Myxovirus resistance protein A (MxA) expression in myositides: Sarcoplasmic expression is common in both dermatomyositis and lupus myositis.

INTRODUCTION/AIMS: Myxovirus resistance protein A (MxA) is a type I interferon (IFN1) pathway activation marker and MxA sarcoplasmic expression is currently recognized as a highly specific marker for dermatomyositis (DM). However, we have frequently observed endothelial tubuloreticular inclusions (TRI), another surrogate IFN1 activation marker, in a variety of overlap myositides. The aim of this study was to examine MxA expression in those myositides. METHODS: We retrospectively performed MxA immunostaining on a wide range of myositides. RESULTS: MxA sarcoplasmic expression was present in DM (94.4%, 17/18), active lupus myositis (LM, 80%,16/20), inactive LM (36%, 4/11), antisynthetase syndrome (ASyS, 20%, 2/10), systemic sclerosis (13%, 2/15), Sjogren's syndrome (7.7%, 1/13), and human immunodeficiency virus (HIV) myositis (5.6%, 1/18) and was absent in immune-mediated necrotizing myopathy (IMNM, 0/16) and hydroxychloroquine myopathy (0/5). The sensitivity and specificity of MxA sarcoplasmic expression for LM and DM combined compared with all other myositides were 84.6% (95% CI: 69.5-94.1) and 92.1 (95% CI: 83.6-97.0), respectively, and superior to TRIs. MxA capillary expression was nonspecific. Histologically, 35% of LM cases demonstrated a unique panfascicular necrotizing myopathy pattern. The remainder of the LM cases had significant morphological overlap with DM/ASyS (20%), IMNM (20%), or polymyositis (15%). DISCUSSION: MxA sarcoplasmic expression is highly prevalent in LM and DM and is a useful marker in differentiating DM and LM from other myositides. LM can manifest in various pathology patterns that need to be differentiated from DM, IMNM, ASyS, and polymyositis.

Ectopic PLAG1 induces muscular dystrophy in the mouse.

Even though various genetic mutations have been identified in muscular dystrophies (MD), there is still a need to understand the biology of MD in the absence of known mutations. Here we reported a new mouse model of MD driven by ectopic expression of PLAG1. This gene encodes a developmentally regulated transcription factor known to be expressed in developing skeletal muscle, and implicated as an oncogene in certain cancers including rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma composed of myoblast-like cells. By breeding loxP-STOP-loxP-PLAG1 (LSL-PLAG1) mice into the MCK-Cre line, we achieved ectopic PLAG1 expression in cardiac and skeletal muscle. The Cre/PLAG1 mice died before 6 weeks of age with evidence of cardiomyopathy significantly limiting left ventricle fractional shortening. Histology of skeletal muscle revealed dystrophic features, including myofiber necrosis, fiber size variation, frequent centralized nuclei, fatty infiltration, and fibrosis, all of which mimic human MD pathology. QRT-PCR and Western blot revealed modestly decreased Dmd mRNA and dystrophin protein in the dystrophic muscle, and immunofluorescence staining showed decreased dystrophin along the cell membrane. Repression of Dmd by ectopic PLAG1 was confirmed in dystrophic skeletal muscle and various cell culture models. In vitro studies showed that excess IGF2 expression, a transcriptional target of PLAG1, phenocopied PLAG1-mediated down-regulation of dystrophin. In summary, we developed a new mouse model of a lethal MD due to ectopic expression of PLAG1 in heart and skeletal muscle. Our data support the potential contribution of excess IGF2 in this model. Further studying these mice may provide new insights into the pathogenesis of MD and perhaps lead to new treatment strategies.

Molecular markers of brain cholesterol homeostasis are unchanged despite a smaller brain mass in a mouse model of cholesteryl ester storage disease.

