SBDSR126T rescues survival of sbds -/- zebrafish in a dose-dependent manner independently of Tp53.

Defects in ribosomal biogenesis profoundly affect organismal development and cellular function, and these ribosomopathies produce a variety of phenotypes. One ribosomopathy, Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, pancreatic exocrine insufficiency, and skeletal anomalies. SDS results from biallelic mutations in SBDS, which encodes a ribosome assembly factor. Some individuals express a missense mutation, SBDS R126T , along with the common K62X mutation. We reported that the sbds-null zebrafish phenocopies much of SDS. We further showed activation of Tp53-dependent pathways before the fish died during the larval stage. Here, we expressed SBDS R126T as a transgene in the sbds -/- background. We showed that one copy of the SBDS R126T transgene permitted the establishment of maternal zygotic sbds-null fish which produced defective embryos with cdkn1a up-regulation, a Tp53 target involved in cell cycle arrest. None survived beyond 3 dpf. However, two copies of the transgene resulted in normal development and lifespan. Surprisingly, neutropenia persisted. The surviving fish displayed suppression of female sex differentiation, a stress response in zebrafish. To evaluate the role of Tp53 in the pathogenesis of sbds -/- fish phenotype, we bred the fish with a DNA binding deficient allele, tp53 M214K Expression of the loss-of-function tp53 M214K did not rescue neutropenia or survival in sbds-null zebrafish. Increased expression of cdkn1a was abrogated in the tp53 M214K/M214K ;sbds -/- fish. We conclude that the amount of SBDSR126T protein is important for development, inactivation of Tp53 fails to rescue neutropenia or survival in the sbds-null background, and cdkn1a up-regulation was dependent on WT tp53 We hypothesize that additional pathways are involved in the pathophysiology of SDS.

Shwachman-Diamond syndromes: clinical, genetic, and biochemical insights from the rare variants.

Shwachman-Diamond syndrome is a rare inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. In 10-30% of cases, transformation to a myeloid neoplasm occurs. Approximately 90% of patients have biallelic pathogenic variants in the SBDS gene located on human chromosome 7q11. Over the past several years, pathogenic variants in three other genes have been identified to cause similar phenotypes; these are DNAJC21, EFL1, and SRP54. Clinical manifestations involve multiple organ systems and those classically associated with the Shwachman-Diamond syndrome (bone, blood, and pancreas). Neurocognitive, dermatologic, and retinal changes may also be found. There are specific gene-phenotype differences. To date, SBDS, DNAJC21, and SRP54 variants have been associated with myeloid neoplasia. Common to SBDS, EFL1, DNAJC21, and SRP54 is their involvement in ribosome biogenesis or early protein synthesis. These four genes constitute a common biochemical pathway conserved from yeast to humans that involve early stages of protein synthesis and demonstrate the importance of this synthetic pathway in myelopoiesis.

Stromal transdifferentiation drives lipomatosis and induces extensive vascular remodeling in the aging human lymph node.

Lymph node (LN) lipomatosis is a common but rarely discussed phenomenon associated with aging that involves a gradual exchange of the LN parenchyma into adipose tissue. The mechanisms behind these changes and the effects on the LN are unknown. We show that LN lipomatosis starts in the medullary regions of the human LN and link the initiation of lipomatosis to transdifferentiation of LN fibroblasts into adipocytes. The latter is associated with a downregulation of lymphotoxin beta expression. We also show that isolated medullary and CD34+ fibroblasts, in contrast to the reticular cells of the T-cell zone, display an inherently higher sensitivity for adipogenesis. Progression of lipomatosis leads to a gradual loss of the medullary lymphatic network, but at later stages, collecting-like lymphatic vessels are found inside the adipose tissue. The stromal dysregulation includes a dramatic remodeling and dilation of the high endothelial venules associated with reduced density of naïve T-cells. Abnormal clustering of plasma cells is also observed. Thus, LN lipomatosis causes widespread stromal dysfunction with consequences for the immune contexture of the human LN. Our data warrant an increased awareness of LN lipomatosis as a factor contributing to decreased immune functions in the elderly and in disease. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Mutations in the SRP54 gene cause severe congenital neutropenia as well as Shwachman-Diamond-like syndrome.

