Alexander S. Whitehead

Polymorphism of the human complement C4 and steroid 21-hydroxylase genes. Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants.

Several autoimmune disorders as well as congenital adrenal hyperplasia (CAH) are either associated or closely linked with genetic variants of the fourth component of complement (C4A and C4B) and the enzyme steroid 21-hydroxylase (21-OH). These proteins are encoded by genes that are located downstream from the genes for complement proteins, C2 and factor B (BF) between HLA-B and -DR in the major histocompatibility complex (MHC). Previous studies of variants and null alleles were based on electrophoretic mobility of C4 protein and linkage with disease phenotypes. These data did not permit analysis of the basis for the observed null alleles and duplicated variants. We studied this region of the MHC in 126 haplotypes for a structural analysis of the four adjacent loci, C4A, 21-OHA, C4B, and 21-OHB. About half of the C4 genes typed as C4 null are deleted and several unrecognized homoduplicated C4 alleles were detected. Hence the frequencies of different C4 structural variants must be recalculated based on a direct analysis of the genes. Analysis of the C4/21-OH genes of patients with the classical (salt-wasting) form of CAH showed that some involve a deletion of the C4B and 21-OHB genes; whereas for two only the 21-OHB gene is deleted, i.e., the C4B gene is present. Together, these data provide a better understanding of the mechanisms generating and importance of deleted C4 and 21-OH null alleles in human disease.

DNA polymorphism of the C4 genes. A new marker for analysis of the major histocompatibility complex

Polymorphisms of the proteins encoded by genes that lie within the major histocompatibility complex (MHC) have served as useful markers for organ transplantation and in genetic analysis of a large number of MHC-linked diseases. To extend the range of MHC polymorphic markers, we used a complementary-DNA probe specific for the fourth component of human complement (C4) to identify a new variant within the MHC. Polymorphic variants at the DNA level were detected among subjects with identical phenotypes of the corresponding protein. C4 genomic polymorphisms are inherited with the segment of the short arm of chromosome 6 that carries the HLA-DR and complement loci. The autosomal codominant mode of inheritance of this genetic marker and its utility for evaluation of 21-hydroxylase-deficiency congenital adrenal hyperplasia, one of the many MHC-linked diseases, were established.

Nucleotide sequence and expression of the human gene encoding apolipoprotein H (β2-glycoprotein I)

Human apolipoprotein H (ApoH), also called beta 2-glycoprotein I, is a 50-kDa serum glycoprotein whose function is not clearly defined. We have cloned and sequenced ApoH cDNAs both from human liver and from a human hepatoma cell line (HepG2). Both cDNA sequences predict a protein 345 amino acids (aa) in length. This sequence includes a 19-aa hydrophobic, N-terminal signal sequence which is not present in the mature protein [Lozier et al., Proc. Natl. Acad. Sci. USA 81 (1984) 3640-3644]. It differs from this previously reported aa sequence at two positions, both of which strengthen the conservation among the four short consensus repeats within the ApoH molecule. COS-1 cells transiently transfected with the ApoH cDNA in a eukaryotic expression vector produced a single species of ApoH mRNA and secreted in the ApoH protein. The level of ApoH mRNA expressed by HepG2 cells is downregulated by incubation with inflammatory mediators, implying that ApoH is a negative acute-phase protein.

Haplotypic polymorphisms of the TNFB gene

The NTFB genes from two major histocomptibility complex (MHC) ancestral haplotypes have been compared. The genes carried by the ancestral haplotypes 8.1 (A1,B8,BfS,C4AQ0, C4B1,DR3) and 57.1 (A1,B57, BfS,C4A6,C4B1,DR7) were cloned and sequenced to determine the degree of polymorphism. In this report we show that the r e spective TNF genes are allelic and have unique nucleotide sequences. The data demonstrate the presence of three nucleotide differences between the TNFB alleles of 8.1 and 57.1. Two of the differences occur in untranslated regions of the gene but the third nucleotide change results in amino acid differences in the mature TNFB protein. These polymorphisms may have implications with respect to differential regulation in disease-and nondisease-associated haplotypes.

The human C-reactive protein gene (CRP) and serum amyloid P component gene (APCS) are located on the proximal long arm of chromosome 1.

The genes encoding two pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are located on the proximal long arm of human chromosome 1. Mapping of the CRP and SAP genes between the centromere and band q32 was achieved by Southern blot analysis of DNA from a panel of human × Chinese hamster somatic cell hybrids carrying defined fragments of human chromosome 1. Both genes were localized more precisely between bands q12 and q23 by in situ hybridization to human metaphase chromosomes.

