Pierre Chagnon

Specific Pathological Tau Protein Variants Characterize Pick's Disease

Picks disease (PiD) is characterized by a pan-laminar frontotemporal cortical atrophy, widespread degeneration of the white matter, chromatolytic neurons, and Pick bodies (PB). Microtubule-associated Tau proteins are the main cytoskeletal components modified during these neurodegenerative changes. In the present study, pathological alterations of Tau proteins were investigated in the brains of five PiD cases at both neuropathological and biochemical levels, using the monoclonal antibody AD2 which recognizes a phosphorylation-dependent Tau epitope and strongly labeled PB. A large number of cortical and subcortical regions were studied on frozen materials. Tau proteins were analyzed on mono- and two-dimensional gel electrophoreses using a quantitative western blot approach. In all specimens, a 55 and 64 kDa Tau doublet was observed in limbic, frontal, and temporal cortices as well as in striatum and substantia nigra. In contrast, Alzheimers disease (AD) brains are characterized by the presence of the 55, 64, and 69 kDa Tau triplet whereas the 64 and 69 kDa doublet is more typical of progressive supranuclear palsy and corticobasal degeneration. Thus, the 55 and 64 kDa doublet appears to be specific to PiD, less acidic than AD Tau proteins, and well correlated with the presence of PB.

A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia.

In this study, we show the high frequency of spontaneous γδ T-cell leukemia (T-ALL) occurrence in mice with biallelic deletion of enhancer of zeste homolog 2 (Ezh2). Tumor cells show little residual H3K27 trimethylation marks compared with controls. EZH2 is a component of the PRC2 Polycomb group protein complex, which is associated with DNA methyltransferases. Using next-generation sequencing, we identify alteration in gene expression levels of EZH2 and acquired mutations in PRC2-associated genes (DNMT3A and JARID2) in human adult T-ALL. Together, these studies document that deregulation of EZH2 and associated genes leads to the development of mouse, and likely human, T-ALL.

Phylogenetic analysis of the mitochondrial genome indicates significant differences between patients with Alzheimer disease and controls in a French-Canadian founder population

The activity of cytochrome oxidase (CO), the terminal enzyme of the mitochondrial electron transport chain, has been reported to be lower in the brains of Alzheimer disease (AD) patients. This suggests that a modification of mitochondrial DNA (mtDNA) may be responsible for this decrease of CO activity. Many mtDNA variants were found by different studies at a higher frequency in AD patients, suggesting that mtDNA variants could confer a genetic susceptibility to AD. In this study, we sequenced the entire mitochondrial genome region that encompasses the three CO genes and the 22 mitochondrial tRNA in 69 AD patients and 83 age-matched controls. We detected a total of 95 mtDNA variants. The allele frequencies of the majority of these variants were similar in patients and controls. However, a haplotype composed of three different modifications (positions: 5633, 7476, and 15812) was present in three of the 69 late-onset AD patients (4.3%) and also in 1 of 16 early-onset AD patients (6.2%) but not in control individuals. Given that one of these variants (15812) has already been shown to be associated with another neurodegenerative disease and that all three modifications are relatively conserved and their frequencies in the general population is only 0.1%, our data suggest that the presence of this haplotype may represent a risk factor for AD. We also found a significant association (P < 0.05) of two other variants at positions 709 (rRNA 12S) and 15928 (tRNA(Thr)). These two mtDNA variants are three times more frequent in control individuals compared with AD patients, suggesting that they may be protective against AD.

A missense mutation (R565W) in Cirhin (FLJ14728) in North American Indian Childhood Cirrhosis

North American Indian childhood cirrhosis (CIRH1A, or NAIC), a severe autosomal recessive intrahepatic cholestasis described in Ojibway-Cree children from northwestern Quebec, is one of several familial cholestases with unknown molecular etiology. It typically presents with transient neonatal jaundice, in a child who is otherwise healthy, and progresses to biliary cirrhosis and portal hypertension. Clinical and physiological investigations have not revealed the underlying cause of the disease. Currently, liver transplantation is the only effective therapy for patients with advanced disease. We previously identified the NAIC locus by homozygosity mapping to chromosome 16q22. Here we report that an exon 15 mutation in gene FLJ14728 (alias Cirhin) causes NAIC: c.1741C-->T in GenBank cDNA sequence NM_032830, found in all NAIC chromosomes, changes the conserved arginine 565 codon to a tryptophan, altering the predicted secondary structure of the protein. Cirhin is preferentially expressed in embryonic liver, is predicted to localize to mitochondria, and contains WD repeats, which are structural motifs frequently associated with molecular scaffolds.

Prediction of graft-versus-host disease in humans by donor gene-expression profiling.

