Marie-Claude Gingras

Whole-Genome Sequencing for Optimized Patient Management

A disease mutation identified by whole-genome sequencing of twins with dystonia allowed optimization of treatment, resulting in clinical improvements. Guiding Treatment with Genomics Whole-genome sequencing of DNA from patients with different diseases is proving useful for identifying new disease-causing mutations, but can it help physicians make better decisions about treatment options for these patients? A new study by Bainbridge and colleagues suggests that it can. Bainbridge et al. sequenced the complete genomes of a male and female fraternal twin pair, who had been diagnosed 9 years earlier with the movement disorder dopa (3,4-dihydroxyphenylalanine)–responsive dystonia (DRD). This complex disorder is difficult to diagnose and may be mistaken for other movement disorders involving loss of the neurotransmitter dopamine. The standard treatment for DRD is to replace dopamine by providing a dopamine precursor called l-dopa, the drug that is also used to treat the common movement disorder Parkinson disease. When the twins were diagnosed with DRD, they seemed to fit the classic description of DRD and were given l-dopa, which did help to alleviate many of their symptoms. When Bainbridge and colleagues analyzed the full genome sequences of the twins, they were surprised to discover no mutations in the two genes most commonly mutated in DRD. Instead, they pinpointed a mutation in the SPR gene encoding sepiapterin reductase, which synthesizes a cofactor needed for the action of enzymes that make not only dopamine but also the neurotransmitter serotonin. This finding suggested to the authors that supplementing the twin’s current l-dopa treatment with a serotonin precursor, 5-hydroxytryptophan, might provide further improvement in their symptoms. Sure enough, when the twins were given both l-dopa and 5-hydroxytryptophan instead of l-dopa alone, they showed improvement in their symptoms after 1 to 2 weeks, including greater attention in school, better motion and coordination, and reduced hand tremor as evidenced by more legible handwriting. Although this study involved only one twin pair, it does demonstrate how whole-genome sequencing could be applied to glean more detailed information about a patient’s disease, leading to more optimized treatment and a better outcome. Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)–responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins.

Trans-ancestry mutational landscape of hepatocellular carcinoma genomes

Diverse epidemiological factors are associated with hepatocellular carcinoma (HCC) prevalence in different populations. However, the global landscape of the genetic changes in HCC genomes underpinning different epidemiological and ancestral backgrounds still remains uncharted. Here a collection of data from 503 liver cancer genomes from different populations uncovered 30 candidate driver genes and 11 core pathway modules. Furthermore, a collaboration of two large-scale cancer genome projects comparatively analyzed the trans-ancestry substitution signatures in 608 liver cancer cases and identified unique mutational signatures that predominantly contribute to Asian cases. This work elucidates previously unexplored ancestry-associated mutational processes in HCC development. A combination of hotspot TERT promoter mutation, TERT focal amplification and viral genome integration occurs in more than 68% of cases, implicating TERT as a central and ancestry-independent node of hepatocarcinogenesis. Newly identified alterations in genes encoding metabolic enzymes, chromatin remodelers and a high proportion of mTOR pathway activations offer potential therapeutic and diagnostic opportunities.

Comparison of cell adhesion molecule expression between glioblastoma multiforme and autologous normal brain tissue

We investigated glioblastoma multiforme (GBM) for a pattern of consistent alterations in cell adhesion molecules (CAM) expression that might distinguish tumor from normal autologous brain tissue. We used frozen section immunohistochemistry with anti-CAM and computerized image analysis to quantify staining intensity which we expressed as relative intensity units (RIU). Our results showed that normal brain tissue generally did not express alpha 1 beta 1, intercellular CAM-1 (ICAM-1), and sialylated Lewisx, slightly expressed alpha 2, alpha 4, alpha 5, alpha 6 beta 1, alpha v beta 3, lymphocyte function-associated antigen-3 (LFA-3), Lewisx, sialylated LewisLewisx, had a good expression of alpha 3 beta 1 and CD44, and strongly expressed neural CAM (NCAM). GBM expressed alpha 2, alpha 3, alpha 5, alpha 6 beta 1, alpha v beta 3, ICAM-1, LFA-3, CD44, Lewisx, sialylated Lewisx, and sialylated LewisLewisx significantly higher (2-11-fold RIU) than normal brain tissue. ICAM-1 and LFA-3 were the most distinctive markers of GBM. The small blood vessel endothelial cells of the normal brain and the GBM showed a few differences. The tumor endothelium expression of alpha 2 beta 1, alpha 4 beta 1, and LFA-3 RIU appeared twice higher than in normal endothelium and alpha 6 beta 1 showed an average of 40% RIU decrease in comparison to normal. These results show that the expression of several CAM is consistently altered in GBM and its microvasculature when compared with autologous normal brain tissue.

