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Krabbe disease (KD) is a rare lysosomal storage disorder caused by biallelic pathogenic variants in GALC. Most patients manifest the severe classic early-infantile form, while a small percentage of cases have later-onset types. We present two siblings with atypical clinical and neuroimaging phenotypes, compared to the classification of KD, who were found to carry biallelic loss-of-function GALC variants, including a recurrent 30 kb deletion and a previously unreported deep intronic variant that was identified by mRNA sequencing. This family represents a unique description in the KD literature and contributes to expanding the clinical and molecular spectra of this rare disorder.

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy and movement disorder. We evaluated a large cohort of patients (n = 25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor and ataxia. Later in the disease course, they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibres and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders.

The RASopathies are a family of clinically related disorders caused by mutations affecting genes participating in the RAS-MAPK signaling cascade. Among them, Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML) are allelic conditions principally associated with dominant mutations in PTPN11, which encodes the nonreceptor SH2 domain-containing protein tyrosine phosphatase SHP2. Individual PTPN11 mutations are specific to each syndrome and have opposite consequences on catalysis, but all favor SHP2's interaction with signaling partners. Here, we report on a subject with NS harboring biallelic variants in PTPN11. While the former (p.Leu261Phe) had previously been reported in NS, the latter (p.Thr357Met) is a novel change impairing catalysis. Members of the family carrying p.Thr357Met, however, did not show any obvious feature fitting NSML or within the RASopathy phenotypic spectrum. A major impact of this change on transcript processing and protein stability was excluded. These findings further support the view that NSML cannot be ascribed merely to impaired SHP2's catalytic activity and suggest that PTPN11 mutations causing this condition act through an alternative dominant mechanism.

Prompt diagnosis of complex phenotypes is a challenging task in clinical genetics. Whole exome sequencing has proved to be effective in solving such conditions. Here, we report on an unpredictable presentation of Werner Syndrome (WRNS) in a 12-year-old girl carrying a homozygous truncating variant in RECQL2, the gene mutated in WRNS, and a de novo activating missense change in PTPN11, the major Noonan syndrome gene, encoding SHP2, a protein tyrosine phosphatase positively controlling RAS function and MAPK signaling, which have tightly been associated with senescence in primary cells. All the major WRNS clinical criteria were present with an extreme precocious onset and were associated with mild intellectual disability, severe growth retardation and facial dysmorphism. Compared to primary fibroblasts from adult subjects with WRNS, proband's fibroblasts showed a dramatically reduced proliferation rate and competence, and a more accelerated senescence, in line with the anticipated WRNS features occurring in the child. In vitro functional characterization of the SHP2 mutant documented its hyperactive behavior and a significantly enhanced activation of the MAPK pathway. Based on the functional interaction of WRN and MAPK signaling in processes relevant to replicative senescence, these findings disclose a unique phenotype likely resulting from negative genetic interaction.

Dominant GNAO1 mutations cause an emerging group of childhood-onset neurological disorders characterized by developmental delay, intellectual disability, movement disorders, drug-resistant seizures and neurological deterioration. GNAO1 encodes the α-subunit of an inhibitory GTP/GDP-binding protein regulating ion channel activity and neurotransmitter release. The pathogenic mechanisms underlying GNAO1-related disorders remain largely elusive and there are no effective therapies. Here, we assessed the functional impact of two disease-causing variants associated with distinct clinical features, c.139A > G (p.S47G) and c.662C > A (p.A221D), using Caenorhabditis elegans as a model organism. The c.139A > G change was introduced into the orthologous position of the C. elegans gene via CRISPR/Cas9, whereas a knock-in strain carrying the p.A221D variant was already available. Like null mutants, homozygous knock-in animals showed increased egg laying and were hypersensitive to aldicarb, an inhibitor of acetylcholinesterase, suggesting excessive neurotransmitter release by different classes of motor neurons. Automated analysis of C. elegans locomotion indicated that goa-1 mutants move faster than control animals, with more frequent body bends and a higher reversal rate and display uncoordinated locomotion. Phenotypic profiling of heterozygous animals revealed a strong hypomorphic effect of both variants, with a partial dominant-negative activity for the p.A221D allele. Finally, caffeine was shown to rescue aberrant motor function in C. elegans harboring the goa-1 variants; this effect is mainly exerted through adenosine receptor antagonism. Overall, our findings establish a suitable platform for drug discovery, which may assist in accelerating the development of new therapies for this devastating condition, and highlight the potential role of caffeine in controlling GNAO1-related dyskinesia.

