Position statement on the role of healthcare professionals, patient organizations and industry in European Reference Networks

A call from the EU for the set-up of European Reference Networks (ERNs) is expected to be launched in the first quarter of 2016. ERNs are intended to improve the care for patients with low prevalent or rare diseases throughout the EU by, among other things, facilitating the pooling and exchange of experience and knowledge and the development of protocols and guidelines. In the past, for example where costly orphan drugs have been concerned, industry has played an important role in facilitating consensus meetings and publication of guidelines. The ERNs should provide a unique opportunity for healthcare professionals and patients to lead these activities in an independent way. However, currently costs for networking activities are not to be covered by EU funds and alternative sources of funding are being explored. There is growing concern that any involvement of the industry in the funding of ERNs and their core activities may create a risk of undue influence. To date, the European Commission has not been explicit in how industry will be engaged in ERNs. We believe that public funding and a conflict of interest policy are needed at the level of the ERNs, Centers of Expertise (CEs), healthcare professionals and patient organizations with the aim of maintaining scientific integrity and independence. Specific attention is needed where it concerns the development of clinical practice guidelines. A proposal for a conflict of interest policy is presented, which may support the development of a framework to facilitate collaboration, safeguard professional integrity and to establish and maintain public acceptability and trust among patients, their organizations and the general public

Expanding the genetic and phenotypic spectrum of branched-chain amino acid transferase 2 deficiency

The first step in branched-chain amino acid (BCAA) catabolism is catalyzed by the two BCAA transferase isoenzymes, cytoplasmic branched-chain amino acid transferase (BCAT) 1, and mitochondrial BCAT2. Defects in the second step of BCAA catabolism cause maple syrup urine disease (MSUD), a condition which has been far more extensively investigated. Here, we studied the consequences of BCAT2 deficiency, an ultra-rare condition in humans. We present genetic, clinical, and functional data in five individuals from four different families with homozygous or compound heterozygous BCAT2 mutations which were all detected following abnormal biochemical profile results or familial mutation segregation studies. We demonstrate that BCAT2 deficiency has a recognizable biochemical profile with raised plasma BCAAs and, in contrast with MSUD, low-normal branched-chain keto acids (BCKAs) with undetectable l-allo-isoleucine. Interestingly, unlike in MSUD, none of the individuals with BCAT2 deficiency developed acute encephalopathy even with exceptionally high BCAA levels. We observed wide-ranging clinical phenotypes in individuals with BCAT2 deficiency. While one adult was apparently asymptomatic, three individuals had presented with developmental delay and autistic features. We show that the biochemical characteristics of BCAT2 deficiency may be amenable to protein-restricted diet and that early treatment may improve outcome in affected individuals. BCAT2 deficiency is an inborn error of BCAA catabolism. At present, it is unclear whether developmental delay and autism are parts of the variable phenotypic spectrum of this condition or coincidental. Further studies will be required to explore this.Fundación Isabel Gemio; MINECO-FEDER; Fondo Europeo de Desarrollo Regional, Grant/Award Number: PI12/02078; Ministerio de Economía y Competitivida

Stromal cells modulate innate immune cell phenotype and function in colorectal cancer via the Sialic acid/Siglec axis