TREM-1, MDL-1, and DAP12 expression is associated with a mature stage of myeloid development

The triggering receptor expressed on myeloid cells (TREM-1) and the myeloid DAP12-associating lectin (MDL-1) are two recently identified receptors which associate non-covalently with DAP12 to form receptor complexes involved in monocytic activation and inflammatory response. In this study, we investigated whether the expression of TREM-1, MDL-1, and DAP12 correlated with myelomonocytic differentiation. Northern and RT-PCR revealed a strong expression of TREM-1, MDL-1, and DAP12 in peripheral blood-derived CD14(+) mature monocytes in contrast to undifferentiated bone marrow CD34(+) stem cells, and in the differentiated versus undifferentiated U937 cells. TREM-1 and MDL-1 RNA expression was also more elevated in adult than fetal tissues and in normal than malignant cells. These findings suggest that the TREM-1/DAP12 and MDL-1/DAP12 signaling pathways are features of mature differentiated myelomonocytic cells. In addition, expression of an alternative mRNA TREM-1 splice variant (TREM-1sv) was detected that might translate into a soluble receptor with potential as a regulator of myeloid activation.

Predominance of a type 2 intratumoural immune response in fresh tumour-infiltrating lymphocytes from human gliomas.

Increasing evidence suggests the existence of polarized human T cell responses described as Th1‐type (promoting cell‐mediated immunity) and Th2‐type (promoting humoral immunity), characterized by a dominant production of either interferon‐gamma (IFN‐γ) or IL‐4, respectively. Little is known about the intratumoural activation of infiltrating lymphocytes (TIL) in human gliomas. Therefore, we assessed fresh TIL at cellular and molecular levels to find out if they were activated and polarized into a type 1 or 2 immune response. Flow cytometry analysis of TIL revealed that the major subset was made of T lymphocytes. Double labelling with α‐CD3 and adhesion/activation markers revealed T cell subsets expressing CD49a, CD49b, CD54, and CD15, some of which were almost absent in autologous T peripheral blood lymphocytes (T‐PBL). Furthermore, the proportions of T‐TIL expressing CD56, CD65, or CD25 were several‐fold higher than in T‐PBL. Intratumoural functional activation of TIL was tested by semiquantitative assessment in relative units (RU) of lymphokine gene activation with mRNA reverse transcriptase‐polymerase chain reaction (RT‐PCR). All TIL populations except one significantly expressed IL‐4 1 to 2 logs of RU above healthy PBL baseline. Similarly, all patients expressed granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in a range comparable to IL‐4. However, most TIL populations did not express IFN‐γ, IL‐2, and tumour necrosis factor‐beta (TNF‐β) at higher levels than healthy normal PBL. The increased proportion of T cells expressing activation markers and the consistent detection of significant IL‐4 and GM‐CSF lymphokine gene activation in TIL populations suggested a predominant type 2 intratumoural immune response that does not promote cell‐mediated tumouricidal activity and may contribute to the inefficiency of the antiglioma immune response.

Mutations in KAT6B, encoding a histone acetyltransferase, cause Genitopatellar syndrome.

Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs.

Differential expression of multiple unexpected genes during U937 cell and macrophage differentiation detected by suppressive subtractive hybridization

OBJECTIVE The objective of this study was to identify new markers of myelomonocytic differentiation using a sensitive technique that permits detection of rare differential gene expression. MATERIALS AND METHODS [corrected] Suppressive subtractive hybridization (SSH) was performed between the human myelomonocytic U937 cell line and 1 alpha, 25-dihydroxyvitamin D3 and transforming growth factor beta 1 differentiated U937 cells. cDNA clones with significant increased expression in differentiated U937 cells over nondifferentiated U937 cells were characterized by sequencing. [corrected] The pattern of differential gene expression obtained by SSH was confirmed by cDNA Southern and Northern blots on the undifferentiated vs. differentiated U937 cells, and by reverse transcriptase polymerase chain reaction on undifferentiated human CD34(+) stem cells isolated from bone marrow vs. peripheral blood CD14(+) mature monocytes. RESULTS Seven cDNAs never associated with in vitro U937 cell myelomonocytic differentiation (prolactin, 11-beta hydroxysteroid dehydrogenase [11 beta-HSD)] haptoglobin alpha (2FS)-beta precursor, GLIPR, RTVP, the RNA helicase P68, and spermidine-spermine N1-acetyltransferase) were identified. The first five of these genes previously were associated with immune function and the last two are important for intermediary metabolism. Differential expression was confirmed in CD34(+)/CD14(+) monocyte differentiation for all genes but 11 beta-HSD. CONCLUSIONS We identified six new markers of U937 cell differentiation, which also are differentially expressed during normal human myelomonocytic differentiation.

Aberrant TGF-β Production and Regulation in Metastatic Malignancy

AbstractWe have examined the possible role of transforming growth factor-β (TGF-β) in metastatic malignancy by analyzing the production and activation of TGF-β, and -β2 and the regulation of TGF-β-responsive genes in oncogene-transformed metastatic fibrosarcomas. All transformed lines derived from either 10T1/2; or N1H 3T3 bv either H-ras or protein-kinase encoding oncogenes produced more TGF-β than parental cells. However, onlv highlv metastatic fibrosarcomas secreted activated TGF-β at rates that were greater than parental fibroblasts. Immunohistochemical staining for TGF-β, showed widespread intra- and extracellular distribution in metastatic lung nodules and adjacent tissue. Cells isolated from tumors successfully metastasizing to the lung had TGF-β1, mRNA levels which were increased 19-fold over in ritro controls. Despite the greatly enhanced rate of secretion of activated TGF-β, metastatic cells exhibited markedly altered responses of TGF-β1, and TGF-β2., being unable to either increase collagen sec...

Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein

In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane–cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.

Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles

Summary Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.