Prenatal diagnosis and treatment for fetal angiotensin converting enzyme deficiency.

OBJECTIVE: To elucidate an etiology in a case with persistent oligohydramnios by prenatal diagnosis and actively treat the case to achieve good prognosis. METHODS: We performed whole exome sequencing (WES) of DNA from the fetus and parents. Serial amnioinfusions were conducted until birth. Pressors were required to maintain normal blood pressure. The infant angiotensin-converting enzyme (ACE) activity, angiotensin II (Ang II, a downstream product of ACE), and compensatory enzymes (CEs) activities were measured. Compensatory enzyme activities in plasma from age-matched healthy controls were also detected. RESULTS: We identified a fetus with a severe ACE mutation prenatally. The infant was born prematurely without pulmonary dysplasia. Hypotension and anuria resolved spontaneously. He had almost no ACE activity, but his Ang II level and CE activity exceeded the upper limit of the normal range and the upper limit of the 95% confidence interval of controls, respectively. His renal function also largely recovered. CONCLUSION: Fetuses with ACE mutations can be diagnosed prenatally through WES. Serial amnioinfusion permits the continuation of pregnancy in fetal ACE deficiency. Compensatory enzymes for defective ACE appeared postnatally. Renal function may be spared by preterm delivery; furthermore, for postnatal vasopressor therapy to begin, improving renal perfusion pressure before nephrogenesis has been completed.

Parainfluenza virus infections in patients with hematological malignancies or stem cell transplantation: Analysis of clinical characteristics, nosocomial transmission and viral shedding.

To assess morbidity and mortality of parainfluenza virus (PIV) infections in immunocompromised patients, we analysed PIV infections in a hematology and stem cell transplantation (SCT) unit over the course of three years. Isolated PIV strains were characterized by sequence analysis and nosocomial transmission was assessed including phylogenetic analysis of viral strains. 109 cases of PIV infection were identified, 75 in the setting of SCT. PIV type 3 (n = 68) was the most frequent subtype. PIV lower respiratory tract infection (LRTI) was observed in 47 patients (43%) with a mortality of 19%. Severe leukopenia, prior steroid therapy and presence of co-infections were significant risk factors for development of PIV-LRTI in multivariate analysis. Prolonged viral shedding was frequently observed with a median duration of 14 days and up to 79 days, especially in patients after allogeneic SCT and with LRTI. Nosocomial transmission occurred in 47 patients. Phylogenetic analysis of isolated PIV strains and combination with clinical data enabled the identification of seven separate clusters of nosocomial transmission. In conclusion, we observed significant morbidity and mortality of PIV infection in hematology and transplant patients. The clinical impact of co-infections, the possibility of long-term viral shedding and frequent nosocomial transmission should be taken into account when designing infection control strategies.

Single-cell RNA sequencing deconvolutes the in vivo heterogeneity of human bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of the transcriptomes suggest that osteoblast precursors induce angiogenesis coupled with osteogenesis, and chondrocyte precursors have the potential to differentiate into myocytes. We also discovered transcripts for several clusters of differentiation (CD) markers that were either highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs, representing potential novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing an insight into the extent of their cellular heterogeneity and roles in maintaining bone homeostasis.

Inter-Laboratory Comparison of Extracellular Vesicle Isolation Based on Ultracentrifugation.