Lysosomal acid lipase (LAL), encoded by the gene LIPA, facilitates the intracellular processing of lipids by hydrolyzing cholesteryl esters and triacylglycerols present in newly internalized lipoproteins. Loss-of-function mutations in LIPA result in cholesteryl ester storage disease (CESD) or Wolman disease when mutations cause complete loss of LAL activity. Although the phenotype of a mouse CESD model has been extensively characterized, there has not been a focus on the brain at different stages of disease progression. In the current studies, whole-brain mass and the concentrations of cholesterol in both the esterified (EC) and unesterified (UC) fractions were measured in Lal-/- and matching Lal+/+ mice (FVB-N strain) at ages ranging from 14 up to 280 days after birth. Compared to Lal+/+ controls at 50, 68-76, 140-142, and 230-280 days of age, Lal-/- mice had brain weights that averaged approximately 6%, 7%, 18%, and 20% less, respectively. Brain EC levels were higher in the Lal-/- mice at every age, being elevated 27-fold at 230-280 days. Brain UC concentrations did not show a genotypic difference at any age. The elevated brain EC levels in the Lal-/- mice did not reflect EC in residual blood. An mRNA expression analysis for an array of genes involved in the synthesis, catabolism, storage, and transport of cholesterol in the brains of 141-day old mice did not detect any genotypic differences although the relative mRNA levels for several markers of inflammation were moderately elevated in the Lal-/- mice. The possible sites of EC accretion in the central nervous system are discussed.

Distinct tissue injury patterns in juvenile dermatomyositis auto-antibody subgroups.

INTRODUCTION: Juvenile dermatomyositis (JDM) can be classified into clinical serological subgroups by distinct myositis-specific antibodies (MSAs). It is incompletely understood whether different MSAs are associated with distinct pathological characteristics, clinical disease activities, or response to treatment. METHODS: We retrospectively reviewed clinicopathological data from consecutive JDM patients followed in the pediatric rheumatology clinic at a single center between October 2016 and November 2018. Demographics, clinical data, and laboratory data were collected and analyzed. Detailed muscle biopsy evaluation of four domains (inflammation, myofiber, vessels, and connective tissue) was performed, followed by statistical analysis. RESULTS: Of 43 subjects included in the study, 26 (60.5%) had a detectable MSA. The most common MSAs were anti-NXP-2 (13, 30.2%), anti-Mi-2 (7, 16.3%), and anti-MDA-5 (5, 11.6%). High titer anti-Mi-2 positively correlated with serum CK > 10,000 at initial visit (r = 0.96, p = 0.002). Muscle biopsied from subjects with high titer anti-Mi-2 had prominent perifascicular myofiber necrosis and perimysial connective tissue damage that resembled perifascicular necrotizing myopathy, but very little capillary C5b-9 deposition. Conversely, there was no positive correlation between the levels of the anti-NXP-2 titer and serum CK (r = - 0.21, p = 0.49). Muscle biopsies from patients with anti-NXP-2 showed prominent capillary C5b-9 deposition; but limited myofiber necrosis. Only one patient had anti-TIF1γ autoantibody, whose muscle pathology was similar as those with anti-NXP2. All patients with anti-MDA-5 had normal CK and near normal muscle histology. CONCLUSIONS: Muscle biopsy from JDM patients had MSA specific tissue injury patterns. These findings may help improve muscle biopsy diagnosis accuracy and inform personalized treatment of JDM.

ASCL1 regulates neurodevelopmental transcription factors and cell cycle genes in brain tumors of glioma mouse models.

Glioblastomas (GBMs) are incurable brain tumors with a high degree of cellular heterogeneity and genetic mutations. Transcription factors that normally regulate neural progenitors and glial development are aberrantly coexpressed in GBM, conferring cancer stem-like properties to drive tumor progression and therapeutic resistance. However, the functional role of individual transcription factors in GBMs in vivo remains elusive. Here, we demonstrate that the basic-helix-loop-helix transcription factor ASCL1 regulates transcriptional targets that are central to GBM development, including neural stem cell and glial transcription factors, oncogenic signaling molecules, chromatin modifying genes, and cell cycle and mitotic genes. We also show that the loss of ASCL1 significantly reduces the proliferation of GBMs induced in the brain of a genetically relevant glioma mouse model, resulting in extended survival times. RNA-seq analysis of mouse GBM tumors reveal that the loss of ASCL1 is associated with downregulation of cell cycle genes, illustrating an important role for ASCL1 in controlling the proliferation of GBM.

Distinct Expression Patterns of Carbonic Anhydrase IX in Clear Cell, Microcystic, and Angiomatous Meningiomas.

