Lethal neonatal respiratory failure due to biallelic variants in BBS1 and monoallelic variant in TTC21B.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a rare, autosomal recessive ciliopathy characterized by early onset retinal dystrophy, renal anomalies, postaxial polydactyly, and cognitive impairment with considerable phenotypic heterogeneity. BBS results from biallelic pathogenic variants in over 20 genes that encode key proteins required for the assembly or primary ciliary functions of the BBSome, a heterooctameric protein complex critical for homeostasis of primary cilia. While variants in BBS1 are most frequently identified in affected individuals, the renal and pulmonary phenotypes associated with BBS1 variants are reportedly less severe than those seen in affected individuals with pathogenic variants in the other BBS-associated genes. CASE-DIAGNOSIS: We report an infant with severe renal dysplasia and lethal pulmonary hypoplasia who was homozygous for the most common BBS1 pathogenic variant (c.1169 T > G; p.M390R) and also carried a predicted pathogenic variant in TTC21B (c.1846C > T; p.R616C), a genetic modifier of disease severity of ciliopathies associated with renal dysplasia and pulmonary hypoplasia. CONCLUSIONS: This report expands the phenotypic spectrum of BBS with the first infant with lethal neonatal respiratory failure associated with biallelic, pathogenic variants in BBS1 and a monoallelic, predicted pathogenic variant in TTC21B. BBS should be considered among the ciliopathies in the differential diagnosis of neonates with renal dysplasia and severe respiratory failure.

Surfactant protein-C promoter variants associated with neonatal respiratory distress syndrome reduce transcription.

Dominant mutations in coding regions of the surfactant protein-C gene, SFTPC, cause respiratory distress syndrome (RDS) in infants. However, the contribution of variants in noncoding regions of SFTPC to pulmonary phenotypes is unknown. By using a case-control group of infants > or =34 weeks gestation (n = 538), we used complete resequencing of SFTPC and its promoter, genotyping, and logistic regression to identify 80 single nucleotide polymorphisms (SNPs). Three promoter SNPs were statistically associated with neonatal RDS among European descent infants. To assess the transcriptional effects of these three promoter SNPs, we selectively mutated the SFTPC promoter and performed transient transfection using MLE-15 cells and a firefly luciferase reporter vector. Each promoter SNP decreased SFTPC transcription. The combination of two variants in high linkage dysequilibrium also decreased SFTPC transcription. In silico evaluation of transcription factor binding demonstrated that the rare allele at g.-1167 disrupts a SOX (SRY-related high mobility group box) consensus motif and introduces a GATA-1 site, at g.-2385 removes a MZF-1 (myeloid zinc finger) binding site, and at g.-1647 removes a potential methylation site. This combined statistical, in vitro, and in silico approach suggests that reduced SFTPC transcription contributes to the genetic risk for neonatal RDS in developmentally susceptible infants.

Alveolar capillary dysplasia with misalignment of pulmonary veins and anterior segment dysgenesis of the eye: a report of a new association and review of the literature.

The association of alveolar capillary dysplasia with misalignment of pulmonary veins (ACD-MPV) and ocular abnormalities has not been previously reported. We present a case of ACD-MPV and anterior segment dysgenesis of the eye in a full-term infant as well as a review of the relevant literature.

Surfactant protein B deficiency: antenatal diagnosis and prospective treatment with surfactant replacement.

An infant with a family history of congenital alveolar proteinosis associated with surfactant protein B (SP-B) deficiency was identified when SP-B was not detected in amniotic fluid obtained at 37, 38, and 40 weeks of gestation. Surfactant replacement with commercially available preparations that contained SP-B was begun soon after delivery. Progressive respiratory failure developed despite continued surfactant replacement, corticosteroid therapy, and extracorporeal membrane oxygenation. The infant died at 54 days of age while awaiting lung transplantation. Surfactant extracted from amniotic fluid, bronchoalveolar lavage fluid, and lung tissue had no phosphatidylglycerol; surface tension was 24 dynes/cm (normal, < 10 dynes/cm) and did not decrease with in vitro addition of exogenous SP-B. Pulmonary vascular permeability measured with positron emission tomography was twice normal. At autopsy the alveolar proteinosis pattern was less prominent than that seen in affected siblings. Immunoreactivity of SP-B was absent in type II cells, but numerous foreign body granulomas with central immunoreactivity for SP-B and surfactant protein C were present. We conclude that exogenous surfactant replacement did not normalize surfactant composition, activity, or pulmonary vascular permeability. These findings suggest that endogenous SP-B synthesis is necessary for mature surfactant metabolism and function.

