A missense mutation in type VII collagen in two affected siblings with recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa is a severe mutilating genodermatosis. Previous ultrastructural demonstrations of altered anchoring fibrils, and recent genetic linkage analyses have suggested that type VII collagen, the major component of anchoring fibrils, is a candidate gene. We have identified a homozygous methionine-to-lysine mutation in two affected siblings, while their unaffected mother and half-brother are heterozygous carriers. The mutation resides in a highly conserved region of the C-terminus of type VII collagen, strongly suggesting that it is the cause of the disease in this family.

The role of an absolutely conserved tryptophan residue in octamer formation and stability in mitochondrial creatine kinases.

In most organisms, mitochondrial creatine kinase (MtCK) is present as dimers and octamers with the latter predominating under physiological conditions. An absolutely conserved tryptophan residue (Trp-264 in chicken sarcomeric MtCK) appears to play a key role in octamer stability. Recently, it has been shown that the sponge Tethya aurantia, a member of the most ancient group of living multi-cellular animals, expresses an obligate, dimeric MtCK that lacks this absolutely conserved tryptophan residue, instead possessing a tyrosine in this position. In the present study we confirm that the absolutely conserved tryptophan residue is lacking in other sponge MtCKs where it is instead substituted by histidine or asparagine. Site directed mutations of the Trp-264 in expression constructs of chicken sarcomeric MtCK and the octameric MtCK from the marine worm Chaetopterus destabilized the octameric quaternary structure producing only dimers. A Tyr-->Trp mutation in an expression construct of the Tethya MtCK construct failed to produce octamerization; Tyr-->His and Tyr-->Asn mutations also yielded dimers. These results, in conjunction with analysis of homology models of Chaetopterus and Tethya MtCKs, strongly support the view that while the absolutely conserved tryptophan residue is important in octamer stability, octamer formation involves a complex suite of interactions between a variety of residues.

Homology between alpha 2(V) and alpha 1(III) collagen promoters and evidence for negatively acting elements in the alpha 2(V) first intron and 5' flanking sequences.

We have isolated a 17 kilobase pair (kb) genomic clone containing the 5' portion of the human alpha 2(V) collagen gene. Nucleotide sequence was determined for 1671 base pairs (bp) comprising the promoter region, first exon and 334 bp of the first intron, and the major transcriptional start site determined by primer extension and S1 nuclease analysis. Sequence comparison revealed the alpha 2(V) promoter to be similar in structure to the promoter of the alpha 1(III) collagen gene. This is the first instance of such similarities between promoter regions of genes encoding different fibrillar collagen chains. Homology, in 5' flanking sequences, extends upstream to about nucleotide -120 in each gene and is particularly striking near the TATTTA sequence (TATA box) present in each promoter. Some homology also surrounds the two transcription start sites. The 5' untranslated regions of the two genes also show strong homology. Chimeric chloramphenicol acetyltransferase (CAT) constructs were prepared with various fragments from the 5' portion of the alpha 2(V) gene. Transient expression assays, in human fibroblasts, localized the functional alpha 2(V) promoter to the region of 5' flanking sequence conserved between the alpha 2(V) and alpha 1(III) genes. Expression assays also identified negatively acting elements, in intron and 5' flanking sequences, which inhibit transcription from the alpha 2(V) promoter.

The accuracy of frozen-section diagnoses in 34 hospitals.

We compared the diagnoses from intraoperative frozen-section consultation with the final diagnosis using permanent tissue sections from 34 hospitals throughout North America. Participating pathologists who provided data from their practices volunteered for a pilot study of Q-Probes, a modular quality assurance program of the College of American Pathologists, Skokie, III. During 4 weeks, 186 pathologists evaluated 1952 frozen-section cases and deferred diagnoses to the final permanent sections in 77 cases (3.9%). Concordance between frozen-section and the final histologic diagnoses was 96.5%; whereas the mean and median concordance rates for participating institutions were 96.8% and 97.4%, respectively. Of 67 discordant diagnoses, 29 occurred from sampling of nonrepresentative tissue specimens, and 29 from diagnostic misinterpretations. The pathologists indicated that the diagnostic discordance had little or no effect on patient care in 94% of cases. We conclude that the North American pathologists studied interpret frozen sections with exceedingly high accuracy, approximating that reported from teaching hospitals.