The Molecular Basis for Genetic Deficiency of the Second Component of Human Complement

Genetic deficiency of the second component of complement (C2) is the most common complement-deficiency state among Western Europeans and is frequently associated with autoimmune diseases. To examine the molecular basis of this deficiency, we established cultures of blood monocytes from four families with C2-deficient members. Using a hemolytic-plaque assay, [35S]methionine metabolic labeling of proteins in tissue culture and immunoprecipitation, RNA extraction and Northern blot analysis, and DNA restriction-enzyme digestion and Southern blot analysis, we found that C2 deficiency is not due to a major gene deletion or rearrangement but is the result of a specific and selective pretranslational regulatory defect in C2 gene expression. This leads to a lack of detectable C2 mRNA and a lack of synthesis of C2 protein. The approach used in this study should prove useful in examination of other plasma protein deficiencies, especially those in which the deficient gene is normally expressed in peripheral-blood monocytes or tissue macrophages and in which ethical considerations preclude the use of liver or other tissue for study.

Molecular genetics of mouse serum amyloid P component (SAP): cloning and gene mapping.

The pentraxins are serum proteins found in a wide range of species. In mammals, they have a discoid native configuration comprising five noncovalently associated monomeric subunits. There are two pentraxins: serum amyloid P component (SAP) and C-reactive protein (CRP); sequence similarity (Woo et al. 1985) and close genetic linkage in humans (Floyd-Smith et al. 1986) suggest that both genes were produced by an ancestral gene-duplication event. SAP is a precursor of amyloid P component which is associated with amyloid deposits in secondary amyloidosis (reviewed by Pepys and Baltz 1983). Human SAP (Pepys and Butler 1987) and human CRP (Robey et al. 1984) both bind chromatin. In the mouse, SAP is an acute phase reactant, increasing in serum concentration during inflammation. Different strains display characteristic endogenous SAP levels; C3H and DBA mice have high levels, and C57BL mice have low levels. In studies of BXD and BXH recombinant inbred (RI) strains derived from the above progenitor strains, Mortensen and co-workers (1985) demonstrated that 91% and 46%, respectively, of the progenitor-specific variation of endogenous SAP levels cosegregated with an allele at the Ly-9 locus on chromosome 1; these studies defined a locus (designated Sap) that is principally, but not exclusively, responsible for controlling strain-specific noninflammatory SAP levels. To determine whether the SAP structural gene maps to the same region as the Sap locus, we generated a SAP cDNA probe for analysis of N-strain DNA samples. The amino acid sequence of both human (Mantzouranis et al. 1985) and Syrian hamster (Dowton et al. 1985) SAP between residues 185 and 192 are encoded by identical nucleotide sequences. As this nucleotide sequence reflects the theoretically preferred codon usage (Lathe 1985) for all but one residue, a complementary oligonucleotide, WQA, was synthesized and used to screen an acute phase mouse liver cDNA library. A SAP-specific cDNA clone,

DNA polymorphism of MHC III genes in inbred and wild mouse strains.

Genes encoding the second component (C2), factor B, and complement protein C4 and Slp (sex-limited protein) are members of the major histocompatibility complex class III gene cluster. In this report we describe isolation of a mouse C2 cDNA clone and its use together with factor B and C4 cDNA clones to examine the S region in a panel of 42 haplotypes in laboratory and wild mice representing 5 species and subspecies of Mus. Conservation of the C2 factor B gene duplex was evidenced by relatively limited polymorphism associated with speciation and nucleotide sequence homology between mouse and human C2 and factor B The C4-Slp gene duplex, on the other hand, showed extensive polymorphism by DNA blot analysis. This polymorphism correlated poorly with the C2/factor B restriction fragment length polymorphism, suggesting independent evolution of these two segments of the S region. Taken together, these data will be of particular importance in studies of mouse strains with abnormal regulation of immune effector systems since the class III gene products are essential for activation of the complement cascade.

Molecular Biology of the Human and Mouse MHC Class III Genes: Phylogenetic Conservation, Genetics and Regulation of Expression

The generation of complementary and genomic DNA clones for the human and mouse MHC class III genes has advanced the study of the organization, structure, genetics and expression of these loci. These clones have been useful in defining new polymorphic markers in each species and therefore permit a more complete genetic analysis of the complement cluster and the MHC as a whole. The coding sequences of the factor B and C4 genes are extensively conserved both within and between species, in contrast to the coding sequences of other MHC products. In human and mouse, the organization of the class III genes is similar with respect to order and position between the class II and class I regions of the MHC. However, these inter-species similarities in the organization and products of the class III genes does not extend to their regulation. In addition to complement gene expression being regulated differently between tissue sites within a species, expression is apparently regulated differently in analogous tissues between species. The considerable progress which has been made in the molecular analysis of C2, factor B and C4 using DNA clones forms the basis for the future study of the biology of the class III genes and the role of complement in inflammatory processes and in the immune system.