmids. Comparative analysis of the pNDM-MAR sequence identified a novel type of IncH plasmid.

An overlook on the current registries for rare and complex connective tissue diseases and the future scenario of TogethERN ReCONNET.

Background: Patient registries play a crucial role in supporting clinical practice, healthcare planning and medical research, offering a real-world picture on rare and complex connective tissue diseases (rCTDs). ERN ReCONNET launched the first European Registry Infrastructure with the aim to plan, upgrade and link registries for rCTDs, with the final goal to promote a harmonized data collection approach all over Europe for rCTDs. Methods: An online survey addressed to healthcare professionals and patients' representatives active in the field of rCTDs was integrated by an extensive database search in order to build a mapping of existing registries for rCTDs. Findings: A total of 140 registries were found, 38 of which include multiple diseases. No disease-specific registry was identified for relapsing polychondritis, mixed connective tissue disease and undifferentiated connective tissue disease. Discussion: This overview on the existing registries for rCTDs provides a useful starting point to identify the gaps and the strengths of registries on the coverage of rCTDs, and to develop a common data set and data collection approach for the establishment of the TogethERN ReCONNET Infrastructure.

Shaping the Future of Rare Diseases after a Global Health Emergency: Organisational Points to Consider.

The unexpected outbreak of the COVID-19 disease had significant and enormous repercussions on the healthcare systems, such as the need to reorganise healthcare organisations in order to concentrate resources needed to the care of COVID-19 patients and to respond in general to this health emergency. Due to these challenges, the care of several chronic conditions was in many cases discontinued and patients and healthcare professionals treating these conditions had to cope with this new scenario. This was the case of the world rare diseases (RDs) that had to face this global emergency despite the vulnerability of people with RDs and the well-known need for high expertise required to treat and manage them. The numerous lessons learned so far regarding health emergencies and RDs should represent the basis for the establishment of new healthcare policies and plans aimed at ensuring the preparedness of our health systems in providing appropriate care to people living with RDs in the case of eventual new emergencies. This paper aims at providing pragmatic considerations that might be useful in designing future actions to create or optimise existing organisational models for the care of RDs in case of future emergencies or any other situation that might threaten the provision of routine care. These policies and plans should benefit from the multi-stakeholder RDs networks (such as the European Reference Networks), that should join forces at European, national, and local levels to minimise the economic, organisational, and health-related impact and the negative effects of potential emergencies on the RDs community. In order to design and develop these policies and plans, a decalogue of points to consider were developed to ensure appropriate care for people living with RDs in the case of eventual future health emergencies.

Germline BRAF mutations in Noonan, LEOPARD, and cardiofaciocutaneous syndromes: molecular diversity and associated phenotypic spectrum.

Noonan, LEOPARD, and cardiofaciocutaneous syndromes (NS, LS, and CFCS) are developmental disorders with overlapping features including distinctive facial dysmorphia, reduced growth, cardiac defects, skeletal and ectodermal anomalies, and variable cognitive deficits. Dysregulated RAS-mitogen-activated protein kinase (MAPK) signal traffic has been established to represent the molecular pathogenic cause underlying these conditions. To investigate the phenotypic spectrum and molecular diversity of germline mutations affecting BRAF, which encodes a serine/threonine kinase functioning as a RAS effector frequently mutated in CFCS, subjects with a diagnosis of NS (N=270), LS (N=6), and CFCS (N=33), and no mutation in PTPN11, SOS1, KRAS, RAF1, MEK1, or MEK2, were screened for the entire coding sequence of the gene. Besides the expected high prevalence of mutations observed among CFCS patients (52%), a de novo heterozygous missense change was identified in one subject with LS (17%) and five individuals with NS (1.9%). Mutations mapped to multiple protein domains and largely did not overlap with cancer-associated defects. NS-causing mutations had not been documented in CFCS, suggesting that the phenotypes arising from germline BRAF defects might be allele specific. Selected mutant BRAF proteins promoted variable gain of function of the kinase, but appeared less activating compared to the recurrent cancer-associated p.Val600Glu mutant. Our findings provide evidence for a wide phenotypic diversity associated with mutations affecting BRAF, and occurrence of a clinical continuum associated with these molecular lesions.

Genomic duplication of PTPN11 is an uncommon cause of Noonan syndrome.

Noonan syndrome (NS) is a genetically heterogeneous disorder caused most commonly by activating mutations in PTPN11. We report a patient with hypotonia, developmental delay and clinical features suggestive of NS. High-resolution chromosome analysis was normal, and sequence analyses of PTPN11, SOS1, KRAS, BRAF, RAF1, MEK, and MEK2 were also normal. Array CGH revealed a single copy gain of 9 BAC clones at 12q24.11q24.21 (8.98 Mb in size), which encompassed the PTPN11 locus at 12q24.13 and was confirmed by FISH analysis. Shchelochkov et al. [Shchelochkov et al. (2008); Am J Med Genet Part A 146A:1042-1048] reported a similar case and speculated that such duplications might account for 15-30% of NS cases with no detectable mutation in NS genes. We screened more than 250 NS cases without mutation in known NS disease-causing genes by quantitative PCR, and none of these studies produced results in the duplicated range. We also explored the possibility that de novo changes affecting the untranslated region (UTR) of the PTPN11 transcript might represent an alternative event involved in SHP2 enhanced expression. DHPLC analysis and direct sequencing of the entire 3' UTR in 36 NS patients without mutation in known genes did not show any disease-associated variant. These findings indicate that duplications of PTPN11 represent an uncommon cause of NS, and functionally relevant variations within the 3'UTR of the gene do not appear to play a major role in NS. However, recurrent observations of NS in individuals with duplications involving the PTPN11 locus suggest that increased dosage of SHP2 may have dysregulating effects on intracellular signaling.

