A problem-based learning approach to teaching medical genetics.

A newly developed problem-based medical genetics course that was integrated into the fourth-year medical school curriculum of the University of Texas Health Science Center at San Antonio is described. To provide a basic genetic background for the clinical rotations, a supplemental computer tutorial is required during the second year. These two formats prepare the medical students to recognize genetic diseases, to provide basic genetic counseling in their daily practice, and to appropriately refer patients to genetic specialists.

Linkage of cystic fibrosis locus and polymorphic DNA markers in 14 families.

Linkage relationships between the cystic fibrosis (CF) locus and three polymorphic DNA markers were examined in 14 families, five of which were of Hispanic origin. Tight linkage was found between the CF locus and MET (maximum lod score = 7.16 at theta = .001), and between CF and pJ3.11 (maximum lod score = 3.87 at theta = .001). We observed two recombinations between CF and collagen, yielding a maximum lod score of 0.359 at theta = .125, and one recombination in the cluster CF-MET-pJ3.11. Analysis by the seriation method indicates the order COL-pJ3.11-CF-MET.

Characterization, mapping, and expression of the human ceruloplasmin gene.

Ceruloplasmin (CP) is a copper-binding protein in vertebrate plasma. It is the product of an intragenic triplication and is composed of three homologous domains. Oligonucleotide probes constructed according to published amino acid sequences were used to identify cDNA clones encoding human CP. Two clones, CP-1 and CP-2, differed from each other by the presence or absence, respectively, of a deduced sequence of four amino acids. The two clones provided 81% of the sequence encoding CP. Comparison of the nucleotides of the three domains of the CP coding sequence revealed internal domain homology with identity of sequences ranging from 50.1% to 56%. The nucleotide sequence of CP-2 cDNa was compared to that of a homologous human protein, clotting factor VIII, and was found to be 48% identical overall. The CP gene was mapped to human chromosome 3 by somatic-cell-hybrid analysis and to 3q25 by in situ hybridization; however, sites of hybridization to DNA on other chromosomal sites suggested additional CP-like DNA sequences in the human genome. A DNA polymorphism was detected with CP cDNA after endonuclease digestion of human DNA by Pst I. CP mRNA was detected in human liver, macrophages, and lymphocytes by in situ histohybridization.

Evolution of haptoglobin: comparison of complementary DNA encoding Hp alpha 1S and Hp alpha 2FS.

Haptoglobin is a transport glycoprotein which removes free hemoglobin from the circulation of vertebrates. In human populations haptoglobin is polymorphic due to three alleles, Hp alpha 1F, Hp alpha 1S and Hp alpha 2. The Hp alpha 2 allele is roughly twice the length of the Hp alpha 1 alleles and is the product of a partial gene duplication possibly resulting from an unequal crossover event in a heterozygous genotype Hp alpha 1F/Hp alpha 1S. In the study described here we compare the cDNA encoding Hp alpha 1S to that encoding Hp alpha 2FS . Both have a leader sequence followed by the genotypic alpha chain sequence, a beta sequence and an untranslated sequence in the 3' end. The cDNA encoding Hp alpha 2FS is composed of alpha 1F and alpha 1S domains differing by four nucleotide replacements. Hp alpha 1S cDNA contains the same replacement site mutations found in the alpha 1S domain of Hp alpha 2FS , indicating that this coding region has sustained few, if any, mutations since its incorporation into the Hp alpha 2FS gene.

Duplication within the haptoglobin Hp2 gene.

DNA sequencing shows that the intragenic duplication within the human haptoglobin Hp2 allele was formed by a non-homologous, probably random, crossing-over within different introns of two Hp1 genes, probably in an Hp1F / Hp1S heterozygote.

Localization of the haptoglobin alpha and beta genes (HPA and HPB) to human chromosome 16q22 by in situ hybridization.

Human haptoglobin (Hp) is a protein that binds free hemoglobin and circulates in plasma of vertebrates as a tetrachain (alpha beta)2 structure. This study maps HPA and HPB, the genes encoding the Hp alpha and beta chains to human chromosome band 16q22 by in situ hybridization.

Identification and characterization of human haptoglobin cDNA.