Clear cell, microcytic, and angiomatous meningiomas are 3 vasculature-rich variants with overlapping morphological features but different prognostic and treatment implications. Distinction between them is not always straightforward. We compared the expression patterns of the hypoxia marker carbonic anhydrase IX (CA-IX) in meningiomas with predominant clear cell (n = 15), microcystic (n = 9), or angiomatous (n = 11) morphologies, as well as 117 cases of other World Health Organization recognized histological meningioma variants. Immunostaining for SMARCE1 protein, whose loss-of-function has been associated with clear cell meningiomas, was performed on all clear cell meningiomas, and selected variants of meningiomas as controls. All clear cell meningiomas showed absence of CA-IX expression and loss of nuclear SMARCE1 expression. All microcystic and angiomatous meningiomas showed diffuse CA-IX immunoreactivity and retained nuclear SMARCE1 expression. In other meningioma variants, CA-IX was expressed in a hypoxia-restricted pattern and was highly associated with atypical features such as necrosis, small cell change, and focal clear cell change. In conclusion, CA-IX may serve as a useful diagnostic marker in differentiating clear cell, microcystic, and angiomatous meningiomas.

Cell-of-origin susceptibility to glioblastoma formation declines with neural lineage restriction.

The contribution of lineage identity and differentiation state to malignant transformation is controversial. We have previously shown that adult neural stem and early progenitor cells give origin to glioblastoma. Here we systematically assessed the tumor-initiating potential of adult neural populations at various stages of lineage progression. Cell type-specific tamoxifen-inducible Cre recombinase transgenes were used to target glioblastoma-relevant tumor suppressors Nf1, Trp53 and Pten in late-stage neuronal progenitors, neuroblasts and differentiated neurons. Mutant mice showed cellular and molecular defects demonstrating the impact of tumor suppressor loss, with mutant neurons being the most resistant to early changes associated with tumor development. However, we observed no evidence of glioma formation. These studies show that increasing lineage restriction is accompanied by decreasing susceptibility to malignant transformation, indicating a glioblastoma cell-of-origin hierarchy in which stem cells sit at the apex and differentiated cell types are least susceptible to tumorigenesis.

BRAF mutation leading to central nervous system rosai-dorfman disease.

Rosai-Dorfman disease (RDD) is an uncommon histiocytic proliferative disorder that can present in nodal, extranodal, or, extremely rarely, in central nervous system (CNS)-restricted form. RDD is characterized histologically as a non-Langerhans cell histiocytosis composed of atypical CD68+ /S-100+ /CD1a- macrophages demonstrating prominent emperipolesis and effacement of the surrounding tissue. Previously thought to represent a reactive process, recent studies have raised the possibility that RDD and other histiocytic lesions, including Erdheim-Chester and Langerhans cell histiocytosis, are clonal processes linked to somatic mutations in the mitogen-activated protein (MAP) kinase pathway. Herein, we present a fatal case of RDD isolated to the CNS and used a next-generation targeted gene panel and Sanger sequencing to uncover a pathogenic deletion in the ß3-αC loop of the kinase domain in exon 12 of BRAF. This mutation, previously described in melanoma and Langerhans cell histiocytosis, represents the first BRAF mutation of this kind identified in RDD. These findings support the idea that RDD is a neoplastic condition and raise the possibility that inhibitors of the MAP kinase pathway may be effective in RDD. Ann Neurol 2018;83:147-152.

Diffuse microvascular C5b-9 deposition is a common feature in muscle and nerve biopsies from diabetic patients.

