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Background: Adverse cardiovascular events are a leading cause of perioperative morbidity and mortality. The definitions of perioperative cardiovascular adverse events are heterogeneous. As part of the international Standardized Endpoints in Perioperative Medicine initiative, this study aimed to find consensus amongst clinical trialists on a set of standardised and valid cardiovascular outcomes for use in future perioperative clinical trials.Methods: We identified currently used perioperative cardiovascular outcomes by a systematic review of the anaesthesia and perioperative medicine literature (PubMed/Ovid, Embase, and Cochrane Library). We performed a three-stage Delphi consensus-gaining process that involved 55 clinician researchers worldwide. Cardiovascular outcomes were first shortlisted and the most suitable definitions determined. These cardiovascular outcomes were then assessed for validity, reliability, feasibility, and clarity.Results: We identified 18 cardiovascular outcomes. Participation in the three Delphi rounds was 100% (n=19), 71% (n=55), and 89% (n=17), respectively. A final list of nine cardiovascular outcomes was elicited from the consensus: myocardial infarction, myocardial injury, cardiovascular death, non-fatal cardiac arrest, coronary revascularisation, major adverse cardiac events, pulmonary embolism, deep vein thrombosis, and atrial fibrillation. These nine cardiovascular outcomes were rated by the majority of experts as valid, reliable, feasible, and clearly defined.Conclusions: These nine consensus cardiovascular outcomes can be confidently used as endpoints in clinical trials designed to evaluate perioperative interventions with the goal of improving perioperative outcomes.

The Duchenne muscular dystrophy (DMD) locus has been localized to the short arm of the human X chromosome (Xp21) by detection of structural abnormalities and by genetic linkage studies. A library highly enriched for human DNA from Xp21 was constructed using DNA isolated from a male patient who had a visible deletion and three X-linked disorders (DMD, retinitis pigmentosa and chronic granulomatous disease). Seven cloned DNA probes from this library and the probe 754 (refs 5, 8) are used in the present study to screen for deletions in the DNA isolated from 57 unrelated males with DMD. Five of these DMD males are shown to exhibit deletions for one of the cloned DNA segments and at least 38 kb of surrounding DNA. In addition, two subclones from the same region detect four restriction fragment length polymorphisms which exhibit no obligate recombination with DMD in 34 meiotic events. These new DNA segments will complement the existing Xp21 probes for use in carrier detection and prenatal diagnosis of DMD. Elucidation of the end points of the five deletions will help delineate the extent of the DMD locus and ultimately lead to an understanding of the specific sequences involved in DMD.

Type 1, or insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease associated with loss of tolerance to several pancreatic islet cell molecules, including insulin, glutamic acid decarboxylase (GAD), ICA69 and the tyrosine phosphatase IA-2 (refs 1-3). Among several predisposing loci, IDDM2 maps to the insulin gene (INS) VNTR (variable number of tandem repeats) minisatellite on chromosome 11p15 (refs 4-9). Allelic variation at this VNTR locus correlates with steady-state levels of INS mRNA in pancreas and transfected rodent cell lines, but it is difficult to reconcile the association of lower INS mRNA levels in the pancreas with class III VNTRs that are dominantly protective from IDDM. We show that during fetal development and childhood, mRNAs for insulin and other islet cell autoantigens (GAD, ICA69, IA-2) are expressed at low levels in the human thymus. Critically, we also detect proinsulin and insulin protein. VNTR alleles correlate with differential INS mRNA expression in the thymus where, in contrast to the pancreas, protective class III VNTRs are associated with higher steady-state levels of INS mRNA expression. This finding provides a plausible explanation for the dominant protective effect of class III VNTRs, and suggests that diabetes susceptibility and resistance associated with IDDM2 may derive from the VNTR influence on INS transcription in the thymus. Higher levels of (pro)insulin in the thymus may promote negative selection (deletion) of insulin-specific T-lymphocytes which play a critical role in the pathogenesis of type-1 diabetes.

Expression of a fluorescent reporter gene has been studied using two alternate promoters to transcribe the green fluorescent protein (gfp) from Aequorea victoria. The human cytomegalovirus (CMV) enhancer/ promoter or the human muscle-specific creatine kinase promoter (CKM) were inserted along with the gfp cDNA into a plasmid expression vector based on a modified adeno-associated virus genome. Naked plasmid DNA was injected into the hamstring muscle of mdx mice and gfp gene expression determined from frozen muscle sections taken at 4, 14, and 42 days postinjection. Fluorescence patterns obtained by photomicroscopy and quantitative fluorescence measurements indicated a near-linear increase in the accumulation of the gfp in skeletal muscle during the length of the study, with gfp expression at 42 days being roughly four times the values obtained at 4 days. The levels of expression of gfp from the CKM construct were consistantly higher than for the CMV construct. The CKM promoter/expression vector combination demonstrates significant potential for simple, direct delivery and long-term, high-level expression of genes in skeletal muscle.