Neonatal and infantile pulmonary hemorrhage: an autopsy study with clinical correlation.

We studied the clinicopathologic features of pulmonary hemorrhage in autopsied infants less than 1 year of age for a 12-month period. There were 70 liveborns (LB) and 24 stillborns (SB). The percentage of LB with pulmonary hemorrhage (PH), pulmonary interstitial emphysema (PIE), hyaline membrane disease (HMD), acute bronchopneumonia (ABP), congenital malformations (CM), and surgery (SUR) were analyzed according to weeks of estimated gestational age (EGA) and as an entire group. Overall, 74% of LB and 24% of SB had histologic evidence of PH. A semiquantitative evaluation of the extent of PH among the LB infants disclosed that hemorrhage involved less than one-third of the observed lung tissue in 42%, one-third to two-thirds of the lung was hemorrhagic in 15%, and the remaining 42% had more than two-thirds hemorrhagic lung parenchyma. A total of 31 clinical and pathologic factors were evaluated for their possible association or relationship to PH. Statistical analysis revealed that hyaline membranes and hemorrhage in other extrapulmonary sites were the only significant associations with PH. PH was more frequent in premature infants born between 24 and 39 weeks EGA and was always associated with multiple other conditions. Although the autopsy finding of hemorrhage in the lungs is relatively frequent in the population we studied, it appears that PH as a primary phenomenon in infants is extremely rare, if it occurs at all.

Biosynthesis and processing of the mannose receptor in human macrophages.

The biosynthesis and processing of the human mannose receptor has been studied in monocyte-derived macrophages. Adherent cells were labeled for 60 min with Trans35S (a mixture of 35S-labeled methionine and cysteine), chased, and subjected to immunoprecipitation by antibody raised against the human placental receptor. The antibody immunoprecipitated a single protein of molecular mass 162 kDa; precipitation of the labeled receptor could be inhibited by placental receptor. The results presented demonstrate that the receptor is synthesized as a 154-kDa precursor which is processed to 162 kDa in 90 min. The precursor is a glycoprotein bearing endoglycosidase H-sensitive oligosaccharides; the 162-kDa form is endoglycosidase H-resistant but peptide:N-glycanase-sensitive. Desialylation of the mannose receptor with neuraminidase generates a protein which is recognized by peanut agglutinin, a lectin that specifically binds desialylated O-linked oligosaccharides. Thus, the human macrophage mannose receptor bears both N- and O-linked oligosaccharide chains. Newly synthesized mannose receptor exhibits a half-life of 33 h as determined by pulse-chase studies. This indicates that on the average, each molecule of receptor recycles between the cell surface and endosomes hundreds of times before degradation.

Cloning the gene for an inherited human disorder--chronic granulomatous disease--on the basis of its chromosomal location.

The gene that is abnormal in the X-linked form of the phagocytic disorder chronic granulomatous disease has been cloned without reference to a specific protein by relying on its chromosomal map position. The transcript of the gene is expressed in the phagocytic lineage of haematopoietic cells and is absent or structurally abnormal in four patients with the disorder. The nucleotide sequence of complementary DNA clones predicts a polypeptide of at least 468 amino acids with no homology to proteins described previously.

Regulation of complement protein biosynthesis in mononuclear phagocytes.