Terminal complement complex deposition in endomysial capillaries detected by a C5b-9 immunostain is considered a diagnostic feature for dermatomyositis. However, we found widespread microvascular C5b-9 reactivity in a substantial subset of muscle biopsies with denervation changes, and in nerve biopsies of peripheral neuropathies, particularly in patients with diabetes. It is unclear whether the presence of C5b-9 deposition signifies active immune-mediated vascular injury that requires immune suppression therapy. We retrospectively identified 63 nerve biopsies in patients with a documented history of diabetes, 26 of which had concomitant muscle biopsies, as well as 54 control nerve biopsies in patients without a documented diabetes history, 18 of which had concomitant muscle biopsies. C5b-9 immunostain was performed on all cases. 87% of the nerve biopsies and 92% of the muscle biopsies from diabetic patients showed microvascular C5b-9 reactivity, compared to 34% and 50% in non-diabetic patients. The differences were statistically significant (p < 0.0001 for nerve and p = 0.002 for muscle). The C5b-9 reactivity was generally proportional to the extent of microvascular sclerosis in diabetic patients, but unrelated to inflammation or vasculitis. C5b-9 deposition in micro-vasculature in both muscle and nerve is therefore a common feature in patients with diabetic neuropathies and may have diagnostic utility. Precaution needs to be taken before using muscle capillary C5b-9 reactivity as evidence of myositis.

Aggressive Behavior in Silent Subtype III Pituitary Adenomas May Depend on Suppression of Local Immune Response: A Whole Transcriptome Analysis.

Silent subtype III pituitary adenomas (SS-3) are clinically nonfunctional adenomas that are more aggressive in terms of invasion and risk of recurrence than their conventional null cell counterparts. We previously showed that these tumors can be distinguished by immunohistochemistry based on the identification of a markedly enlarged and fragmented Golgi apparatus. To understand the molecular correlates of differential aggressiveness, we performed whole transcriptome sequencing (RNAseq) on 4 SS-3 and 4 conventional null cell adenomas. The genes that were highly upregulated in all the SS-3 adenomas included 2 secreted proteins involved in the suppression of T-lymphocyte activity, i.e., ARG2 (multiple testing adjusted padj = 1.5 × 10-3) and SEMA3A (padj = 3.3 × 10-3). Highly downregulated genes in all the SS-3 adenomas included HLA-B (padj = 3.3 × 10-6), suggesting reduced antigen presentation by the adenoma to cytotoxic T-cells. Quantitative RT-PCR of these genes performed on the adenoma samples supported the RNAseq results. We also found a relative decrease in the overall concentration of T-lymphocytes in the SS-3 tumors. These results suggest that SS-3 adenomas actively suppress the immune system and raise the possibility that they may be treatable with immune checkpoint inhibitors or nonspecific cancer immunotherapies.

Morin Stain Detects Aluminum-Containing Macrophages in Macrophagic Myofasciitis and Vaccination Granuloma With High Sensitivity and Specificity.

Macrophagic myofasciitis (MMF) is an inflammatory condition associated with the intramuscular (i.m.) injection of aluminum adjuvant-containing vaccines. It is clinically characterized by myalgia, weakness, and chronic fatigue and histologically by aggregates of cohesive macrophages with abundant basophilic, periodic acid-Schiff (PAS)-positive, diastase-resistant granules that percolate through the peri- and endomysium without eliciting substantial myofiber damage. The definitive diagnosis of MMF requires demonstration of aluminum within these macrophages. We evaluated the Morin stain, a simple, 2-step histochemical stain for aluminum, as a confirmatory diagnostic tool for MMF. Among 2270 muscle biopsies processed at UTSW between 2010 and 2015, a total of 12 MMF cases and 1 subcutaneous vaccination granuloma case were identified (11 pediatric, 2 adults). With the Morin stain, all 13 cases showed strong granular reactivity within the cytoplasm of macrophages but not in myofibers or connective tissue. Three cases of inflammatory myopathy with abundant macrophages (IMAM), 8 cases of granulomatous inflammation and 23 other deltoid muscle biopsies used as controls were all negative. Morin stain could be used in both formalin-fixed paraffin-embedded and cryostat sections. Thus, Morin stain detects aluminum with high sensitivity and specificity in human muscle and soft tissue and may improve the diagnostic yield of MMF and vaccination granuloma.

Non-Invasive Targeted Peripheral Nerve Ablation Using 3D MR Neurography and MRI-Guided High-Intensity Focused Ultrasound (MR-HIFU): Pilot Study in a Swine Model.

