Identification of three novel ECEL1 mutations in three families with distal arthrogryposis type 5D

Arthrogryposis refers to congenital contracture in at least two different body parts. When distal joints are primarily involved, the term distal arthrogryposis (DA) is used. The recognition of clinically distinct subtypes of DA has proven very useful in mapping the disease genes for this genetically heterogeneous condition. DA5D is characterized by ocular involvement usually in the form of ptosis and incomitant strabismus, but extraocular manifestations have also been reported. In a multiplex consanguineous family with DA5D, we combined autozygosity mapping and exome sequencing to identify a novel mutation in ECEL1. This was followed by targeted sequencing of this gene in another two extended consanguineous family with the same phenotype, which revealed two additional novel homozygous mutations. Our results support the recent identification of mutations in ECEL1 as a disease gene in DA5D and expand the clinical and allelic spectrum of this condition.     

A novel ECEL1 mutation expands the phenotype of distal arthrogryposis multiplex congenita type 5D to include pretibial vertical skin creases

Arthrogryposis multiplex congenita (AMC) is a heterogeneous disorder characterized by multiple joint contractures often in association with other congenital abnormalities. Pretibial linear vertical creases are a rare finding associated with arthrogryposis, and the etiology of the specific condition is unknown. We aimed to genetically and clinically characterize a boy from a consanguineous family, presenting with AMC and pretibial vertical linear creases on the shins. Whole exome sequencing and variant analysis revealed homozygous novel missense variants of ECEL1 (c.1163T > C, p.Leu388Pro, NM_004826) and MUSK (c.2572C > T, p.Arg858Cys, NM_005592). Both variants are predicted to have deleterious effects on the protein function, with amino acid positions highly conserved among species. The variants segregated in the family, with healthy mother, father, and sister being heterozygous carriers and the index patient being homozygous for both mutations. We report on a unique patient with a novel ECEL1 homozygous mutation, expanding the phenotypic spectrum of Distal AMC Type 5D to include vertical linear skin creases. The homozygous mutation in MUSK is of unknown clinical significance. MUSK mutations have previously shown to cause congenital myasthenic syndrome, a neuromuscular disorder with defects in the neuromuscular junction.     

The stimulatory effect of α‐melanotropin on progesterone release from rat granulosa cells is inhibited by interleukin‐1β and by tumour necrosis factor‐α

Aim: Several studies have shown that a variety of peptides and cytokines are involved in ovarian regulatory mechanisms; however, their exact function is still unclear. In this work we study whether the administration of peptide α‐melanotropin and the cytokines interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) on their own modify the release of progesterone in cultured granulosa cells (GC) from pro‐oestrous rats. We also investigate an interaction between these cytokines and α‐melanotropin in the modulation of progesterone secretion. Methods: Granulosa cells were collected from the ovaries of female Wistar rats and cultured for up to 24 h in the presence of different concentrations of α‐melanotropin, cytokines or a combination of both. Progesterone concentration was measured by radioimmunoassay. Results: The addition of α‐melanotropin in a dose of 0.01 and 0.1 mm had no effect on progesterone release, whereas a dose of 1 mm significantly increased progesterone release (P < 0.01) compared with the control culture. Progesterone release was not modified when different concentrations of interleukin‐1β or TNF‐α were added to the cell cultures. However, when interleukin‐1β or TNF‐α were added simultaneously with 1 μm α‐melanotropin, a significant reduction (P < 0.01 for interleukin‐1β and P < 0.05 for TNF‐α) of the steroid release was found with respect to the α‐melanotropin‐treated group. Conclusions: These results lead us to suggest that, although α‐melanotropin stimulates progesterone release in pre‐ovulatory GC, this effect is blocked by the presence of interleukin‐1β or TNF‐α.     

Gut immune deficits in LEW.1AR1‐iddm rats partially overcome by feeding a diabetes‐protective diet

