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Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and intestinal features of 6 unrelated MIA-CID patients. All patients displayed a profound, generalized lymphocytopenia, with few lymphocytes present in the lymph nodes. The thymus was hypoplastic and exhibited an abnormal distribution of epithelial cells. Patients also had profound disruption of the epithelial barrier along the entire gastrointestinal tract. Using linkage analysis and whole-exome sequencing, we identified 10 mutations in tetratricopeptide repeat domain-7A (TTC7A), all of which potentially abrogate TTC7A expression. Intestinal organoid cultures from patient biopsies displayed an inversion of apicobasal polarity of the epithelial cells that was normalized by pharmacological inhibition of Rho kinase. Our data indicate that TTC7A deficiency results in increased Rho kinase activity, which disrupts polarity, growth, and differentiation of intestinal epithelial cells, and which impairs immune cell homeostasis, thereby promoting MIA-CID development.

Background Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes may overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. Objective To investigate the ability of whole-exome screening methods to detect disease-causing variants in individuals with PIDDs. Methods Individuals with PIDDs from 278 families from 22 countries were investigated using whole-exome sequencing (WES). Computational CNV prediction pipelines and an exome-tiling chromosomal microarray were also applied to identify intragenic copy number variants (CNVs). Analytic approaches initially focused on 475 known or candidate PIDD genes, but were non-exclusive and were further tailored based upon clinical data, family history and immunophenotyping. Results A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on the molecular findings. Twelve PIDD-causing CNVs were detected, including seven smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. Conclusion This high-throughput genomic approach enabled detection of disease-related variants in unexpected genes, permitted detection of low-grade constitutional, somatic and revertant mosaicism, and provided evidence of a mutational burden in mixed PIDD immunophenotypes.

ABSTRACT. Objectives. To investigate the occurrence of and risk factors for late-onset septicemia (LOS) in a national cohort of extremely premature infants who received very early full human milk feeding.Methods. A prospective study of all infants born in Norway in 1999 and 2000 with gestational age of <28 weeks or birth weight of <1000 g was performed. Extensive clinical information, including data on feeding practices and episodes of septicemia, was collected on predefined forms. LOS was defined as growth of bacteria or fungi in blood cultures in conjunction with clinical symptoms consistent with systemic infection occurring after day 6 of life. Cox regression models, including models allowing for time-dependent covariates, were applied in the analysis of LOS.Results. Of 464 eligible infants, 462 (99.6%) were enrolled and 405 (87.7%) survived until day 7. LOS was diagnosed for 80 (19.7%). The predominant pathogens were coagulase-negative staphylococci, followed by Candida spp. Case fatality rates associated with septicemia were 10% in general and 43% for Candida spp septicemia. Necrotizing enterocolitis or bowel perforation was diagnosed for 19 infants (4%). Enteral feeding with human milk was initiated within the third day for 98% of patients, and 92% were receiving full enteral feeding (FEF) with human milk within the third week. Both high Clinical Risk Index for Babies scores and an umbilical venous catheter in situ at 7 days of age significantly predicted LOS. However, the overall most influential risk factor for LOS was the number of days without establishment of FEF with human milk, with an adjusted relative risk of 3.7 (2.0 -6.9) for LOS if FEF was not established within the second week of life.Conclusions. The incidence and case fatality rate of septicemia for this cohort of extremely preterm infants were lower than values in comparable studies. The main difference, compared with other studies, was the feeding practice, and the data suggest that very early FEF with human milk significantly reduces the risk of LOS among extremely premature infants. L ate-onset septicemia (LOS) remains an important cause of death, morbidity, and long-term sequelae among premature infants. 1-9 The reported incidence of LOS among infants with birth weight (BW) of Ͻ1500 g ranges between 16 and 30%, [1][2][3][4]8,9 approaching 50% among infants with BW of Ͻ1000 g, 8 and case fatality rates are high (17-21%). [1][2][3]8 Gram-positive bacterial flora, mainly coagulase-negative staphylococci (CONS), has dominated bloodstream recovery for some years. 1,5,10,11 However, among the smallest infants, systemic fungal infections have become a major problem, with high case fatality rates and high rates of severe sequelae among survivors. 7 For these patients, the risk of invasive infection is high, dependent on a number of clinical factors, and inversely related to BW and gestational age (GA). [1][2][3][4]8,9 Enteral feeding with human milk is generally regarded as beneficial 12,13 and may reduce the incidence of necrotizing enterocoli...

Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to Gram-negative bacteria were here studied taking advantage of human genetic complement-deficiencies-nature's own knockouts-including a previously undescribed C5 defect. Such deficiencies provide a unique tool for investigating the biological role of proteins. The experimental conditions allowed cross-talk between the different inflammatory pathways using a whole blood model based on the anticoagulant lepirudin, which does not interfere with the complement system. Expression of tissue factor, cell adhesion molecules, and oxidative burst depended highly on C5, mediated through the activation product C5a, whereas granulocyte enzyme release relied mainly on C3 and was C5a-independent. Release of cytokines and chemokines was mediated to varying degrees by complement and CD14; for example, interleukin (IL)-1␤ and IL-8 were more dependent on complement than IFN-␥ and IL-6, which were highly dependent on CD14. IL-1 receptor antagonist (IL-1ra) and IFN-␥ inducible protein 10 (IP-10) were fully dependent on CD14 and inversely regulated by complement, that is, complement deficiency and complement inhibition enhanced their release. Granulocyte responses were mainly complement-dependent, whereas monocyte responses were more dependent on CD14. Notably, all responses were abolished by combined neutralization of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to Gram-negative bacteria.

Human phosphoglucomutase 3 (PGM3) catalyzes the conversion of N-acetyl-glucosamine (GlcNAc)-6-phosphate into GlcNAc-1-phosphate during the synthesis of uridine diphosphate (UDP)-GlcNAc, a sugar nucleotide critical to multiple glycosylation pathways. We identified three unrelated children with recurrent infections, congenital leukopenia including neutropenia, B and T cell lymphopenia, and progression to bone marrow failure. Whole-exome sequencing demonstrated deleterious mutations in PGM3 in all three subjects, delineating their disease to be due to an unsuspected congenital disorder of glycosylation (CDG). Functional studies of the disease-associated PGM3 variants in E. coli cells demonstrated reduced PGM3 activity for all mutants tested. Two of the three children had skeletal anomalies resembling Desbuquois dysplasia: short stature, brachydactyly, dysmorphic facial features, and intellectual disability. However, these additional features were absent in the third child, showing the clinical variability of the disease. Two children received hematopoietic stem cell transplantation of cord blood and bone marrow from matched related donors; both had successful engraftment and correction of neutropenia and lymphopenia. We define PGM3-CDG as a treatable immunodeficiency, document the power of whole-exome sequencing in gene discoveries for rare disorders, and illustrate the utility of genomic analyses in studying combined and variable phenotypes.

There has been an increase in data suggesting that besides air, hospital water is a potential source of transmission of filamentous fungi, and in particular Aspergillus fumigatus. Molecular characterization of environmental and clinical A. fumigatus isolates, collected prospectively during an 18-month period, was performed to establish if waterborne fungi play a role in the pathogenesis of invasive aspergillosis. Isolates recovered from water (n ‫؍‬ 54) and air (n ‫؍‬ 21) at various locations inside and outside the hospital and from 15 patients (n ‫؍‬ 21) with proven, probable, or possible invasive aspergillosis were genotyped by amplified fragment length polymorphism analysis. Based on genomic fingerprints, the environmental A. fumigatus isolates could be grouped into two major clusters primarily containing isolates recovered from either air or water. The genotypic relatedness between clinical and environmental isolates suggests that patients with invasive aspergillosis can be infected by strains originating from water or from air. In addition, 12 clusters with genetically indistinguishable or highly related strains were differentiated, each containing two to three isolates. In two clusters, clinical isolates recovered from patients matched those recovered from water sources, while in another cluster the clinical isolate was indistinguishable from one cultured from air. This observation might open new perspectives in the development of infection control measures to prevent invasive aspergillosis in high-risk patients. The genetic variability found between airborne and waterborne A. fumigatus strains might prove to be a powerful tool in understanding the transmission of invasive aspergillosis and in outbreak control.With the continuing increase in the number of severely immunocompromised patients, hospitals are faced with the growing problem of invasive aspergillosis and other opportunistic fungal infections (17). Since treatment of these infections is difficult and the outcome is often fatal, preventive measures are of major importance in the control of invasive filamentous fungal infections. Nosocomial invasive aspergillosis is thought to be primarily airborne; however, despite the use of appropriate hospital air filtration systems, the incidence of this infection continues to increase. Together with the lack of genetic relatedness between airborne strains and those causing invasive disease (10, 12), it is tempting to speculate about the existence of other routes of transmission.As early as 1985, Aspergillus species and other filamentous fungi were shown to inhabit water distribution systems that deliver water to hospitals in Europe and the United States (13). Recently, there has been an increase in data supporting water as a potential source of filamentous fungi and, in particular, Aspergillus fumigatus (1, 2, 4, 28). However, direct evidence that waterborne A. fumigatus strains are involved in human infection is lacking. To test the hypothesis that a wet route of transmission might exist, we performed molecu...