Recombinant plasmids containing human cDNA encoding haptoglobin, a plasma protein that binds free hemoglobin, have been isolated by screening an adult human liver library with a mixed oligonucleotide probe. Four cDNA clones containing inserts have been obtained that span 1,218 nucleotides of the haptoglobin coding sequence, including the 3' end of the haptoglobin cDNA. The cDNA sequence included a leader sequence followed by alpha 2-chain and beta-chain sequences. A heretofore unseen arginine residue was deduced between the human alpha- and beta-chain sequences. This is a probable site of limited proteolysis leading to the formation of the alpha and beta polypeptides in mature haptoglobin. A comparison of the haptoglobin alpha-beta-junction region and the heavy-light-chain junction of tissue-type plasminogen activator strengthens the evolutionary homology found in haptoglobin and the serine proteases. The Hp alpha 2 gene, which was shown earlier to be a partial duplication produced by unequal crossing-over between Hp alpha 1 genes, has been impossible to align by protein characterization. The cDNA sequence establishes the alignment of Hp alpha 2FS in the Hp alpha 2 gene studied here.

Cystic fibrosis. II. The urinary mucociliary inhibitor.

In the current study, the cystic fibrosis cationic mucociliary inhibitor has been purified from urine by ion exchange chromatography, gel filtration, lectin affinity chromatography, isoelectric focusing, and high performance liquid chromatography. The molecular size of the cationic mucociliary inhibitor was estimated to be in the range of 4,000 to 13,500 MW, by its elution on Sephadex G-50, and between 7,500 and 2,750 MW, by urea-sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition to the cationic mucociliary inhibitor, an anionic mucociliary inhibitor was also detected in the urinary fraction isoelectrically focused between pH 4.5 and 4.9. The identity of the mucociliary inhibitor as a glycoprotein was established in the current study by affinity chromatography on Phaseolus lunatus lectin, by radiolabeling the carbohydrate with galactose oxidase and tritiated sodium borohydride, and by determining the presence of a large concentration of glucosamine and small amounts of galactosamine by amino acid analysis. The amino acid analysis of the purified major component of the cationic mucociliary inhibitor reveals that the glucosamine concentrations represents a high percentage of the composition of the glycoprotein.

Serum concentrations of vitamin D-binding protein (group-specific component) in cystic fibrosis.

Vitamin D-binding protein (DBP) concentrations were determined in the sera of 90 cystic fibrosis homozygotes, 57 obligate heterozygotes, and 46 normal controls. Very significantly lower mean concentrations were found in the sera of CF homozygotes compared with both heterozygotes and controls (P less than 0.01, Wilcoxon Rank Sums Test). Subdivision of the samples by Gc phenotype showed that this relationship held true both in the Gc1 and Gc2-1 phenotypes. The small sample size of the Gc2 genotype makes the significance levels of limited usefulness, but the pattern of variation of DBP levels among CF homozygotes, heterozygotes, and controls was consistent with that observed for the Gc1 and Gc2-1 classes. Haptoglobin levels showed high coefficients of variation when compared among CF homozygotes, obligate heterozygotes, and controls, presumably because of nonspecific elevation in the acute-phase response. Alpha 2-macroglobulin levels were, if anything, slightly elevated in CF homozygotes compared with controls, while albumin levels showed no significant mean differences between these groups. Since the DBP concentration does not vary with age nor with levels of vitamin D and its metabolites, we interpret our results to mean that DBP levels are specifically decreased in cystic fibrosis, perhaps as the result of impaired glycosylation of the protein.

A transferrin variant in a kindred with cystic fibrosis.

An electrophoretically fast transferrin (Tf) variant, TfB, was found in a cystic fibrosis homozygote. Since neither cystic fibrosis nor the transferrin structural gene has been mapped on human chromosomes, a study was made of this kindred. Genetic markers including ABO, MN, Rh, Fy blood groups and Tf, group-specific component, and haptoglobin serum protein polymorphisms were determined in available members of the kindred. The genes for cystic fibrosis and Tf appear to segregate independently in the kindred, although crossing-over between linked genes in the homozygous cystic fibrosis brother of the propositus could account for the genotypes observed.

Studies of cystic fibrosis utilizing mucociliary activity in oyster gills.

