Heterozygous mis-sense mutations in Prkcb as a critical determinant of anti-polysaccharide antibody formation

To identify rate-limiting steps in T cell-independent type 2 (TI-2) antibody production against polysaccharide antigens, we performed a genome-wide screen by immunizing several hundred pedigrees of C57BL/6 mice segregating ENU-induced mis-sense mutations. Two independent mutations, Tilcara and Untied, were isolated that semi-dominantly diminished antibody against polysaccharide but not protein antigens. Both mutations resulted from single amino acid substitutions within the kinase domain of Protein Kinase C Beta (PKCβ). In Tilcara, a Ser552>Pro mutation occurred in helix G, in close proximity to a docking site for the inhibitory N-terminal pseudosubstrate domain of the enzyme, resulting in almost complete loss of active, autophosphorylated PKCβI whereas the amount of alternatively spliced PKCβII protein was not markedly reduced. Circulating B cell subsets were normal and acute responses to BCR-stimulation such as CD25 induction and initiation of DNA synthesis were only measurably diminished in Tilcara homozygotes, whereas the fraction of cells that had divided multiple times was decreased to an intermediate degree in heterozygotes. These results, coupled with evidence of numerous mis-sense PRKCB mutations in the human genome, identify Prkcb as a genetically sensitive step likely to contribute substantially to population variability in anti-polysaccharide antibody levels

Computational analysis of genomic variants affecting predicted microRNA:target interactions in prostate cancer.

Prostate cancer (PCa) is the most common cancer of men in the United States and is third only to lung and colon as a cause of cancer death. Clinical behavior of the disease is variable and the combination of prostate-specific antigen (PSA) screening and Gleason score staging are currently the best available molecular and pathology tools to predict outcomes. Cancer biology research establishes microRNAs (miRNAs) as key molecular components in both normal and pathological states. Thus, elucidating miRNAs perturbed by genomic alterations will expand our understanding of the molecular taxonomy of PCa with the aim to complement current practices in the diagnosis, prognosis, and treatment of the disease. This study reports the computational analysis of genomic variants affecting the seed sequence of five miRNAs, changing the prediction of microRNA:target interactions in PC3, an androgen-independent cell line that closely resembles prostatic small cell neuroendocrine carcinoma (SCNC). Genomic variants were detected via deep-sequencing of PC3 and further computational work focused on mapping changes within the seed sequence of predicted mature miRNAs. Five microRNA candidates (from now on denominated microRNA*) with changes in the g2-g8 seed region were selected: miR-3161*-5p with rs35834266 G\u3einsA; miR-3620*-5p with rs2070960 C\u3eT; miR-1178*-5p with rs7311975 T\u3eG; miR-4804*-5pwith rs266435 C\u3eG; and miR-449c*-3p with rs35770269 A\u3eT. Subsequently, the computational prediction of miRNA*:target interactions revealed 643 new relationships. After functional enrichment analysis of new targets, seven genes were associated with endocrine resistance (ABCB11, CDKN1B, NOTCH2, SHC4, CCND1, SP1, ADCY2) and five genes with endocrine and other factor regulated calcium reabsorption (ATP1A2, ESR1, PRRKCB, AP2B1, SLC8A1) categories. A gene-disease association literature search was performed for each of the aforementioned genes in order to understand if they have been implicated in cancer, where CDKN1B, NOTCH2, CCND1 have been reported to participate in prostate cancer progression. Microarray gene expression analyses showed that few predicted microRNA* targets were underexpressed in untreated PC3 samples versus prostate epithelial cells from the GEO database. However, after assessing the frequency of observed underexpressed genes per candidate microRNA* using a Fisher’s exact test, miR-4804*-5p target genes (TNKS and GUCY1A3) were statistically significant. Next steps included the comparison between groups of genes subject to non-mutated microRNA and mutated microRNA* regulation using a Kruskal-Wallis non-parametric test. Results were consistent with the microRNA-gene expression regulation model despite the genomic variant in the seed region, nevertheless the effect of miR-3161*-5p, miR-3620*-5p, miR-1178*-5p, miR-4804*-5p, and miR-449c*-3p cannot be predicted solely with the indirect experimental approach that microarray gene expression platforms provide. For this reason, the assessment of recurrent pairwise microRNA-mRNA expression associations was performed using CancerMiner, an online tool from The Cancer Genome Atlas (TCGA) based on a multivariate linear model and rank transformations. Only the relationship of miRNA-3161:CDKN1B was retrieved as a recurrent expression association in uterine corpus endometrial carcinoma (UCEC). In the context of this study, results suggest that CDKN1B (p27Kip1)dysregulation by miR-3161*-5p would be leading to PC3 super proliferation due to the lack of cell cycle arrest from phase G1 to S. Prostate cancer cell line PC3 has shown to share features with prostatic small cell neuroendocrine carcinomas (SCNC) with the implication that molecular mechanisms and therapeutic efficacies observed with PC3 cells are likely applicable to SCNC1. Prostatic small cell neuroendocrine carcinoma is a variant form of prostate cancer often characterized by an aggressive course with a poor response to conventional androgen deprivation therapy (ADT), consistent with the lack of the androgen receptor in prostatic small cell carcinoma (SCC)2. In some men treated with ADT, development of small cell carcinoma might represent the “escape” of a subpopulation of hormone-independent cells resulting from the selective pressure of hormonal therapy3. Hence, the suggestion of CDKN1B dysregulation by miR-3161*-5p might go beyond the idiosyncrasy of the PC3 cell line, but rather an interesting future direction to investigate prostate cancer patients with SCNC rendering to an adverse disease outcome due to uncontrolled cell proliferation