Proteins of the complement system (with the exception of the terminal components C6-9) are synthesized in mononuclear phagocytes. The extrahepatic macrophage is therefore an important local source of the complement proteins which may serve as a first-line host defence mechanism. Net synthesis and secretion of complement by these cells is a function of maturation of the mononuclear phagocytic series, the tissue from which the cells are isolated, and the state of macrophage activation. To define some of the mechanisms for regulation of complement gene expression in mononuclear phagocytes, the major histocompatibility complex class III genes and C3 have been investigated. These genes are expressed constitutively in hepatocytes and monocytes/macrophages. In the mononuclear phagocyte, interferon-gamma, at physiological concentrations, effects a dose- and time-dependent increase in factor B and C2 mRNA and a corresponding increase in factor B and C2 biosynthesis. This effect is specific inasmuch as the expression of other genes (e.g. C3) is decreased by interferon-gamma, and interferon-alpha and beta at concentrations one to two logs greater have only a minimal effect on C2 and factor B gene expression. Endotoxin acting directly on monocytes has qualitatively different effects on expression of the complement genes. These complex regulatory mechanisms are being investigated with the use of murine fibroblasts transfected with human DNA bearing the relevent complement genes.

gamma-Interferon increases expression of class III complement genes C2 and factor B in human monocytes and in murine fibroblasts transfected with human C2 and factor B genes.

gamma-Interferon (IFN-gamma) is a well characterized lymphokine known to regulate many mononuclear phagocyte functions, including expression of class I and class II major histocompatibility complex genes. The second component of complement (C2) and factor B are major histocompatibility complex class III gene products synthesized in mononuclear phagocytes. Recombinant IFN-gamma increased the synthesis of C2 and factor B in primary cultures of human mononuclear phagocytes and in murine fibroblasts transfected with cosmid DNA bearing the human C2 and factor B genes. In both cell types the increases in C2 and factor B protein synthesis were detected at concentrations of IFN-gamma less than 1 unit/ml and the regulation of each was pretranslational. The IFN-gamma-induced increases in C2 and factor B mRNA did not require new protein synthesis. In primary cultures of human monocytes, the kinetics of induction of C2 and factor B synthesis differed, but in the transfected L-cells the kinetics were similar, suggesting differences in transduction of the IFN-gamma signal, transcriptional, and/or post-transcriptional events in the two cell types. The small size of the factor B 5' flanking region, which is bounded by the 3' terminus of the IFN-gamma-regulated C2 gene, provides a well defined region to probe the structural basis for IFN-gamma regulation of gene expression.

mRNA and apolipoprotein E synthesis abnormalities in peripheral blood monocyte macrophages in familial apolipoprotein E deficiency.

We have studied synthesis of apolipoprotein E (apo-E) and apo-E mRNA in cultures of peripheral blood human monocyte macrophages (M-M cultures) obtained from a patient with familial apolipoprotein E deficiency. We have found that the M-M cultures of the apo-E-deficient patients contained two apo-E mRNA species with slightly different molecular weight as compared to normal apo-E mRNA. The apo-E mRNA concentration of the apo-E-deficient cultures was approximately 50-fold reduced as compared to the normal cultures, whereas the actin mRNA concentrations were identical in both M-M cultures. Genomic blotting analysis using a full-length apo-E cDNA clone as hybridization probe did not show gross differences between the restriction patterns of the DNA obtained from the apo-E-deficient patient and two normal controls. When normal M-M cultures were grown in media containing [35S]methionine they synthesized and secreted apo-E into the culture media. In contrast we could not detect any intracellular or extracellular apo-E in the patient's M-M cultures grown under identical conditions. These observations are consistent with the hypothesis that familial apo-E deficiency results from structural apo-E gene mutation(s). The putative mutation(s) affect either the transcription of the apo-E gene or the processing of the primary apo-E mRNA transcript. These abnormalities are associated with low levels of synthesis of aberrant apo-E mRNA forms which are either very unstable or cannot be translated into protein.

Distribution of apolipoprotein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction of apolipoprotein E mRNA by cultures of human monocyte-macrophages.

