DNAJC12 deficiency: A new strategy in the diagnosis of hyperphenylalaninemias

Patients with hyperphenylalaninemia (HPA) are detected through newborn screening for phenylketonuria (PKU). HPA is known to be caused by deficiencies of the enzyme phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH₄). Current guidelines for the differential diagnosis of HPA would, however, miss a recently described DNAJC12 deficiency. The co-chaperone DNAJC12 is, together with the 70 kDa heat shock protein (HSP70), responsible for the proper folding of PAH. All DNAJC12-deficient patients investigated to date responded to a challenge with BH₄ by lowering their blood phenylalanine levels. In addition, the patients presented with low levels of biogenic amine in CSF and responded to supplementation with BH₄, L-dopa/carbidopa and 5-hydroxytryptophan. The phenotypic spectrum ranged from mild autistic features or hyperactivity to severe intellectual disability, dystonia and parkinsonism. Late diagnosis result in permanent neurological disability, while early diagnosed and treated patients develop normally. Molecular diagnostics for DNAJC12 variants are thus mandatory in all patients in which deficiencies of PAH and BH₄ are genetically excluded.     

Pathogenic variants of DNAJC12 and evaluation of the encoded cochaperone as a genetic modifier of hyperphenylalaninemia

Biallelic variants of the gene DNAJC12, which encodes a cochaperone, were recently described in patients with hyperphenylalaninemia (HPA). This paper reports the retrospective genetic analysis of a cohort of unsolved cases of HPA. Biallelic variants of DNAJC12 were identified in 20 patients (generally neurologically asymptomatic) previously diagnosed with phenylalanine hydroxylase (PAH) deficiency (phenylketonuria [PKU]). Further, mutations of DNAJC12 were identified in four carriers of a pathogenic variant of PAH. The genetic spectrum of DNAJC12 in the present patients included four new variants, two intronic changes c.298‐2A>C and c.502+1G>C, presumably affecting the splicing process, and two exonic changes c.309G>T (p.Trp103Cys) and c.524G>A (p.Trp175Ter), classified as variants of unknown clinical significance (VUS). The variant p.Trp175Ter was detected in 83% of the mutant alleles, with 14 cases homozygous, and was present in 0.3% of a Spanish control population. Functional analysis indicated a significant reduction in PAH and its activity, reduced tyrosine hydroxylase stability, but no effect on tryptophan hydroxylase 2 stability, classifying the two VUS as pathogenic variants. Additionally, the effect of the overexpression of DNAJC12 on some destabilizing PAH mutations was examined and a mutation‐specific effect on stabilization was detected suggesting that the proteostasis network could be a genetic modifier of PAH deficiency and a potential target for developing mutation‐specific treatments for PKU.     

Dominant high expression of wild‐type HSP110 defines a poor prognostic subgroup of colorectal carcinomas with microsatellite instability: a whole‐section immunohistochemical analysis

The aim of this study is to establish heat shock protein 110 (HSP110) as a prognostic biomarker of colorectal carcinomas (CRCs) with microsatellite instability‐high (MSI‐H) by considering the intratumoral heterogeneity of HSP110 expression. We performed whole‐section immunohistochemistry (IHC) for wild‐type HSP110 (HSP110wt) in 164 MSI‐H CRCs. The intensity of the HSP110wt expression in tumor cells was semiquantitatively scored (0/1/2/3), and the HSP110wt expression status of each tumor was classified as low or high using the following four scoring criteria: H‐score, dominant intensity score, lowest intensity score, and highest intensity score. Among the four criteria, only the dominant intensity score‐based dichotomous classification of HSP110wt expression was significantly associated with a difference in disease‐free survival (log‐rank p = 0.035) in 164 MSI‐H CRCs. The HSP110wt‐low MSI‐H CRCs were significantly correlated with larger deletions in the HSP110 T₁₇ mononucleotide repeat (≥4 bp; p < 0.001). In conclusion, the dominant intensity score‐based assessment of HSP110wt IHC can be a simple and useful method for the prognostic stratification of MSI‐H CRCs. It is expected that HSP110wt IHC may be used to identify a subgroup of MSI‐H CRCs with poor prognosis and/or candidates for further treatment, such as immunotherapy using immune checkpoint inhibitors in MSI‐H CRCs.     

