Population-based estimates of surfactant protein B deficiency.

OBJECTIVE: Surfactant protein B deficiency is a lethal cause of respiratory distress in infancy that results most commonly from a homozygous frameshift mutation (121ins2). Using independent clinical ascertainment and molecular methods in different populations, we sought to determine allele frequency. STUDY DESIGN: Using clinical characteristics of the phenotype of affected infants, we screened the Missouri linked birth-death database (n = 1 052 544) to ascertain potentially affected infants. We used molecular amplification and restriction enzyme digestion of DNA samples from a metropolitan New York birth cohort (n = 6599) to estimate allele frequency. RESULTS: The point estimate and 95% confidence interval of the 121ins2 allele frequency in the Missouri cohort are 1/1000 individuals (.03-5.6/1000) and in the New York cohort are.15/1000 (. 08-.25/1000). These estimates are not statistically different. CONCLUSIONS: The close approximation of these independent estimates suggests accurate gene frequency (approximately one 121ins2 mutation per 1000-3000 individuals) despite its rare occurrence and that this mutation does not account for the majority of full-term infants with lethal respiratory distress.

Modulation of renal disease in MRL/lpr mice genetically deficient in the alternative complement pathway factor B.

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B-/-), and heterozygous (B+/-) MRL/lpr mice. Compared with B+/- or B+/+ mice, MRL/lpr B-/- mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B-/- mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.

Complement: a critical test of its biological importance.

The biological activities of the more than 30 proteins that comprise the complement system have been elucidated in parallel lines of investigation that resulted in the purification of the proteins and studies of their function in vitro. Twenty years ago, the first complement cDNA clones were generated. Subsequently the structure and chromosomal localization of the complement genes and the primary sequences of their gene products were revealed. For some, even their higher order structure was solved. This work, coupled with studies of complement gene expression, biosynthesis, post-synthetic processing and secretion, contributed to an analysis of the relatively rare naturally occurring genetic deficiencies of complement proteins discovered fortuitously in humans sand experimental animals. Not until the past 5 years, with the application of methods for manipulating genes in vivo (targeted deletion and overexpression), has it been possible to definitively assign specific functions to complement proteins and to assess their importance in the intact organism. These relatively recent studies have confirmed the in vitro work or revealed unexpected roles for complement effector and regulatory proteins in host defenses, specific immunity, immunopathology, metabolism and reproductive biology This work is reviewed and the implications for understanding human diseases and the design of novel pharmaceutical agents are discussed. The promise of this line of investigation is certain but the context imposed by gender, developmental stage, other genes and environment must be taken into account before the practical implications of this deeper understanding of complement biology are fully realized.

The role of complement activation in tumour necrosis factor-induced lethal hepatitis.

Injection of tumour necrosis factor (TNF) in animals causes severe liver cell toxicity, especially when D-(+)-galactosamine (GalN) is co-administered. After challenge with TNF/GalN, serum complement activity (CH50 and APCH50) decreased dramatically, suggesting strong activation of both the classical and the alternative pathways. TNF or GalN alone had no such effect. A cleavage product of complement protein C3 [C3(b)] was deposited on the surface of hepatocytes of TNF/GalN-treated mice. Intravenous administration of cobra venom factor (CVF), which depletes complement, inhibited the development of hepatitis. However, CVF pretreatment also protected C3-deficient mice. Pretreatment of mice with a C1q-depleting antibody did not prevent TNF/GalN lethality, although the anti-C1q antibody had depleted plasma C1q. Factor B-deficient and C3-deficient mice, generated by gene targeting, proved to be as sensitive to TNF/GalN as control mice. Furthermore, induction of lethal shock by platelet-activating factor, an important mediator in TNF-induced hepatic failure, was not reduced in C3-deficient mice. These data indicate that complement, although activated, plays no major role in the generation of acute lethal hepatic failure in this model and that CVF-induced protection is independent of complement depletion.

Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA.

Genetic deficiencies of the complement protein C3 occur naturally in humans and animal models and have been induced in mice by targeted deletion of the C3 gene. The study of these deficiencies has provided evidence for C3 functions in immune responses. C3 deficient mice were generated by replacing the 5'-flanking region of the C3 gene with the neomycin-resistance (neo) gene. Serum from these mice had no detectable C3 protein or complement activity. Challenge with Streptococcus pneumoniae revealed approximately 2000-fold increase in bacteremia as compared to littermate controls. C3 mRNA was absent in the liver, but it was detected in the lung, kidney, fat tissue, heart and spleen. Metabolic labeling of the lung tissue and peritoneal macrophages showed synthesis of pro-C3, but no post-synthetic intracellular processing of the protein and no secretion of mature C3. cDNA analysis at the cap site indicated that extrahepatic transcription of the targeted gene was initiated in the neo cassette, close to the C3/neo junction and predicted a primary translation product lacking the leader peptide. The data indicate that these mice provide a good animal model for the study of complete C3 deficiencies and a potential probe for tissue-specific C3 gene regulatory elements.