Interinstitutional comparison of frozen-section consultation. A College of American Pathologists Q-Probe study of 79,647 consultations in 297 North American institutions.

In 1989, the College of American Pathologists Q-Probes Quality Assurance Program studied intraoperative frozen-section consultations performed in 297 institutions with mean bed size of 316 (range, 0 to 1351 beds) in North America during 5 consecutive months. The aggregate database was composed of 933,751 surgical cases (mean, 3144 per institution); 52,464 frozen-section cases (mean, 177); and 79,647 individual frozen sections performed (mean, 268). The rate of frozen sections per all surgical case accessions was 5.6% (cases with frozen section) and 7.3% (individual frozen sections performed), with an average of 1.5 frozen sections per case. Frozen-section rate increased proportional to bed size, from less than 5% in institutions with bed size below 150 to 15% in institutions with bed size above 600. Of all frozen sections performed, 4.2% were deferred. Deferrals to paraffin sections in pathologists' opinions were 92.6% appropriate, 1.2% inappropriate, and 6.2% not stated. When frozen-section diagnoses were compared with permanent section diagnoses, there was a 98.3% diagnostic concordance, adjusted for deferred diagnoses, but including the performance of frozen sections on mammographically directed biopsy specimens with no gross abnormalities in 80% of institutions. This practice accounted for 11.8% of the discordant frozen-section diagnoses. The reasons for diagnostic discordances were gross tissue sampling (44.8%); misinterpretation (40%); sectioning (12.7%); inadequate history (5.6%); staining (1.5%); labeling (0.5%); and other (3%). Assessment of diagnostic discordance on patient outcomes by the reviewing pathologist showed that patient management was unaffected in 74%, minimally affected in 20%, and greatly affected in 2.5%.

The pro-alpha 1(V) collagen chain. Complete primary structure, distribution of expression, and comparison with the pro-alpha 1(XI) collagen chain.

We have isolated overlapping cDNA clones from human and hamster libraries which comprise the entire coding sequences for the prepro-alpha 1(V) collagen chains of both species. The translated polypeptide has a signal peptide of 36 amino acids, a central triple helical domain of 338 uninterrupted Gly-X-Y triplets, and 266 amino acids which comprise the C-telopeptide and propeptide. The N-propeptide and telopeptide are comprised of 522 residues in humans and 524 residues in hamsters. The cDNA-derived pro-alpha 1(V) amino acid sequences exhibit a variety of structural features characteristic of fibrillar collagens. Pro-alpha 1(V) is found to be unique among fibrillar collagen chains, however, in lacking potential cross-linking lysyl residues in either telopeptide, and in possessing potential N-asparaginyl-linked carbohydrate attachment sites in its N-propeptide. Of particular interest is the strong homology found between the pro-alpha 1(V) and pro-alpha 1(XI) collagen chains in most domains, with the notable exception of a subdomain in the globular region of the N-propeptide. RNase protection analysis of RNA with a variety of pro-alpha 1(V) cDNA-derived riboprobes indicates a broad distribution of expression of the pro-alpha 1(V) chain in tissues and suggests that transcripts encoding the pro-alpha 1(V) chain and the putative pro-alpha 1'(V) chain are not products of the same gene.

High levels of expression of full length human pro-alpha 2(V) collagen cDNA in pro-alpha 2(V)-deficient hamster cells.