B cell deficiency and anaemia caused by mutations in the murine atp11c gene

Organization of the plasma membrane into specialised substructures in lymphocytes facilitates important biological functions including the initiation of crucial intracellular signalling cascades at the plasma membrane. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. Membrane-bound lipid transporters are believed to generate and dynamically maintain the lipid asymmetry between the two leaflets of the cell membranes, but not much is known about the role of these transporters in a variety of biological systems in mammals. This thesis examines the effect of two ENU-induced mutations of the murine Atp11c gene, which encodes a member of the P4-type ATPase family thought to serve as 'flippases' that mediate the translocation of specific aminophospholipids to the cytoplasmic leaflet of cell membranes. Loss of ATP11C in mice led to a severe B cell deficiency due to a developmental arrest at the pro-B cell stage during early B cell development in the bone marrow. The number of splenic follicular B cells and peritoneal B1 cells was also severely reduced in mutant mice. However, marginal zone B cells as well as other haematopoietic lineages including T, NK and myeloid cells appeared to accumulate normally in mutant mice. Moreover, the requirement for ATP11C in B cells was cell autonomous, and could not be corrected by the expression of pre-rearranged immunoglobulin transgenes or enforced expression of the pro-survival protein BCL-2 or by transgenic expression of IL-7. Further analysis of mutant mice revealed that while the IL-7R-mediated signalling pathway appears mostly intact, the ATP11Camb mutation leads to a defect in the expression and/or signalling through the pre-BCR, which provides essential signals for the development of pre-B cells. In contrast, B cells from B cell receptor (BCR) transgenic ATP11C-deficient mice were able to form germinal centres upon immunisation when adoptively transferred into wild-type host, indicating an intact in vivo signalling through the mature BCR in mutant animals. Functional analysis using fluorescently labelled phospholipid derivatives revealed that cells of the immune system from ATP11C-deficient mice exhibit impaired aminophospholipid flippase activity compared to those from control animals, indicating for the first time that ATP11C functions as a phospholipid flippase in biological membranes. Although the first phospholipid translocase activity was described in erythrocytes about 30 years ago, the characterisation of enzyme activity and its effect on the membrane asymmetry, development and survival of erythrocytes remains to be unveiled. Intriguingly, ATP11C-deficient mice developed anaemia due to a shortened erythrocyte lifespan, exhibited a large proportion of abnormal-shaped erythrocytes, and increased phosphatidylserine exposure on their surface due to impaired flippase activity. Thus, these findings identified ATP11C as a candidate for aminophospholipid translocation activity in erythrocytes. In conclusion, the findings of this thesis provide novel insights into the role of the putative phospholipid transporter ATP11C in B cell development and erythrocyte survival, and suggest a new candidate for inherited B cell deficiency and anaemia in humans

Heterozygous mis-sense mutations in Prkcb as a critical determinant of anti-polysaccharide antibody formation