Congenital neutropenias (CNs) are rare heterogeneous genetic disorders, with about 25% of patients without known genetic defects. Using whole-exome sequencing, we identified a heterozygous mutation in the SRP54 gene, encoding the signal recognition particle (SRP) 54 GTPase protein, in 3 sporadic cases and 1 autosomal dominant family. We subsequently sequenced the SRP54 gene in 66 probands from the French CN registry. In total, we identified 23 mutated cases (16 sporadic, 7 familial) with 7 distinct germ line SRP54 mutations including a recurrent in-frame deletion (Thr117del) in 14 cases. In nearly all patients, neutropenia was chronic and profound with promyelocytic maturation arrest, occurring within the first months of life, and required long-term granulocyte colony-stimulating factor therapy with a poor response. Neutropenia was sometimes associated with a severe neurodevelopmental delay (n = 5) and/or an exocrine pancreatic insufficiency requiring enzyme supplementation (n = 3). The SRP54 protein is a key component of the ribonucleoprotein complex that mediates the co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER). We showed that SRP54 was specifically upregulated during the in vitro granulocytic differentiation, and that SRP54 mutations or knockdown led to a drastically reduced proliferation of granulocytic cells associated with an enhanced P53-dependent apoptosis. Bone marrow examination of SRP54-mutated patients revealed a major dysgranulopoiesis and features of cellular ER stress and autophagy that were confirmed using SRP54-mutated primary cells and SRP54 knockdown cells. In conclusion, we characterized a pathological pathway, which represents the second most common cause of CN with maturation arrest in the French CN registry.

Pancreatic Lipomatous Hamartoma: A Hitherto Unrecognized Variant.

Pancreatic masses consisting of lipomatous components clinically include lipoma, liposarcoma, lipomatous pseudohypertrophy of the pancreas, fat-containing neoplasms such as perivascular epithelioid cell tumor, and malignant neoplasm with lipoid degeneration. We present pancreatic lipomatous hamartoma, which has not been reported hitherto. A solid pancreatic mass was detected from a computed tomographic scan check-up in each of 3 cases of Japanese men. Macroscopically, well-demarcated solid lipomatous masses were detected at the uncus, body, and tail of the pancreas, respectively. Microscopically, the masses predominantly consisted of mature adipocytes with no atypia, but contained characteristics components of pancreatic hamartoma, such as small ducts, a well-preserved acinar structure, and/or fibrous stroma. On the basis of the unique features, lack of islets and absence of periductal elastic fibers, these tumors are a distinct variant of pancreatic hamartoma. Furthermore, high-mobility group AT-hook 2 expression in the fibro-adipocytes of this tumor indicated that these cells are an integral component of the pancreatic lipomatous hamartoma. Consequently, the unique tumors described herein are pancreatic lipomatous hamartoma, which must be discriminated from other lipomatous lesions of the pancreas.

Shwachman-Diamond Syndrome Protein SBDS Maintains Human Telomeres by Regulating Telomerase Recruitment.

Shwachman-Diamond syndrome (SDS) is a rare pediatric disease characterized by various systemic disorders, including hematopoietic dysfunction. The mutation of Shwachman-Bodian-Diamond syndrome (SBDS) gene has been proposed to be a major causative reason for SDS. Although SBDS patients were reported to have shorter telomere length in granulocytes, the underlying mechanism is still unclear. Here we provide data to elucidate the role of SBDS in telomere protection. We demonstrate that SBDS deficiency leads to telomere shortening. We found that overexpression of disease-associated SBDS mutants or knockdown of SBDS hampered the recruitment of telomerase onto telomeres, while the overall reverse transcriptase activity of telomerase remained unaffected. Moreover, we show that SBDS could specifically bind to TPP1 during the S phase of cell cycle, likely functioning as a stabilizer for TPP1-telomerase interaction. Our findings suggest that SBDS is a telomere-protecting protein that participates in regulating telomerase recruitment.

Ataluren-driven restoration of Shwachman-Bodian-Diamond syndrome protein function in Shwachman-Diamond syndrome bone marrow cells.

Shwachman-Diamond syndrome (SDS) is a rare inherited recessive disease mainly caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene, which encodes for the homonymous protein SBDS, whose function still remains to be fully established. SDS affects several organs causing bone marrow failure, exocrine pancreatic insufficiency, skeletal malformations, and cognitive disorders. About 15% of SDS patients develop myelodysplastic syndrome (MDS) and are at higher risk of developing acute myeloid leukemia (AML). Deficiency in SBDS expression has been associated with increased apoptosis and lack of myeloid differentiation in bone marrow hematopoietic progenitors. Importantly, most SDS patients carry nonsense mutations in SBDS. Since ataluren is a well-characterized small molecule inhibitor that can suppress nonsense mutations, here, we have assessed the efficacy of this drug in restoring SBDS expression in hematopoietic cells obtained from a cohort of SDS patients. Remarkably, we show that ataluren treatment readily restores SBDS protein expression in different cell types, particularly bone marrow stem cells. Furthermore, ataluren promotes myeloid differentiation in hematopoietic progenitors, reduces apoptotic rate in primary PBMCs, and brings mammalian target of rapamycin phosphorylation levels back to normal in both lymphoblasts and bone marrow mesenchymal stromal cells (BM-MSCs). Since a specific therapy against SDS is currently lacking, these results provide the rationale for ataluren repurposing clinical trials.