A full length cDNA encoding human pro-alpha 2(V) collagen was constructed. Partial sequencing of the cDNA and primer extension analysis of mRNA from fibroblasts found that pro-alpha 2(V) mRNA differs from the mRNAs of other fibrillar collagens in the increased length of its 5'-untranslated region. The pro-alpha 2(V) cDNA was placed downstream of the human cytomegalovirus immediate early promoter/regulatory sequences for expression studies in cultured Chinese hamster lung cells. These cells have been shown previously to synthesize large quantities of pro-alpha 1(V) homotrimers as their only collagenous product. Transfection resulted in a number of clonal cell lines that express human alpha 2(V) RNA at levels comparable to, and in some cases greater than, levels found in normal human skin fibroblasts. Pro-alpha 2(V) chains produced in the majority of clonal lines were of sufficient quantity to complex all available endogenous pro-alpha 1(V) chains. Chimeric heterotrimers, composed of hamster alpha 1(V) and human alpha 2(V) chains in a 2:1 ratio, were stable to pepsin digestion and were found predominantly associated with the cell layer. Surprisingly, pro-alpha 2(V) chains, in excess to pro-alpha 1(V) chains, were found in the extracellular matrix and, in much greater abundance, in media. These chains were pepsin sensitive, indicating that pro-alpha 2(V) chains can be secreted as nonstable homotrimers or as free chains.

Complete structural organization of the human alpha 1 (V) collagen gene (COL5A1): divergence from the conserved organization of other characterized fibrillar collagen genes.

Genes that encode the vertebrate fibrillar collagen types I-III have previously been shown to share a highly conserved intron/exon organization, thought to reflect common ancestry and evolutionary pressures at the protein level. We report here the complete intron/exon organization of COL5A1, the human gene that encodes the alpha 1 chain of fibrillar collagen type V. The structure of COL5A1 is shown to be considerably diverged from the conserved structure of the genes for fibrillar collagen types I-III. COL5A1 has 66 exons, which is greater than the number of exons found in the genes for collagen types I-III. The increased number of exons is partly due to the increased size of the pro-alpha 1(V) N-propeptide, relative to the sizes of the N-propeptides of the types I-III procollagen molecules. In addition, however, the increased number of exons is due to differences in the intron/exon organization of the triple-helix coding region of COL5A1 compared to the organization of the triple-helix coding regions of the genes for collagen types I-III. Of particular interest is the increase of 54 bp exons in this region of COL5A1, strongly supporting the proposal that the triple-helix coding regions of fibrillar collagen genes evolved from duplication of a 54 bp primordial genetic element. Moreover, comparison of the structure of COL5A1 to the highly conserved structure of the genes of collagen types I-III provides insights into the probable structure of the ancestral gene that gave rise to what appears to be two classes of vertebrate fibrillar collagen genes.

Spectinomycin resistance and associated DNA amplification in Streptomyces achromogenes subsp. rubradiris.

Streptomyces achromogenes subsp. rubradiris plated at low density on 1,000 micrograms of spectinomycin per ml initially produces slow-growing, bald colonies from which arise, in a spatially and temporally random fashion, foci of rapidly growing aerial mycelium-forming cells whose DNA contains an approximately 200- to 300-fold amplification of an 8-kilobase (kb) sequence. This sequence was cloned in Escherichia coli on pBR322 and physically characterized. It was separately cloned also in Streptomyces lividans as a BglII fragment and shown to impart high-level resistance to spectinomycin in an orientation-independent manner when present in either the high-copy-number vector pIJ702 or the unit-copy-number vector pIJ943. A spectinomycin resistance determinant was shown to reside on a 1.7-kb SphI-BglII subfragment. Analysis of Southern blots of restriction enzyme digests of wild-type S. achromogenes DNA probed with the labeled 8-kb DNA sequence resulted in the identification and subsequent cloning in S. lividans of a 10.4-kb BamHI fragment which probably includes the complete 8.8-kb amplifiable unit of DNA. This unit is present in wild-type S. achromogenes and in the initially slow-growing, bald colonies arising on 1,000 micrograms of spectinomycin per ml as a single copy. It carries two 0.8-kb direct repeats at its termini as well as the spectinomycin resistance determinant close to one of these termini. About 5% of protoplast regenerants from wild-type S. achromogenes and 77% of protoplast regenerants from the rapidly growing strains lost both the ability to grow on spectinomycin at 10 micrograms/ml and the sequences that hybridize with the 8-kb probe DNA. The 1.7-kb Bg/II-SphI resistance fragment, when introduced via the vector pIJ702 into an S. achromogenes strain sensitive to 10 microgram of spectinomycin per ml, permitted its vigorous growth on 1,000 micrograms of the antibiotic per ml.