Background: The immunosuppressive tumor microenvironment reduces immune response effectiveness in stromal-rich tumors, including consensus molecular subtype 4 colorectal cancer (CRC). Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote cancer progression by suppressing anti-tumor immune responses. Hypersialylation of glycans on tumors engages Siglec receptors on immune cells, driving immune dysfunction, but its role in stromal-mediated suppression of innate immunity remains unclear. Methods: Sialylation, Sialic acids and Siglec ligands were measured on CRC tissue, primary human normal-associated fibroblasts (NAFs), CAFs, and tumor-conditioned MSCs (MSCTCS) using transcriptional profiles, immunohistochemistry and flow cytometry, respectively. The effect of stromal cell sialylation on macrophages and NK cells was assessed in ex vivo human primary stromal and immune cell co-cultures, and expression of Siglec-10 and immune cell phenotype markers and function was measured by flow cytometry and real-time imaging. Using an immunocompetent Balb/c CT26 mouse model, we induced tumors with/without conditioned stromal cells, with/without pretreatment of stromal cells with sialyltransferase inhibitor (3FAX) or sialidase (E610). We assessed the effect of stromal cell sialylation on macrophages and NK cells in the tumor and secondary lymphoid tissues by flow cytometry. Results: Stromal cells, including CAFs, in CRC tumors are highly sialylated compared with epithelial cancer cells and are associated with high expression of the sialyltransferase ST6GALNAC6. Genetic knockdown of ST6GALNAC6 reduced the expression of stromal cell Siglec-10 ligands in MSCs. CAFs and MSCTCS induced Siglec-10 on macrophages and NK cells and impaired macrophage phagocytosis and NK cell cytotoxicity. Sialidase treatment reduced Siglec-10 expression, restoring macrophage and NK cell antitumor functions. In vivo and ex vivo, desialylation of stromal cells increased macrophage activation (CD11b+CD80+) and reduced immunosuppressive marker expression (CD206, PD-L1, Siglec-G) in lymphoid tissues, indicating sustained systemic anti-tumor immunity. Intratumoral NK cells exhibited high Siglec-G expression and impaired cytotoxicity, and granzyme B expression significantly increased with sialidase treatment of stromal cells. In an inflammatory tumor model, inflammatory tumor-conditioned MSCs (MSCiTCS) promoted metastasis and Siglec-G induction on NK cells and macrophages, both reversed by sialyltransferase inhibition, underscoring the effects of stromal modulation of innate immune cell function in inflammatory tumors. Conclusions: Stromal cell sialylation modulates innate immune suppression in CRC via the sialic acid/Siglec axis. Targeting stromal sialylation restores NK cytotoxicity and macrophage activation, offering novel insights that may shape therapeutic strategies for reversing immunosuppression in stromal-rich tumors

Capturing phenotypic heterogeneity in MPS I: results of an international consensus procedure

<p>Abstract</p> <p>Background</p> <p>Mucopolysaccharidosis type I (MPS I) is traditionally divided into three phenotypes: the severe Hurler (MPS I-H) phenotype, the intermediate Hurler-Scheie (MPS I-H/S) phenotype and the attenuated Scheie (MPS I-S) phenotype. However, there are no clear criteria for delineating the different phenotypes. Because decisions about optimal treatment (enzyme replacement therapy or hematopoietic stem cell transplantation) need to be made quickly and depend on the presumed phenotype, an assessment of phenotypic severity should be performed soon after diagnosis. Therefore, a numerical severity scale for classifying different MPS I phenotypes at diagnosis based on clinical signs and symptoms was developed.</p> <p>Methods</p> <p>A consensus procedure based on a combined modified Delphi method and a nominal group technique was undertaken. It consisted of two written rounds and a face-to-face meeting. Sixteen MPS I experts participated in the process. The main goal was to identify the most important indicators of phenotypic severity and include these in a numerical severity scale. The correlation between the median subjective expert MPS I rating and the scores derived from this severity scale was used as an indicator of validity.</p> <p>Results</p> <p>Full consensus was reached on six key clinical items for assessing severity: age of onset of signs and symptoms, developmental delay, joint stiffness/arthropathy/contractures, kyphosis, cardiomyopathy and large head/frontal bossing. Due to the remarkably large variability in the expert MPS I assessments, however, a reliable numerical scale could not be constructed. Because of this variability, such a scale would always result in patients whose calculated severity score differed unacceptably from the median expert severity score, which was considered to be the 'gold standard'.</p> <p>Conclusions</p> <p>Although consensus was reached on the six key items for assessing phenotypic severity in MPS I, expert opinion on phenotypic severity at diagnosis proved to be highly variable. This subjectivity emphasizes the need for validated biomarkers and improved genotype-phenotype correlations that can be incorporated into phenotypic severity assessments at diagnosis.</p

Bi-allelic loss-of-function CACNA1B mutations in progressive epilepsy-dyskinesia

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment

Prevalence and architecture of de novo mutations in developmental disorders

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

A 3D View of Colorectal Cancer Models in Predicting Therapeutic Responses and Resistance