BACKGROUND/AIMS: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioactive cargo mediating intercellular communication in physiological and pathological conditions. EVs are increasingly investigated as therapeutic agents and targets, but also as disease biomarkers. However, a definite consensus regarding EV isolation methods is lacking, which makes it intricate to standardize research practices and eventually reach a desirable level of data comparability. In our study, we performed an inter-laboratory comparison of EV isolation based on a differential ultracentrifugation protocol carried out in 4 laboratories in 2 independent rounds of isolation. METHODS: Conditioned medium of colorectal cancer cells was prepared and pooled by 1 person and distributed to each of the participating laboratories for isolation according to a pre-defined protocol. After EV isolation in each laboratory, quantification and characterization of isolated EVs was collectively done by 1 person having the highest expertise in the respective test method: Western blot, flow cytometry (fluorescence-activated cell sorting [FACS], nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). RESULTS: EVs were visualized with TEM, presenting similar cup-shaped and spherical morphology and sizes ranging from 30 to 150 nm. NTA results showed similar size ranges of particles in both isolation rounds. EV preparations showed high purity by the expression of EV marker proteins CD9, CD63, CD81, Alix, and TSG101, and the lack of calnexin. FACS analysis of EVs revealed intense staining for CD63 and CD81 but lower levels for CD9 and TSG101. Preparations from 1 laboratory presented significantly lower particle numbers (p < 0.0001), most probably related to increased processing time. However, even when standardizing processing time, particle yields still differed significantly between groups, indicating inter-laboratory differences in the efficiency of EV isolation. Importantly, no relation was observed between centrifugation speed/k-factor and EV yield. CONCLUSIONS: Our findings demonstrate that quantitative differences in EV yield might be due to equipment- and operator-dependent technical variability in ultracentrifugation-based EV isolation. Furthermore, our study emphasizes the need to standardize technical parameters such as the exact run speed and k-factor in order to transfer protocols between different laboratories. This hints at substantial inter-laboratory biases that should be assessed in multi-centric studies.

Lineage-associated connexin 43 expression in bisphosphonate-exposed rat bones.

Expression of signaling proteins in bone cells depends on their embryological mesoderm-derived (e.g. tibia) or cranial neural crest (CNC)-derived (e.g. jaw) origin. Connexin 43 (Cx43) is a gap junction protein that plays an essential role in the mode of action of bisphosphonates (BP). This study aimed to investigate Cx43 expression and the influence of BP application on mesoderm- and CNC-derived bone. Using a rat model, molar extraction and tibia osteotomy with (Group 4) or without (Group 3) previous BP application was performed. Untreated (Group 1) and animals selectively treated with BPs (Group 2) served as controls. Cx43 expression was immunohistochemically determined 12 and 16 weeks postoperatively via a labeling index. Cx43 expression in CNC-derived bone was significantly higher compared with mesodermal bone. BP application decreased Cx43 expression; however, detected expression levels were still higher in jawbone (Group 2 tibia vs jaw: 5.83 ± 5.06 vs 23.52 ± 6.42; p = 0.007). During bone healing after surgical intervention (Group 3) there were no expression differences between tibia and jawbone. BP treatment prior to surgery resulted in significantly lower Cx43 expression in CNC-derived compared with tibia bone (Group 4 tibia vs jaw: 56.84 ± 15.57 vs 16.40 ± 5.66; p < 0.01). Increased Cx43 expression in jaw compared with tibia bone is in line with their embryological origins. A significant Cx43 suppression in jawbone after BP application and surgery might contribute to the selectively altered osseous turnover and development of MRONJ in CNC-derived bone.

Iron isotope evidence for very rapid accretion and differentiation of the proto-Earth.

Nucleosynthetic isotope variability among solar system objects provides insights into the accretion history of terrestrial planets. We report on the nucleosynthetic Fe isotope composition (µ54Fe) of various meteorites and show that the only material matching the terrestrial composition is CI (Ivuna-type) carbonaceous chondrites, which represent the bulk solar system composition. All other meteorites, including carbonaceous, ordinary, and enstatite chondrites, record excesses in µ54Fe. This observation is inconsistent with protracted growth of Earth by stochastic collisional accretion, which predicts a µ54Fe value reflecting a mixture of the various meteorite parent bodies. Instead, our results suggest a rapid accretion and differentiation of Earth during the ~5-million year disk lifetime, when the volatile-rich CI-like material is accreted to the proto-Sun via the inner disk.

Prioritization of Variants for Investigation of Genotype-Directed Nutrition in Human Superpopulations.