Pathogenic missense variants in cell division control protein 42 (CDC42) differentially affect protein function, causing a clinically wide phenotypic spectrum variably affecting neurodevelopment, hematopoiesis, and immune response. More recently, 3 variants at the C-terminus of CDC42 were proposed to similarly impact protein function and cause a novel autoinflammatory disorder.We sought to clinically and functionally classify these variants to improve patient management.Comparative analysis of the available clinical data and medical history of patients was performed. In vitro and in vivo studies were carried out to functionally characterize individual variants.Differently from what had previously been observed for the p.R186C change causing neonatal-onset cytopenia, autoinflammation, and recurrent hemophagocytic lymphohistiocytosis, p.C188Y and p.∗192Cext∗24 promoted accelerated protein degradation. Unprenylated CDC42Pathogenic variants at the CDC42 C-terminus differently impact protein stability, localization, and function, and cause different diseases, with p.R186C specifically associated with neonatal-onset pancytopenia and severe autoinflammation/hemophagocytic lymphohistiocytosis requiring emapalumab and bone marrow transplantation, and p.C188Y and p.∗192Cext∗24 causing anakinra-sensitive autoinflammation.

BackgroundDysfunction in non-motile cilia is associated with a broad spectrum of developmental disorders characterised by clinical heterogeneity. While over 100 genes have been associated with primary ciliopathies, with wide phenotypic overlap, some patients still lack a molecular diagnosis.ObjectiveTo investigate and functionally characterise the molecular cause of a malformation disorder observed in two sibling fetuses characterised by microphthalmia, cleft lip and palate, and brain anomalies.MethodsA trio-based whole exome sequencing (WES) strategy was used to identify candidate variants in the TOGARAM1 gene. In silico, in vitro and in vivo (Caenorhabditis elegans) studies were carried out to explore the impact of mutations on protein structure and function, and relevant biological processes.ResultsTOGARAM1 encodes a member of the Crescerin1 family of proteins regulating microtubule dynamics. Its orthologue in C. elegans, che-12, is expressed in a subset of sensory neurons and localises in the dendritic cilium where it is required for chemosensation. Nematode lines harbouring the corresponding missense variant in TOGARAM1 were generated by CRISPR/Cas9 technology. Although chemotaxis ability on a NaCl gradient was not affected, che-12 point mutants displayed impaired lipophilic dye uptake, with shorter and altered cilia in sensory neurons. Finally, in vitro analysis of microtubule polymerisation in the presence of wild-type or mutant TOG2 domain revealed a faster polymerisation associated with the mutant protein, suggesting aberrant tubulin binding.ConclusionsOur data are in favour of a causative role of TOGARAM1 variants in the pathogenesis of this novel disorder, connecting this gene with primary ciliopathy.

Germline PTPN11 mutations cause Noonan syndrome (NS), the most common disorder among RASopathies. PTPN11 encodes SHP2, a protein tyrosine-phosphatase controlling signaling through the RAS-MAPK and PI3K-AKT pathways. Generally, NS-causing PTPN11 mutations are missense changes destabilizing the inactive conformation of the protein or enhancing its binding to signaling partners. Here, we report on two PTPN11 variants resulting in the deletion or duplication of one of three adjacent glutamine residues (Gln255 -to-Gln257 ). While p.(Gln257dup) caused a typical NS phenotype in carriers of a first family, p.(Gln257del) had incomplete penetrance in a second family. Missense mutations involving Gln256 had previously been reported in NS. This poly-glutamine stretch is located on helix B of the PTP domain, a region involved in stabilizing SHP2 in its autoinhibited state. Molecular dynamics simulations predicted that changes affecting this motif perturb the SHP2's catalytically inactive conformation and/or substrate recognition. Biochemical data showed that duplication and deletion of Gln257 variably enhance SHP2's catalytic activity, while missense changes involving Gln256 affect substrate specificity. Expression of mutants in HEK293T cells documented their activating role on MAPK signaling, uncoupling catalytic activity and modulation of intracellular signaling. These findings further document the relevance of helix B in the regulation of SHP2's function.