Notexin, a myotoxic phospholipase, was used to induce focal necrosis in the sartorius muscles of normal mixed-breed adult dogs and in 12-week-old beagles. Notexin injury caused pathologic changes similar to those of Duchenne muscular dystrophy (DMD) and its canine homologue, golden retriever muscular dystrophy (GRMD). All three conditions are characterized by increased serum creatine kinase (CK) levels, sarcolemmal defects, delta lesions, hyaline degeneration of myofibers, calcium-positive myofibers, and minimal effects on neurovascular structures. Four and 24 h after exposure to notexin, serum CK levels were elevated, and many myofibers were necrotic. In addition, by 24 h the necrotic areas were heavily invaded by mononuclear cells, and calcium-positive myofibers were prominent. Capillaries appeared intact even in areas of marked myonecrosis. Massive cellular infiltrate and myotube formation was evident at 3 days post injury. By 7 days, most affected fascicles were occupied by small immature myofibers. Regeneration was largely complete at 21 days. Our results suggest that notexin-induced muscle injury in dogs will be useful in the evaluation of potential therapies for DMD such as myoblast transplantation.

We describe a partial TaqI map of the dystrophin gene, obtained mainly by analysis of 87 overlapping DMD/BMD deletions with small fragments of the dystrophin cDNA probes; exon 6 of the dystrophin gene was identified on the TaqI map using the polymerase chain reaction. The cDNA probes detect five polymorphisms with TaqI, more than with HindIII (one), BglII (four), or PstI (three). The five polymorphisms are analysed concomitant with screening for deletions on the TaqI map, and in the one-third of DMD/BMD cases with no detected deletion the polymorphism information may be used for counselling. Correlation of the TaqI map with the HindIII map in the region of probes 5b-7 and 8 has allowed the establishment of reading frame. In this region of the dystrophin gene, all of 41 DMD deletions resulted in a shift of reading frame and all of 10 BMD patients maintained reading frame, in agreement with the 'reading frame hypothesis'.

The majority of Duchenne and Becker muscular dystrophy cases are caused by deletions observable in Southern blots with cDNA probes for the gene. When the deletion includes polymorphic probes, they may be used to determine carrier status by deletion segregation analysis: non-inheritance of parental alleles, or heterozygosity. The polymorphic genomic probe P20 is deleted in a large percentage of probands. P20 hybridizes with two constant fragments of 6.7 and 0.8 kb in Taql digests. In a number of probands, only the larger P20 Taq1 fragment is deleted. This study demonstrates that this fragment corresponds with the polymorphic EcoRV and Mspl fragments of P20. Families in which the upper Taql fragment is deleted may be screened for carrier status using non-inheritance of parental alleles or heterozygosity of P20 in EcoRV or Mspl digests.

We undertook linkage analysis in four large North Carolina kindreds with X-linked hypophosphatemic rickets (HYP) using a newly defined polymorphic probe, derived from the 5' untranslated portion of the human glycine receptor (GLR). Two-point linkage analysis established linkage between GLR and HYP [Z(theta) = 7.91 at theta = 0.07] and confirmed linkage between HYP and DXS41 [Z(theta) = 8.31 at theta = 0.06] and DXS43 [Z(theta) = 5.94 at theta = 0.05]. Additionally, we found GLR tightly linked to DXS43 [Z(theta) = 5.40 at theta = 0.0]. Multipoint analysis indicated that GLR is on the telomeric side of HYP with a map order of Xpcen-DXS41-HYP-(GLR/DSX43).

There are many phenotypic features that can be monitored to, in the end, assess the success of any form of treatment for muscular dystrophy (Table 1). Of course, in this setting, we are speaking principally of myoblast transplantation.