The gut immune system and its modification by diet have been implicated in the pathogenesis of type 1 diabetes (T1D). Therefore, we investigated gut immune status in non‐diabetes‐prone LEW.1AR1 and diabetes‐prone LEW.1AR1‐iddm rats and evaluated the effect of a low antigen, hydrolysed casein (HC)‐based diet on gut immunity and T1D. Rats were weaned onto a cereal‐based or HC‐based diet and monitored for T1D. Strain and dietary effects on immune homeostasis were assessed in non‐diabetic rats (50–60 days old) and rats with recent‐onset diabetes using flow cytometry and immunohistochemistry. Immune gene expression was analysed in mesenteric lymph nodes (MLN) and jejunum using quantitative RT‐PCR and PCR arrays. T1D was prevented in LEW.1AR1‐iddm rats by feeding an HC diet. Diabetic LEW.1AR1‐iddm rats had fewer lymphoid tissue T cells compared with LEW.1AR1 rats. The percentage of CD4⁺ Foxp3⁺ regulatory T (Treg) cells was decreased in pancreatic lymph nodes (PLN) of diabetic rats. The jejunum of 50‐day LEW.1AR1‐iddm rats contained fewer CD3⁺ T cells, CD163⁺ M2 macrophages and Foxp3⁺ Treg cells. Ifng expression was increased in MLN and Foxp3 expression was decreased in the jejunum of LEW.1AR1‐iddm rats; Ifng/Il4 was decreased in jejunum of LEW.1AR1‐iddm rats fed HC. PCR arrays revealed decreased expression of M2‐associated macrophage factors in 50‐day LEW.1AR1‐iddm rats. Wheat peptides stimulated T‐cell proliferation and activation in MLN and PLN cells from diabetic LEW.1AR1‐iddm rats. LEW.1AR1‐iddm rats displayed gut immune cell deficits and decreased immunoregulatory capacity, which were partially corrected in animals fed a low antigen, protective HC diet consistent with other models of T1D.     

Altered BAFF‐receptor signaling and additional modifier loci contribute to systemic autoimmunity in A/WySnJ mice

Systemic lupus erythematosus pathology reflects autoantibody‐mediated damage due to a failure of B‐lymphocyte tolerance. We previously reported that B‐lymphopenic A/WySnJ mice develop a lupus‐like syndrome and linked this syndrome to the B‐cell maturation defect‐1 (Bcmd‐1) mutant allele of the B‐cell‐activating factor belonging to the TNF family‐receptor (Baffr) gene. Here, we further evaluate the genetic basis for autoimmunity in A/WySnJ mice. We produced B6.Bcmd‐1 and AW.Baffr^?/? congenic mice (N5), and compared them with B6.Baffr^?/? and A/WySnJ mice with respect to B‐lymphocyte development. Bcmd‐1‐expressing mice had more B cells with greater maturity than Baffr^?/? mice regardless of genetic background, indicating that Bcmd‐1 encodes a partially functional BAFF‐R. We also compared these mice for lupus phenotypes to determine whether Bcmd‐1 is necessary and sufficient for disease, or whether the Baffr^?/?‐ allele can also cause autoimmunity. The Baffr^?/? allele did not lead to autoimmunity on either genetic background. In contrast, the Bcmd‐1 allele was necessary and sufficient for development of low levels of IgM autoantibodies in B6.Bcmd‐1 mice. However, Bcmd‐1 plus unidentified A/WySnJ modifier genes were necessary for development of IgG autoantibodies and renal pathology. We propose that in A/WySnJ mice an excess of BAFF per B cell rescues self‐reactive B cells through a partially functional BAFF‐R in a B‐lymphopenic environment.     

SFPQ‐ABL1‐positive B‐cell precursor acute lymphoblastic leukemias

In recent years, a subgroup of B‐cell precursor acute lymphoblastic leukemia (BCP ALL) without an established abnormality (“B‐other”) has been shown to be characterized by rearrangements of ABL1, ABL2, CSF1R, or PDGFRB (a.k.a. ABL‐class genes). Using FISH with probes for these genes, we screened 55 pediatric and 50 adult B‐other cases. Three (6%) of the adult but none of the childhood B‐other cases were positive for ABL‐class aberrations. RT‐PCR and sequencing confirmed a rare SFPQ‐ABL1 fusion in one adult B‐other case with t(1;9)(p34;q34). Only six SFPQ‐ABL1‐positive BCP ALLs have been reported, present case included. A review of these shows that all harbored fusions between exon 9 of SFPQ and exon 4 of ABL1, that the fusion is typically found in adolescents/younger adults without hyperleukocytosis, and that IKZF1 deletions are recurrent. The few patients not treated with tyrosine kinase inhibitors (TKIs) and/or allogeneic stem cell transplantation relapsed, strengthening the notion that TKI should be added to the therapy of SFPQ‐ABL1‐positive BCP ALL.     

