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Primary bone diffuse large B-cell lymphoma: a retrospective evaluation on 76 cases from French institutional and LYSA studies

Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is a rare DLBCL location variant. We treated 76 PB-DLBCL patients by immuno-chemotherapy, resulting in an 84% sustained complete remission rate and a 78.9% survival over a 4.7-year median follow-up period. Ann Arbor stage IV and high age-adjusted international prognostic index were predictive of adverse outcome in univariate analysis. In multivariate analysis using a Cox model, only aa-IPI predicted long-term survival. While based on a limited number of cases, we suggested that radiotherapy may be useful as a consolidation modality in PB-DLBCL. We also suggested that positron emission tomography/CT scan should be interpreted with caution due to a persistent [18F]fluorodeoxyglucose [18FDG] uptake of bone lesions even after remission in some in PB-DLBCL patients. Our study based on a homogeneous cohort of PB-DLBCL patients confirmed the favorable outcome of this DLBCL variant and support the implementation of prospective clinical trials in this disease

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.This study was supported by the CNRS (Centre National de la Recherche Scientifique), the Brittany Region (France) (PhD funding for JP), the European Commission (FP7-LUPA, GA-201370), the Rosembloom Family and the Companion animal health fund from the Faculté de Médecine Vétérinaire, Université de Montréal, and the CRB-Anim infrastructure, ANR-11-INBS-0003, funded by the French National Research Agency in the frame of the ‘Investing for the Future’ program.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.This study was supported by the CNRS (Centre National de la Recherche Scientifique), the Brittany Region (France) (PhD funding for JP), the European Commission (FP7-LUPA, GA-201370), the Rosembloom Family and the Companion animal health fund from the Faculté de Médecine Vétérinaire, Université de Montréal, and the CRB-Anim infrastructure, ANR-11-INBS-0003, funded by the French National Research Agency in the frame of the ‘Investing for the Future’ program.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Summary of the SNPs found in the targeted sequencing.

<p>Only variants that are homozygous for a rare allele in all affected dogs and not homozygous in controls were considered to perfectly segregate. Nb SNPs: Number of SNPs; Hom: Nb Homozygous variants; HTZ: Nb Heterozygous variants.</p

The AMS variant affect binding of nuclear complex.

<p>EMSAs were performed with wild type (WT) or mutated (Mut) duplex and nuclear extract (NE). (A) Detection of a mobility shift after incubation of 40 fmoles of radiolabelled WT-duplex (lane 1 to 4) or Mut-duplex (lane 5 to 8) with increasing amounts (0,3–0,6–1,2–2,4 μl) of HeLa NE. (B) 40 fmoles of radiolabelled WT-duplex were incubated without (lane 9 and 13) or with 1μl of HeLa NE (lane 10 to 12) or 5 μl of SY5Y (lane 14 to 16) NE and in the presence of 2 pmol of competitor WT-duplex (lane 11 and 15) or Mut-duplex (lane 12 and 16). Black bar: specific complex associated with the radioactive duplex, asterisk: non specific complex.</p

qRT-PCR results in Dorsal Root Ganglia using the ΔΔCt methodology (cases in grey, controls in white).

<p>Measures obtained from two DRG by individual were pooled, and we also pooled affected dogs and controls. We used <i>PPIB</i> (<i>Peptidylprolyl Isomerase B</i>) as control and all genes of the locus (<i>GDNF-AS</i>, <i>GDNF</i>, <i>WDR70</i>, <i>NUP155</i>, <i>SLC1A3</i>) were tested 3 times with 3 replicates for each (t-test: p-value *<0.0001).</p

Summary of the variants obtained in the targeted sequencing before and after filtering.

<p>Summary of the variants obtained in the targeted sequencing before and after filtering.</p

Best-SNPs obtained by GWAS using 21 affected <i>vs</i> 28 unaffected French spaniels.

<p>Best-SNPs obtained by GWAS using 21 affected <i>vs</i> 28 unaffected French spaniels.</p