Functional consequences and rescue potential of pathogenic missense mutations in tripeptidyl peptidase I

There are 35 missense mutations among 68 different mutations in the TPP1 gene, which encodes tripeptidyl peptidase I (TPPI), a lysosomal aminopeptidase associated with classic late‐infantile neuronal ceroid lipofuscinosis (CLN2 disease). To elucidate the molecular mechanisms underlying TPPI deficiency in patients carrying missense mutations and to test the amenability of mutant proteins to chemical chaperones and permissive temperature treatment, we introduced individually 14 disease‐associated missense mutations into human TPP1 cDNA and analyzed the cell biology of these TPPI variants expressed in Chinese hamster ovary cells. Most TPPI variants displayed obstructed transport to the lysosomes, prolonged half‐life of the proenzyme, and residual or no enzymatic activity, indicating folding abnormalities. Protein misfolding was produced by mutations located in both the prosegment (p.Gly77Arg) and throughout the length of the mature enzyme. However, the routes of removal of misfolded proteins by the cells varied, ranging from their efficient degradation by the ubiquitin/proteasome system to abundant secretion. Two TPPI variants demonstrated enhanced processing in response to folding improvement treatment, and the activity of one of them, p.Arg447His, showed a fivefold increase under permissive temperature conditions, which suggests that folding improvement strategies may ameliorate the function of some misfolding TPPI mutant proteins. Hum Mutat 31:1–12, 2010. © 2010 Wiley‐Liss, Inc.     

Histone methyltransferase Suv39h1 deficiency prevents Myc‐induced chromosomal instability in murine myeloid leukemias

Suv39h1 mediates heterochromatin formation in pericentric and telomeric regions by trimethylation of lysine 9 of histone 3 (H3K9me3). Yet, its role in the induction of chromosomal instability is poorly understood. We established a leukemia model by retrovirally expressing Myc in wild‐type and histone methyltransferase Suv39h1‐deficient hematopoietic cells and characterized the resulting leukemias for chromosomal instability. All mice that received cells overexpressing Myc developed myeloid leukemia with a median survival of 44 days posttransplantation. Myc‐overexpressing wild‐type leukemias demonstrated clones with numerical chromosomal aberrations (5/16). In secondary transplantations of these leukemic cells, structural changes, mostly end‐to‐end fusions of chromosomes, appeared (10/12). In contrast, leukemic cells overexpressing Myc with reduced or no Suv39h1 expression had a normal karyotype in primary, secondary, and tertiary transplantations (16/16). Myc‐transduced Suv39h1‐deficient cells showed less critically short telomeres (P < 0.05) compared with Myc‐transduced wild‐type bone marrow cells. Gene expression analysis showed upregulation of genes involved in the alternative lengthening of telomeres (ALT) mechanism. Thus, we hypothesize that loss of Suv39h1 implies activation of the ALT mechanism, in turn ensuring telomere length and stability. Our data show for the first time that Suv39h1 deficiency may prevent chromosomal instability by more efficient telomere stabilization in hematopoietic bone marrow cells overexpressing Myc. © 2013 Wiley Periodicals, Inc.     