Recently developed molecular probes for human apolipoprotein (apo) genes have been used to study the specificity of human tissue expression of the apo A-I, apo C-II, apo C-III, and apo E genes. We have found that apo E mRNA was present in all tissues examined. On the basis of total RNA concentration the relative abundance of apo E mRNA expressed as a percentage of the liver value is as follows: adrenal gland and macrophages, 74-100%; gonads and kidney, 12-15%; spleen, brain, thymus, ovaries, intestine, and pancreas, 3-9%; heart, 1.5%; stomach, striated muscle, and lung, less than 1%. The relative concentration of apo E mRNA in cultures of human peripheral blood monocyte-macrophages increases dramatically as a function of time in culture, and after 5 days, it compares to that of liver. The human tissues shown to synthesize apo E mRNA were also examined for their ability to synthesize apo A-I, apo C-II, and apo C-III mRNA. The relative abundance of apo A-I, apo C-III, and apo C-II mRNA expressed as a percentage of the liver value is as follows: apo A-I, intestine, 50%; apo A-I, pancreas and gonads, 12%; apo A-I, kidney, 4%; apo A-I, adrenal, 2.5%; apo A-I, ovaries and heart, 1%; apo A-I, stomach and thymus, less than 1%; apo C-III, intestine, 62%; apo C-III, pancreas, 7%; apo C-II, intestine, 3%; apo C-II, pancreas, less than 1%. The knowledge of tissue specificities in the synthesis of apolipoproteins is important for our understanding of the regulation of apolipoproteins and lipoprotein metabolism.

Intrapulmonary chemotaxins in the normal and in the cyclophosphamide-treated host.

The kinetics of intrapulmonary chemotactic activity and the generation of a pulmonary polymorphonuclear leukocytic (PMN) response during experimental Pseudomonas aeruginosa pneumonia were studied in normal and in cyclophosphamide-treated guinea pigs. In normal animals, chemotactic activity for PMN appeared in airways promptly (2 h) after infection and preceded the influx of PMN to infected airways. Week-long regimens of intraperitoneally administered cyclophosphamide, in dosages of 7.5 mg/kg/day (low-dose) or 15 mg/kg/day (high-dose), resulted in systemic myelosuppression accompanied by a dose-related decrease in recruitment of PMN to infected airways. The chemotactic activity assayed in bronchoalveolar fluids obtained from low-dose-treated animals was not affected by cyclophosphamide. However, chemotactic activity in bronchoalveolar fluids was significantly reduced (p less than 0.01) in animals receiving the high-dose cyclophosphamide regimen. Gel chromatography of bronchoalveolar fluids from infected animals revealed that a high molecular weight (20,000 daltons or greater) and a low molecular weight (5,000 daltons) chemotactic factor were present in normal specimens, and that both were absent from specimens obtained from animals receiving the high-dose treatment. Hemolytically active C5 was detected in infected bronchial fluids, but cyclophosphamide treatment did not reduce amounts of C5 in infected airways. These data suggest that in addition to myelosuppression, cyclophosphamide treatment impairs the capacity for pulmonary inflammation by reducing the normal intrapulmonary chemotactic gradient during infection.

Tissue-specific pretranslational regulation of complement production in human mononuclear phagocytes.

The net production of the complement protein C2, C4, and factor B differ among mononuclear phagocytes from peripheral blood and different tissues in experimental animals and in humans. To examine the mechanisms that regulate these differences in humans, the proportions of C2-producing cells, the average single-cell production rate of C2, the posttranslational glycosylation and kinetics of secretion of C2 and factor B, and the amounts of C2 and factor B mRNA were examined in freshly isolated peripheral blood monocytes, monocytes maintained up to 2 wk in culture, and freshly isolated tissue macrophages from breast milk and bronchoalveolar lavage. In addition, the biosynthesis of two other proteins synthesized and secreted by mononuclear phagocytes, C3 and lysozyme, were examined. We report that despite comparable rates of C3 and lysozyme synthesis and similar processing and kinetics of secretion of C2 and factor B, the freshly isolated tissue macrophage differs from the monocyte-derived macrophage in the proportion of C2-producing cells, in the average single-cell production rate of complement, and in the amounts of specific C2 and factor B mRNA. These differences are tissue specific, because C2-specific mRNA content in bronchoalveolar macrophages is considerably greater than in breast milk macrophages, although the amounts of factor B mRNA are comparable. These data suggest that tissue-specific regulation of complement production in human mononuclear phagocytes occurs at a pretranslational level. These studies now provide a basis for investigation of the molecular effects of agents that modulate the biologic functions of monocytes and macrophages.

Expression of the alpha 1-proteinase inhibitor gene in human monocytes and macrophages.