Golden retriever muscular dystrophy (GRMD) has been proposed to be an animal model for Duchenne muscular dystrophy (DMD) (Kornegay et al., 1989). We have been studying GRMD to determine the underlying defect in this model using methodology and cDNA probes developed for the study of DMD (Koenig et al., 1987). Observed molecular differences between normal and affected animals may be useful for monitoring myoblast transfer in this model similar to what Ron Worton proposed as a means of defining tissue-specific markers for myoblast transplantation in DMD families (Worton reference this book). To illustrate the potential use of these markers of transplantation, an example of a human pedigree with a restriction fragment polymorphism which differs between individuals is illustrated in Figure 1. In this particular pedigree, there are two patients with deletions. Two potential markers would be useful for testing persistence of donor myoblasts after transplantation. The molecular markers in this case would be the presence of the deleted portion of the gene found in normal donor myoblasts, and if the donor was a non-carrier female, a gene dosage analysis which discriminates between the relative dosage of the X and Y-specific sequences would identify the donor cells.

The myotonic dystrophy (DM) gene is localized to the proximal long arm of chromosome 19. There have been reports of tight linkage to a number of chromosome 19 markers, including APOC2 and creatine kinase muscle type (CKMM), but they did not establish orientation of the 2 markers to DM. We screened several large multi-generational DM families for linkage to a series of chromosome 19 markers including CKMM. CKMM is tightly linked to DM in these data with z(theta) = 28.41; theta = 0.01. Analysis of cross-over data indicates CKMM is on the same side and closer to DM than APOC2. Thus, CKMM is a useful probe for carrier detection studies in presymptomatic individuals as well as for prenatal diagnosis.

Sensitivity to nonmodified poliovirus infection is an autosomal dominant trait, specific to primates. The gene for poliovirus sensitivity (PVS) is encoded on human chromosome 19. In order to sublocalize the PVS gene, we infected rodent-human hybrid cell lines that divide human chromosome 19 into four regions with poliovirus 1 and/or 3. When infected, these hybrid cell lines showed the typical cytopathic effect of poliovirus infection only if they contained 19q12→q13.2 as the smallest region of overlap. Appropriate negative and positive controls were used. PVS may be of relevance to myotonic dystrophy (DM) and the inherited motor neuron diseases: to DM because it localizes to the same region of chromosome 19 and to the inherited motor neuron diseases because it encodes a cell-surface receptor expressed on motor neurons.

The two major androgen-dependent secretory proteins of rat dorsal prostate and coagulating gland, DP I and DP II, were compared by in vitro translation of total poly(A)RNA and by pulse-chase techniques by means of [35S]methionine and tissue minces of coagulating gland. DP I is a major in vitro translation product of isolated poly(A)RNA, whereas DP II is undetectable in a mouse embryo fibroblast cell-free system where glycosylation does not occur. DP I is synthesized within 20 min in minces of coagulating gland incubated in the presence of [35S]methionine and is secreted in 40 min. DP II is detectable in the medium only after 8 hr of labeling. Inhibition of asparagine-linked protein glycosylation with tunicamycin (10 micrograms/ml) blocked the synthesis and secretion of DP II with an apparent increase in DP I secretion. Inhibition of DP II synthesis by monensin implicates the Golgi in the processing of DP II oligosaccharides. The data are consistent with the proposal that DP I enters a pathway of rapid secretion that is enhanced by the absence of core glycosylation, whereas DP II follows a slow pathway through the Golgi that involves extensive glycosylation.

Accurate carrier testing and prenatal diagnosis in Duchenne muscular dystrophy (DMD) families is facilitated when an Xp21 deletion is found to be segregating within a family. We discuss the results of the DNA testing in two families, one in which DNA from affected males was available for study and the other in which no DNA from an affected male was available. Factors complicating the counselling of DMD deletion families are outlined.

RNA sequence relationships between the genomes of the Kirsten murine sarcoma virus (MSV-K) complex, the Kirsten murine leukemia virus (MuLV-K) complex, the Gross murine leukemia virus (MuLV-G), and the Moloney murine leukemia virus (MuLV-M) were investigated. Sedimentation analyses revealed the expected 30 and 34 S RNA subunits in the MSV-K complex and a previously undetected 30 S RNA subunit accompanying the 34 S RNA subunit in the MuLV-K complex. Nucleic acid hybridization data indicated that each Kirsten virus 30 S RNA subunit had about 40% sequence homology with the RNA genome of MuLV-G, although these sequences were only partially homologous between the two 30 S subunits. In contrast, the MuLV-K 34 S RNA subunit had 96% sequence homology with the MuLV-G genome, whereas the MSV-K 34 S RNA subunit displayed only 71% sequence homology with the MuLV-G genome. Similar relationships were indicated by oligonucleotide fingerprinting. The oligonucleotide data, taken with published sequence data on the MuLV-G and MuLV-M genomes, enabled us to construct partial sequence maps of the MuLV-K 34 S RNA subunit and the MSV-K 34 and 30 S RNA subunits. The sequence arrangements indicated that (1) the MuLV-K 34 S RNA subunit is a variant of the MuLV-G genome; (2) the MSV-K 34 S RNA subunit is a recombinant molecule, which maintains the lenght of its leukemia virus parent; and (3) the MSV-K 30 S RNA subunit may have been generated from the MuLV-K 34 S genome by a two-stage process, culminating in the retention of parental sequences only within the U5 and U3 noncoding segments and within several amino-terminal coding segments. Further examination of published retrovirus genome sequences revealed several strategically situated sets of potential recognition signals for transcription and translation and suggested a model for genetic recombination based on mRNA splicing signals and areas of limited sequence homology. This model may explain how foreign gene elements can be inserted into retrovirus genomes to generate either functional or defective recombinant retroviruses.