To identify rate-limiting steps in T cell-independent type 2 antibody production against polysaccharide antigens, we performed a genome-wide screen by immunizing several hundred pedigrees of C57BL/6 mice segregating N-ethyl-N-nitrosurea-induced mis-sense mutations. Two independent mutations, Tilcara and Untied, were isolated that semi-dominantly diminished antibody against polysaccharide but not protein antigens. Both mutations resulted from single-amino-acid substitutions within the kinase domain of protein kinase C-β (PKCβ). In Tilcara, a Ser552>Pro mutation occurred in helix G, in close proximity to a docking site for the inhibitory N-terminal pseudosubstrate domain of the enzyme, resulting in almost complete loss of active, autophosphorylated PKCβI, whereas the amount of alternatively spliced PKCβII protein was not markedly reduced. Circulating B cell subsets were normal and acute responses to B-cell receptor stimulation such as CD25 induction and initiation of DNA synthesis were only measurably diminished in Tilcara homozygotes, whereas the fraction of cells that had divided multiple times was decreased to an intermediate degree in heterozygotes. These results, coupled with evidence of numerous mis-sense PRKCB mutations in the human genome, identify Prkcb as a genetically sensitive step likely to contribute substantially to population variability in anti-polysaccharide antibody levels

Gene Expression Profiling of Peripheral Tissues in Amyotrophic Lateral Sclerosis

Background: Amyotrophic Lateral Sclerosis, in which cortical and spinal motor neurons degenerate, is a late onset neurodegenerative condition that accounts for ~1 in 400 UK deaths, typically within 3-5 years from the initial manifestations of disease. It forms part of a broad spectrum of clinically, genetically as well as pathologically heterogeneous disorders that include behavioural variant frontotemporal lobar degeneration (bvFTLD). A large intronic hexanucleotide G4C2 repeat expansion of >30 copies was recently identified, in 2011, in the previously uncharacterised chromosome 9 open reading frame 72 (C9ORF72) gene which is now thought to explain up to 43% of familial ALS (~20-30% of familial FTLD) and around 7% of sporadic cases. Rationale & Hypothesis: The principle aim of the PhD was to perform gene expression profiling of peripheral tissues in ALS. In the first instance whole blood was trialled. However, this proved unreliable, owing to the shear abundance of erythrocyte derived alpha and beta haemoglobin transcripts that are contained within the sample and the variability in the efficiency of its removal using the Ambion® GLOBINClear or NuGEN Ovation® WB reduction strategies. Instead disease related changes in transcription/alternative splicing were detected in a large bank (n=820) of patient and control lymphoblastoid cell lines (LCL’s) with the main purpose of: 1) elucidating further the mechanism(s) of neurotoxicity associated with the C9ORF72 G4C2 repeat expansion and, 2) establishing within this specific genetic subtype, modifiers of a fast (<2yrs) versus slow (≥4yrs) disease progression in order to identify potential new areas of therapeutic research. Methodology: Biotinylated, sense-strand cDNA targets of ~40 -70nt were hybridized onto Human Exon 1.0ST GeneChip® Arrays. A GC-RMA normalisation procedure was carried out in Partek® Genomics Suite and differentially expressed or alternatively spliced transcripts were detected at the 5% significance level (p<0.05) with a fold-change threshold of ≥ ±1.20 applied. Findings: Overall a marginal increase in gene transcription was observed with respect to C9ORF72 (59.3%, n=650/1,096) and nonC9ORF72-related_SALS patients (63.9%, n=1,148/1,796) compared to neurologically healthy controls. DAVID enriched gene ontology terms included translation, which was specific to carriers of the G4C2 repeat, in addition to RNA processing, DNA metabolism, RNP complex biogenesis and the cell cycle which reflect more common features of the broader ALS phenotype. A number of key validation targets, including several RNA binding partners of the G4C2 repeat (FUS, RPL22, NUDT2, PURA, EIF4H and HNRNPA0/F) were subsequently confirmed in a qRT-PCR assay. Isoform A/B specific transcripts of the C9ORF72 gene, itself, were found not to be differentially expressed across the LCL’s in the ECACC discovery and replication cohorts. Conclusions: Whether pathogenicity of the G4C2 expanded allele arises as a consequence of haploinsufficiency or through an aberrant gain of function mechanism has yet to be determined; although emerging evidence favours a role of RNA toxicity. In light of this model, an up-regulation in the expression of C9ORF72 binding partners and other, RNA processing & splicing related transcripts fits with the hypothesis that the cells are attempting to compensate for the sequestration of these proteins into toxic RNA foci in the cytoplasm which leads to disruption of their normal physiological function. Small sample sizes meant limited conclusions could be drawn from the analysis of C9ORF72 specific modifiers of survival in ALS. Clinical data points towards a possible effect of gender which is supported in the literature but other factors such as correlations with expansion length would need to be considered in conducting future work