Inherited IL-17RC deficiency in patients with chronic mucocutaneous candidiasis

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections of the skin, nail, oral, and genital mucosae with Candida species, mainly C. albicans. Autosomal-recessive (AR) IL-17RA and ACT1 deficiencies and autosomal-dominant IL-17F deficiency, each reported in a single kindred, underlie CMC in otherwise healthy patients. We report three patients from unrelated kindreds, aged 8, 12, and 37 yr with isolated CMC, who display AR IL-17RC deficiency. The patients are homozygous for different nonsense alleles that prevent the expression of IL-17RC on the cell surface. The defect is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. However, in contrast to what is observed for the IL-17RA– and ACT1-deficient patients tested, the response to IL-17E (IL-25) is maintained in these IL-17RC–deficient patients. These experiments of nature indicate that human IL-17RC is essential for mucocutaneous immunity to C. albicans but is otherwise largely redundant.In humans, chronic mucocutaneous candidiasis (CMC) is characterized by infections of the skin, nail, digestive, and genital mucosae with Candida species, mainly C. albicans, a commensal of the gastrointestinal tract in healthy individuals (Puel et al., 2012). CMC is frequent in acquired or inherited disorders involving profound T cell defects (Puel et al., 2010b; Vinh, 2011; Lionakis, 2012). Human IL-17 immunity has recently been shown to be essential for mucocutaneous protection against C. albicans (Puel et al., 2010b, 2012; Cypowyj et al., 2012; Engelhardt and Grimbacher, 2012; Huppler et al., 2012; Ling and Puel, 2014). Indeed, patients with primary immunodeficiencies and syndromic CMC have been shown to display impaired IL-17 immunity (Puel et al., 2010b). Most patients with autosomal-dominant (AD) hyper-IgE syndrome (AD-HIES) and STAT3 deficiency (de Beaucoudrey et al., 2008; Ma et al., 2008; Milner et al., 2008; Renner et al., 2008; Chandesris et al., 2012) and some patients with invasive fungal infection and autosomal-recessive (AR) CARD9 deficiency (Glocker et al., 2009; Lanternier et al., 2013) or Mendelian susceptibility to mycobacterial diseases (MSMD) and AR IL-12p40 or IL-12Rβ1 deficiency (de Beaucoudrey et al., 2008, 2010; Prando et al., 2013; Ouederni et al., 2014) have low proportions of IL-17A–producing T cells and CMC (Cypowyj et al., 2012; Puel et al., 2012). Patients with AR autoimmune polyendocrine syndrome type 1 (APS-1) and AIRE deficiency display CMC and high levels of neutralizing autoantibodies against IL-17A, IL-17F, and/or IL-22 (Browne and Holland, 2010; Husebye and Anderson, 2010; Kisand et al., 2010, 2011; Puel et al., 2010a).These findings paved the way for the discovery of the first genetic etiologies of CMC disease (CMCD), an inherited condition affecting individuals with none of the aforementioned primary immunodeficiencies (Puel et al., 2011; Casanova and Abel, 2013; Casanova et al., 2013, 2014). AR IL-17RA deficiency, AR ACT1 deficiency, and AD IL-17F deficiency were described, each in a single kindred (Puel et al., 2011; Boisson et al., 2013). A fourth genetic etiology of CMCD, which currently appears to be the most frequent, has also been reported: heterozygous gain-of-function (GOF) mutations of STAT1 impairing the development of IL-17–producing T cells (Liu et al., 2011; Smeekens et al., 2011; van de Veerdonk et al., 2011; Hori et al., 2012; Takezaki et al., 2012; Tóth et al., 2012; Al Rushood et al., 2013; Aldave et al., 2013; Romberg et al., 2013; Sampaio et al., 2013; Soltész et al., 2013; Uzel et al., 2013; Wildbaum et al., 2013; Frans et al., 2014; Kilic et al., 2014; Lee et al., 2014; Mekki et al., 2014; Mizoguchi et al., 2014; Sharfe et al., 2014; Yamazaki et al., 2014). We studied three unrelated patients with CMCD without mutations of IL17F, IL17RA, ACT1, or STAT1. We used a genome-wide approach based on whole-exome sequencing (WES). We found AR complete IL-17RC deficiency in all three patients.     

Harnessing malE for the study of antigen/antibody recognitions

The construction of hybrids between the variable fragment (Fv) of antibodies and protein MalE of Escherichia coli at the genetic level makes possible their preparation in a functional state, independently of any interaction with the antigen. We used such hybrids and a mutagenesis approach to study the recognition between antibody D1.3 and its antigen lysozyme, and its maturation. We subsequently transformed D1.3 into a reagentless fluorescent biosensor by knowledge-based design.     