CD274 (PDL1) and JAK2 genomic amplifications in pulmonary squamous‐cell and adenocarcinoma patients

Aims

CD274 (PDL1) and JAK2 (9p24.1) gene amplifications have been recently described in pulmonary carcinomas in association with programmed death‐ligand 1 (PD‐L1) expression. Furthermore, PTEN loss has been explored preclinically in relation to PD‐L1 expression. Our aim was to determine whether these genomic alterations affect PD‐L1 expression levels in non‐small‐cell lung cancer.

Methods and results

PD‐L1 and PTEN expression determined by immunohistochemistry (IHC), and CD274, JAK2 and PTEN copy number alterations (CNAs) determined by fluorescence in‐situ hybridisation, were studied in 171 pulmonary carcinoma specimens. PD‐L1 expression was positive in 40 cases (23.3%), and CD274 amplification was present in 14 tumours (8.8%). Concordance between both events was found in 12 of 14 amplified cases (P = 0.0001). We found nine JAK2‐amplified cases (5.7%), seven with PD‐L1 expression (P = 0.0006). Moreover, six of the seven cases had JAK2 and CD274 coamplification (9p24.1 genomic amplification). Remarkably, the average PD‐L1 IHC score was higher in these amplified cases (230 versus 80; P = 0.001). Non‐statistical associations were observed between PD‐L1 expression and PTEN loss and PTEN deletions.

Conclusions

We describe a subset of patients (8.2%) who had 9p24.1 amplifications resulting in high expression of PD‐L1. Our results provide evidence for genomic up‐regulation of PD‐L1 expression in non‐small‐cell lung cancer.     

Neuronally expressed Ras-family GTPase Di-Ras modulates synaptic activity in Caenorhabditis elegans

Ras‐family GTPases regulate a wide variety of cellular functions including cell growth and differentiation. Di‐Ras, which belongs to a distinct subfamily of Ras‐family GTPases, is expressed predominantly in brain, but the role of Di‐Ras in nervous systems remains totally unknown. Here, we report that the Caenorhabditis elegans Di‐Ras homologue drn‐1 is expressed specifically in neuronal cells and involved in synaptic function at neuromuscular junctions. Loss of function of drn‐1 conferred resistance to the acetylcholinesterase inhibitor aldicarb and partially suppressed the aldicarb‐hypersensitive phenotypes of heterotrimeric G‐protein mutants, in which acetylcholine release is up‐regulated. drn‐1 mutants displayed no apparent defects in the axonal distribution of the membrane‐bound second messenger diacylglycerol (DAG), which is a key stimulator of acetylcholine release. Finally, we have identified EPAC‐1, a C. elegans Epac homologue, as a binding partner for DRN‐1. Deletion mutants of epac‐1 displayed an aldicarb‐resistant phenotype as drn‐1 mutants. Genetic analysis of drn‐1 and epac‐1 showed that they acted in the same pathway to control acetylcholine release. Furthermore, DRN‐1 and EPAC‐1 were co‐immunoprecipitated. These findings suggest that DRN‐1 may function cooperatively with EPAC‐1 to modulate synaptic activity in C. elegans.     

NF1 microdeletions in neurofibromatosis type 1: from genotype to phenotype

In 5‐10% of patients, neurofibromatosis type 1 (NF1) results from microdeletions that encompass the entire NF1 gene and a variable number of flanking genes. Two recurrent microdeletion types are found in most cases, with microdeletion breakpoints located in paralogous regions flanking NF1 (proximal NF1‐REP‐a and distal NF1‐REP–c for the 1.4 Mb type‐1 microdeletion, and SUZ12 and SUZ12P for the 1.2 Mb type‐2 microdeletion). A more severe phenotype is usually associated with NF1 microdeletion patients than in those with intragenic mutations. We characterized NF1 microdeletions in 70 unrelated NF1 microdeleted patients using a high‐resolution NF1 custom array comparative genomic hybridization (CGH). Genotype‐phenotype correlations were studied in 58 of these microdeletion patients and compared to 389 patients with intragenic truncating NF1 mutations and phenotyped in the same standardized way. Our results confirmed in an unbiased manner the existence of a contiguous gene syndrome with a significantly higher incidence of learning disabilities and facial dysmorphism in microdeleted patients compared to patients with intragenic NF1 mutations. Microdeleted NF1 patients also showed a trend toward significance for childhood overgrowth. High‐resolution array‐CGH identified a new recurrent ～1.0 Mb microdeletion type, designated as type‐3, with breakpoints in the paralogous regions middle NF1‐REP‐b and distal NF1‐REP–c. © 2010 Wiley‐Liss, Inc.     