Disruption of disulfide bonds is responsible for impaired secretion in human complement factor H deficiency.

Factor H, a secretory glycoprotein composed of 20 short consensus repeat modules, is an inhibitor of the complement system. Previous studies of inherited factor H deficiency revealed single amino acid substitutions at conserved cysteine residues, on one allele arginine for cysteine 518 (C518R) and on the other tyrosine for cysteine 941 (C941Y) (Ault, B. H., Schmidt, B. Z., Fowler, N. L., Kashtan, C. E., Ahmed, A. E., Vogt, B. A., and Colten, H. R. (1997) J. Biol. Chem. 272, 25168-25175). To ascertain if the phenotype, impaired secretion of factor H, is due to the C518R substitution or the C941Y substitution and to ascertain the mechanism by which secretion is impaired, we studied COS-1 and HepG2 cells transfected with wild type and several mutant factor H molecules. The results showed markedly impaired secretion of both C518R and C941Y factor H as well as that of factor H molecules bearing alanine or arginine substitutions at the Cys518-Cys546 disulfide bond (C518A, C546A, C546R, C518A-C546A). In each case, mutant factor H was retained in the endoplasmic reticulum and degraded relatively slowly as compared with most other mutant secretory and membrane proteins that are retained in the endoplasmic reticulum. These data indicate that impaired secretion of the naturally occurring C518R and C941Y mutant factor H proteins is due to disruption of framework-specific disulfide bonds in factor H short consensus repeat modules.

Deficiency of human complement protein C4 due to identical frameshift mutations in the C4A and C4B genes.

The complement protein C4, encoded by two genes (C4A and C4B) on chromosome 6p, is the most polymorphic among the MHC III gene products. We investigated the molecular basis of C4 deficiency in a Finnish woman with systemic lupus erythematosus. C4-specific mRNA was present at low concentrations in C4-deficient (C4D) patient fibroblasts, but no pro-C4 protein was detected. This defect in C4 expression was specific in that synthesis of two other complement proteins was normal. Analysis of genomic DNA showed that the proposita had both deleted and nonexpressed C4 genes. Each of her nonexpressed genes, a C4A null gene inherited from the mother, a C4A null gene, and a C4B null gene inherited from the father, all contained an identical 2-bp insertion (TC) after nucleotide 5880 in exon 29, providing the first confirmatory proof of the C4B pseudogene. This mutation has been previously found only in C4A null genes. Although the exon 29/30 junction is spliced accurately, this frameshift mutation generates a premature stop at codon 3 in exon 30. These truncated C4A and C4B gene products were confirmed through RT-PCR and sequence analysis. Among the possible genetic mechanisms that produce identical mutations is both genes, the most likely is a mutation in C4A followed by a gene conversion to generate the mutated C4B allele.

Identification of a novel alternatively spliced mRNA of murine pulmonary surfactant protein B.

An alternatively spliced mRNA of pulmonary surfactant protein B (SP-B) was identified in murine lung. Sequencing analysis revealed a 69 base-pair (bp) deletion at the beginning of exon 7 of SP-B, presumably the result of an alternative splicing event. Reverse transcription-polymerase chain reaction (RT-PCR) of mouse, rat, and rabbit lung RNA revealed the existence of full-length and the 69-bp deleted short form. Ribonuclease protection assay of the SP-B messenger RNA (mRNA) demonstrated expression of both isoforms in five strains of adult and fetal mice with different genetic backgrounds, as well as in rabbit, but not in human. Splice junction sequences in exon 6 and at the exon 7 splice boundary for the two isoforms are similar, including AG doublet identity, but sequence differences do not account for species variation in isoform abundance. The abundance of the short SP-B mRNA isoform was approximately 30% of total SP-B mRNA in mouse and rabbit. Analysis of precursor SP-B protein in mouse lung suggested that the two mRNA species are expressed as stable protein isoforms.

Molecular heterogeneity in deficiency of complement protein C2 type I.