Although there have been many advances in recent years for the treatment of colorectal cancer (CRC), it still remains the third most common cause of cancer-related deaths worldwide. Many patients with late stage CRC display resistance to multiple different therapeutics. An important aspect in developing effective therapeutics for CRC patients is understanding the interactions that take place in the tumor microenvironment (TME), as it has been shown to contribute to drug resistance in vivo. Much research over the past 100 years has focused on 2D monolayer cultures or in vivo studies, however, the efficacy in translating these to the clinic is very low. More recent studies are turning towards developing an effective 3D model of CRC that is clinically relevant, that can recapitulate the TME in vitro and bridge the gap between 2D cultures and in vivo studies, with the aim of reducing the use of animal models in the future. This review summarises the advantages and limitations of different 3D CRC models. It emphasizes how different 3D models may be optimised to study cellular and extracellular interactions that take place in the TME of CRC in an effort to allow the development of more translatable effective treatment options for patients.</jats:p

A 3D View of Colorectal Cancer Models in Predicting Therapeutic Responses and Resistance

Although there have been many advances in recent years for the treatment of colorectal cancer (CRC), it still remains the third most common cause of cancer-related deaths worldwide. Many patients with late stage CRC display resistance to multiple different therapeutics. An important aspect in developing effective therapeutics for CRC patients is understanding the interactions that take place in the tumor microenvironment (TME), as it has been shown to contribute to drug resistance in vivo. Much research over the past 100 years has focused on 2D monolayer cultures or in vivo studies, however, the efficacy in translating these to the clinic is very low. More recent studies are turning towards developing an effective 3D model of CRC that is clinically relevant, that can recapitulate the TME in vitro and bridge the gap between 2D cultures and in vivo studies, with the aim of reducing the use of animal models in the future. This review summarises the advantages and limitations of different 3D CRC models. It emphasizes how different 3D models may be optimised to study cellular and extracellular interactions that take place in the TME of CRC in an effort to allow the development of more translatable effective treatment options for patients

The hypergonadotropic hypogonadism conundrum of classic galactosemia

BACKGROUND: Hypergonadotropic hypogonadism is a burdensome complication of classic galactosemia (CG), an inborn error of galactose metabolism that invariably affects female patients. Since its recognition in 1979, data have become available regarding the clinical spectrum, and the impact on fertility. Many women have been counseled for infertility and the majority never try to conceive, yet spontaneous pregnancies can occur. Onset and mechanism of damage have not been elucidated, yet new insights at the molecular level are becoming available that might greatly benefit our understanding. Fertility preservation options have expanded, and treatments to mitigate this complication either by directly rescuing the metabolic defect or by influencing the cascade of events are being explored. OBJECTIVE AND RATIONALE: The aims are to review: the clinical picture and the need to revisit the counseling paradigm; insights into the onset and mechanism of damage at the molecular level; and current treatments to mitigate ovarian damage. SEARCH METHODS: In addition to the work on this topic by the authors, the PubMed database has been used to search for peer-reviewed articles and reviews using the following terms: 'classic galactosemia', 'gonadal damage', 'primary ovarian insufficiency', 'fertility', 'animal models' and 'fertility preservation' in combination with other keywords related to the subject area. All relevant publications until August 2022 have been critically evaluated and reviewed. OUTCOMES: A diagnosis of premature ovarian insufficiency (POI) results in a significant psychological burden with a high incidence of depression and anxiety that urges adequate counseling at an early stage, appropriate treatment and timely discussion of fertility preservation options. The cause of POI in CG is unknown, but evidence exists of dysregulation in pathways crucial for folliculogenesis such as phosphatidylinositol 3-kinase/protein kinase B, inositol pathway, mitogen-activated protein kinase, insulin-like growth factor-1 and transforming growth factor-beta signaling. Recent findings from the GalT gene-trapped (GalTKO) mouse model suggest that early molecular changes in 1-month-old ovaries elicit an accelerated growth activation and burnout of primordial follicles, resembling the progressive ovarian failure seen in patients. Although data on safety and efficacy outcomes are still limited, ovarian tissue cryopreservation can be a fertility preservation option. Treatments to overcome the genetic defect, for example nucleic acid therapy such as mRNA or gene therapy, or that influence the cascade of events are being explored at the (pre-)clinical level. WIDER IMPLICATIONS: Elucidation of the molecular pathways underlying POI of any origin can greatly advance our insight into the pathogenesis and open new treatment avenues. Alterations in these molecular pathways might serve as markers of disease progression and efficiency of new treatment options