Objective To investigate the molecular cause(s) underlying a severe form of infantile-onset parkinsonism and characterize functionally the identified variants. Methods A trio-based whole exome sequencing (WES) approach was used to identify the candidate variants underlying the disorder. In silico modeling, and in vitro and in vivo studies were performed to explore the impact of these variants on protein function and relevant cellular processes. Results WES analysis identified biallelic variants in WARS2, encoding the mitochondrial tryptophanyl tRNA synthetase (mtTrpRS), a gene whose mutations have recently been associated with multiple neurological phenotypes, including childhood-onset, levodopa-responsive or unresponsive parkinsonism in a few patients. A substantial reduction of mtTrpRS levels in mitochondria and reduced OXPHOS function was demonstrated, supporting their pathogenicity. Based on the infantile-onset and severity of the phenotype, additional variants were considered as possible genetic modifiers. Functional assessment of a selected panel of candidates pointed to a de novo missense mutation in CHRNA6, encoding the α6 subunit of neuronal nicotinic receptors, which are involved in the cholinergic modulation of dopamine release in the striatum, as a second event likely contributing to the phenotype. In silico, in vitro (Xenopus oocytes and GH4C1 cells) and in vivo (C. elegans) analyses demonstrated the disruptive effects of the mutation on acetylcholine receptor structure and function. Conclusion Our findings consolidate the association between biallelic WARS2 mutations and movement disorders, and suggest CHRNA6 as a genetic modifier of the phenotype.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in multiple signaling pathways and a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective on disease-associated SHP2 mutants. Based on the consideration that pathogenic lesions cause signaling hyperactivation due to increased SHP2 association with cognate proteins, we developed peptide-based molecules with low nM affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation and an affinity for pathogenic variants of SHP2 up to 20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool to investigate the role of protein-protein interactions in the function of SHP2.

Context Genes causing familial forms of diabetes mellitus are only partially known. Objective We set out to identify the genetic cause of hyperglycemia in multigenerational families with an apparent autosomal dominant form of adult-onset diabetes not due to mutations in known monogenic diabetes genes. Methods Existing whole-exome sequencing (WES) data were used to identify exonic variants segregating with diabetes in 60 families from the United States and Italy. Functional studies were carried out in vitro (transduced MIN6-K8 cells) and in vivo (Caenorhabditis elegans) to assess the diabetogenic potential of 2 variants in the malate dehydrogenase 2 (MDH2) gene linked with hyperglycemia in 2 of the families. Results A very rare mutation (p.Arg52Cys) in MDH2 strongly segregated with hyperglycemia in 1 family from the United States. An infrequent MDH2 missense variant (p.Val160Met) also showed disease cosegregation in a family from Italy, although with reduced penetrance. In silico, both Arg52Cys and Val160Met were shown to affect MDH2 protein structure and function. In transfected HepG2 cells, both variants significantly increased MDH2 enzymatic activity, thereby decreasing the NAD+/NADH ratio—a change known to affect insulin signaling and secretion. Stable expression of human wild-type MDH2 in MIN6-K8 cell lines enhanced glucose- and GLP-1-stimulated insulin secretion. This effect was blunted by the Cys52 or Met160 substitutions. Nematodes carrying equivalent changes at the orthologous positions of the mdh-2 gene showed impaired glucose-stimulated insulin secretion. Conclusion Our findings suggest a central role of MDH2 in human glucose homeostasis and indicate that gain of function variants in this gene may be involved in the etiology of familial forms of diabetes.

Chemosensory receptors play a crucial role in distinguishing the wide range of volatile/soluble molecules by binding them with high accuracy. Chemosensation is the main sensory modality in organisms lacking long-range sensory mechanisms like vision/hearing. Despite its low number of sensory neurons, the nematode Caenorhabditis elegans possesses several chemosensory receptors, allowing it to detect about as many odorants as mammals. Here, we show that C. elegans displays attraction towards urine samples of women with breast cancer, avoiding control ones. Behavioral assays on animals lacking AWC sensory neurons demonstrate the relevance of these neurons in sensing cancer odorants: calcium imaging on AWC increases the accuracy of the discrimination (97.22%). Also, chemotaxis assays on animals lacking GPCRs expressed in AWC allow to identify receptors involved in binding cancer metabolites, suggesting that an alteration of a few metabolites is sufficient for the cancer discriminating behavior of C. elegans, which may help identify a fundamental fingerprint of breast cancer.