Heme oxygenase-1 expression in the ovary dictates a proper oocyte ovulation, fertilization, and corpora lutea maintenance

Citation Zenclussen ML, Jensen F, Rebelo S, El‐Mousleh T, Casalis PA, Zenclussen AC. Heme oxygenase‐1 expression in the ovary dictates a proper oocyte ovulation, fertilization, and corpora lutea maintenance. Am J Reprod Immunol 2012; 67: 376–382 Problem Animals deficient in Heme oxygenase‐1 (HO‐1, Hmox1^?/? mice) have impaired pregnancies, characterized by intrauterine fetal death. HO‐1 expression has been shown to be essential for pregnancy by dictating placentation and intrauterine fetal development. Its absence leads to intrauterine fetal growth restriction and fetal loss, which is independent of the immune system. Defect in previous steps, e.g., ovulation, may, however, also count for their poor reproductive outcome. Method of study Here, we investigated ovulation after hormonal hyperstimulation in Hmox1 wild‐type and knockout animals. Results and Conclusions We observed that animals lacking HO‐1 produced significantly less oocytes after hormonal stimulation than wild type animals and this was mirrored by the number of corpora lutea in the ovary. Furthermore, ovulated oocytes from Hmox1^?/? animals were poorly fertilized compared with those from wild‐type animals. In conclusion, we demonstrate here that HO‐1 plays a pivotal role in the process of oocyte ovulation as well as fertilization, bringing to light a new and unsuspected role for HO‐1.     

Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

Phenylketonuria (PKU) is a genetic disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine to neurotoxic levels. Here, we analyzed the cellular stability, localization, and interaction with wild‐type PAH of 20 selected PKU‐linked PAH protein missense variants. Several were present at reduced levels in human cells, and the levels increased in the presence of a proteasome inhibitor, indicating that proteins are proteasome targets. We found that all the tested PAH variants retained their ability to associate with wild‐type PAH, and none formed aggregates, suggesting that they are only mildly destabilized in structure. In all cases, PAH variants were stabilized by the cofactor tetrahydrobiopterin (BH₄), a molecule known to alleviate symptoms in certain PKU patients. Biophysical calculations on all possible single‐site missense variants using the full‐length structure of PAH revealed a strong correlation between the predicted protein stability and the observed stability in cells. This observation rationalizes previously observed correlations between predicted loss of protein destabilization and disease severity, a correlation that we also observed using new calculations. We thus propose that many disease‐linked PAH variants are structurally destabilized, which in turn leads to proteasomal degradation and insufficient amounts of cellular PAH protein.     

Increased FXYD1 and PGC‐1α mRNA after blood flow‐restricted running is related to fibre type‐specific AMPK signalling and oxidative stress in human muscle

Aim

This study explored the effects of blood flow restriction (BFR) on mRNA responses of PGC‐1α (total, 1α1, and 1α4) and Na⁺,K⁺‐ATPase isoforms (NKA; α_1‐3, β_1‐3, and FXYD1) to an interval running session and determined whether these effects were related to increased oxidative stress, hypoxia, and fibre type‐specific AMPK and CaMKII signalling, in human skeletal muscle.

Methods

In a randomized, crossover fashion, 8 healthy men (26 ± 5 year and 57.4 ± 6.3 mL kg^?1 min^?1) completed 3 exercise sessions: without (CON) or with blood flow restriction (BFR), or in systemic hypoxia (HYP, ~3250 m). A muscle sample was collected before (Pre) and after exercise (+0 hour, +3 hours) to quantify mRNA, indicators of oxidative stress (HSP27 protein in type I and II fibres, and catalase and HSP70 mRNA), metabolites, and α‐AMPK Thr¹⁷²/α‐AMPK, ACC Ser²²¹/ACC, CaMKII Thr²⁸⁷/CaMKII, and PLBSer¹⁶/PLB ratios in type I and II fibres.

Results

Muscle hypoxia (assessed by near‐infrared spectroscopy) was matched between BFR and HYP, which was higher than CON (~90% vs ~70%; P < .05). The mRNA levels of FXYD1 and PGC‐1α isoforms (1α1 and 1α4) increased in BFR only (P < .05) and were associated with increases in indicators of oxidative stress and type I fibre ACC Ser²²¹/ACC ratio, but dissociated from muscle hypoxia, lactate, and CaMKII signalling.