Background Graft-versus-host disease (GVHD) results from recognition of host antigens by donor T cells following allogeneic hematopoietic cell transplantation (AHCT). Notably, histoincompatibility between donor and recipient is necessary but not sufficient to elicit GVHD. Therefore, we tested the hypothesis that some donors may be “stronger alloresponders” than others, and consequently more likely to elicit GVHD. Methods and Findings To this end, we measured the gene-expression profiles of CD4+ and CD8+ T cells from 50 AHCT donors with microarrays. We report that pre-AHCT gene-expression profiling segregates donors whose recipient suffered from GVHD or not. Using quantitative PCR, established statistical tests, and analysis of multiple independent training-test datasets, we found that for chronic GVHD the “dangerous donor” trait (occurrence of GVHD in the recipient) is under polygenic control and is shaped by the activity of genes that regulate transforming growth factor-β signaling and cell proliferation. Conclusions These findings strongly suggest that the donor gene-expression profile has a dominant influence on the occurrence of GVHD in the recipient. The ability to discriminate strong and weak alloresponders using gene-expression profiling could pave the way to personalized transplantation medicine.

No evidence that skewing of X chromosome inactivation patterns is transmitted to offspring in humans

Skewing of X chromosome inactivation (XCI) can occur in normal females and increases in tissues with age. The mechanisms underlying skewing in normal females, however, remain controversial. To better understand the phenomenon of XCI in nondisease states, we evaluated XCI patterns in epithelial and hematopoietic cells of over 500 healthy female mother-neonate pairs. The incidence of skewing observed in mothers was twice that observed in neonates, and in both cohorts, the incidence of XCI was lower in epithelial cells than hematopoietic cells. These results suggest that XCI incidence varies by tissue type and that age-dependent mechanisms can influence skewing in both epithelial and hematopoietic cells. In both cohorts, a correlation was identified in the direction of skewing in epithelial and hematopoietic cells, suggesting common underlying skewing mechanisms across tissues. However, there was no correlation between the XCI patterns of mothers and their respective neonates, and skewed mothers gave birth to skewed neonates at the same frequency as nonskewed mothers. Taken together, our data suggest that in humans, the XCI pattern observed at birth does not reflect a single heritable genetic locus, but rather corresponds to a complex trait determined, at least in part, by selection biases occurring after XCI.

RNA-seq analysis of 2 closely related leukemia clones that differ in their self-renewal capacity

The molecular mechanisms regulating self-renewal of leukemia stem cells remain poorly understood. Here we report the generation of 2 closely related leukemias created through the retroviral overexpression of Meis1 and Hoxa9. Despite their apparent common origin, these clonal leukemias exhibit enormous differences in stem cell frequency (from 1 in 1.4, FLA2; to 1 in 347, FLB1), suggesting that one of these leukemias undergoes nearly unlimited self-renewal divisions. Using next-generation RNA-sequencing, we characterized the transcriptomes of these phenotypically similar, but biologically distinct, leukemias, identifying hundreds of differentially expressed genes and a large number of structural differences (eg, alternative splicing and promoter usage). Focusing on ligand-receptor pairs, we observed high expression levels of Sdf1-Cxcr4; Jagged2-Notch2/1; Osm-Gp130; Scf-cKit; and Bmp15-Tgfb1/2. Interestingly, the integrin beta 2-like gene (Itgb2l) is both highly expressed and differentially expressed between our 2 leukemias (∼ 14-fold higher in FLA2 than FLB1). In addition, gene ontology analysis indicated G-protein-coupled receptor had a much higher proportion of differential expression (22%) compared with other classes (∼ 5%), suggesting a potential role regulating subtle changes in cellular behavior. These results provide the first comprehensive transcriptome analysis of a leukemia stem cell and document an unexpected level of transcriptome variation between phenotypically similar leukemic cells.

Localization of a Recessive Gene for North American Indian Childhood Cirrhosis to Chromosome Region 16q22—and Identification of a Shared Haplotype

North American Indian childhood cirrhosis (NAIC, or CIRH1A) is an isolated nonsyndromic form of familial cholestasis reported in Ojibway-Cree children and young adults in northwestern Quebec. The pattern of transmission is consistent with an autosomal recessive mode of inheritance. To map the NAIC locus, we performed a genomewide scan on three DNA pools of samples from 13 patients, 16 unaffected siblings, and 22 parents from five families. Analysis of 333 highly polymorphic markers revealed 3 markers with apparent excess allele sharing among affected individuals. Additional mapping identified a chromosome 16q segment shared by all affected individuals. When the program FASTLINK/LINKAGE was used and a completely penetrant autosomal recessive mode of inheritance was assumed, a maximum LOD score of 4.44 was observed for a recombination fraction of 0, with marker D16S3067. A five-marker haplotype (D16S3067, D16S752, D16S2624, D16S3025, and D16S3106) spanning 4.9 cM was shared by all patients. These results provide significant evidence of linkage for a candidate gene on chromosome 16q22.

Overexpression of PRDM16 in the presence and absence of the RUNX1/PRDM16 fusion gene in myeloid leukemias.