Neurofibromatosis 1 (NF1) is a disorder characterized by variable expressivity caused by loss-of-function variants in NF1, encoding neurofibromin, a protein negatively controlling RAS signaling. We evaluated whether concurrent variation in proteins functionally linked to neurofibromin contribute to the variable expressivity of NF1. Parallel sequencing of a RASopathy gene panel in 138 individuals with molecularly confirmed clinical diagnosis of NF1 identified missense variants in PTPN11, encoding SHP2, a positive regulator of RAS signaling, in four subjects from three unrelated families. Three subjects were heterozygous for a gain-of-function variant and showed a severe expression of NF1 (developmental delay, multiple cerebral neoplasms and peculiar cortical MRI findings), and features resembling Noonan syndrome (a RASopathy caused by activating variants in PTPN11). Conversely, the fourth subject, who showed an attenuated presentation, carried a previously unreported PTPN11 variant that had a hypomorphic behavior in vitro. Our findings document that functionally relevant PTPN11 variants occur in a small but significant proportion of subjects with NF1 modulating disease presentation, suggesting a model in which the clinical expression of pathogenic NF1 variants is modified by concomitant dysregulation of protein(s) functionally linked to neurofibromin. We also suggest targeting of SHP2 function as an approach to treat evolutive complications of NF1.

The Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations inWe re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape ofWe identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652AG; p.(Asp551Gly)) or a 39 kb deletion encompassingOur study identified novel

Dysfunction in non-motile cilia is associated with a broad spectrum of developmental disorders characterised by clinical heterogeneity. While over 100 genes have been associated with primary ciliopathies, with wide phenotypic overlap, some patients still lack a molecular diagnosis.To investigate and functionally characterise the molecular cause of a malformation disorder observed in two sibling fetuses characterised by microphthalmia, cleft lip and palate, and brain anomalies.A trio-based whole exome sequencing (WES) strategy was used to identify candidate variants in theOur data are in favour of a causative role of

Signal transduction through the RAF-MEK-ERK pathway, the first described mitogen-associated protein kinase (MAPK) cascade, mediates multiple cellular processes and participates in early and late developmental programs. Aberrant signaling through this cascade contributes to oncogenesis and underlies the RASopathies, a family of cancer-prone disorders. Here, we report that de novo missense variants in MAPK1, encoding the mitogen-activated protein kinase 1 (i.e., extracellular signal-regulated protein kinase 2, ERK2), cause a neurodevelopmental disease within the RASopathy phenotypic spectrum, reminiscent of Noonan syndrome in some subjects. Pathogenic variants promote increased phosphorylation of the kinase, which enhances translocation to the nucleus and boosts MAPK signaling in vitro and in vivo. Two variant classes are identified, one of which directly disrupts binding to MKP3, a dual-specificity protein phosphatase negatively regulating ERK function. Importantly, signal dysregulation driven by pathogenic MAPK1 variants is stimulus reliant and retains dependence on MEK activity. Our data support a model in which the identified pathogenic variants operate with counteracting effects on MAPK1 function by differentially impacting the ability of the kinase to interact with regulators and substrates, which likely explains the minor role of these variants as driver events contributing to oncogenesis. After nearly 20 years from the discovery of the first gene implicated in Noonan syndrome, PTPN11, the last tier of the MAPK cascade joins the group of genes mutated in RASopathies. Sin financiación 11.025 JCR (2020) Q1, 11/176 Genetics & Heredity 6.661 SJR (2020) Q1, 11/340 Genetics No data IDR 2020 UEM

Background We previously associated HIST1H1E mutations causing Rahman syndrome with a specific genome-wide methylation pattern. Results Methylome analysis from peripheral blood samples of six affected subjects led us to identify a specific hypomethylated profile. This “episignature” was enriched for genes involved in neuronal system development and function. A computational classifier yielded full sensitivity and specificity in detecting subjects with Rahman syndrome. Applying this model to a cohort of undiagnosed probands allowed us to reach diagnosis in one subject. Conclusions We demonstrate an epigenetic signature in subjects with Rahman syndrome that can be used to reach molecular diagnosis.