Deficiency of the complement protein C2 (C2D), one of the most common genetic deficiencies of the complement system, is associated with rheumatological disorders and increased susceptibility to infection. Two types of C2D have been recognized, each in the context of specific major histocompatibility complex (MHC) haplotypes; type I, a deletion, frameshift and premature stop codon resulting in absence of detectable C2 protein synthesis, and type II, missense mutations resulting in a block in secretion of C2 proteins. Analysis of C2 expression in a child with C2 deficiency, a MHC haplotype different from those associated with type I or II C2D, and recurrent infections revealed additional molecular heterogeneity among C2 deficient patients. No detectable C2 protein was synthesized in the child's fibroblasts under conditions supporting C2 synthesis and secretion in normals and the child's C2 mRNA was reduced to 42% of normal. Nucleotide sequencing of RT-PCR fibroblast mRNA and genomic DNA revealed a type I C2 deficiency (28 base-pair deletion) on one allele and a previously unrecognized two base-pair deletion in exon 2 on the other. Expression of the closely linked factor B gene was markedly decreased (Bf mRNA 25% of normal), though Bf was up-regulated appropriately by interferon-gamma and the flanking sequence containing the Bf promoter was normal in this C2-deficient patient. Moreover, the concentration of Bf protein was normal in the patient's plasma.

Human factor H deficiency. Mutations in framework cysteine residues and block in H protein secretion and intracellular catabolism.

The synthesis and secretion of factor H, a regulatory protein of the complement system, were studied in skin fibroblasts from an H-deficient child who has chronic hypocomplementemic renal disease. In normal fibroblasts, factor H transcripts of 4.3 and 1.8 kilobase pairs (kb) encode a 155-kDa protein containing short consensus repeat (SCR) domains 1-20 and a 45-kDa protein which contains SCRs 1-7, respectively. The patient's fibroblasts expressed normal amounts of the 4.3- and 1.8-kb messages constitutively and after tumor necrosis factor-alpha/interferon-gamma stimulation. Lysates of [35S]methionine-labeled fibroblasts from the patient contained the 155- and 45-kDa H polypeptides, but secretion of the 155-kDa protein was blocked; the 45-kDa protein was secreted with normal kinetics. The patient's plasma lacked the 155-kDa protein but contained the small form of H. Moreover, in fibroblasts the retained 155-kDa factor H protein was not degraded, even after 12 h. Immunoflourescent staining and confocal microscopic imaging of the patient's fibroblasts indicated that factor H was retained in the endoplasmic reticulum. Sequence analysis of reverse transcription-polymerase chain reaction products (the entire coding region) and genomic DNA revealed a T1679C substitution on one allele and a G2949A substitution on the other (C518R mutation in SCR 9 and C991Y mutation in SCR 16, respectively). Both mutations affect conserved cysteine residues characteristic of SCR modules and therefore predict profound changes in the higher order structure of the 155-kDa factor H protein. These data provide the first description of a molecular mechanism for factor H deficiency and yield important insights into the normal secretory pathway for this and other plasma proteins with SCR motifs.

Abrogation of the alternative complement pathway by targeted deletion of murine factor B.

To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P = 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

Lung transplantation for treatment of infants with surfactant protein B deficiency.

OBJECTIVE: To evaluate lung transplantation for treatment of surfactant protein B (SP-B) deficiency. STUDY DESIGN: We compared surfactant composition and function from pretransplantation and posttransplantation samples of bronchoalveolar lavage fluid, somatic and lung growth, neurodevelopmental progress, pulmonary function, and pulmonary immunohistology in 3 infants with SP-B deficiency who underwent bilateral lung transplantation at 2 months of age and 3 infants who underwent lung transplantation for other reasons. RESULTS: Two years after transplantation, the 2 surviving infants with SP-B deficiency exhibited comparable somatic growth and cognitive development to the comparison infants. All infants had delays in gross motor development that improved with time. Both groups have exhibited normal gas exchange, lung growth, and pulmonary function. The SP-B-deficient infants have also exhibited normal SP-B expression and pulmonary surfactant function after lung transplantation. In two SP-B-deficient infants antibody to SP-B developed. No pathologic consequences of this antibody were identified. CONCLUSIONS: Apart from the development of anti-SP-B antibody, the outcomes for SP-B-deficient infants after lung transplantation are similar to those of infants who undergo lung transplantation for other reasons. Lung transplantation offers a successful interim therapy until gene replacement for this disease is available.

Constitutive expression of murine complement factor B gene is regulated by the interaction of its upstream promoter with hepatocyte nuclear factor 4.