Null alleles of the COL5A1 gene of type V collagen are a cause of the classical forms of Ehlers-Danlos syndrome (types I and II).

Ehlers-Danlos syndrome (EDS) types I and II, which comprise the classical variety, are well characterized from the clinical perspective, but it has been difficult to identify the molecular basis of the disorder in the majority of affected individuals. Several explanations for this failure to detect mutations have been proposed, including genetic heterogeneity, failure of allele expression, and technical difficulties. Genetic heterogeneity has been confirmed as an explanation for such failure, since causative mutations have been identified in the COL5A1, COL5A2, and tenascin X genes and since they have been inferred in the COL1A2 gene. Nonetheless, in the majority of families with autosomal dominant inheritance of EDS, there appears to be linkage to loci that contain the COL5A1 or COL5A2 genes. To determine whether allele-product instability could explain failure to identify some mutations, we analyzed polymorphic variants in the COL5A1 gene in 16 individuals, and we examined mRNA for the expression of both alleles and for alterations in splicing. We found a splice-site mutation in a single individual, and we determined that, in six individuals, the mRNA from one COL5A1 allele either was not expressed or was very unstable. We identified small insertions or deletions in five of these cell strains, but we could not identify the mutation in the sixth individual. Thus, although as many as one-half of the mutations that give rise to EDS types I and II are likely to lie in the COL5A1 gene, a significant portion of them result in very low levels of mRNA from the mutant allele, as a consequence of nonsense-mediated mRNA decay.

Structural organization and expression patterns of the human and mouse genes for the type I procollagen COOH-terminal proteinase enhancer protein.

The procollagen C-proteinase enhancer (PCPE) is a glycoprotein that potentiates enzymatic cleavage of the type I procollagen C-propeptide by bone morphogenetic protein-1 (BMP-1). The human PCPE gene (PCOLCE) was previously mapped to 7q22, an area frequently disrupted in uterine leiomyomata, while disruption of the rat PCPE gene leads to anchorage-independent growth and loss of contact inhibition in rat fibroblasts. Here we describe the entire intron/exon organizations of PCOLCE and the mouse PCPE gene (Pcolce) and analyze expression of PCOLCE RNA in various human adult and fetal tissues and of Pcolce RNA at various stages of mouse development. PCOLCE and Pcolce are shown to be small genes 6.0 and 6.5 kb, respectively, with a conserved intron/exon structure comprising 9 exons. A notable difference between the two genes derives from insertion of multiple Alu sequences immediately upstream and downstream and within PCOLCE. Temporal expression of PCPE mRNA is shown to differ from that of BMP-1 and type I procollagen during mouse development, consistent with possible additional functions for PCPE beyond enhancement of C-proteinase activity. Consistent with a possible role in leiomyomata, PCOLCE is shown to be expressed at relatively high levels in uterus.

Characterization of a novel gene product (mammalian tolloid-like) with high sequence similarity to mammalian tolloid/bone morphogenetic protein-1.

Bone morphogenetic protein-1 (BMP-1), a metalloprotease isolated from osteogenic extracts of demineralized bone, is capable of cleaving the C-propeptides of procollagen types I, II, and III. A single mammalian gene produces alternatively spliced RNA transcripts for BMP-1 and for a second longer protein, designated mammalian tolloid (mTld) due to a domain structure identical to that of the Drosophila dorsal-ventral patterning gene product tolloid (Tld). Here we report the use of a cDNA library, prepared from BMP-1/mTld-null mouse embryos, to isolate cDNA clones for a novel mammalian protein with a domain structure identical to that of mTld. The new protein, designated mammalian tolloid-like (mTll), has 76% identity with mTld for amino acid residues in all domains downstream of, and including, the protease domain. In contrast, the N-terminal activation domains of the two proteins show little similarity. In situ hybridizations show the distribution of mTll RNA to overlap extensively that previously shown for the BMP-1 and mTld RNA forms. However, mTll shows additional strong expression in structures of the developing, neonatal, and adult brain in which expression of BMP-1 and mTld has not been observed. The murine mTll gene (Tll) is mapped to central chromosome 8, which is a different chromosomal location than that of the BMP-1/mTld gene. Loci for some developmental abnormalities map to the same general chromosomal location as Tll.

