Szymon M. Kielbasa

Mutations in PIGO, a member of the GPI-anchor-synthesis pathway, cause hyperphosphatasia with mental retardation

Hyperphosphatasia with mental retardation syndrome (HPMRS), an autosomal-recessive form of intellectual disability characterized by facial dysmorphism, seizures, brachytelephalangy, and persistent elevated serum alkaline phosphatase (hyperphosphatasia), was recently shown to be caused by mutations in PIGV, a member of the glycosylphosphatidylinositol (GPI)-anchor-synthesis pathway. However, not all individuals with HPMRS harbor mutations in this gene. By exome sequencing, we detected compound-heterozygous mutations in PIGO, a gene coding for a membrane protein of the same molecular pathway, in two siblings with HPMRS, and we then found by Sanger sequencing further mutations in another affected individual; these mutations cosegregated in the investigated families. The mutant transcripts are aberrantly spliced, decrease the membrane stability of the protein, or impair enzyme function such that GPI-anchor synthesis is affected and the level of GPI-anchored substrates localized at the cell surface is reduced. Our data identify PIGO as the second gene associated with HPMRS and suggest that a deficiency in GPI-anchor synthesis is the underlying molecular pathomechanism of HPMRS.

Disease variants alter transcription factor levels and methylation of their binding sites

Most disease-associated genetic variants are noncoding, making it challenging to design experiments to understand their functional consequences. Identification of expression quantitative trait loci (eQTLs) has been a powerful approach to infer the downstream effects of disease-associated variants, but most of these variants remain unexplained. The analysis of DNA methylation, a key component of the epigenome, offers highly complementary data on the regulatory potential of genomic regions. Here we show that disease-associated variants have widespread effects on DNA methylation in trans that likely reflect differential occupancy of trans binding sites by cis-regulated transcription factors. Using multiple omics data sets from 3,841 Dutch individuals, we identified 1,907 established trait-associated SNPs that affect the methylation levels of 10,141 different CpG sites in trans (false discovery rate (FDR) < 0.05). These included SNPs that affect both the expression of a nearby transcription factor (such as NFKB1, CTCF and NKX2-3) and methylation of its respective binding site across the genome. Trans methylation QTLs effectively expose the downstream effects of disease-associated variants.

Identification of context-dependent expression quantitative trait loci in whole blood

Genetic risk factors often localize to noncoding regions of the genome with unknown effects on disease etiology. Expression quantitative trait loci (eQTLs) help to explain the regulatory mechanisms underlying these genetic associations. Knowledge of the context that determines the nature and strength of eQTLs may help identify cell types relevant to pathophysiology and the regulatory networks underlying disease. Here we generated peripheral blood RNA–seq data from 2,116 unrelated individuals and systematically identified context-dependent eQTLs using a hypothesis-free strategy that does not require previous knowledge of the identity of the modifiers. Of the 23,060 significant cis-regulated genes (false discovery rate (FDR) ≤ 0.05), 2,743 (12%) showed context-dependent eQTL effects. The majority of these effects were influenced by cell type composition. A set of 145 cis-eQTLs depended on type I interferon signaling. Others were modulated by specific transcription factors binding to the eQTL SNPs.

Two Plasmodium 6-Cys family-related proteins have distinct and critical roles in liver-stage development

