NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation.

The Nck-associated protein 1-like (NCKAP1L) gene, alternatively called hematopoietic protein 1 (HEM-1), encodes a hematopoietic lineage-specific regulator of the actin cytoskeleton. Nckap1l-deficient mice have anomalies in lymphocyte development, phagocytosis, and neutrophil migration. Here we report, for the first time, NCKAP1L deficiency cases in humans. In two unrelated patients of Middle Eastern origin, recessive mutations in NCKAP1L abolishing protein expression led to immunodeficiency, lymphoproliferation, and hyperinflammation with features of hemophagocytic lymphohistiocytosis. Immunophenotyping showed an inverted CD4/CD8 ratio with a major shift of both CD4+ and CD8+ cells toward memory compartments, in line with combined RNA-seq/proteomics analyses revealing a T cell exhaustion signature. Consistent with the core function of NCKAP1L in the reorganization of the actin cytoskeleton, patients' T cells displayed impaired early activation, immune synapse morphology, and leading edge formation. Moreover, knockdown of nckap1l in zebrafish led to defects in neutrophil migration. Hence, NCKAP1L mutations lead to broad immune dysregulation in humans, which could be classified within actinopathies.

Delirium and encephalopathy in severe COVID-19: a cohort analysis of ICU patients.

BACKGROUND: Neurotropism of SARS-CoV-2 and its neurological manifestations have now been confirmed. We aimed at describing delirium and neurological symptoms of COVID-19 in ICU patients. METHODS: We conducted a bicentric cohort study in two French ICUs of Strasbourg University Hospital. All the 150 patients referred for acute respiratory distress syndrome due to SARS-CoV-2 between March 3 and May 5, 2020, were included at their admission. Ten patients (6.7%) were excluded because they remained under neuromuscular blockers during their entire ICU stay. Neurological examination, including CAM-ICU, and cerebrospinal fluid analysis, electroencephalography, and magnetic resonance imaging (MRI) were performed in some of the patients with delirium and/or abnormal neurological examination. The primary endpoint was to describe the incidence of delirium and/or abnormal neurological examination. The secondary endpoints were to describe the characteristics of delirium, to compare the duration of invasive mechanical ventilation and ICU length of stay in patients with and without delirium and/or abnormal neurological symptoms. RESULTS: The 140 patients were aged in median of 62 [IQR 52; 70] years old, with a median SAPSII of 49 [IQR 37; 64] points. Neurological examination was normal in 22 patients (15.7%). One hundred eighteen patients (84.3%) developed a delirium with a combination of acute attention, awareness, and cognition disturbances. Eighty-eight patients (69.3%) presented an unexpected state of agitation despite high infusion rates of sedative treatments and neuroleptics, and 89 (63.6%) patients had corticospinal tract signs. Brain MRI performed in 28 patients demonstrated enhancement of subarachnoid spaces in 17/28 patients (60.7%), intraparenchymal, predominantly white matter abnormalities in 8 patients, and perfusion abnormalities in 17/26 patients (65.4%). The 42 electroencephalograms mostly revealed unspecific abnormalities or diffuse, especially bifrontal, slow activity. Cerebrospinal fluid examination revealed inflammatory disturbances in 18/28 patients, including oligoclonal bands with mirror pattern and elevated IL-6. The CSF RT-PCR SARS-CoV-2 was positive in one patient. The delirium/neurological symptoms in COVID-19 patients were responsible for longer mechanical ventilation compared to the patients without delirium/neurological symptoms. Delirium/neurological symptoms could be secondary to systemic inflammatory reaction to SARS-CoV-2. CONCLUSIONS AND RELEVANCE: Delirium/neurological symptoms in COVID-19 patients are a major issue in ICUs, especially in the context of insufficient human and material resources. TRIAL REGISTRATION: NA.

FACS - based isolation of human eosinophils allows purification of high quality RNA.

Here we extensively describe a FACS-based protocol for isolating intact non-stained human eosinophils from peripheral blood; a stop forward from our recently published initial study. This method of purification could be accomplished in <3 h with only small volumes of whole blood necessary, even in healthy subjects generally exhibiting low levels of circulating eosinophils. Eosinophil activation during the isolation steps appeared to be minimal and this purification procedure yielded high quality RNA. Moreover, these FACS-isolated eosinophils had prolonged viability in culture and were suitable for further activation assays.