Dietary guidelines recommended by key health agencies are generally designed for a global population. However, ethnicity affects human disease and environment-gene interactions, including nutrient intake. Historically, isolated human populations with different genetic backgrounds have adapted to distinct environments with varying food sources. Ethnicity is relevant to the interaction of food intake with genes and disease susceptibility; yet major health agencies generally do not recommend food and nutrients codified by population genotypes and their frequencies. In this paper, we have consolidated published nutrigenetic variants and examine their frequencies in human superpopulations to prioritize these variants for future investigation of population-specific genotype-directed nutrition. The nutrients consumed by individuals interact with their genome and may alter disease risk. Herein, we searched the literature, designed a data model, and manually curated hundreds of papers. The resulting database houses 101 variants that reached significance (p < 0.05), from 35 population studies. Nutrigenetic variants associated with modified nutrient intake have the potential to reduce the risk of colorectal cancer, obesity, metabolic syndrome, type 2 diabetes, and several other diseases. Since many nutrigenetic studies have identified a major variant in some populations, we suggest that superpopulation-specific genotype-directed nutrition modifications be prioritized for future study and evaluation. Genotype-directed nutrition approaches to dietary modification have the potential to reduce disease risk in select human populations.

Minimotifs dysfunction is pervasive in neurodegenerative disorders.

Minimotifs are modular contiguous peptide sequences in proteins that are important for posttranslational modifications, binding to other molecules, and trafficking to specific subcellular compartments. Some molecular functions of proteins in cellular pathways can be predicted from minimotif consensus sequences identified through experimentation. While a role for minimotifs in regulating signal transduction and gene regulation during disease pathogenesis (such as infectious diseases and cancer) is established, the therapeutic use of minimotif mimetic drugs is limited. In this review, we discuss a general theme identifying a pervasive role of minimotifs in the pathomechanism of neurodegenerative diseases. Beyond their longstanding history in the genetics of familial neurodegeneration, minimotifs are also major players in neurotoxic protein aggregation, aberrant protein trafficking, and epigenetic regulation. Generalizing the importance of minimotifs in neurodegenerative diseases offers a new perspective for the future study of neurodegenerative mechanisms and the investigation of new therapeutics.

Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo.

Extracellular vesicles (EVs) are released from nearly all mammalian cells and different EV populations have been described. Microvesicles represent large EVs (LEVs) released from the cellular surface, while exosomes are small EVs (SEVs) released from an intracellular compartment. As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation Vs. apoptosis induction). We could clearly separate two EV populations, namely SEVs (average diameter <200 nm) and LEVs (diameter range between 200 and 1000 nm). Morphology and size were analyzed by electron microscopy and nanoparticle tracking analysis. Apoptosis induction caused a massive release of LEVs, while activated T-cells released SEVs and LEVs in considerably lower amounts. The release of SEVs from apoptotic T-cells was comparable with LEV release from activated ones. LEVs contained signaling proteins and proteins of the actin-myosin cytoskeleton. SEVs carried cytoplasmic/endosomal proteins like the 70-kDa heat shock protein 70 (HSP70) or tumor susceptibility 101 (TSG101), microtubule-associated proteins, and ubiquitinated proteins. The protein expression profile of SEVs and LEVs changed substantially after the induction of apoptosis. After apoptosis induction, HSP70 and TSG101 (often used as exosome markers) were highly expressed within LEVs. Interestingly, in contrast to HSP70 and TSG101, gelsolin and eps15 homology domain-containing protein 3 (EHD3) turned out to be specific for SEVs irrespective of the stimulus causing the EV release. Finally, we detected several subunits of the proteasome (PSMB9, PSMB10) as well as the danger signal HMGB1 exclusively within apoptotic cell-released LEVs. Thus, we were able to identify new marker proteins that can be useful to discriminate between distinct LEV subpopulations. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD009074.