Activation of human neutrophils by electronically transmitted phorbol-myristate acetate

We report the transfer of the activity of 4-phorbol-12-beta-myristate-13-acetate (PMA) by electronic means. Neutrophils were placed at 37 degrees C on one coil attached to an oscillator, while PMA was placed on another coil at room temperature. The oscillator was then turned on for 15 min, after which cells were usually further incubated for up to 45 min at 37 degrees C before measurement of reactive oxygen metabolites (ROMs) production. In 20 blind experiments, PMA thus 'transmitted' induced ROM production. ROM were not induced when: (1) PMA vehicle or 4-alpha-phorbol 12,13-didecanoate (an inactive PMA analogue) were transmitted; (2) the oscillator was switched off; (3) superoxide dismutase or protein kinase C inhibitors were added to cells before transmission. These results suggest that PMA molecules emit signals that can be transferred to neutrophils by artificial physical means in a manner that seems specific to the source molecules.     

Single nucleotide variant counts computed from RNA sequencing and cellular traffic into human kidney allografts

Advances in bioinformatics allow identification of single nucleotide polymorphisms (variants) from RNA sequence data. In an allograft biopsy, 2 genomes contribute to the RNA pool, 1 from the donor organ and the other from the infiltrating recipient's cells. We hypothesize that imbalances in genetic variants of RNA sequence data of kidney allograft biopsies provide an objective measure of cellular infiltration of the allograft. We performed mRNA sequencing of 40 kidney allograft biopsies, selected to represent a comprehensive range of diagnostic categories. We analyzed the sequencing reads of these biopsies and of 462 lymphoblastoid cell lines from the 1000 Genomes Project, for RNA variants. The ratio of heterozygous to nonreference genome homozygous variants (Het/Hom ratio) on all autosomes was determined for each sample, and the estimation of stromal and immune cells in malignant tumors using expression data (ESTIMATE) score was computed as a complementary estimate of the degree of cellular infiltration into biopsies. The Het/Hom ratios (P = .02) and the ESTIMATE scores (P < .001) were associated with the biopsy diagnosis. Both measures correlated significantly (r = .67, P < .0001), even though the Het/Hom ratio is based on mRNA sequence variation, while the ESTIMATE score uses mRNA expression. Het/Hom ratio and the ESTIMATE score may offer unbiased and quantitative parameters for characterizing cellular traffic into human kidney allografts.     

The mutation spectrum in RECQL4 diseases

Mutations in the RECQL4 gene can lead to three clinical phenotypes with overlapping features. All these syndromes, Rothmund-Thomson (RTS), RAPADILINO and Baller-Gerold (BGS), are characterized by growth retardation and radial defects, but RAPADILINO syndrome lacks the main dermal manifestation, poikiloderma that is a hallmark feature in both RTS and BGS. It has been previously shown that RTS patients with RECQL4 mutations are at increased risk of osteosarcoma, but the precise incidence of cancer in RAPADILINO and BGS has not been determined. Here, we report that RAPADILINO patients identified as carriers of the c.1390+2delT mutation (p.Ala420_Ala463del) are at increased risk to develop lymphoma or osteosarcoma (6 out of 15 patients). We also summarize all the published RECQL4 mutations and their associated cancer cases and provide an update of 14 novel RECQL4 mutations with accompanying clinical data.     

The human gene damage index as a gene-level approach to prioritizing exome variants

The protein-coding exome of a patient with a monogenic disease contains about 20,000 variants, only one or two of which are disease causing. We found that 58% of rare variants in the protein-coding exome of the general population are located in only 2% of the genes. Prompted by this observation, we aimed to develop a gene-level approach for predicting whether a given human protein-coding gene is likely to harbor disease-causing mutations. To this end, we derived the gene damage index (GDI): a genome-wide, gene-level metric of the mutational damage that has accumulated in the general population. We found that the GDI was correlated with selective evolutionary pressure, protein complexity, coding sequence length, and the number of paralogs. We compared GDI with the leading gene-level approaches, genic intolerance, and de novo excess, and demonstrated that GDI performed best for the detection of false positives (i.e., removing exome variants in genes irrelevant to disease), whereas genic intolerance and de novo excess performed better for the detection of true positives (i.e., assessing de novo mutations in genes likely to be disease causing). The GDI server, data, and software are freely available to noncommercial users from lab.rockefeller.edu/casanova/GDI.Germ-line mutations can contribute to the long-term adaptation of humans, but at the expense of causing a large number of genetic diseases (1). The advent of next-generation sequencing (NGS)-based approaches, including whole-exome sequencing (WES), whole-genome sequencing (WGS), and RNA-Seq, has facilitated the large-scale detection of gene variants at both the individual and population levels (2–6). In patients suffering from a monogenic disease, at most two variants are disease causing [true positives (TP)], and the other 20,000 or so protein-coding exome variants are false positives (FP; type I error). Several variant-level metrics predicting the biochemical impact of DNA mutations (7–9) can be used to prioritize candidate variants for a phenotype of interest (10, 11). Gene-level metrics aim to prioritize the genes themselves, providing information that can be used for the further prioritization of variants. There are currently fewer gene-level than variant-level computational methods. They provide complementary information, as it is best to predict the impact of a variant by also taking into account population genetics data for its locus. Current gene-level methods include genic intolerance, as measured by the residual variation intolerance score (RVIS) (12) and de novo excess (DNE) (13). These metrics are particularly useful for determining whether a given gene (and, by inference, its variants) is a plausible candidate for involvement in a particular genetic disease (i.e., for the selection of a short list of candidate genes and variants, which include the TPs). However, owing to the large number and diversity of variants, the selection of a single candidate gene from the NGS data for a given patient with a specific disease remains challenging.We reasoned that genes frequently mutated in healthy populations would be unlikely to cause inherited and rare diseases, but would probably make a disproportionate contribution to the variant calls observed in any given patient. Conversely, mutations in genes that are never or only rarely mutated under normal circumstances are more likely to be disease-causing. Leading gene-level strategies are based on selective pressure (12) and de novo mutation rate estimates (13). These methods are tailored to detect genes likely to harbor TPs. However, these methods do not directly calculate quantitatively the mutational load for human genes in the general (i.e., “healthy”) population or the frequencies of mutant alleles. These methods may, therefore, not be optimal for filtering out highly mutated genes, which are likely to harbor many FPs. Moreover, there has been no formal comparison of the power of these gene-level methods and their combinations for maximizing the discovery of FPs and TPs by NGS. We therefore aimed to generate a robust metric of the cumulative mutational damage to each human protein-coding gene, to make it easier to distinguish the FP variants harbored by highly damaged genes (e.g., under relaxed constraint or positive selection) from potential candidate genes and variants, including the TPs. By damaged genes, we refer to genes displaying many nonsynonymous mutations, which are not necessarily damaging biochemically or evolutionarily. We developed the gene damage index (GDI), which defines, in silico, the mutational damage accumulated by each protein-coding human gene in the general population, and reflecting the combined influences of drifts and selections. We then tested this approach with the WES data for 84 patients in our in-house database, each of these patients having a known primary immunodeficiency (PID). Finally, we used receiver operating characteristic (ROC) curves for formal comparisons of performance between GDI and the existing gene-level RVIS and DNE approaches, and to assess the power of the gene-level methods for detecting enrichment in de novo mutations in cases versus controls. We also tested whether these methods could act in synergy to filter out FPs and select TPs.     