Localization of the human collagen gene COL7A1 to 3p21.3 by fluorescence in situ hybridization.

An 8-kb genomic probe, containing 34 collagen-encoding exons, was localized to 3p21.3 by fluorescence in situ hybridization. The genomic probe encoded a previously uncharacterized carboxyl terminal portion of the alpha 1(VII) collagen chain. This mapping result confirms the previous assignment of the alpha 1(VII) gene (COL7A1) to 3p21 and offers a finer subregional localization than was previously available.

A nationwide quality assurance program can describe standards for the practice of pathology and laboratory medicine.

An important component of quality assessment is the analysis of peer group comparisons, although little data are available for evaluation. We developed and tested six interinstitutional quality indicators related to Pathology and Laboratory Medicine among 36 institutions. Results showed that the mean frequency of intraoperative frozen section consultations (6.0%), sensitivity of fine needle aspiration cytology diagnosis (87%), nosocomial infections (5.0%) and average cross-match to transfusion ratio (2.1%) was comparable with previous studies, but the range of values was large. The median stat laboratory turnaround time of approximately 1 hr for CSF cell count, glucose, protein and gram smear was considerably longer than expected from previous investigations, and was longer for larger institutions. Analysis of serious laboratory reporting errors showed the lowest number detected by individuals working in transfusion medicine, and highest numbers among hematology workers. We conclude that interinstitutional comparison of data from quality assurance programs can be used to describe performance standards related to the quality and effectiveness of care.

Deletion of the pro-alpha 1(I) N-propeptide affects secretion of type I collagen in Chinese hamster lung cells but not in Mov-13 mouse cells.

We have introduced two mutations into a full-length human pro-alpha 1(I) cDNA that delete 114 amino acids or the entire 139 amino acids of the N-propeptide domain. Wild-type and mutated versions of the cDNA were introduced into cultured Chinese hamster lung (CHL) cells, which do not produce endogenous type I collagen, and into Mov-13 mouse cells, which produce endogenous pro-alpha 2(I) chains but not pro-alpha 1(I) chains. As judged by resistance to proteases, neither mutation impaired intracellular triple helical assembly of human alpha 1(I) homotrimers in CHL cells, or of chimeric type I collagen comprised of human alpha 1(I) and mouse alpha 2(I) chains in Mov-13 cells. Thus, the N-propeptide is not necessary for intracellular assembly of the main helical collagen domain of type I collagen. In CHL cells the rate of secretion of the mutant homotrimers was greatly reduced as compared to wild type homotrimers, and by immunofluorescence and immunoelectron microscopy, the mutant chains were shown to be accumulated in large vesicular expansions of the rough endoplasmic reticulum. When such cells were retransfected with cDNA encoding wild-type human alpha 2(I) chains, mutant alpha 1(I) chains were not rescued and heterotrimers containing the mutant chains were also retained in the intracellular vesicles. By contrast, deletion of the N-propeptide did not affect secretion of heterotrimers containing mutant chains from Mov-13 cells. Thus, an intact N-propeptide appears necessary for efficient secretion of type I collagen from some but not all cell types.

Strategy for identification of sequence variants in COL7A1 and a novel 2-bp deletion mutation in recessive dystrophic epidermolysis bullosa.

The diagnostic hallmark of the dystrophic forms of epidermolysis bullosa (DEB), a group of heritable blistering skin diseases, is abnormalities in the anchoring fibrils at the dermal-epidermal basement membrane zone. Since type VII collagen is the major, if not the exclusive, component of the anchoring fibrils, the corresponding gene (COL7A1) is the candidate gene in DEB. Recent cloning of the type VII collagen cDNA and elucidation of the exon-intron organization of the gene have provided the basis for us to develop a novel strategy for identification of sequence variants in COL7A1. Optimization of 72 balanced primer pairs corresponding to flanking intronic sequences allowed PCR amplification of all 118 exons directly from genomic DNA. The PCR products were examined by heteroduplex analysis followed by comparative nucleotide sequencing. More than 100 sequence variants have been identified thus far in COL7A1 using this method, some of which are single base pair polymorphisms and many of which are pathogenetic mutations contributing to the blistering phenotype in DEB. The comprehensive method described is useful for rapid, reliable, and sensitive detection of sequence variants in COL7A1. We demonstrate the utility of this novel strategy in mutation detection and prenatal exclusion of RDEB in a consanguineous family at risk for recurrence.