Mutations in signal recognition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond-like features.

Shwachman-Diamond syndrome (SDS) (OMIM #260400) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. Seventy-five to ninety percent of patients have compound heterozygous loss-of-function mutations in the Shwachman-Bodian-Diamond syndrome (sbds) gene. Using trio whole-exome sequencing (WES) in an sbds-negative SDS family and candidate gene sequencing in additional SBDS-negative SDS cases or molecularly undiagnosed IBMFS cases, we identified 3 independent patients, each of whom carried a de novo missense variant in srp54 (encoding signal recognition particle 54 kDa). These 3 patients shared congenital neutropenia linked with various other SDS phenotypes. 3D protein modeling revealed that the 3 variants affect highly conserved amino acids within the GTPase domain of the protein that are critical for GTP and receptor binding. Indeed, we observed that the GTPase activity of the mutated proteins was impaired. The level of SRP54 mRNA in the bone marrow was 3.6-fold lower in patients with SRP54-mutations than in healthy controls. Profound reductions in neutrophil counts and chemotaxis as well as a diminished exocrine pancreas size in a SRP54-knockdown zebrafish model faithfully recapitulated the human phenotype. In conclusion, autosomal dominant mutations in SRP54, a key member of the cotranslation protein-targeting pathway, lead to syndromic neutropenia with a Shwachman-Diamond-like phenotype.

Next-Generation Sequencing of the HLA locus: Methods and impacts on HLA typing, population genetics and disease association studies.

The human Major Histocompatibility Complex, known as the "Human Leukocyte Antigen (HLA)", could be defined as a "super locus" (historically called "supergene") governing the adaptive immune system in vertebrates. It also harbors genes involved in innate immunity. HLA is the most gene-dense, polymorphic and disease-associated region of the human genome. It is of critical medical relevance given its involvement in the fate of the transplanted organs/tissues and its association with more than 100 diseases. However, despite these important roles, comprehensive sequence analysis of the 4 megabase HLA locus has been limited due to technological challenges. Thanks to recent improvements in Next-Generation Sequencing (NGS) technologies however, one is now able to handle the peculiarities of the MHC notably the tight linkage disequilibrium between genes as well as their high degree of polymorphism (and hence heterozygosity). Increased read lengths, throughput, accuracy, as well as development of new bioinformatics tools now enable to efficiently generate complete and accurate full-length HLA haplotypes without phase ambiguities. The present report reviews current NGS approaches to capture, sequence and analyze HLA genes and loci. The impact of these new methodologies on various applications including HLA typing, population genetics and disease association studies are discussed.

On the genetics of the Silk Route: association analysis of HLA, IL10, and IL23R-IL12RB2 regions with Behçet's disease in an Iranian population.

Despite that the association of Behçet's disease (BD) with the HLA-B5 was first established in the 1970s, a number of recent genome-wide association studies have both confirmed and furthered this association--in various populations--to individual SNPs both inside and outside the HLA. The former include HLA-B, MICA, and HLA-A, while the latter encompass IL10 and IL23R-IL12RB2 regions. The present study examined whether some of these SNPs could be replicated in an Iranian population, where the prevalence of disease is amply documented. Eight SNPs were selected and tested in 552 patients and 417 controls. These were rs7539328, rs12119179, rs1495965, rs1518111, and rs1800871 in IL10 and IL23R-IL12RB2 regions and rs114854070, rs12525170, and rs76546355 (formerly rs116799036) in the HLA locus. The well-known BD-associated genes HLA-B and MICA were independently genotyped. Although we were not able to formally replicate the association with IL10 and IL23R-IL12RB2, we do report that BD in Iran is strongly associated with HLA-B*51, MICA-A6, and the three HLA-linked SNPs (odds ratio (OR) = 3.38, P = 6.21 × 10(-14); OR = 2.08, P = 1.58 × 10(-13); and OR = 1.67-4.05, P = 1.45 × 10(-04) to 4.79 × 10(-34), respectively). Our data further indicate that the robust HLA-B/MICA association may be explained by a single variant (rs76546355) between the two genes. Overall, these data contribute to a better appraisal of the intriguing linkage between BD and the ancient Silk Route, spanning from the Mediterranean shores to Japan.