Factor B (Bf) is a constituent of the alternative pathway of complement activation encoded within the major histocompatibility complex. Transcription of the murine gene from two initiation sites generates two Bf mRNA species differing in size and tissue distribution. Striking genetic, tissue-specific differences in Bf mRNA levels at extrahepatic sites (kidney and intestine) among mouse strains correlate with a DNA sequence polymorphism in the 5'-flanking region of the gene and differential nuclear protein binding at the Bf upstream transcriptional initiation site (UIS). To ascertain the functional consequences of this polymorphism in the Bf promoter, we analyzed the effects of strain-specific sequences in the Bf 5' region on the expression of a chloramphenicol acetyltransferase (CAT) reporter gene transfected in human and mouse hepatoma cells. The CAT activity and mRNA level produced when transcription was driven by the sequence of strains with high extrahepatic expression were reduced to background levels when the sequence specific to the low expressor strains was used. Eighty percent of this difference was accounted for by a point substitution that affects DNA-protein interaction at the UIS, the sequence of higher affinity conferring higher expression. Hepatocyte nuclear factor 4 (HNF-4), derived from HepG2, mouse liver and kidney or cell-free translation of HNF-4 RNA, is the nuclear protein that preferentially binds to the high expressor UIS. Bf-CAT is not expressed in cells that lack HNF-4 (CV-1). However, co-transfection of HNF-4 into CV-1 cells drives Bf-CAT expression and reproduces the differences derived from the substitution that affect HNF-4 binding in vitro. These data show that interaction of HNF-4 with polymorphic variants of the upstream Bf promoter is the major determinant of strain-specific extrahepatic factor B expression.

Type II human complement C2 deficiency. Allele-specific amino acid substitutions (Ser189 --> Phe; Gly444 --> Arg) cause impaired C2 secretion.

Type II complement protein C2 deficiency is characterized by a selective block in C2 secretion. The Type II C2 null allele (C2Q0) is linked to two major histocompatibility haplotypes (MHC) that differ from the MHC of the more common Type I C2 deficiency. To determine the molecular basis of Type II deficiency the two Type II C2Q0 genes were isolated and transfected separately into L-cells. Subsequent molecular biology, biosynthetic, and immunofluorescence studies demonstrated that C2 secretion is impaired in Type II C2 deficiency because of different missense mutations at highly conserved residues in each of the C2Q0 alleles. One is in exon 5 (nucleotide C566 --> T; Ser189 --> Phe) of the C2Q0 gene linked to the MHC haplotype A11,B35,DRw1,BFS, C4A0B1. The other is in exon 11 (G1930 --> A; Gly444 --> Arg) of the C2Q0 gene linked to the MHC haplotype A2,B5, DRw4,BFS,C4A3B1. Each mutant C2 gene product is retained early in the secretory pathway. These mutants provide models for elucidating the C2 secretory pathway.

Genetic deficiencies of complement.

Genetic deficiencies of proteins of the complement system are associated with diverse clinical phenotypes. These clinical manifestations vary as a function of the specific component that is missing. Molecular and cellular biological methods, coupled with more intensive clinical studies, have defined the pathophysiological basis for this set of genetic disorders. Insights into the normal function of complement and its role in immunopathology have been derived from the extensive work in this field during the past few years.

Translational regulation of murine complement factor B alternative transcripts by upstream AUG codons.

Factor B (Bf), a constituent of the alternative pathway of complement activation, is encoded by a single gene that is located within the MHC. In murine kidney and intestine, two Bf transcripts (Bf short and Bf long), generated from distinct transcriptional initiation sites, are expressed in approximately equal amounts. In the liver, > 95% of Bf mRNA is the short transcript. To ascertain the biologic consequences of this tissue-specific mRNA polymorphism, we quantitated the effect of structural differences between the two transcripts on net Bf protein synthesis. Cell-free translation of Bf mRNA in vitro revealed that the rate of translation of Bf short is about twice that of Bf long. The 5' extension of Bf long includes four short open reading frames upstream of the authentic translational initiation codons. Mutation of all four upstream AUGs generates a Bf long transcript with a translational rate about equal to that of Bf short. This effect was primarily accounted for by mutation of the second AUG from the 5' end. Similar studies of Bf expression in vivo showed an approximately twofold difference in translational rate between Bf long and the transcript mutated at the upstream AUGs. Because systemic and local inflammation in kidney alters the ratio of Bf long and short, net production of complement protein Bf in extrahepatic tissues is regulated by both transcriptional and translational control mechanisms.

Human complement protein C2. Alternative splicing generates templates for secreted and intracellular C2 proteins.

Alternative splicing of the primary transcript for human complement protein C2 generates templates for translation of a secreted (C2 long) protein and an intracellular (C2 short) form in liver, bronchoalveolar macrophages, and fibroblasts. The approximate ratio of C2 long to C2 short mRNA is 2:1. The C2 short mRNA does not contain the 396-base pair encompassed by exons 2 and 3 of the full-length C2 long and thus lacks codons for the 5 carboxyl-terminal residues of the signal peptide. Synthesis of C2 in cells transfected with full-length RNA corresponding to each of the transcripts show that C2 long is secreted within a half-time of approximately 1 h and that C2 short is not secreted. Cell-free biosynthesis in the presence of microsomes demonstrate that this intracellular C2 protein (70 kDa) is apparently capable of traversing the membrane of the endoplasmic reticulum. Though the function of the intracellular C2 protein is unknown, it is abundant in all cell types that express the C2 gene.