Crassostrea virginica, the oyster native to the gulf coast, has served as a source of ciliated epithelium for studies on the inhibitory factor in cystic fibrosis. In these studies, protein molecules with biological activity, obtained from serum, urine, saliva, and cells from cystic fibrosis homozygotes and heterozygotes, were detected, purified, and characterized. Properties of these factors are similar to those detected by other ciliated systems in mussel gills and rabbit trachea. Although none of the ciliated assays in their present stage of development offer a reliable means for heterozygote screening or for prenatal diagnosis, they represent powerful tools for characterizing biologically active molecules related to cystic fibrosis. The importance of purifying and characterizing the various cystic fibrosis factors described by several laboratories is based on evidence that their biological activities observed in vitro mimic some of the expressions of the disease observed in vivo and that the concentration of the mucociliary inhibitor in serum and fibroblast medium preparations from cystic fibrosis heterozygotes is approximately one-half of that in serum and fibroblast medium preparations from homozygotes.

Somatic cell genetic studies of the cystic fibrosis mucociliary inhibitor.

A series of cystic fibrosis homozygous fibroblast X mouse cell hybrids were constructed in order to determine the chromosome which carries the gene controlling the cystic fibrosis mucociliary inhibitor (CFMI) phenotype. Several hybrids and their subclones retained the CFMI phenotype for long periods in cell culture. Correlation of the CFMI phenotype with the presence of specific human chromosomes indicated that possible linkage existed between CFMI and human chromosomes 2, 4, 6, 10, and 18. The strongest chance of linkage existed for chromosome 4.

Novel urinary fragments from human basement membrane collagen.

Five fragments of collagen have been purified from human urine. The fragments cross-reacted with basement membrane collagen from pig kidney cortices when tested against antiserum to basement membrane collagen, type IV, from murine tumor. The concentrations of these fragments were sufficient for amino acid analysis, immunological analysis, and in one case, preliminary analytical amino acid sequencing. Compositional analysis of the fragments indicated concentrations of hydroxylysine, hydroxyproline, and glycine that are typical of collagen. High values of hexosamines and neutral sugars were also found. An NH2-terminal sequence of 18 residues was obtained for one fragment of Mr approximately 6,500. This sequence was unique among proteins reported to date, but was obviously collagen-like. A urinary source of basement membrane collagen fragments will permit molecular characterization of basement membrane in inherited or acquired diseases affecting this structure.

Covalent structure of human haptoglobin: a serine protease homolog.

The complete amino acid sequences and the disulfide arrangements of the two chains of human haptoglobin 1-1 were established. The alpha 1 and beta chains of haptoglobin contain 83 and 245 residues, respectively. Comparison of the primary structure of haptoglobin with that of the chymotrypsinogen family of serine proteases revealed a significant degree of chemical similarity. The probability was less than 10(-5) that the chemical similarity of the beta chain of haptoglobin to the proteases was due to chance. The amino acid sequence of the beta chain of haptoglobin is 29--33% identical to bovine trypsin, bovine chymotrypsin, porcine elastase, human thrombin, or human plasmin. Comparison of haptoglobin alpha 1 chain to activation peptide regions of the zymogens revealed an identity of 25% to the fifth "kringle" region of the activation peptide of plasminogen. The probability was less than 0.014 that this similarity was due to chance. These results strongly indicate haptoglobin to be a homolog of the chymotrypsinogen family of serine proteases. Alignment of the beta-chain sequence of haptoglobin to the serine proteases is remarkably consistent except for an insertion of 16 residues in the region corresponding to the methionyl loop of the serine proteases. The active-site residues typical of the serine proteases, histidine-57 and serine-195, are replaced in haptoglobin by lysine and alanine, respectively; however, aspartic acid-102 and the trypsin specificity, residue, aspartic acid-189, do occur in haptoglobin. Haptoglobin and the serine proteases represent a striking example of homologous proteins with different biological functions.

Altered polyamine metabolism in cystic fibrosis.

Children with cystic fibrosis excreted elevated urinary levels of all three polyamines--putrescine, spermidine, and spermine. Heterozygote parents excreted intermediate concentrations of the polyamines, but not levels significantly different from levels in normal controls. Patients with cystic fibrosis who were administered a tracer amount of [14C]spermidine excreted 11--13% of the radiolabel within 72 hr whereas normal controls excreted 60--76% of the radiolabel within 72 hr. Spermine excretion was positively correlated with increased pathology as assessed by the National Institutes of Health (NIH) clinical score, whereas urinary putrescine and spermidine levels were negatively correlated with increased pathology.