Molecular basis of the human ribosomopathy Shwachman-Diamond syndrome.

Mutations that target the ubiquitous process of ribosome assembly paradoxically cause diverse tissue-specific disorders (ribosomopathies) that are often associated with an increased risk of cancer. Ribosomes are the essential macromolecular machines that read the genetic code in all cells in all kingdoms of life. Following pre-assembly in the nucleus, precursors of the large 60S and small 40S ribosomal subunits are exported to the cytoplasm where the final steps in maturation are completed. Here, I review the recent insights into the conserved mechanisms of ribosome assembly that have come from functional characterisation of the genes mutated in human ribosomopathies. In particular, recent advances in cryo-electron microscopy, coupled with genetic, biochemical and prior structural data, have revealed that the SBDS protein that is deficient in the inherited leukaemia predisposition disorder Shwachman-Diamond syndrome couples the final step in cytoplasmic 60S ribosomal subunit maturation to a quality control assessment of the structural and functional integrity of the nascent particle. Thus, study of this fascinating disorder is providing remarkable insights into how the large ribosomal subunit is functionally activated in the cytoplasm to enter the actively translating pool of ribosomes.

Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca(2+)]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

Shwachman-Diamond syndrome presenting with early ichthyosis, associated dermal and epidermal intracellular lipid droplets, hypoglycemia, and later distinctive clinical SDS phenotype.

Shwachman-Diamond syndrome (SDS) is a recessive ribosomopathy, characterized by bone marrow failure and exocrine pancreatic insufficiency (ePI) often associated with neurodevelopmental and skeletal abnormalities. The aim of this report is to describe a SDS patient with early ichthyosis associated with dermal and epidermal intracellular lipid droplets (iLDs), hypoglycemia and later a distinctive clinical SDS phenotype. At 3 months of age, she had ichthyosis, growth retardation, and failure to thrive. She had not cytopenia. Ultrasonography (US) showed pancreatic diffuse high echogenicity. Subsequently fasting hypoketotic hypoglycemia occurred without permanent hepatomegaly or hyperlipidemia. Continuous gavage feeding was followed by clinical improvement including ichthyosis and hypoglycemia. After 14 months of age, she developed persistent neutropenia and ePI consistent with SDS. The ichthyotic skin biopsy, performed at 5 months of age, disclosed iLDs in all epidermal layers, in melanocytes, eccrine sweat glands, Schwann cells and dermal fibroblasts. These iLDs were reminiscent of those described in Dorfman-Chanarin syndrome (DCS) or Wolman's disease. Both LIPA and CGI-58 analysis did not revealed pathogenic mutation. By sequencing SBDS, a compound heterozygous for a previously reported gene mutation (c.258 + 2T>C) and a novel mutation (c.284T>G) were found. Defective SBDS may hypothetically interfere as in DCS, with neutral lipid metabolism and play a role in the SDS phenotype such as ichthyosis with dermal and epidermal iLDs and hypoglycemia. This interference with neutral lipid metabolism must most likely occur in the cytoplasm compartment as in DCS and not in the lysosomal compartment as in Wolman's disease. © 2016 Wiley Periodicals, Inc.

A specific mutation in TBL1XR1 causes Pierpont syndrome.

BACKGROUND: The combination of developmental delay, facial characteristics, hearing loss and abnormal fat distribution in the distal limbs is known as Pierpont syndrome. The aim of the present study was to detect and study the cause of Pierpont syndrome. METHODS: We used whole-exome sequencing to analyse four unrelated individuals with Pierpont syndrome, and Sanger sequencing in two other unrelated affected individuals. Expression of mRNA of the wild-type candidate gene was analysed in human postmortem brain specimens, adipose tissue, muscle and liver. Expression of RNA in lymphocytes in patients and controls was additionally analysed. The variant protein was expressed in, and purified from, HEK293 cells to assess its effect on protein folding and function. RESULTS: We identified a single heterozygous missense variant, c.1337A>G (p.Tyr446Cys), in transducin ß-like 1 X-linked receptor 1 (TBL1XR1) as disease-causing in all patients. TBL1XR1 mRNA expression was demonstrated in pituitary, hypothalamus, white and brown adipose tissue, muscle and liver. mRNA expression is lower in lymphocytes of two patients compared with the four controls. The mutant TBL1XR1 protein assembled correctly into the nuclear receptor corepressor (NCoR)/ silencing mediator for retinoid and thyroid receptors (SMRT) complex, suggesting a dominant-negative mechanism. This contrasts with loss-of-function germline TBL1XR1 deletions and other TBL1XR1 mutations that have been implicated in autism. However, autism is not present in individuals with Pierpont syndrome. CONCLUSIONS: This study identifies a specific TBL1XR1 mutation as the cause of Pierpont syndrome. Deletions and other mutations in TBL1XR1 can cause autism. The marked differences between Pierpont patients with the p.Tyr446Cys mutation and individuals with other mutations and whole gene deletions indicate a specific, but as yet unknown, disease mechanism of the TBL1XR1 p.Tyr446Cys mutation.