Minimotif Miner 4: a million peptide minimotifs and counting.

Minimotif Miner (MnM) is a database and web system for analyzing short functional peptide motifs, termed minimotifs. We present an update to MnM growing the database from ∼300 000 to >1 000 000 minimotif consensus sequences and instances. This growth comes largely from updating data from existing databases and annotation of articles with high-throughput approaches analyzing different types of post-translational modifications. Another update is mapping human proteins and their minimotifs to know human variants from the dbSNP, build 150. Now MnM 4 can be used to generate mechanistic hypotheses about how human genetic variation affect minimotifs and outcomes. One example of the utility of the combined minimotif/SNP tool identifies a loss of function missense SNP in a ubiquitylation minimotif encoded in the excision repair cross-complementing 2 (ERCC2) nucleotide excision repair gene. This SNP reaches genome wide significance for many types of cancer and the variant identified with MnM 4 reveals a more detailed mechanistic hypothesis concerning the role of ERCC2 in cancer. Other updates to the web system include a new architecture with migration of the web system and database to Docker containers for better performance and management. Weblinks:minimotifminer.org and mnm.engr.uconn.edu.

Apoptotic-cell-derived membrane microparticles and IFN-α induce an inflammatory immune response.

A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-α (IFN-α), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-α on professional phagocytes. In the presence of IFN-α, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-α concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-α and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-α, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material.

Damaging the Integrated HIV Proviral DNA with TALENs.

HIV-1 integrates its proviral DNA genome into the host genome, presenting barriers for virus eradication. Several new gene-editing technologies have emerged that could potentially be used to damage integrated proviral DNA. In this study, we use transcription activator-like effector nucleases (TALENs) to target a highly conserved sequence in the transactivation response element (TAR) of the HIV-1 proviral DNA. We demonstrated that TALENs cleave a DNA template with the HIV-1 proviral target site in vitro. A GFP reporter, under control of HIV-1 TAR, was efficiently inactivated by mutations introduced by transfection of TALEN plasmids. When infected cells containing the full-length integrated HIV-1 proviral DNA were transfected with TALENs, the TAR region accumulated indels. When one of these mutants was tested, the mutated HIV-1 proviral DNA was incapable of producing detectable Gag expression. TALEN variants engineered for degenerate recognition of select nucleotide positions also cleaved proviral DNA in vitro and the full-length integrated proviral DNA genome in living cells. These results suggest a possible design strategy for the therapeutic considerations of incomplete target sequence conservation and acquired resistance mutations. We have established a new strategy for damaging integrated HIV proviral DNA that may have future potential for HIV-1 proviral DNA eradication.

The HIVToolbox 2 web system integrates sequence, structure, function and mutation analysis.

There is enormous interest in studying HIV pathogenesis for improving the treatment of patients with HIV infection. HIV infection has become one of the best-studied systems for understanding how a virus can hijack a cell. To help facilitate discovery, we previously built HIVToolbox, a web system for visual data mining. The original HIVToolbox integrated information for HIV protein sequence, structure, functional sites, and sequence conservation. This web system has been used for almost 40,000 searches. We report improvements to HIVToolbox including new functions and workflows, data updates, and updates for ease of use. HIVToolbox2, is an improvement over HIVToolbox with new functions. HIVToolbox2 has new functionalities focused on HIV pathogenesis including drug-binding sites, drug-resistance mutations, and immune epitopes. The integrated, interactive view enables visual mining to generate hypotheses that are not readily revealed by other approaches. Most HIV proteins form multimers, and there are posttranslational modification and protein-protein interaction sites at many of these multimerization interfaces. Analysis of protease drug binding sites reveals an anatomy of drug resistance with different types of drug-resistance mutations regionally localized on the surface of protease. Some of these drug-resistance mutations have a high prevalence in specific HIV-1 M subtypes. Finally, consolidation of Tat functional sites reveals a hotspot region where there appear to be 30 interactions or posttranslational modifications. A cursory analysis with HIVToolbox2 has helped to identify several global patterns for HIV proteins. An initial analysis with this tool identifies homomultimerization of almost all HIV proteins, functional sites that overlap with multimerization sites, a global drug resistance anatomy for HIV protease, and specific distributions of some DRMs in specific HIV M subtypes. HIVToolbox2 is an open-access web application available at [http://hivtoolbox2.bio-toolkit.com].