Whole-genome sequencing is more powerful than whole-exome sequencing for detecting exome variants

We compared whole-exome sequencing (WES) and whole-genome sequencing (WGS) in six unrelated individuals. In the regions targeted by WES capture (81.5% of the consensus coding genome), the mean numbers of single-nucleotide variants (SNVs) and small insertions/deletions (indels) detected per sample were 84,192 and 13,325, respectively, for WES, and 84,968 and 12,702, respectively, for WGS. For both SNVs and indels, the distributions of coverage depth, genotype quality, and minor read ratio were more uniform for WGS than for WES. After filtering, a mean of 74,398 (95.3%) high-quality (HQ) SNVs and 9,033 (70.6%) HQ indels were called by both platforms. A mean of 105 coding HQ SNVs and 32 indels was identified exclusively by WES whereas 692 HQ SNVs and 105 indels were identified exclusively by WGS. We Sanger-sequenced a random selection of these exclusive variants. For SNVs, the proportion of false-positive variants was higher for WES (78%) than for WGS (17%). The estimated mean number of real coding SNVs (656 variants, ∼3% of all coding HQ SNVs) identified by WGS and missed by WES was greater than the number of SNVs identified by WES and missed by WGS (26 variants). For indels, the proportions of false-positive variants were similar for WES (44%) and WGS (46%). Finally, WES was not reliable for the detection of copy-number variations, almost all of which extended beyond the targeted regions. Although currently more expensive, WGS is more powerful than WES for detecting potential disease-causing mutations within WES regions, particularly those due to SNVs.Whole-exome sequencing (WES) is routinely used and is gradually being optimized for the detection of rare and common genetic variants in humans (1–8). However, whole-genome sequencing (WGS) is becoming increasingly attractive as an alternative, due to its broader coverage and decreasing cost (9–11). It remains difficult to interpret variants lying outside the protein-coding regions of the genome. Diagnostic and research laboratories, whether public or private, therefore tend to search for coding variants, most of which can be detected by WES, first. Such variants can also be detected by WGS, and several studies previously compared WES and WGS for different types of variations and/or in different contexts (9, 11–16), but none of them in a really comprehensive manner. Here, we compared WES and WGS, in terms of detection rates and quality, for single-nucleotide variants (SNVs), small insertions/deletions (indels), and copy-number variants (CNVs) within the regions of the human genome covered by WES, using the most recent next-generation sequencing (NGS) technologies. We aimed to identify the most efficient and reliable approach for identifying these variants in coding regions of the genome, to define the optimal analytical filters for decreasing the frequency of false-positive variants, and to characterize the genes that were either hard to sequence by either approach or were poorly covered by WES kits.     

Collaboration based on product lifecycles interoperability for extended enterprise

The paper proposes a new approach for managing two products lifecycles in the context of collaboration between aircraft OEM and their equipment suppliers. The concept of entities and specific roles are used to improve the semantic interoperability between both aircraft and equipment projects’ data. These concepts are also used to formalize the relation between various processes that guarantee the connection between the aircraft and the assembly equipment lifecycles. The aim of this integration approach is to enable the information sharing between OEM and suppliers. It intends to enlarge the role of suppliers in the development process of fixture equipment, which are used in the aircraft assembly process.