Both plasmacytoid dendritic cells and monocytes stimulate natural killer cells early during human herpes simplex virus type 1 infections

Herpes simplex virus type 1 (HSV‐1), a member of the herpes virus family, is characterized by a short replication cycle, high cytopathogenicity and distinct neurotropism. Primary infection and reactivation may cause severe diseases in immunocompetent and immunosuppressed individuals. This study investigated the role of human plasmacytoid dendritic cells (pDC) in the activation of natural killer (NK) cells for the control of herpesviral infections. Within peripheral blood mononuclear cells, UV‐inactivated HSV‐1 and CpG‐A induced CD69 up‐regulation on NK cells, whereas infectious HSV‐1 was particularly active in inducing NK cell effector functions interferon‐γ (IFN‐γ) secretion and degranulation. The pDC‐derived IFN‐α significantly contributed to NK cell activation, as evident from neutralization and cell depletion experiments. In addition, monocyte‐derived tumour necrosis factor‐α (TNF‐α) induced after exposure to infectious HSV‐1 was found to stimulate IFN‐γ secretion. A minority of monocytes was shown to be non‐productively infected in experiments using fluorescently labelled viruses and quantitative PCR analyses. HSV‐1‐exposed monocytes up‐regulated classical HLA‐ABC and non‐classical HLA‐E molecules at the cell surface in an IFN‐α‐dependent manner, whereas stress molecules MICA/B were not induced. Notably, depletion of monocytes reduced NK cell effector functions induced by infectious HSV‐1 (P < 0·05). Altogether, our data suggest a model in which HSV‐1‐stimulated pDC and monocytes activate NK cells via secretion of IFN‐α and TNF‐α. In addition, infection of monocytes induces NK cell effector functions via TNF‐α‐dependent and TNF‐α‐independent mechanisms. Hence, pDC and monocytes, which are among the first cells infiltrating herpetic lesions, appear to have important bystander functions for NK cells to control these viral infections.     

sTREM‐1 in patients with chronic kidney disease on hemodialysis

The triggering receptor expressed on myeloid cells‐1 (TREM‐1) is a member of the immunoglobulin superfamily. TREM‐1 has been implicated as an amplifier of inflammation. Soluble TREM‐1 (sTREM‐1) was investigated in different clinical conditions, but not in hemodialysis (HD) patients. We aimed to investigate sTREM‐1 as a marker of inflammation in HD patients. We investigated 40 CKD patients undergoing chronic HD treatment and 15 controls. Routine laboratory investigations in addition to CRP measured by immunoturbidimetry, TNF‐ α, and sTREM‐1 measured by ELISA were assayed in post–hemodialysis patients’ blood samples and in controls’ blood samples. CRP, TNF‐α, and sTREM‐1 levels were significantly higher in HD patients than in controls (p < 0.001 for all). sTREM‐1 was positively correlated with CRP and TNF‐α (r = +0.50, p < 0.001 and r = +0.53, p < 0.001 respectively). It was negatively correlated with hemoglobin concentration (r = ?0.69, p < 0.001). Hemoglobin concentration was the significant predictor of sTREM‐1 level (p < 0.001). In conclusion, sTREM‐1 level is significantly increased in HD patients as are other pro‐inflammatory markers.     

Copy number alterations determined by single nucleotide polymorphism array testing in the clinical laboratory are indicative of gene fusions in pediatric cancer patients

Gene fusions resulting from structural rearrangements are an established mechanism of tumorigenesis in pediatric cancer. In this clinical cohort, 1,350 single nucleotide polymorphism (SNP)‐based chromosomal microarrays from 1,211 pediatric cancer patients were evaluated for copy number alterations (CNAs) associated with gene fusions. Karyotype or fluorescence in situ hybridization studies were performed in 42% of the patients. Ten percent of the bone marrow or solid tumor specimens had SNP array‐associated CNAs suggestive of a gene fusion. Alterations involving ETV6, ABL1‐NUP214, EBF1‐PDGFRB, KMT2A(MLL), LMO2‐RAG, MYH11‐CBFB, NSD1‐NUP98, PBX1, STIL‐TAL1, ZNF384‐TCF3, P2RY8‐CRLF2, and RUNX1T1‐RUNX1 fusions were detected in the bone marrow samples. The most common alteration among the low‐grade gliomas was a 7q34 tandem duplication resulting in a KIAA1549‐BRAF fusion. Additional fusions identified in the pediatric brain tumors included FAM131B‐BRAF and RAF1‐QKI. COL1A1‐PDGFB, CRTC1‐MAML2, EWSR1, HEY1, PAX3‐ and PAX7‐FOXO1, and PLAG1 fusions were determined in a variety of solid tumors and a novel potential gene fusion, FGFR1‐USP6, was detected in an aneurysmal bone cyst. The identification of these gene fusions was instrumental in tumor diagnosis. In contrast to hematologic and solid tumors in adults that are predominantly driven by mutations, the majority of hematologic and solid tumors in children are characterized by CNAs and gene fusions. Chromosomal microarray analysis is therefore a robust platform to identify diagnostic and prognostic markers in the clinical setting.     