A de novo ADCY5 mutation causes early-onset autosomal dominant chorea and dystonia.

IMPORTANCE: Apart from Huntington's disease, little is known of the genetics of autosomal dominant chorea associated with dystonia. Here we identify adenylate cyclase 5 (ADCY5) as a likely new causal gene for early-onset chorea and dystonia. OBSERVATIONS: Whole exome sequencing in a three-generation family affected with autosomal dominant chorea associated with dystonia identified a single de novo mutation­c.2088+1G>A in a 5' donor splice-site of ADCY5­segregating with the disease. This mutation seeming leads to RNA instability and therefore ADCY5 haploinsufficiency. CONCLUSIONS AND RELEVANCE: Our finding confirms the genetic/clinical heterogeneity of the disorder; corroborated by previous identification of ADCY5 mutations in one family with dyskinesia-facial myokymia and in two unrelated sporadic cases of paxoysmal choreic/dystonia-facial myokymia; ADCY5's high expression in the striatum and movement disorders in ADCY5-deficient mice. Hence ADCY5 genetic analyses may be relevant in the diagnostic workup of unexplained early-onset hyperkinetic movement disorders.

High diversity of MIC genes in non-human primates.

The human MHC class I (MHC-I) chain-related genes A and B (MICA and MICB) encode stress-induced glycoproteins, ligands for the activating receptor NKG2D. They display an unusually high degree of polymorphism, next only to that of classical MHC-I. The functional relevance and selective pressure behind this peculiar polymorphism, which is quite distinct from that of classical MHC-I, remain largely unknown. This study increases the repertoire of allelic sequences determined for the MIC genes of non-human primates. Sequencing (mainly exons 2, 3, 4, 5) MIC genes of 72 Macaca fascicularis (Mafa), 63 Pan troglodytes (Patr), and 18 Gorilla gorilla (Gogo) individuals led to the identification of 35, 14, and 3 new alleles, respectively. Additionally, we confirm the existence of three independent MIC genes in M. fascicularis, i.e., Mafa-MICA, Mafa-MICB, and Mafa-MICB/A, the latter being a hybrid of Mafa-MICB and Mafa-MICA. By multiple sequence alignment and phylogenetic analysis, we further demonstrate that the present day MIC genes most likely derive from a single human MICB-like ancestral gene.

The non-conventional MHC class I MR1 molecule controls infection by Klebsiella pneumoniae in mice.

As opposed to the well established role of MHC-linked, polymorphic, class I (MHC-I) genes in adaptive immunity, a universal role for non-conventional MHC-I is unknown, thus requiring a case-by-case study. The MHC unlinked, monomorphic, but ß2microglobulin (ß2m)-associated "MHC class I related" MR1 molecule interacts with a semi-invariant TCR. The pathophysiology of this interaction or more importantly of this peculiar MHC-I remains mostly unknown. Recently it was shown that ß2m deficient mice were more susceptible to infection by Klebsiella pneumoniae, a widely spread Gram-negative bacteria that causes diverse and often severe ailments in man. Here we demonstrate, using both an in vivo imaging system and survival tests, the increased susceptibility to K. pneumoniae (but not to several other Gram negative bacteria) of MR1 deficient mice. This is accompanied by a consequent change in body temperature and systemic cytokine profile. Hence MR1 controls K. pneumoniae infection in vivo.

Lipolysis is altered in MHC class I zinc-alpha(2)-glycoprotein deficient mice.

Non-conventional major histocompatibility complex class I molecules are involved in a variety of physiological functions, most at the periphery of the immune system per se. Zinc-alpha(2)-glycoprotein (ZAG), the sole soluble member of this superfamily has been implicated in cachexia, a poorly understood yet life-threatening, severe wasting syndrome. To further ascertain the role of ZAG in lipid metabolism and perhaps the immune system, we inactivated both ZAG alleles by gene targeting in mice. Subjecting these ZAG deficient animals to standard or lipid rich food regimens led to increased body weight in comparison to identically treated wild-type mice. This phenotype appeared to correlate with a significant decrease in adipocytic lipolysis that could not be rescued by several pharmacological agents including beta(3)-adrenoreceptor agonists. Furthermore, in contrast to previously reported data, ZAG was found to be ubiquitously and constitutively expressed, with an especially high level in the mouse liver. No overt immunological phenotype was identified in these animals.