Structural organization of the human type VII collagen gene (COL7A1), composed of more exons than any previously characterized gene.

The human type VII collagen (COL7A1) gene is the locus for mutations in at least some cases of dystrophic epidermolysis bullosa. Here we describe the entire intron/exon organization of COL7A1, which is shown to have 118 exons, more than any previously described gene. Despite this complexity, COL7A1 is compact. Consisting of 31,132 bp from transcription start site to polyadenylation site, it is only about three times the size of type VII collagen mRNA. Thus, COL7A1 introns are small. A 71-nucleotide COL7A1 intron is the smallest intron yet reported in a collagen gene, and only one COL7A1 intron is greater than 1 kb in length. All exons in the COL7A1 triple helix coding region that do not begin with sequences corresponding to imperfections of the triple helix begin with intact codons for Gly residues of Gly-X-Y repeats. This is reminiscent of the structure of fibrillar rather than other nonfibrillar collagen genes. In addition, the COL7A1 triple helix coding region contains many exons of recurring sizes (e.g., 25 exons are 36 bp, 12 exons are 45 bp, 8 exons are 63 bp), suggesting an evolutionary origin distinct from those of other nonfibrillar collagen genes. Sequences from the 5' portion of COL7A1 are presented along with the 3766-bp intergenic sequence, which separates COL7A1 from the upstream gene encoding the core I protein of the cytochrome bc1 complex. The COL7A1 promoter region is found to lack extensive homologies with promoter regions of other genes expressed primarily in skin.

Complete coding sequence, intron/exon organization, and chromosomal location of the gene for the core I protein of human ubiquinol-cytochrome c reductase.

Core I protein is a nuclear-encoded component of the ubiquinol-cytochrome c reductase complex of the mitochondrial respiratory chain. We have located the gene for the human core I protein in the p21 region of chromosome 3, just upstream of the COL7A1 gene which encodes type VII collagen. The core I gene, which has been sequenced in its entirety, is comprised of 10,417 base pairs, from the major transcription start site to the polyadenylation signal, and contains 13 exons. The predicted polypeptide contains 480 amino acids, of which the first 34 are predicted to constitute a typical mitochondrial leader peptide containing 6 positively charged arginine residues. The predicted human protein shows significant homology with core I protein from Saccharomyces cerevisiae, rather high homology (64% similarity, 46% identity) with the processing enhancing protein, which functions as core I protein in Neurospora crassa, and, surprisingly, highest homology with the small subunit of the mitochondrial processing peptidase of rat (74% similarity, 55% identity). The predicted human sequence is 87% identical to the reported bovine core I sequence predicted from cDNA cloning, up to residue 298, but the two predicted sequences are widely divergent after that point.

Mouse and human homologues of the yeast origin of replication recognition complex subunit ORC2 and chromosomal localization of the cognate human gene ORC2L.

ORC2 is a subunit of the origin recognition complex in yeast and has been implicated in the initiation of DNA replication and transcriptional silencing. We have isolated mouse and human cDNA clones encoding proteins with 47.9 and 46.3% similarity, respectively, to yeast ORC2. This degree of similarity and the alignment of sequences suggest that these clones may represent a mammalian ORC2 homologue. The existence of such a homologue would, in turn, suggest the existence of a mammalian origin recognition complex, similar to that found in yeast. Although Northern blot analysis of various adult mouse tissues found the highest levels of expression of ORC2-like (ORC2L) RNA in testes, strong signals did not always correspond to tissues in which high levels of DNA replication would be expected. This finding may reflect functional roles of ORC2L distinct from those that it may play in DNA replication. Analyses of somatic cell hybrid DNA and fluorescence in situ hybridization were employed to map the human ORC2L gene to chromosome 2q33.