PURPOSE: Ultrasound (US)-guided high intensity focused ultrasound (HIFU) has been proposed for noninvasive treatment of neuropathic pain and has been investigated in in-vivo studies. However, ultrasound has important limitations regarding treatment guidance and temperature monitoring. Magnetic resonance (MR)-imaging guidance may overcome these limitations and MR-guided HIFU (MR-HIFU) has been used successfully for other clinical indications. The primary purpose of this study was to evaluate the feasibility of utilizing 3D MR neurography to identify and guide ablation of peripheral nerves using a clinical MR-HIFU system. METHODS: Volumetric MR-HIFU was used to induce lesions in the peripheral nerves of the lower limbs in three pigs. Diffusion-prep MR neurography and T1-weighted images were utilized to identify the target, plan treatment and immediate post-treatment evaluation. For each treatment, one 8 or 12 mm diameter treatment cell was used (sonication duration 20 s and 36 s, power 160-300 W). Peripheral nerves were extracted < 3 hours after treatment. Ablation dimensions were calculated from thermal maps, post-contrast MRI and macroscopy. Histological analysis included standard H&E staining, Masson's trichrome and toluidine blue staining. RESULTS: All targeted peripheral nerves were identifiable on MR neurography and T1-weighted images and could be accurately ablated with a single exposure of focused ultrasound, with peak temperatures of 60.3 to 85.7°C. The lesion dimensions as measured on MR neurography were similar to the lesion dimensions as measured on CE-T1, thermal dose maps, and macroscopy. Histology indicated major hyperacute peripheral nerve damage, mostly confined to the location targeted for ablation. CONCLUSION: Our preliminary results indicate that targeted peripheral nerve ablation is feasible with MR-HIFU. Diffusion-prep 3D MR neurography has potential for guiding therapy procedures where either nerve targeting or avoidance is desired, and may also have potential for post-treatment verification of thermal lesions without contrast injection.

Clinical Outcome of Silent Subtype III Pituitary Adenomas Diagnosed by Immunohistochemistry.

Silent subtype III pituitary adenomas (SS-3) are nonfunctioning radiosensitive adenomas that may be associated with an increased risk of recurrence and invasion. The features that have been proposed to be diagnostically important are identifiable by electron microscopy (EM) and include an enlarged Golgi apparatus, along with several other ultrastructural features. The often limited availability of EM and the uncertainty about the relative importance of individual features pose practical challenges to the diagnosis. We hypothesized that it may be possible to diagnose SS-3 based solely on a markedly enlarged Golgi apparatus identified at the light microscopic level. In this prospective study, we used immunohistochemistry (IHC) for the Golgi apparatus with the MG-160/GLG-1 antibody to identify 10 cases with features suggestive of SS-3. Electron microscopy was performed for confirmation on 1 case. Compared with a control group of 20 conventional null cell adenomas, the SS-3 adenomas showed an increased MIB-1 proliferation index (p < 0.01), a higher risk of invasion (p < 0.01), and a higher incidence of recurrence (p < 0.01). Thus, in this first controlled study, we demonstrate that SS-3 is clinically aggressive and identifiable by IHC, without the need for EM. The routine diagnostic workup of nonsecreting adenomas should rule out SS-3, which can be done quickly and efficiently by IHC.

Adult Lineage-Restricted CNS Progenitors Specify Distinct Glioblastoma Subtypes.

A central question in glioblastoma multiforme (GBM) research is the identity of the tumor-initiating cell, and its contribution to the malignant phenotype and genomic state. We examine the potential of adult lineage-restricted progenitors to induce fully penetrant GBM using CNS progenitor-specific inducible Cre mice to mutate Nf1, Trp53, and Pten. We identify two phenotypically and molecularly distinct GBM subtypes governed by identical driver mutations. We demonstrate that the two subtypes arise from functionally independent pools of adult CNS progenitors. Despite histologic identity as GBM, these tumor types are separable based on the lineage of the tumor-initiating cell. These studies point to the cell of origin as a major determinant of GBM subtype diversity.

The G protein α subunit Gαs is a tumor suppressor in Sonic hedgehog-driven medulloblastoma.

Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Herein, we identify GNAS, encoding the G protein Gαs, as a potent tumor suppressor gene that, when expressed at low levels, defines a subset of aggressive Sonic hedgehog (SHH)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically distinct progenitors in mice is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gαs is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation in levels of a Gαs effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas-ablated mice. Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gαs that can be found consistently across Shh-group medulloblastomas of disparate cellular and anatomical origins, highlighting G protein modulation as a potential therapeutic avenue.