MIC and other NKG2D ligands: from none to too many.

NKG2D, a prime activatory receptor on human NK, CD8(+) alphabeta and gammadelta cells, has a variety of ligands, which, despite sharing membership of the MHC class I structural club, display an array of unique features. Chronologically, human MIC molecules were the first NKG2D ligands to be identified. Then came RAET1 (ULBP) molecules, which were identified in both man and mouse, as well as H60 and MULT1, which have no counterparts in man to date. The question remains as to why, more than how, the evolutionary conserved, apparently monomorphic, single copy, NKG2D, can/should adapt to this variety of ligands, and when it does, what is the evolutionary advantage of this profusion of ligands for a single receptor?

In vivo immunogenetics: from MIC to RAET1 loci.

The major histocompatibility complex (MHC) comprises approximately one thousandth of the genome and encompasses its most polymorphic members. This diversity enables the MHC, at the population level, to counteract the extraordinarily diverse microbiological threats. Reviewed here are two separate sets of MHC class I genes: MIC and RAET1. Whilst the former are encoded within the MHC (6p21.3), the latter are located on the opposite arm of the same chromosome (6q24.2-q25.3). Differing from the prototypical class I genes in structure, transcription, diversity and potential function, they both exemplify the versatility of the MHC fold, despite convergence onto a single ligand, the activatory C-type lectin-like receptor, NKG2D. Why the immune system uses two distinct gene families to interact with a unique ligand remains a fascinating question. To answer this question, the reader will be chronologically exposed to the field whilst following a single thread, i.e. genomics and gene diversity.

Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1.

The evolutionary conservation of T lymphocyte subsets bearing T-cell receptors (TCRs) using invariant alpha-chains is indicative of unique functions. CD1d-restricted natural killer T (NK-T) cells that express an invariant Valpha14 TCRalpha chain have been implicated in microbial and tumour responses, as well as in auto-immunity. Here we show that T cells that express the canonical hValpha7.2-Jalpha33 or mValpha19-Jalpha33 TCR rearrangement are preferentially located in the gut lamina propria of humans and mice, respectively, and are therefore genuine mucosal-associated invariant T (MAIT) cells. Selection and/or expansion of this population requires B lymphocytes, as MAIT cells are absent in B-cell-deficient patients and mice. In addition, we show that MAIT cells are selected and/or restricted by MR1, a monomorphic major histocompatibility complex class I-related molecule that is markedly conserved in diverse mammalian species. MAIT cells are not present in germ-free mice, indicating that commensal flora is required for their expansion in the gut lamina propria. This indicates that MAIT cells are probably involved in the host response at the site of pathogen entry, and may regulate intestinal B-cell activity.

Iron overload in mice expressing HFE exclusively in the intestinal villi provides evidence that HFE regulates a functional cross-talk between crypt and villi enterocytes.

Hereditary hemochromatosis (HH), a common autosomal recessive disorder due to a mutation in HFE, which encodes an atypical MHC class I glycoprotein, is characterized by excessive absorption of dietary iron. Little is known however of the apparently complex pathophysiology of HFE involvement in the process of iron influx. Here, in order to tackle the issue in vivo, we decided to target HFE expression exclusively to the relevant tissue, intestinal epithelium. This was achieved by putting HFE under transcriptional control of the rat fatty acid binding protein (Fabpi) promoter. Quite unexpectedly, Fabpi-HFE mice had significantly elevated serum transferrin saturation levels in comparison to those of normal littermates. By a careful, layer by layer analysis of transgene expression along the crypt-villus axis, we were able to affirm that the ectopic expression of transgenic HFE in the differentiated villi enterocytes was responsible for ferric hyperabsorption, a phenomenon exacerbated in the absence of endogenous HFE expression, which we assessed by crossing the transgene onto an HFE(-/-) (knockout) background. This forced dichotomy between the absence of HFE in the crypt and expression in the villi provides experimental support that HFE functions as a "gatekeeper," regulating the cross-talk between the crypt and villi enterocytes and thereby modulating the avidity of mature enterocytes for dietary iron.