Genetic analysis of the interleukin‐1 receptor antagonist and its homologue IL‐1L1 in alopecia areata: strong severity association and possible gene interaction‡

Alopecia areata is an inflammatory hair loss disease with a major genetic component. The presence of focal inflammatory lesions with perifollicular T‐cell infiltrates reflects the importance of local cytokine production in the pathogenesis. In addition to its fundamental pro‐inflammatory role, the interleukin‐1 (IL‐1) system has major effects on hair growth regulation in vitro, with the inhibitory actions of IL‐1α and IL‐1β being opposed by the receptor antagonist IL‐1ra. The novel interleukin‐1 like molecule 1 (IL‐1L1) which has greatest gene sequence homology with IL1RN, the gene encoding IL‐1ra, is another potential IL‐1 antagonist. In view of previous studies suggesting a significant role for IL1RN polymorphisms in the pathogenesis of autoimmune/inflammatory disease, we have analysed polymorphisms of IL‐1ra (IL1RN+2018) and its homologue IL‐1L1 (IL1L1+4734) in a case–control association study on 165 patients and a large number of matched controls. Homozygosity for the rare allele of IL1RN (IL1RN*2) was significantly associated with alopecia areata [odds ratio (OR) = 1.89, 95% CI (1.09, 3.28); P = 0.02], confirming our previous findings of significant association with the IL1RN variable number tandem repeat (VNTR). The results also revealed a novel association involving a polymorphism of the interleukin‐1 receptor antagonist homologue IL1L1 at position + 4734, IL1RN+2018, and alopecia areata. The effect of a genotype combining three copies of the rare alleles at the IL1RN and IL1L1 loci conferred a more than additive increase in the risk of disease compared to IL1RN+2018 or IL1L1+4734 alone [OR 3.37 (1.60, 7.06); P = 0.002], suggesting possible synergy between the IL1RN and IL1L1 genes. This effect was stronger in patients with severe disease (alopecia totalis/universalis) [OR 4.62 (1.87, 11.40), P = 0.0022], and in those with early age at onset (< 20 years) [OR = 6.38 (2.64, 15.42), P = 0.0002]. Our results suggest that these polymorphisms within IL1RN and IL1L1 themselves or a gene in linkage disequilibrium with IL1RN and IL1L1 predispose to the more severe forms of alopecia areata.     

Integrons, β‐lactamase and qnr genes in multidrug resistant clinical isolates of Proteus mirabilis and P. vulgaris

Thirty‐three isolates of Proteus mirabilis and two P. vulgaris were examined for their antimicrobial resistance, the presence of integrons with regard to gene cassette content, and genetic determinants of β‐lactam and low‐level quinolone resistance. Integrons were detected in 23 (69.7%) P. mirabilis isolates; six (18.2%) of them had class 1 integrons, 11 (33.3%) possessed class 2 integrons and six (18.2%) carried integrons of both classes. One P. vulgaris strain possessed class 1 and class 2 integrons. The presence of integrons was associated with increased frequency of resistance to gentamicin, ciprofloxacin, sulfamethoxazole and co‐trimoxazole. Moreover, integron presence was associated with increased resistance range in terms of both the number of antimicrobials and the number of classes of antimicrobials to which a strain was resistant. Class 1 integrons contained aadA1, aadB‐aadA1, dfrA1‐aadA1, bla_PSE‐1‐aadA1 and aacA4‐orfA‐orfB‐aadA1 gene cassette arrays, whereas all class 2 integrons had a dfrA1‐sat2‐aada1 array. β‐lactamase genes not associated with integrons comprised bla_TEM‐2, bla_DHA‐1 and bla_CMY‐15. Plasmid‐mediated fluoroquinolone resistance was determined by qnrD and qnrS1 genes. This is the first report of P. vulgaris strains harbouring qnrD genes in Europe.