Conclusion

Blood flow restriction augmented exercise‐induced increases in muscle FXYD1 and PGC‐1α mRNA in men. This effect was related to increased oxidative stress and fibre type‐dependent AMPK signalling, but unrelated to the severity of muscle hypoxia, lactate accumulation, and modulation of fibre type‐specific CaMKII signalling.

     

Elevated core and muscle temperature to levels comparable to exercise do not increase heat shock protein content of skeletal muscle of physically active men

Aim: Exercise‐associated hyperthermia is routinely cited as the signal responsible for inducing an increased production of heat shock proteins (HSPs) following exercise. This hypothesis, however, has not been tested in human skeletal muscle. The aim of the present study was to therefore investigate the role of increased muscle and core temperature in contributing to the exercise‐induced production of the major HSP families in human skeletal muscle. Methods: Seven physically active males underwent a passive heating protocol of 1 h duration during which the temperature of the core and vastus lateralis muscle were increased to similar levels to those typically occurring during moderately demanding aerobic exercise protocols. One limb was immersed in a tank containing water maintained at approximately 45 °C whilst the contra‐lateral limb remained outside the tank and was not exposed to heat stress. Muscle biopsies were obtained from the vastus lateralis of both legs immediately prior to and at 48 h and 7 days post‐heating. Results: The heating protocol induced significant increases (P < 0.05) in rectal (1.5 ± 0.2 °C) and muscle temperature of the heated leg (3.6 ± 0.5 °C). Muscle temperature of the non‐heated limb showed no significant change (P > 0.05) following heating (pre: 36.1 ± 0.5, post: 35.7 ± 0.2 °C). Heating failed to induce a significant increase (P > 0.05) in muscle content of HSP70, HSC70, HSP60, HSP27, αB‐crystallin, MnSOD protein content or in the activity of superoxide dismutase and catalase. Conclusions: These data demonstrate that increases in both systemic and local muscle temperature per se do not appear to mediate the exercise‐induced production of HSPs in human skeletal muscle and suggest that non‐heat stress factors associated with contractile activity are of more importance in mediating this response.     

Up-regulation of inducible heat shock protein-70 expression in multiple sclerosis patients

Abstract

Inducible heat shock protein (HSP)70 (HSP70-1A and HSP70-1B proteins) is a chaperone responsible for assisting proper protein folding. Following stress conditions, HSP70 is highly up-regulated to mediate cytoprotective functions. In addition, HSP70 is able to trigger innate and adaptive immune responses that promote the immune recognition of antigens and to act as a cytokine when it is released. The data in the literature are controversial with regard to expression studies in peripheral blood mononuclear cells (PBMCs). In the present study, we aimed to examine if alterations of HSP70-1A/B expression are involved in the autoimmune pathogenesis of multiple sclerosis (MS). We determined both mRNA and protein expression in PBMCs of MS patients and healthy donors (HDs). We found a baseline increased expression of the HSPA1A gene in PBMCs from MS patients compared with HDs. Gene expression findings were associated with an increased protein expression of HSP70-1A/B in T lymphocytes (CD4+ and CD8+) and monocytes from MS patients under basal conditions that may reflect the immunological activation occurring in MS patients. We also provided evidence that heat shock (HS) stimulus induced HSP70-1A/B protein expression in HDs and MS patients, and that?HS-induced HSP70-1A/B protein expression in monocytes correlated with the number of T2 lesions at baseline in MS patients. However, after lipopolysaccharide inflammatory stimulus, monocytes from MS patients failed to induce HSP70-1A/B protein expression. Our data hint at altered immune responses in MS and may indicate either a state of chronic stress or increased vulnerability to physiological immune responses in MS patients.     