The RD-Connect Registry & Biobank Finder: a tool for sharing aggregated data and metadata among rare disease researchers.

In rare disease (RD) research, there is a huge need to systematically collect biomaterials, phenotypic, and genomic data in a standardized way and to make them findable, accessible, interoperable and reusable (FAIR). RD-Connect is a 6 years global infrastructure project initiated in November 2012 that links genomic data with patient registries, biobanks, and clinical bioinformatics tools to create a central research resource for RDs. Here, we present RD-Connect Registry & Biobank Finder, a tool that helps RD researchers to find RD biobanks and registries and provide information on the availability and accessibility of content in each database. The finder concentrates information that is currently sparse on different repositories (inventories, websites, scientific journals, technical reports, etc.), including aggregated data and metadata from participating databases. Aggregated data provided by the finder, if appropriately checked, can be used by researchers who are trying to estimate the prevalence of a RD, to organize a clinical trial on a RD, or to estimate the volume of patients seen by different clinical centers. The finder is also a portal to other RD-Connect tools, providing a link to the RD-Connect Sample Catalogue, a large inventory of RD biological samples available in participating biobanks for RD research. There are several kinds of users and potential uses for the RD-Connect Registry & Biobank Finder, including researchers collaborating with academia and the industry, dealing with the questions of basic, translational, and/or clinical research. As of November 2017, the finder is populated with aggregated data for 222 registries and 21 biobanks.

Recommendations for Improving the Quality of Rare Disease Registries.

Rare diseases (RD) patient registries are powerful instruments that help develop clinical research, facilitate the planning of appropriate clinical trials, improve patient care, and support healthcare management. They constitute a key information system that supports the activities of European Reference Networks (ERNs) on rare diseases. A rapid proliferation of RD registries has occurred during the last years and there is a need to develop guidance for the minimum requirements, recommendations and standards necessary to maintain a high-quality registry. In response to these heterogeneities, in the framework of RD-Connect, a European platform connecting databases, registries, biobanks and clinical bioinformatics for rare disease research, we report on a list of recommendations, developed by a group of experts, including members of patient organizations, to be used as a framework for improving the quality of RD registries. This list includes aspects of governance, Findable, Accessible, Interoperable and Reusable (FAIR) data and information, infrastructure, documentation, training, and quality audit. The list is intended to be used by established as well as new RD registries. Further work includes the development of a toolkit to enable continuous assessment and improvement of their organizational and data quality.

Diversity, parental germline origin, and phenotypic spectrum of de novo HRAS missense changes in Costello syndrome.

Activating mutations in v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS) have recently been identified as the molecular cause underlying Costello syndrome (CS). To further investigate the phenotypic spectrum associated with germline HRAS mutations and characterize their molecular diversity, subjects with a diagnosis of CS (N = 9), Noonan syndrome (NS; N = 36), cardiofaciocutaneous syndrome (CFCS; N = 4), or with a phenotype suggestive of these conditions but without a definitive diagnosis (N = 12) were screened for the entire coding sequence of the gene. A de novo heterozygous HRAS change was detected in all the subjects diagnosed with CS, while no lesion was observed with any of the other phenotypes. While eight cases shared the recurrent c.34G>A change, a novel c.436G>A transition was observed in one individual. The latter affected residue, p.Ala146, which contributes to guanosine triphosphate (GTP)/guanosine diphosphate (GDP) binding, defining a novel class of activating HRAS lesions that perturb development. Clinical characterization indicated that p.Gly12Ser was associated with a homogeneous phenotype. By analyzing the genomic region flanking the HRAS mutations, we traced the parental origin of lesions in nine informative families and demonstrated that de novo mutations were inherited from the father in all cases. We noted an advanced age at conception in unaffected fathers transmitting the mutation.

Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2.

Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.

Linked Registries: Connecting Rare Diseases Patient Registries through a Semantic Web Layer.

Patient registries are an essential tool to increase current knowledge regarding rare diseases. Understanding these data is a vital step to improve patient treatments and to create the most adequate tools for personalized medicine. However, the growing number of disease-specific patient registries brings also new technical challenges. Usually, these systems are developed as closed data silos, with independent formats and models, lacking comprehensive mechanisms to enable data sharing. To tackle these challenges, we developed a Semantic Web based solution that allows connecting distributed and heterogeneous registries, enabling the federation of knowledge between multiple independent environments. This semantic layer creates a holistic view over a set of anonymised registries, supporting semantic data representation, integrated access, and querying. The implemented system gave us the opportunity to answer challenging questions across disperse rare disease patient registries. The interconnection between those registries using Semantic Web technologies benefits our final solution in a way that we can query single or multiple instances according to our needs. The outcome is a unique semantic layer, connecting miscellaneous registries and delivering a lightweight holistic perspective over the wealth of knowledge stemming from linked rare disease patient registries.