Pathogen-triggered activation of plasmacytoid dendritic cells induces IL-10-producing B cells in response to Staphylococcus aureus.

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.

Apoptotic-cell-derived membrane vesicles induce an alternative maturation of human dendritic cells which is disturbed in SLE.

The clearance of apoptotic cells occurs in a non-inflammatory context. Defects in this clearance process have been linked to the emergence of human autoimmune diseases like systemic lupus erythematosus (SLE). A characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surfaces. Those vesicles have recently been recognized as mediators of intercellular communication or as adjuvant in the pathogenesis of autoimmune diseases. We analyzed the interactions between these apoptotic cell-derived membrane vesicles and professional antigen presenting cells. These vesicles were engulfed by monocyte-derived dendritic cells (mDC) and stimulated their maturation towards a phenotype comprising an upregulation of CD80, CD83, CD86, and a remarkable downregulation of MHC class II molecules. We observed only a minor release of proinflammatory cytokines from these mDC when compared to LPS stimulation. mDC stimulated by apoptotic vesicles did not cause significant T-cell expansion. Interestingly, when compared to normal healthy donors SLE patients-derived dendritic cells showed a significantly different phenotype lacking the downregulation of MHC class II, which correlated to disease activity.

Arginine deficiency leads to impaired cofilin dephosphorylation in activated human T lymphocytes.

The amino acid arginine is fundamentally involved in the regulation of the immune response during infection, inflammatory diseases and tumor growth. Arginine deficiency (e.g. due to the myeloid cell enzyme arginase) inhibits proliferation and effector functions of activated T lymphocytes. Here, we studied intracellular mechanisms mediating this suppression of human T lymphocytes. Our proteomic analysis revealed an impaired dephosphorylation of the actin-binding protein cofilin upon T-cell activation in the absence of arginine. We show that this correlates with alteration of actin polymerization and impaired accumulation of CD2 and CD3 in the evolving immunological synapse in T cell-antigen presenting cells conjugates. In contrast, T-cell cytokine synthesis is differentially regulated in human T lymphocytes in the absence of arginine. While the production of certain cytokines (e.g. IFN-γ) is severely reduced, T lymphocytes produce other cytokines (e.g. IL-2) independent of extracellular arginine. MEK and PI3K activity are reciprocally regulated in association with impaired cofilin dephosphorylation. Finally, we show that impaired cofilin dephosphorylation is also detectable in human T cells activated in a granulocyte-dominated purulent micromilieu due to arginase-mediated arginine depletion. Our novel results identify cofilin as a potential regulator of human T-cell activation under conditions of inflammatory arginine deficiency.

The human phosphotyrosine signaling network: evolution and hotspots of hijacking in cancer.

Phosphotyrosine (pTyr) signaling, which plays a central role in cell-cell and cell-environment interactions, has been considered to be an evolutionary innovation in multicellular metazoans. However, neither the emergence nor the evolution of the human pTyr signaling system is currently understood. Tyrosine kinase (TK) circuits, each of which consists of a TK writer, a kinase substrate, and a related reader, such as Src homology (SH) 2 domains and pTyr-binding (PTB) domains, comprise the core machinery of the pTyr signaling network. In this study, we analyzed the evolutionary trajectories of 583 literature-derived and 50,000 computationally predicted human TK circuits in 19 representative eukaryotic species and assigned their evolutionary origins. We found that human TK circuits for intracellular pTyr signaling originated largely from primitive organisms, whereas the inter- or extracellular signaling circuits experienced significant expansion in the bilaterian lineage through the "back-wiring" of newly evolved kinases to primitive substrates and SH2/PTB domains. Conversely, the TK circuits that are involved in tissue-specific signaling evolved mainly in vertebrates by the back-wiring of vertebrate substrates to primitive kinases and SH2/PTB domains. Importantly, we found that cancer signaling preferentially employs the pTyr sites, which are linked to more TK circuits. Our work provides insights into the evolutionary paths of the human pTyr signaling circuits and suggests the use of a network approach for cancer intervention through the targeting of key pTyr sites and their associated signaling hubs in the network.

Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles.

Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

Loss-of-function mutations of ILDR1 cause autosomal-recessive hearing impairment DFNB42.

By using homozygosity mapping in a consanguineous Pakistani family, we detected linkage of nonsyndromic hearing loss to a 7.6 Mb region on chromosome 3q13.31-q21.1 within the previously reported DFNB42 locus. Subsequent candidate gene sequencing identified a homozygous nonsense mutation (c.1135G>T [p.Glu379X]) in ILDR1 as the cause of hearing impairment. By analyzing additional consanguineous families with homozygosity at this locus, we detected ILDR1 mutations in the affected individuals of 10 more families from Pakistan and Iran. The identified ILDR1 variants include missense, nonsense, frameshift, and splice-site mutations as well as a start codon mutation in the family that originally defined the DFNB42 locus. ILDR1 encodes the evolutionarily conserved immunoglobulin-like domain containing receptor 1, a putative transmembrane receptor of unknown function. In situ hybridization detected expression of Ildr1, the murine ortholog, early in development in the vestibule and in hair cells and supporting cells of the cochlea. Expression in hair cell- and supporting cell-containing neurosensory organs is conserved in the zebrafish, in which the ildr1 ortholog is prominently expressed in the developing ear and neuromasts of the lateral line. These data identify loss-of-function mutations of ILDR1, a gene with a conserved expression pattern pointing to a conserved function in hearing in vertebrates, as underlying nonsyndromic prelingual sensorineural hearing impairment.

Clinical manifestations but not cytokine profiles differentiate adult-onset Still's disease and sepsis.

OBJECTIVE: To analyze clinical manifestations, serum ferritin, and serum cytokine levels in patients with adult-onset Still's disease (AOSD) or bacterial sepsis and to evaluate their potential use for differential diagnosis. METHODS: Twenty-two consecutive patients with the first flare of AOSD and 6 patients with an established diagnosis of AOSD under immunosuppressive therapy were compared with 14 patients with bacterial sepsis. Clinical manifestations were scored in a Pouchot AOSD activity score including elevated serum ferritin levels to obtain a modified Pouchot score. Serum cytokine profiles were analyzed from each patient. RESULTS: The scores of clinical manifestations using a modified Pouchot activity score were significantly higher in patients with active untreated AOSD (mean 5.60 ± 1.93) compared with patients with chronic AOSD (mean 1.16 ± 0.98; p < 0.001) and patients with sepsis (mean 2.38 ± 1.19; p < 0.001). A modified Pouchot score ≥ 4 shows a sensitivity of 92% and a specificity of 93% for active AOSD. Serum cytokine levels of interleukin 1ß (IL-1ß), IL-6, IL-8, IL-10, IL-12, IL-18, interferon-γ, tumor necrosis factor-α, and calprotectin were elevated in acute AOSD and sepsis. Significant differences were detected only in patients with sepsis who had higher levels of IL-6 and IL-8. The overlap of the 2 groups limits the use of cytokines for differential diagnosis in individual patients. CONCLUSION: A modified Pouchot AOSD activity score including elevated serum ferritin levels was more useful to confirm the diagnosis of AOSD compared to patients with sepsis. Elevated serum cytokines correlate with inflammation but are of limited use to differentiate between active AOSD and bacterial sepsis.