Use of multivariate analysis to suggest a new molecular classification of colorectal cancer

Molecular classification of colorectal cancer (CRC) is currently based on microsatellite instability (MSI), KRAS or BRAF mutation and, occasionally, chromosomal instability (CIN). Whilst useful, these categories may not fully represent the underlying molecular subgroups. We screened 906 stage II/III CRCs from the VICTOR clinical trial for somatic mutations. Multivariate analyses (logistic regression, clustering, Bayesian networks) identified the primary molecular associations. Positive associations occurred between: CIN and TP53 mutation; MSI and BRAF mutation; and KRAS and PIK3CA mutations. Negative associations occurred between: MSI and CIN; MSI and NRAS mutation; and KRAS mutation, and each of NRAS, TP53 and BRAF mutations. Some complex relationships were elucidated: KRAS and TP53 mutations had both a direct negative association and a weaker, confounding, positive association via TP53–CIN–MSI–BRAF–KRAS. Our results suggested a new molecular classification of CRCs: (1) MSI⁺ and/or BRAF‐mutant; (2) CIN⁺ and/or TP53^– mutant, with wild‐type KRAS and PIK3CA; (3) KRAS‐ and/or PIK3CA‐mutant, CIN⁺, TP53‐wild‐type; (4) KRAS^– and/or PIK3CA‐mutant, CIN^–, TP53‐wild‐type; (5) NRAS‐mutant; (6) no mutations; (7) others. As expected, group 1 cancers were mostly proximal and poorly differentiated, usually occurring in women. Unexpectedly, two different types of CIN⁺ CRC were found: group 2 cancers were usually distal and occurred in men, whereas group 3 showed neither of these associations but were of higher stage. CIN⁺ cancers have conventionally been associated with all three of these variables, because they have been tested en masse. Our classification also showed potentially improved prognostic capabilities, with group 3, and possibly group 1, independently predicting disease‐free survival. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Stimulation of HSP72 expression following ATP depletion and short‐term exercise training in fast‐twitch muscle

Aim: Previous data have reported increases in HSP72 expression in skeletal muscles after endurance training but the physiological and biochemical signals that induce HSP72 accumulation remain largely unknown. In this study, we tested the hypothesis that energy status is a key regulatory event for HSP72 accumulation in skeletal muscles. Methods: Reduction of high‐energy phosphate levels was induced by supplementation with a creatine analogue, beta‐guanidinopropionic acid (GPA) for 3 weeks while control rats received distilled water in the same conditions. Half of the animals were kept sedentary while the others were submitted to a short‐term (2 weeks) training program on a treadmill (30 m min^?1, 0% slope; 50–70 min day^?1). Results: GPA supplementation resulted in a large drop (～50%) in adenosine triphosphate (ATP) level in both fast and slow muscles whether the animals were trained or remained sedentary. HSP72 level did not change with GPA alone, but the training‐induced increase in HSP72 level was strongly enhanced by superimposition of GPA diet in fast but not in slow skeletal muscles. The changes in HSP72 level were not linked to changes in fibre typology and/or mitochondrial capacities. Conclusions: The results of the present investigation indicate that levels of high‐energy phosphate per se do not play a direct role in determining HSP72 level in skeletal muscles. However, during superimposition of training to GPA, then the adaptive strategy of fast‐twitch muscle (e.g. plantaris) seems to be directed towards appearance of some properties of red, oxidative fibres (increase in oxidative capacities and HSP72 level).     

Impaired removal of Vβ8+ lymphocytes aggravates colitis in mice deficient for B cell lymphoma‐2‐interacting mediator of cell death (Bim)

We investigated the role of B cell lymphoma (BCL)‐2‐interacting mediator of cell death (Bim) for lymphocyte homeostasis in intestinal mucosa. Lymphocytes lacking Bim are refractory to apoptosis. Chronic colitis was induced in Bim‐deficient mice (Bim^–/–) with dextran sulphate sodium (DSS). Weight loss and colonoscopic score were increased significantly in Bim^–/– mice compared to wild‐type mice. As Bim is induced for the killing of autoreactive cells we determined the role of Bim in the regulation of lymphocyte survival at mucosal sites. Upon chronic dextran sulphate sodium (DSS)‐induced colitis, Bim^–/– animals exhibited an increased infiltrate of lymphocytes into the mucosa compared to wild‐type mice. The number of autoreactive T cell receptor (TCR) Vβ8⁺ lymphocytes was significantly higher in Bim^–/– mice compared to wild‐type controls. Impaired removal of autoreactive lymphocytes in Bim^–/– mice upon chronic DSS‐induced colitis may therefore contribute to aggravated mucosal inflammation.     