In a recent study published in Genes, Chromosomes & Cancer, Sakai et al. (2005) reported the identification of a novel RUNX1 partner gene, MDS1/EVI1-like gene 1 (PRDM16), in a patient with acute myeloid leukemia (AML) M4 with the t(1;21) translocation. This fusion gene has also been described recently in a case of t(1;21) positive therapy-related AML (Stevens-Kroef et al., 2006). The t(1;21)(p36;q22) is a rare but recurrent translocation associated with de novo and therapy-related AML and with myelodysplastic syndromes (MDS). We, here, describe the first case of chronic myeloid leukemia (CML) blast crisis cells in which the RUNX1/PRDM16 fusion gene likely contributed to clonal evolution from CML to AML. A 59-year-old woman was referred in July 2000 for a CML. Cytogenetic analysis showed 46,XX,t(9;22)(q34.1;q11.2) in 20 metaphases (UPN 00-H59). She was treated initially with IFN for 20 months with a minor cytogenetic response and then with imatinib mesylate. After 6 months of imatinib, she developed a blastic transformation with 68% blast cells in the peripheral blood. Cells were obtained after an informed consent. Conventional G-banding karyotype showed a possible deletion at band 21q22 in addition to the t(9;22) in all metaphases. Spectral karyotyping of these cells revealed a t(1;21) translocation in addition to the t(9;22) translocation (UPN 02-H056)(Fig. 1A and 1B). The t(1;21) translocation was not present in chronic phase cells isolated from this patient. Fluorescence in situ hybridization (FISH) experiments were performed on peripheral blood metaphases of the blast phase cells using bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) clones obtained from the BACPAC Resource Center (Children’s Hospital Oakland Research Institute in Oakland, California, http://bacpac.chori.org/ home.htm). Clone physical positions and covered genes are available at the UCSC Human Genome Browser (http://genome.ucsc.edu/). The RUNX1 gene located at band 21q22.12 was first studied using two selected BACs, RP11-771C10 (containing intron 6 to exon 8 and the 30 end of RUNX1) and RP11-299D9 (containing intron 1 to exon 8 and the 30 end of RUNX1). Split signals were observed with BAC RP11-299D9 (Fig. 1C). The candidate gene, PRDM16, located at band 1p36.32 was studied using two PACs, RP1-163G9 (containing the 50 end of PRDM16, exon 1 and intron 1) and RP4-785P20 (containing intron 3 to exon 17 of PRDM16) and the BAC RP11-659J6 (containing part of intron 1, exons 2 and 3, part of intron 3 of PRDM16). A split signal was detected using PAC RP1-163G9, indicating that the breakpoint was in intron 1 of PRDM16 (Fig. 1D). On the basis of the results of the FISH studies, we designed PCR primers covering most exons of RUNX1 in both directions (forward and reverse), and for PRDM16 in exon 1 (forward), exons 2–4 (reverse) to assess the expression of PRDM16/RUNX1 and RUNX1/ PRDM16 fusion products. RT-PCR analysis and sequencing of the amplicons revealed eight RUNX1/PRDM16 transcripts joining RUNX1 exons 5 or 6 and PRDM16 exons 2 or 3. Two RUNX1/ PRDM16 cDNAs represented an open reading frame encoding exons 1–5 or exons 1–6 of the RUNX1 gene containing the RUNT domain fused to exon 2 of PRDM16 that contain the PR domain (Fig. 2). However, the reciprocal PRDM16/RUNX1 fusion transcript was not detected. We analyzed the relative expression levels of PRDM16 using real-time PCR (ABI Prism 7900HT Sequence Detection System) in our CML case at the time of chronic phase (UPN 00-H59) and blast crisis (UPN 02-H056), and we compared PRDM16 transcript levels in this case to a series of 88 other myeloid malignancies without evident karyotypic abnormalities of chromosomal band 1p36.3 (including 4 CML-blast crisis derived cell lines, 6 CML blast crisis samples, 14 MDS and MDS transformed to AML samples, and 68 AML samples of different morphological and cytogenetic groups). The Ct (threshold cycle) values of PRDM16 were normalized to an endogenous control gene (GAPDH)

Hb Montreal II: A Novel Elongated β-Globin Variant Caused by a Frameshift Mutation [β142 (−C)]

We report a novel elongated C-terminal β hemoglobin (Hb) variant caused by a single nucleotide (C) deletion at codon 143 (nucleotide 480 of GenBank entry NM_000518). This deletion leads to the substitution of histidine 143 by threonine, and displaces the β Hb gene stop codon from codon 147 to codon 157. It was identified in a 30-year-old man from Montreal, and called Hb Montreal II. This Hb variant differs from its normal counterpart by 14 residues, the latter 10 being identical to those observed in Hbs Tak, Cranston, Saverne, Trento, and Florida. The patient did not present thalassemic features but had a compensated chronic hemolysis with splenomegaly, red cell inclusion bodies, and a positive Kleihauer test.