The 10 Plasmodium 6‐Cys proteins have critical roles throughout parasite development and are targets for antimalaria vaccination strategies. We analyzed the conserved 6‐cysteine domain of this family and show that only the last 4 positionally conserved cysteine residues are diagnostic for this domain and identified 4 additional “6‐Cys family‐related” proteins. Two of these, sequestrin and B9, are critical to Plasmodium liver‐stage development. RT‐PCR and immunofluorescence assays show that B9 is translationally repressed in sporozoites and is expressed after hepatocyte invasion where it localizes to the parasite plasma membrane. Mutants lacking B9 expression in the rodent malaria parasites P. berghei and P. yoelii and the human parasite P. falciparum developmentally arrest in hepatocytes. P. berghei mutants arrest in the livers of BALB/c (100%) and C57BL6 mice (>99.9%), and in cultures of Huh7 human‐hepatoma cell line. Similarly, P. falciparum mutants while fully infectious to primary human hepatocytes abort development 3 d after infection. This growth arrest is associated with a compromised parasitophorous vacuole membrane a phenotype similar to, but distinct from, mutants lacking the 6‐Cys sporozoite proteins P52 and P36. Our results show that 6‐Cys proteins have critical but distinct roles in establishment and maintenance of a parasitophorous vacuole and subsequent liver‐stage development—Annoura, T., van Schaijk, B. C. L., Ploemen, I. H. J., Sajid, M., Lin, J.‐W., Vos, M. W., Dinmohamed, A G., Inaoka, D. K., Rijpma, S. R., van Gemert, G.‐J., Chevalley‐Maurel, S., Kiełbasa, S. M., Scheltinga, F., Franke‐Fayard, B., Klop, O. Hermsen, C. C., Kita, K., Gego, A., Franetich, J.‐F., Mazier, D., Hoffman, S. L., Janse, C. J., Sauerwein, R. W., Khan, S. M. Two Plasmodium 6‐Cys family‐related proteins have distinct and critical roles in liver‐stage development. FASEB J. 28, 2158–2170 (2014). www.fasebj.org

Whole-transcriptome analysis of flow-sorted cervical cancer samples reveals that B cell expressed TCL1A is correlated with improved survival

Cervical cancer is typically well infiltrated by immune cells. Because of the intricate relationship between cancer cells and immune cells, we aimed to identify both cancer cell and immune cell expressed biomarkers. Using a novel approach, we isolated RNA from flow-sorted viable EpCAM+ tumor epithelial cells and CD45+ tumor-infiltrating immune cells obtained from squamous cell cervical cancer samples (n = 24). Total RNA was sequenced and differential gene expression analysis of the CD45+ immune cell fractions identified TCL1A as a novel marker for predicting improved survival (p = 0.007). This finding was validated using qRT-PCR (p = 0.005) and partially validated using immunohistochemistry (p = 0.083). Importantly, TCL1A was found to be expressed in a subpopulation of B cells (CD3−/CD19+/CD10+/CD34−) using multicolor immunofluorescence. A high TCL1A/CD20 (B cell) ratio, determined in total tumor samples from a separate patient cohort using qRT-PCR (n = 52), was also correlated with improved survival (p = 0.027). This is the first study demonstrating the prognostic value of separating tumor epithelial cells from tumor-infiltrating immune cells and determining their RNA expression profile for identifying putative cancer biomarkers. Our results suggest that intratumoral TCL1A+ B cells are important for controlling cervical cancer development.

ARTISAN PCR: rapid identification of full-length immunoglobulin rearrangements without primer binding bias.