Shwachman-Bodian-Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPß mRNAs.

Mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene cause Shwachman-Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -ß mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPß-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5' untranslated regions (5' UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -ß deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype.

Chronic pancreatitis and lipomatosis are associated with defective function of ciliary genes in pancreatic ductal cells.

Genetic diseases associated with defects in primary cilia are classified as ciliopathies. Pancreatic lesions and ductal cysts are found in patients with ciliopathic polycystic kidney diseases suggesting a close connection between pancreatic defects and primary cilia. Here we investigate the role of two genes whose deletion is known to cause primary cilium defects, namely Hnf6 and Lkb1, in pancreatic ductal homeostasis. We find that mice with postnatal duct-specific deletion of Hnf6 or Lkb1 show duct dilations. Cells lining dilated ducts present shorter cilia with swollen tips, suggesting defective intraciliary transport. This is associated with signs of chronic pancreatitis, namely acinar-to-ductal metaplasia, acinar proliferation and apoptosis, presence of inflammatory infiltrates, fibrosis and lipomatosis. Our data reveal a tight association between ductal ciliary defects and pancreatitis with perturbed acinar homeostasis and differentiation. Such injuries can account for the increased risk to develop pancreatic cancer in Peutz-Jeghers patients who carry LKB1 loss-of-function mutations.

Eukaryotic translation initiation factor 6 is a novel regulator of reactive oxygen species-dependent megakaryocyte maturation.

BACKGROUND: Ribosomopathies constitute a class of inherited disorders characterized by defects in ribosome biogenesis and function. Classically, bone marrow (BM) failure is a clinical symptom shared between these syndromes, including Shwachman-Bodian-Diamond syndrome (SBDS). Eukaryotic translation initiation factor 6 (eIF6) is a critical translation factor that rescues the quasilethal effect of the loss of the SBDS protein. OBJECTIVES: To determine whether eIF6 activity is necessary for BM development. METHODS: We used eIF6(+/-) mice and primary BM megakaryocytes to investigate the involvement of eIF6 in the regulation of hematopoiesis. RESULTS: We provide evidence that reduced eIF6 expression negatively impacts on megakaryopoiesis. We show that inhibition of eIF6 leads to a reduction in cell size and mean ploidy level of megakaryocytes and a delay in megakaryocyte maturation by blocking the G1 /S transition. Consistent with this phenotype, only few megakaryocyte-forming proplatelets were found in eIF6(+/-) cells. We also discovered that, in eIF6(+/-) cells, the steady-state abundance of mitochondrial respiratory chain complex I-encoding mRNAs is decreased, resulting in decreased reactive oxygen species (ROS) production. Intriguingly, connectivity map analysis showed that eIF6-mediated changes overlap with specific translational inhibitors. eIF6 is a translation factor acting downstream of insulin/phorbol 12-myristate 13-acetate (PMA) stimulation. PMA treatment significantly restored eIF6(+/-) megakaryocyte maturation, indicating that activation of eIF6 is essential for the rescue of the phenotype. CONCLUSIONS: Taken together, our results show a role for eIF6-driven translation in megakaryocyte development, and unveil the novel connection between translational control and ROS production in this cell subset.

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfß, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfß molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to be p53-dependent. Our findings therefore point to cell/tissue-specific responses to p53-activation that include distinction between apoptosis and senescence pathways, in the context of translation disruption.

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein.

Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1.

Nevus lipomatosus cutaneous superficialis with focal lipocytic pagetoid epidermal spread and secondary calcinosis cutis: a case report.

A case of nevus lipomatosus cutaneous superficialis is presented, wherein the lesion occurred as a gluteal swelling in a 6-year-old boy. On histopathology examination, it consisted of typical superficial expanses of mature adipose tissue in the papillary dermis of the polypoidal lesion. It was accompanied by 2 unusual findings, which consisted of the focal presence of lipocytes at the epidermal­dermal junction in the pagetoid manner and the prominent deposition of calcium resulting in calcinosis cutis­like lesions. Calcinosis cutis in the present case occurred as dystrophic calcification in focal fat necrosis changes of the lesion, whereas the focal pagetoid spread of fat cells is likely related to the same hamartomatous adipose tissue growth that ultimately results in the nevus lipomatosus.