A protein-based film (PBF), obtained from black soldier fly prepupae proteins, was assessed for its agronomic performance as mulch. PBF was investigated in a potting experiment and compared with Mater-Bi (MB), polyethylene (PE) and bare soil. During the experiment, degraded surface area, weight and thickness of the film, water evaporated from the pot, and the soil microbiological content (SMC) were recorded. In addition, films were buried to assess their biodegradability and impact on SMC. During the mulching process, the PBF showed a significant degradation. In terms of evaporated water, the PBF performed similarly to MB and worse than PE. Regarding SMC, film of any nature caused an increase in the Clostridia spp. and a decrease of total mesophilic aerobic bacteria and fungi contents. When buried, only PBF recorded a faster biodegradability, showing a reduction of surface area, thickness and weight. PBF and MB highlighted a significant increase in contents of Clostridia spp., mesophilic aerobic bacteria and fungi. Our results reported, for the first time, the valorization of black soldier fly (BSF) prepupae proteins as a biodegradable film for mulching purposes. However, further study is needed to reduce the PBF biodegradability and allow it to be used for the most important mulched crops.

IntroductionThe Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with ‘Mental Retardation Autosomal Dominant 57’ (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies.MethodsWe re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity.ResultsWe identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2, and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage.ConclusionOur study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders.

Germline PTPN11 mutations cause Noonan syndrome (NS), the most common disorder among RASopathies. PTPN11 encodes SHP2, a protein tyrosine-phosphatase controlling signaling through the RAS-MAPK and PI3K-AKT pathways. Generally, NS-causing PTPN11 mutations are missense changes destabilizing the inactive conformation of the protein or enhancing its binding to signaling partners. Here, we report on two PTPN11 variants resulting in the deletion or duplication of one of three adjacent glutamine residues (Gln

RASopathies are a heterogeneous group of genetic syndromes characterized by mutations in genes that regulate cellular processes, including proliferation, differentiation, survival, migration, and metabolism. Excluding congenital heart defects, hypertrophic cardiomyopathy is the most frequent cardiovascular defect in patients affected by RASopathies. A worse outcome (in terms of surgical risk and/or mortality) has been described in a specific subset of Rasopathy patients with early onset, severe hypertrophic cardiomyopathy presenting with heart failure. New short-term therapy with a mammalian target of rapamycin inhibitor has recently been used to prevent heart failure in these patients with a severe form of hypertrophic cardiomyopathy.

Germline mutations in PTPN11, the gene encoding the Src-homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu261 , Leu262 , and Arg265 in 16 unrelated individuals with clinical diagnosis of NS or with features suggestive for this disorder, specifying a novel disease-causing mutation cluster. Expression of the mutant proteins in HEK293T cells documented their activating role on MAPK signaling. Structural data predicted a gain-of-function role of substitutions at residues Leu262 and Arg265 exerted by disruption of the N-SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu261 , with possible impact on SHP2's catalytic activity/selectivity and proper interaction of the PTP domain with the regulatory SH2 domains. Consistent with that, biochemical data indicated that substitutions at codons 262 and 265 increased the catalytic activity of the phosphatase, while those affecting codon 261 were only moderately activating but impacted substrate specificity. Remarkably, these mutations underlie a relatively mild form of NS characterized by low prevalence of cardiac defects, short stature, and cognitive and behavioral issues, as well as less evident typical facial features.

ObjectiveTo characterize clinically and molecularly an early-onset, variably progressive neurodegenerative disorder characterized by a cerebellar syndrome with severe ataxia, gaze palsy, dyskinesia, dystonia, and cognitive decline affecting 11 individuals from 3 consanguineous families.MethodsWe used whole-exome sequencing (WES) (families 1 and 2) and a combined approach based on homozygosity mapping and WES (family 3). We performed in vitro studies to explore the effect of the nontruncating SQSTM1 mutation on protein function and the effect of impaired SQSTM1 function on autophagy. We analyzed the consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in vivo using zebrafish as a model.ResultsWe identified 3 homozygous inactivating variants, including a splice site substitution (c.301+2T>A) causing aberrant transcript processing and accelerated degradation of a resulting protein lacking exon 2, as well as 2 truncating changes (c.875_876insT and c.934_936delinsTGA). We show that loss of SQSTM1 causes impaired production of ubiquitin-positive protein aggregates in response to misfolded protein stress and decelerated autophagic flux. The consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in zebrafish documented a variable but reproducible phenotype characterized by cerebellum anomalies ranging from depletion of axonal connections to complete atrophy. We provide a detailed clinical characterization of the disorder; the natural history is reported for 2 siblings who have been followed up for >20 years.ConclusionsThis study offers an accurate clinical characterization of this recently recognized neurodegenerative disorder caused by biallelic inactivating mutations in SQSTM1 and links this phenotype to defective selective autophagy.