Expression of the alpha 1-proteinase inhibitor (alpha 1PI) gene was studied in human mononuclear cells. Using RNA blot and dot hybridization, alpha 1PI mRNA was detected in human peripheral blood monocytes, bronchoalveolar and breast milk macrophages, but not in B or T lymphocytes. Using incorporation of a radiolabeled amino acid precursor, synthesis and secretion of alpha 1PI were demonstrated in human monocytes and macrophages, but not in lymphocytes. In addition, alpha 1PI was secreted in functionally active form as shown by complexing with serine proteases. Biosynthesis of alpha 1PI by mononuclear phagocytes was greatest during the first 24 hr in culture and progressively decreased over the next 10 days. The reduction in alpha 1PI biosynthesis in vitro involved a mechanism acting at the pretranslational level as alpha 1PI mRNA content also progressively declined over 10 days in culture. The ease of sampling human monocytes and macrophages now permits examination of the biochemical defect in homozygous PiZ and PiS alpha 1PI deficiencies and study of the functional significance of locally produced alpha 1PI in normal tissues and sites of injury or inflammation.

Distinct primary translation products from human liver mRNA give rise to secreted and cell-associated forms of complement protein C2.

The second component of complement (C2), is a class III major histocompatibility complex gene product and a glycoprotein in the classical complement activating system. Synthesis in the human hepatoma-derived cell line HepG2 results in three intracellular forms: an 84-kDa form secreted in 1-2 h; 79-kDa and 70-kDa forms that remain cell-associated for intervals up to 12 h. All three forms are C2 polypeptides as demonstrated by inhibition of immunoprecipitation with unlabeled C2 and the presence of common major peptide fragments following chymotryptic digestion. The cell-associated forms of C2 are not products of proteolysis as demonstrated by experiments with multiple proteinase inhibitors and by observations of the kinetics of synthesis. Inhibition of core glycosylation by tunicamycin and deglycosylation by acid hydrolysis indicate that the three intracellular C2 polypeptides are glycosylated to a similar extent. Although the 84-kDa form of C2 is susceptible to C1s cleavage, the two cell-associated forms are not. Cell-free biosynthesis by mRNA from HepG2 or human liver results in three primary translation products corresponding to the three unglycosylated forms of C2. These results indicate that HepG2 cells synthesize C2 protein in both secreted and cell-associated forms and that each form is derived from a separate primary translation product.

Structure and biosynthesis of cytoplasmic and secreted variants of gelsolin.

Gelsolin is an actin-fragmenting cytoplasmic protein. A functionally similar protein has also been identified in plasma. We have compared the structure of the cytoplasmic and plasma forms of gelsolin and examined their biosynthetic relationships. Plasma gelsolin is larger than cytoplasmic gelsolin (Mr 93,000 versus 90,000, respectively) and is more positively charged. Partial amino acid sequencing analyses show that the two gelsolins share a common 29 amino acid sequence which lies at the NH2-terminal end of cytoplasmic gelsolin and spans residues 26-55 of plasma gelsolin. Compared with cytoplasmic gelsolin, plasma gelsolin contains an additional peptide of 25 amino acids at its NH2 terminus. The human hepatoma-derived cell line, HepG2, synthesizes both the 90-kDa and the 93-kDa gelsolins but secretes only the 93-kDa form. Pulse-chase experiments demonstrate that the rate of disappearance of the 93-kDa gelsolin from the cells corresponds with the rate of appearance of the 93-kDa gelsolin in the medium, whereas the rate of disappearance of the 90-kDa gelsolin is independent of and slower than that of the secreted plasma protein. We conclude that cytoplasmic and plasma gelsolins are structurally similar but not identical, that after synthesis these proteins are processed independently, and that the fate of each is distinct.

Isolation and characterization of resident macrophages from guinea pig and human intestine.

The evaluation of mucosal cellular immune function in the gastrointestinal tract has focused on properties of lymphocytes. We describe methods for use in guinea pig and human tissue that will now permit the maintenance and in vitro study of intestinal macrophages. This study characterizes the resident gastrointestinal macrophage population in the two species and shows that morphologic and ultra-structural characteristics are similar to macrophages from other tissues. Histochemically, the cells are esterase positive and peroxidase negative. They possess surface receptors for immunoglobulin G and complement, and are phagocytic via the Fc receptor.