High expression of the stem cell marker nestin is an adverse prognostic factor in WHO grade II-III astrocytomas and oligoastrocytomas.

Infiltrating astrocytomas and oligoastrocytomas of low to anaplastic grade (WHO grades II and III), in spite of being associated with a wide range of clinical outcomes, can be difficult to subclassify and grade by the current histopathologic criteria. Unlike oligodendrogliomas and anaplastic oligodendrogliomas that can be identified by the 1p/19q codeletion and the more malignant glioblastomas (WHO grade IV astrocytomas) that can be diagnosed solely based on objective features on routine hematoxylin and eosin sections, no such objective criteria exist for the subclassification of grade II-III astrocytomas and oligoastrocytomas (A+OA II-III). In this study, we evaluated the prognostic and predictive value of the stem cell marker nestin in adult A+OA II-III (n = 50) using immunohistochemistry and computer-assisted analysis on tissue microarrays. In addition, the correlation between nestin mRNA level and total survival was analyzed in the NCI Rembrandt database. The results showed that high nestin expression is a strong adverse prognostic factor for total survival (p = 0.0004). The strength of the correlation was comparable to but independent of the isocitrate dehydrogenase 1/2 (IDH 1/2) mutation status. Histopathological grading and subclassification did not correlate significantly with outcome, although the interpretation of this finding is limited by the fact that grade III tumors were treated more aggressively than grade II tumors. These results suggest that nestin level and IDH 1/2 mutation status are strong prognostic features in A+OA II-III and possibly more helpful for treatment planning than routine histopathological variables such as oligodendroglial component (astrocytoma vs. oligoastrocytoma) and WHO grade (grade II vs. III).

Progressive leg pain and weakness.

A 54-year-old man presented with progressive asymmetric leg pain and weakness. He had a history of invasive squamous cell carcinoma that was fully treated 2 years earlier. His leg symptoms progressed relentlessly during several months. Imaging studies demonstrated enhancement of the cauda equina and leptomeninges of the lower spinal cord. Initial cerebrospinal fluid examination showed an elevated protein concentration and lymphocytic pleocytosis with no malignant cells on cytological analysis. There was short-term improvement in symptoms and cerebrospinal fluid abnormalities with intravenous steroids. Two additional cerebrospinal fluid studies showed normal cytological findings, elevated IgG synthesis, and elevated antibody titers to varicella-zoster virus. Over time, the patient worsened, developed cranial neuropathies, and ultimately died. The pathological diagnosis and the approach to the clinical data are discussed.

Somatic gain-of-function mutations in PIK3CA in patients with macrodactyly.

Macrodactyly is a discrete congenital anomaly consisting of enlargement of all tissues localized to the terminal portions of a limb, typically within a 'nerve territory'. The classic terminology for this condition is 'lipofibromatous hamartoma of nerve' or Type I macrodactyly. The peripheral nerve, itself, is enlarged both in circumference and in length. It is not related to neurofibromatosis (NF1), nor is it associated with vascular malformations, such as in the recently reported CLOVES syndrome. The specific nerve pathophysiology in this form of macrodactyly has not been well described and a genetic etiology for this specific form of enlargement is unknown. To identify the genetic cause of macrodactyly, we used whole-exome sequencing to identify somatic mutations present in the affected nerve of a single patient. We confirmed a novel mutation in PIK3CA (R115P) present in the patient's affected nerve tissue but not in blood DNA. Sequencing PIK3CA exons identified gain-of-function mutations (E542K, H1047L or H1047R) in the affected tissue of five additional unrelated patients; mutations were absent in blood DNA available from three patients. Immunocytochemistry confirmed AKT activation in cultured cells from the nerve of a macrodactyly patient. Additionally, we found that the most abnormal structure within the involved nerve in a macrodactylous digit is the perineurium, with additional secondary effects on the axon number and size. Thus, isolated congenital macrodactyly is caused by somatic activation of the PI3K/AKT cell-signaling pathway and is genetically and biochemically related to other overgrowth syndromes.