Inactivation of the hemochromatosis gene differentially regulates duodenal expression of iron-related mRNAs between mouse strains.

BACKGROUND & AIMS: Hfe knockout mice, like patients with hereditary hemochromatosis, have augmented duodenal iron absorption and increased iron deposition in hepatic parenchymal cells. The goals of the present study were to gain further insight into the control of iron absorption by comparing the transcript levels of iron-related genes in the duodenum of DBA/2 Hfe-/- mice, susceptible to iron loading, and wild-type controls, and to test whether variations in the duodenal expression of these messengers contribute to the DBA/2 and C57BL/6 strain differences in the severity of hepatic iron loading. METHODS: Expression of the different transcripts was quantified by real-time polymerase chain reaction. RESULTS: The 2 strains differ strikingly, not only in the severity of hepatic iron loading, but also in the duodenal expression of iron-related genes. In DBA/2 Hfe-/- mice, increased intestinal iron absorption results from the concomitant up-regulation of the Dcytb, DMT1, and FPN1 messengers. No increase in the expression of these messengers is seen in C57BL/6 Hfe-/- mice. CONCLUSIONS: The up-regulation of these transcripts suggests that an inappropriate iron-deficiency signal is sensed by the duodenal enterocytes, leading to an enhanced ferric reductase activity and the increase of duodenal iron uptake and transfer to the circulation. The genes modifying the hemochromatosis phenotype probably act by modifying the expression of these 3 messengers.

A basolateral sorting motif in the MICA cytoplasmic tail.

The MHC class I chain-related MICA molecule is a stress-induced, highly polymorphic, epithelia-specific, membrane-bound glycoprotein interacting with the activating NK cell receptor NKG2D and/or gut-enriched Vdelta1-bearing gammadelta T cells. We have previously reported the presence of a MICA transmembrane-encoded short-tandem repeat harboring a peculiar allele, A5.1, characterized by a frame shift mutation leading to a premature intradomain stop codon, thus denying the molecule of its 42-aa cytoplasmic tail. Given that this is the most common population-wide MICA allele found, we set out to analyze the functional consequences of cytoplasmic tail deletion. Here, we show native expression of MICA at the basolateral surface of human intestinal epithelium, the site of putative interaction with intraepithelial T and NK lymphocytes. We then demonstrate, in polarized epithelial cells, that although the full-length MICA protein is sorted to the basolateral membrane, the cytoplasmic tail-deleted construct as well as the naturally occurring A5.1 allele are aberrantly transported to the apical surface. Site-directed mutagenesis identified the cytoplasmic tail-encoded leucine-valine dihydrophobic tandem as the basolateral sorting signal. Hence, the physiological location of MICA within epithelial cells is governed by its cytoplasmic tail, implying impairment in A5.1 homozygous individuals, perhaps relevant to the immunological surveillance exerted by NK and T lymphocytes on epithelial malignancies.

A cluster of ten novel MHC class I related genes on human chromosome 6q24.2-q25.3.

We have identified a novel family of human major histocompatibility complex (MHC) class I genes. This MHC class I related gene family is defined by 10 members, among which 6 encode potentially functional glycoproteins. The 180-kb cluster containing them has been generated by serial duplication and minimal diversification of an ancestral prototype. They are not located within the MHC on 6p21.3, but near the tip of its long arm at q24.2-q25.3, close to the human equivalent of the mouse H2-linked t-complex, a subchromosomal region syntenic to a segment of mouse chromosome 10 harboring the orthologous MHC class I related retinoic acid early transcript loci, Raet1a-d. Hence we have named the identified loci RAET1E-N. Human RAET1 products are all devoid of the membrane-proximal immunoglobulin-like alpha3 domain and most, but not all, are predicted to remain membrane-anchored via glycosylphosphatidylinositol linkage and are shown to display an atypical pattern of polymorphism. RAET1 transcripts are absent from hematopoietic tissues, but largely expressed in tumors. The involvement of orthologous mouse RAET1A-D/H60 in natural killer and T-cell activation through NKG2D engagement augurs a similar function for the human RAET1 proteins.