Major improvement in the detection of microsatellite instability in colorectal cancer using HSP110 T17 E‐ice‐COLD‐PCR

Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI) to screen for Lynch syndrome. Evaluation of MSI status involves screening tumor DNA for the presence of somatic deletions in DNA repeats using PCR followed by fragment analysis. While this method may lack sensitivity due to the presence of a high level of germline DNA, which frequently contaminates the core of primary colon tumors, no other method developed to date is capable of modifying the standard PCR protocol to achieve improvement of MSI detection. Here, we describe a new approach developed for the ultra‐sensitive detection of MSI in CRC based on E‐ice‐COLD‐PCR, using HSP110 T17, a mononucleotide DNA repeat previously proposed as an optimal marker to detect MSI in tumor DNA, and an oligo(dT)₁₆ LNA blocker probe complementary to wild‐type genotypes. The HT17 E‐ice‐COLD‐PCR assay improved MSI detection by 20–200‐fold compared with standard PCR using HT17 alone. It presents an analytical sensitivity of 0.1%–0.05% of mutant alleles in wild‐type background, thus greatly improving MSI detection in CRC samples highly contaminated with normal DNA. HT17 E‐ice‐COLD‐PCR is a rapid, cost‐effective, easy‐to‐implement, and highly sensitive method, which could significantly improve the detection of MSI in routine clinical testing.     

Clear cell renal cell carcinoma with wild‐type von Hippel‐Lindau gene: a non‐existent or new tumour entity?

The current World Health Organisation (WHO) classification of renal tumours is based on characteristic histological features or specific molecular alterations. von Hippel‐Lindau (VHL) alteration is the hallmark of clear cell renal cell carcinoma (RCC). After identification of the MiT translocation family of tumours, clear cell papillary renal cancer and others, the group of ccRCC with wild‐type VHL is small. TCEB1 mutation combined with chromosome 8q loss is an emerging tumour entity with wild‐type VHL. Inactivation of TCEB1 increases HIF stabilisation via the same mechanism as VHL inactivation. Importantly, recent molecular analyses suggest the existence of another ‘VHL wild‐type’ evolutionary subtype of clear cell RCC in addition to TCEB1 mutated RCC and clear cell papillary renal cancer. These tumours are characterised by an aggressive behaviour, high tumour cell proliferation rate, elevated chromosomal instability and frequent presence of sarcomatoid differentiation. Future clinicopathological studies will have to provide data to determine whether TCEB1 tumours and clear cell RCC with wild‐type VHL are separate tumour entities or represent variants of a clear cell RCC tumour family.     

Cytoskeletal proteins bound to heat‐shock protein 70 may elicit resistance to simian immunodeficiency virus infection of CD4+ T cells

This study is based on the evidence that immunization of macaques with human CD4⁺ T cells elicits prevention of simian immunodeficiency virus (SIV) infection. We hypothesized that heat‐shock protein 70 (HSP70) isolated from CD4⁺ T cells may act as a chaperone and carry the protective host proteins. Two moieties of HSP70 were affinity‐purified from human CD4⁺ T cells; an ADP preparation with HSP70‐bound proteins (ADP‐HSP) and an ATP control preparation. Immunization of rhesus macaques with these preparations showed significant inhibition of SIVmac251 infectivity ex vivo in CD4⁺ T cells only with the ADP‐HSP (P = 0·01). Proteomic analysis identified three cytoskeletal elements, cofilin, profilin and γ‐actin, exclusively in the ADP‐HSP preparation. Investigation of the mechanism of prevention of SIV replication suggests that antibodies to the cytoskeletal proteins may inhibit actin depolymerization and facilitate viral degradation by the innate antiviral APOBEC3G. As cytoskeletal proteins are critical in the formation of virological and immunological synapses, finding specific antibodies and anti‐SIV/human immunodeficiency virus (HIV) factors suggests a novel insight into HIV‐1 immunopathogenesis.     

