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SUMMARYAmyotrophic lateral sclerosis - frontotemporal dementia spectrum disorder (ALS/FTD) is a complex neurodegenerative disease; up to 10% of cases are familial, usually arising from single dominant mutations in >30 causative genes. Transgenic mouse models that overexpress human ALS/FTD causative genes have been the preferred organism for in vivo modelling. However, while conferring human protein biochemistry, these overexpression models are not ideal for dosage-sensitive proteins such as TDP-43 or FUS.We have created three next-generation genomically humanised knock-in mouse models for ALS/FTD research, by replacing the entire mouse coding region of Sod1, Tardbp (TDP-43) and Fus, with their human orthologues to preserve human protein biochemistry, with exons and introns intact to enable future modelling of coding or non-coding mutations and variants and to preserve human splice variants. In generating these mice, we have established a new-standard of quality control: we demonstrate the utility of indirect capture for enrichment of a region of interest followed by Oxford Nanopore sequencing for robustly characterising large knock-in alleles. This approach confirmed that targeting occurred at the correct locus and to map homologous recombination events. Furthermore, extensive expression data from the three lines shows that homozygous humanised animals only express human protein, at endogenous levels. Characterisation of humanised FUS animals showed that they are phenotypically normal compared to wildtype littermates throughout their lifespan.These humanised mouse strains are critically needed for preclinical assessment of interventions, such as antisense oligonucleotides (ASOs), to modulate expression levels in patients, and will serve as templates for the addition of human ALS/FTD mutations to dissect disease pathomechanisms.

Summary Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context., Graphical abstract, Highlights • Replacement in mice of Sod1, Tardbp, and Fus with orthologous human genomic sequence • Thorough allele QC of knockin alleles using targeted long-read sequencing • Mice are viable, fertile, and provide refined tools for ALS/FTD research • Aging of hFUS mice reveals no overt phenotype and minimal transcriptomic disruption, Neurogenetics; Neuroscience; Model organism

Gain-of-function STAT1 mutations have recently been associated with autosomal dominant chronic mucocutaneous candidiasis (CMC). The purpose of this study was to characterize the three members of a non-consanguineous family, the father and his two sons, who presented with recurrent oral thrush and ocular candidiasis since early childhood. The three patients had reduced levels of IL-17-producing T cells. This reduction affected specifically IL-17 + IFN-γ − T cells, because the levels of IL-17 + IFN-γ + T cells were similar to controls. We found that PBMC (peripheral blood mononuclear cells) from the patients did not respond to Candida albicans ex vivo . Moreover, after polyclonal activation, patients’ PBMC produced lower levels of IL-17 and IL-6 and higher levels of IL-4 than healthy controls. Genetic analyses showed that the three patients were heterozygous for a new mutation in STAT1 (c.894A > C, p.K298N) that affects a highly conserved residue of the coiled-coil domain of STAT1. STAT1 phosphorylation levels were significantly higher in patients’ cells than in healthy controls, both in basal conditions and after IFN-γ stimulation, suggesting a permanent activation of STAT1. Cells from the patients also presented increased IFN-γ-mediated responses measured as MIG and IP-10 production. In conclusion, we report a novel gain-of-function mutation in the coiled-coil domain of STAT1, which increases STAT1 phosphorylation and impairs IL-17-mediated immunity. The mutation is responsible for CMC in this family with autosomal dominant inheritance of the disease.

The role of mannose-binding lectin (MBL) deficiency (MBL2; XA/O and O/O genotypes) in host defences remains controversial. The surfactant proteins (SP)-A1, -A2 and -D, other collectins whose genes are located near MBL2, are part of the first-line lung defence against infection. We analysed the role of MBL on susceptibility to pneumococcal infection and the existence of linkage disequilibrium (LD) among the four genes. We studied 348 patients with pneumococcal community-acquired pneumonia (P-CAP) and 2,110 controls. A meta-analysis of MBL2 genotypes in susceptibility to P-CAP and to invasive pneumococcal disease (IPD) was also performed. The extent of LD of MBL2 with SFTPA1, SFTPA2 and SFTPD was analysed. MBL2 genotypes did not associate with either P-CAP or bacteraemic P-CAP in the case-control study. The MBL-deficient O/O genotype was significantly associated with higher risk of IPD in a meta-analysis, whereas the other MBL-deficient genotype (XA/O) showed a trend towards a protective role. We showed the existence of LD between MBL2 and SP genes. The data do not support a role of MBL deficiency on susceptibility to P-CAP or to IPD. LD among MBL2 and SP genes must be considered in studies on the role of MBL in infectious diseases.

Conflicting results about the role of genetic variability at IL6, particularly the -174 G/C single nucleotide polymorphism (SNP), in sepsis have been reported. We studied the genetic variability at IL6 in patients with community-acquired pneumonia (CAP) and pneumococcal CAP (P-CAP). This was a multicenter, prospective observational study. IL6 -174 was analyzed in 1,227 white Spanish patients with CAP (306 with P-CAP). IL6 1753 C/G (N = 750), 2954 G/C (N = 845), and haplotypes defined by these SNPs were also studied. In CAP patients the genotype -174 GG were associated with protection against acute respiratory distress syndrome (ARDS) (p = 0.008, OR = 0.4, 95% CI 0.2–0.8). No other significant associations were observed. However, in patients with P-CAP multivariate analysis adjusted for age, gender, co-morbidity, hospital of origin, and severity (pneumonia severity index, PSI) showed that the IL6 -174 GG genotype was protective against the development of ARDS (p = 0.002, OR = 0.25, 95% CI 0.07–0.79), septic shock (p = 0.006, OR = 0.46, 95% CI 0.18–0.79), and multiple organ dysfunction syndrome (p = 0.02, OR = 0.53, 95% CI 0.27–0.89). P-CAP patients homozygous for IL6 -174 G also showed a higher survival in a logistic regression analysis adjusted for age, gender, co-morbidity, hospital of origin, and PSI (p = 0.048, OR = 0.27, 95% CI 0.07–0.98). Our results indicate that the IL-6 -174 GG genotype is associated with lower severity and mortality in patients with P-CAP. This effect was higher than that observed in patients with CAP irrespective of the causal pathogen involved. Our results highlight the importance of the causal pathogen in genetic epidemiological studies in sepsis.

The role of mannose-binding lectin (MBL) deficiency (MBL2 XA/O + O/O genotypes) in host defences remains controversial. The surfactant proteins (SP)-A1, SP-A2 and SP-D, and other collectins whose genes are located near MBL2, are part of the first-line lung defence against infection. We analyzed the role of MBL on susceptibility to pneumococcal infection and the existence of linkage disequilibrium (LD) among the four genes.