B cells recognize specific antigens by their membrane-bound B-cell receptor (BCR). Functional BCR genes are assembled in pre-B cells by recombination of the variable (V), diversity (D) and joining (J) genes [V(D)J recombination]. When B cells participate in germinal centre reactions, non-templated point mutations are introduced into BCR genes by somatic hypermutation (SHM) (Rajewsky, 1996). V(D)J recombination and SHM create virtually unlimited BCR repertoires. Many research applications require identification of fulllength BCR sequences expressed in biological samples. BCR are commonly identified by multiplex polymerase chain reaction (PCR) with upstream primers that anneal to conserved stretches in variable or leader gene segments. BIOMED-2 is the current European standard protocol to assess BCR clonality as diagnostic criterion for lymphoma (van Dongen et al, 2003). However, a single point mutation in a primer binding site can obscure a dominant clonal BCR (Wu et al, 2009), whereas reliable identification of functional BCR genes is essential to avoid erroneous conclusions about the BCR (Koning et al, 2014). Therefore, several investigators have emphasized the need for an unbiased method of BCR amplification (Warren et al, 2013; Georgiou et al, 2014; Koning et al, 2014). We here describe ARTISAN PCR (Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR) as a truly unbiased method with excellent applicability. Polyadenylated mRNA (Dynabeads mRNA DIRECT Kit, Invitrogen, Grand Island, NY, USA) was directly isolated from thawed suspensions of cryopreserved cells, or from 10 lm sections of fresh frozen material that was homogenized immediately upon addition of lysis buffer. Isolation of B cells by magnetic beads prior to mRNA isolation was found to be dispensable. Reverse primers (Table SI) were designed to anneal to a consensus sequence from all functional alleles of l, c, a, j, and k Ig constant regions included in the ImMunoGeneTics (IMGT) database (Lefranc et al, 1999). Messenger RNA was mixed with reverse transcription primers and the SA.rt anchor oligonucleotide (Table SII). Synthesis of cDNA was performed with a reverse transcriptase that adds a non-templated 30 polycytosine terminus (Zhu et al, 2001). Annealing of the SA.rt oligonucleotide to this polycytosine stretch creates a template for extension of the cDNA with the complementary SA.rt sequence in the same reaction. Subsequent PCR amplification of anchored cDNA was performed with anchor-specific primer SA.pcr in combination with a nested primer that anneals to one of the constant regions of interest (Cl.pcr, Cc.pcr, Ca.pcr, Cj.pcr, or Ck.pcr; Table SI and Table SII). The efficacy of ARTISAN PCR was compared with a BIOMED-2-based multiplex PCR kit (IGH FR1, IGK tube A and IGL Clonality Assays, Invivoscribe, San Diego, CA, USA). For massive parallel sequencing, column-purified ARTISAN amplicons were barcoded by PCR (Table SII). Samples were sequenced on the PacBio RS system (Pacific Biosciences, Menlo Park, CA, USA). After filtering for a minimum of eight passes (SMRT Portal software, Pacific Biosciences) and correction of indels in polyhomologous stretches (Koren et al, 2012), sequence reads were analysed with Geneious 6.1.6 software and IMGT HighV-QUEST tools (Alamyar et al, 2012). PacBio sequencing of ARTISAN PCR amplicons identified full-length functional V(D)J sequences in all 39 Epstein–Barr virus-transformed lymphoblastoid cell lines (EBV LCL) tested (Table SIII). 10 cells sufficed as starting material. Each PacBio SMRT cell yielded >25 9 10 reads with at least eight passes. Of 52 VDJ sequences, 50 were functional and 2 non-productive. Thirty-two EBV LCL were monoclonal and were six biclonal; one EBV LCL harboured six different B-cell clones. The sequence error rate as calculated for clonal EBV LCL sequences was 0 126 9 10 3 per bp without significant differences between l, c, a, j, and k amplicons. The multiplex protocol failed to amplify seven VDJ, one VJ-j and nine VJ-k, indicating success rates of 87%, 95%, and 78%, respectively, compared to ARTISAN PCR. Complete BCR heterodimers were obtained by the multiplex protocol for only 37 of the 50 functional EBV LCL clones identified by ARTISAN PCR (74%). Dilution experiments with B-cell lines demonstrated proportional BCR sequence representation according to cellular input (Fig 1A). Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR identified complete functional BCR sequences from cryopreserved primary cells in all 24 cases of various B cell (pre)malignancies (3 samples each from Burkitt lymphoma, mantle cell lymphoma, marginal zone lymphoma, follicular lymphoma, multiple myeloma, Waldenstr€ om macroglobulinaemia, chronic lymphocytic leukaemia and monoclonal B-cell lymphocytosis; Table SIV). Multiplex RT-PCR failed to identify four VDJ and eight VJ sequences, and identified the complete BCR heterodimer in only 13 cases (54%). Finally, ARTISAN PCR was applied to 17 fresh frozen excisional biopsies archived between 1988 and 1991 and labelled Correspondence

Integrated Whole Genome and Transcriptome Analysis Identified a Therapeutic Minor Histocompatibility Antigen in a Splice Variant of ITGB2.