Lam et al. characterize a novel hematological/autoinflammatory disorder due to a de novo recurrent missense mutation of CDC42. The authors use in silico, in vitro, and in vivo analyses to correlate the molecular mechanisms altering CDC42 function to the observed phenotype., Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival., Graphical Abstract

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

OBJECTIVE: To characterize clinically and molecularly an early-onset, variably progressive neurodegenerative disorder characterized by a cerebellar syndrome with severe ataxia, gaze palsy, dyskinesia, dystonia, and cognitive decline affecting 11 individuals from 3 consanguineous families. METHODS: We used whole-exome sequencing (WES) (families 1 and 2) and a combined approach based on homozygosity mapping and WES (family 3). We performed in vitro studies to explore the effect of the nontruncating SQSTM1 mutation on protein function and the effect of impaired SQSTM1 function on autophagy. We analyzed the consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in vivo using zebrafish as a model. RESULTS: We identified 3 homozygous inactivating variants, including a splice site substitution (c.301+2T>A) causing aberrant transcript processing and accelerated degradation of a resulting protein lacking exon 2, as well as 2 truncating changes (c.875_876insT and c.934_936delinsTGA). We show that loss of SQSTM1 causes impaired production of ubiquitin-positive protein aggregates in response to misfolded protein stress and decelerated autophagic flux. The consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in zebrafish documented a variable but reproducible phenotype characterized by cerebellum anomalies ranging from depletion of axonal connections to complete atrophy. We provide a detailed clinical characterization of the disorder; the natural history is reported for 2 siblings who have been followed up for >20 years. CONCLUSIONS: This study offers an accurate clinical characterization of this recently recognized neurodegenerative disorder caused by biallelic inactivating mutations in SQSTM1 and links this phenotype to defective selective autophagy.

Item does not contain fulltext Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

Saposin (Sap) C is a small lysosomal disulfide bridge-containing glycoprotein required for glucosylceramide (GC) hydrolysis by glucosylceramidase (GCase). Sap C deficiency causes a variant form of Gaucher disease (GD), a rare genetic disorder characterized by GC accumulation in lysosomes of monocyte/macrophage lineage. Efforts to develop fast and efficient methodologies to express and purify Sap C have been made in the last years. Here, human Sap C was expressed in a bacterial strain that greatly enhances disulfide bond formation, and the recombinant protein was purified in a single chromatographic step using an affinity tag-based protein purification system. Mass spectrometry analysis demonstrated that disulfide bridges required for Sap C stability and functionality were retained. Consistently, the recombinant protein was shown to interact with anionic phospholipids-containing vesicles, and reconstitute GCase activity in vitro. Recombinant Sap C was efficiently endocytosed by Sap C-deficient fibroblasts, and targeted to lysosomes. These findings document that the bacterially purified Sap C exerts biological properties functionally equivalent to those observed for the native protein, indicating its potential use in the development of therapeutic intervention.

Contains fulltext : 88373.pdf (Publisher’s version ) (Closed access) RAS signaling plays a key role in controlling appropriate cell responses to extracellular stimuli and participates in early and late developmental processes. Although enhanced flow through this pathway has been established as a major contributor to oncogenesis, recent discoveries have revealed that aberrant RAS activation causes a group of clinically related developmental disorders characterized by facial dysmorphism, a wide spectrum of cardiac disease, reduced growth, variable cognitive deficits, ectodermal and musculoskeletal anomalies, and increased risk for certain malignancies. Here, we report that heterozygous germline mutations in CBL, a tumor-suppressor gene that is mutated in myeloid malignancies and encodes a multivalent adaptor protein with E3 ubiquitin ligase activity, can underlie a phenotype with clinical features fitting or partially overlapping Noonan syndrome (NS), the most common condition of this disease family. Independent CBL mutations were identified in two sporadic cases and two families from among 365 unrelated subjects who had NS or suggestive features and were negative for mutations in previously identified disease genes. Phenotypic heterogeneity and variable expressivity were documented. Mutations were missense changes altering evolutionarily conserved residues located in the RING finger domain or the linker connecting this domain to the N-terminal tyrosine kinase binding domain, a known mutational hot spot in myeloid malignancies. Mutations were shown to affect CBL-mediated receptor ubiquitylation and dysregulate signal flow through RAS. These findings document that germline mutations in CBL alter development to cause a clinically variable condition that resembles NS and that possibly predisposes to malignancies.

Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3–independent growth in Ba/F3 cells and/or IL-9–independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.