Mice lacking α1,3‐fucosyltransferase 9 exhibit modulation of in vivo immune responses against pathogens

Carbohydrate structures, including Lewis X (Le^x), which is not synthesized in mutant mice that lack α1,3‐fucosyltransferase 9 (Fut9^?/?), are involved in cell–cell recognition and inflammation. However, immunological alteration in Fut9^?/? mice has not been studied. Thus, the inflammatory response of Fut9^?/? mice was examined using the highly neurovirulent mouse hepatitis virus (MHV) JHMV srr7 strain. Pathological study revealed that inflammation induced in the brains of Fut9^?/? mice after infection was more extensive compared with that of wild‐type mice, although viral titers obtained from the brains of mutant mice were lower than those of wild‐type mice. Furthermore, the reduction in cell numbers in the spleens of wild‐type mice after infection was not observed in the infected Fut9^?/? mice. Although there were no clear differences in the levels of cytokines examined in the brains between Fut9^?/? and wild‐type mice except for interferon‐β (IFN‐β) expression, some of those in the spleens, including interferon‐γ (IFN‐γ), interleukin‐6 (IL‐6), and monocyte chemoattractant protein‐1 (MCP‐1), showed higher levels in Fut9^?/? than in wild‐type mice. Furthermore, Fut9^?/? mice were refractory to the in vivo inoculation of endotoxin (LPS) compared with wild‐type mice. These results indicate that Le^x structures are involved in host responses against viral or bacterial challenges.     

Near universal detection of alterations in CTNNB1 and Wnt pathway regulators in desmoid‐type fibromatosis by whole‐exome sequencing and genomic analysis

CTNNB1 mutations or APC abnormalities have been observed in ～85% of desmoids examined by Sanger sequencing and are associated with Wnt/β‐catenin activation. We sought to identify molecular aberrations in “wild‐type” tumors (those without CTNNB1 or APC alteration) and to determine their prognostic relevance. CTNNB1 was examined by Sanger sequencing in 117 desmoids; a mutation was observed in 101 (86%) and 16 were wild type. Wild‐type status did not associate with tumor recurrence. Moreover, in unsupervised clustering based on U133A‐derived gene expression profiles, wild‐type and mutated tumors clustered together. Whole‐exome sequencing of eight of the wild‐type desmoids revealed that three had a CTNNB1 mutation that had been undetected by Sanger sequencing. The mutation was found in a mean 16% of reads (vs. 37% for mutations identified by Sanger). Of the other five wild‐type tumors sequenced, two had APC loss, two had chromosome 6 loss, and one had mutation of BMI1. The finding of low‐frequency CTNNB1 mutation or APC loss in wild‐type desmoids was validated in the remaining eight wild‐type desmoids; directed miSeq identified low‐frequency CTNNB1 mutation in four and comparative genomic hybridization identified APC loss in one. These results demonstrate that mutations affecting CTNNB1 or APC occur more frequently in desmoids than previously recognized (111 of 117; 95%), and designation of wild‐type genotype is largely determined by sensitivity of detection methods. Even true CTNNB1 wild‐type tumors (determined by next‐generation sequencing) may have genomic alterations associated with Wnt activation (chromosome 6 loss/BMI1 mutation), supporting Wnt/β‐catenin activation as the common pathway governing desmoid initiation. © 2015 Wiley Periodicals, Inc.