Purpose: In HLA-matched allogeneic hematopoietic stem cell transplantation (alloSCT), donor T cells recognizing minor histocompatibility antigens (MiHAs) can mediate desired antitumor immunity as well as undesired side effects. MiHAs with hematopoiesis-restricted expression are relevant targets to augment antitumor immunity after alloSCT without side effects. To identify therapeutic MiHAs, we analyzed the in vivo immune response in a patient with strong antitumor immunity after alloSCT. Experimental Design: T-cell clones recognizing patient, but not donor, hematopoietic cells were selected for MiHA discovery by whole genome association scanning. RNA-sequence data from the GEUVADIS project were analyzed to investigate alternative transcripts, and expression patterns were determined by microarray analysis and qPCR. T-cell reactivity was measured by cytokine release and cytotoxicity. Results: T-cell clones were isolated for two HLA-B*15:01–restricted MiHA. LB-GLE1-1V is encoded by a nonsynonymous SNP in exon 6 of GLE1. For the other MiHAs, an associating SNP in intron 3 of ITGB2 was found, but no SNP disparity was present in the normal gene transcript between patient and donor. RNA-sequence analysis identified an alternative ITGB2 transcript containing part of intron 3. qPCR demonstrated that this transcript is restricted to hematopoietic cells and SNP-positive individuals. In silico translation revealed LB-ITGB2-1 as HLA-B*15:01–binding peptide, which was validated as hematopoietic MiHA by T-cell experiments. Conclusions: Whole genome and transcriptome analysis identified LB-ITGB2-1 as MiHAs encoded by an alternative transcript. Our data support the therapeutic relevance of LB-ITGB2-1 and illustrate the value of RNA-sequence analysis for discovery of immune targets encoded by alternative transcripts. Clin Cancer Res; 22(16); 4185–96. ©2016 AACR.

Genes associated with venous thromboembolism in colorectal cancer patients

Essentials The underlying pathophysiological mechanisms behind cancer‐associated thrombosis are unknown. We compared expression profiles in tumor cells from patients with and without thrombosis. Tumors from patients with thrombosis showed significant differential gene expression profiles. Patients with thrombosis had a proinflammatory status and increased fibrin levels in the tumor.

RNA-seq analysis of Lgr6(+) stem cells and identification of an Lgr6 isoform.

We studied Lgr6+ stem cells in experimental UV carcinogenesis in hairless mice. For further characterization through RNA‐seq, these stem cells were isolated by FACS from transgenic hairless mice bearing an EGFP‐Ires‐CreERT2 reporter cassette inserted into exon 1 of the Lgr6 gene (purity confirmed by human ERT2 expression). Between Lgr6/EGFP+ and Lgr6/EGFP− basal cells (Tg/wt), 682 RNAs were differentially expressed, indicating stemness and expression of cancer‐related pathways in Lgr6/EGFP+ cells. We discovered that suspected “Lgr6 null” mice (Tg/Tg) expressed RNA of an Lgr6 isoform (delta‐Lgr6, lacking 74 N‐terminal aa) which could be functional and explain the lack of a phenotype.

Molecular signatures of age-associated chronic degeneration of shoulder muscles

Chronic muscle diseases are highly prevalent in the elderly causing severe mobility limitations, pain and frailty. The intrinsic molecular mechanisms are poorly understood due to multifactorial causes, slow progression with age and variations between individuals. Understanding the underlying molecular mechanisms could lead to new treatment options which are currently limited. Shoulder complaints are highly common in the elderly, and therefore, muscles of the shoulders rotator cuff could be considered as a model for chronic age-associated muscle degeneration. Diseased shoulder muscles were characterized by muscle atrophy and fatty infiltration compared with unaffected shoulder muscles. We confirmed fatty infiltration using histochemical analysis. Additionally, fibrosis and loss of contractile myosin expression were found in diseased muscles. Most cellular features, including proliferation rate, apoptosis and cell senescence, remained unchanged and genome-wide molecular signatures were predominantly similar between diseased and intact muscles. However, we found down-regulation of a small subset of muscle function genes, and up-regulation of extracellular region genes. Myogenesis was defected in muscle cell culture from diseased muscles but was restored by elevating MyoD levels. We suggest that impaired muscle functionality in a specific environment of thickened extra-cellular matrix is crucial for the development of chronic age-associated muscle degeneration.