The two markers most closely linked to the myotonic dystrophy (DM) locus on chromosome 19 are the gene that codes for apolipoprotein CII (APOC2) and the anonymous probe D19S19 (LDR152). Both of these markers show tight linkage to DM, with maximum lod scores of greater than 20 at recombination fractions of less than 0.05. We have identified, in a family in which DM segregates, an affected individual where a meiotic recombination event has occurred in which both of these linked markers have crossed over with the gene defect. This demonstrates that APOC2 and D19S19 are probably on the same side of DM.

Myotonic muscular dystrophy (DM) is the most common muscular dystrophy, affecting adults as well as children. It is inherited as an autosomal dominant trait and is characterized by variable expressivity and late age-of-onset. Linkage studies have established the locus on chromosome 19. In order to identify tightly linked probes for diagnosis as well as to define in detail the DM gene region, chromosome 19 libraries were constructed and screened for restriction fragment length polymorphisms tightly linked to DM. A genomic clone, LDR152 (D19S19), was isolated that is tightly linked to DM; recombination fraction = 0.0 (95% confidence limits 0.0-0.03); lod score, 15.4.

Intracellular RNA complementary to genomic RNA of the Moloney-murine sarcoma virus complex was detected in virus-producing rat cells. Hybrids formed between this novel type of RNA and its homologous viral genomic RNA were very stable exhibiting a T m of 88°. Further characterization of the virus-specific complementary RNA revealed that it represented a minimal 74% of the viral RNA genome. Cross-hybridization data of hybrids formed between this viral complementary RNA and normal rat liver RNA or genomic RNA of several other mammalian retroviruses demonstrated its virus specificity and sequence relatedness. An examination of total cellular RNA from virus-producing cells for the number of intracellular virus-specific RNA copies disclosed 14 copies of viral complementary RNA per cell and 1272 copies of viral genomic RNA per cell.

Screening polymorphic DNA probes for linkage to myotonic dystrophy (DM) and to other reported chromosome 19 (CH19) genes will develop a linkage map for human CH19. We report here the assignment of 3 cloned unique DNA sequences to 3 distinct regions of CH19. The novel use of 35S-labeled probes facilitated the rapid localization of the gene for the third complement factor (C3) to 19pl3.2 by in situ hybridization. Metaphase chromosomes were from normal peripheral lymphocytes as well as from a fibroblast line containing a 15;19 translocation which permitted clear identification of CH19 regions of localization. Two random clones isolated from a plasmid library of human F-group enriched chromosomal DNA (D19S5 and D19S6) were in like manner assigned to 19pl.2 and 19ql3.2 to 19qter, respectively.

Alzheimer’s disease (AD) is a devastating neurological disorder and the leading cause of dementia among the elderly. Recent studies have localized the gene for familial AD to chromosome 21 in a series of early-onset AD families (EOAD; mean age-of-onset, 60) is a more common clinical form of the disorder. Linkage studies undertaken to test the localization of LOAD families to chromosome 21 failed to establish linkage and excluded linkage from a portion of the region where the EOAD gene was localized. These findings suggest that more than one etiology may exist for familial Alzheimer’s disease and indicate the need for continued screening of the genome in LOAD families.

DNA probes are available for Duchenne muscular dystrophy (DMD) carrier detection and prenatal diagnosis. With probes for about 25% of the proximal portion of the gene, we found the proximal probes detected deletions in 23% of nonselected DMD boys, while a single distal probe detected 17% more as deletions. The combined percentage was 39% for all probes tested. Prenatal diagnosis and carrier detection are more accurate if deletions are mapped rather than by use of restriction fragment length polymorphism analysis. The effort involved in screening all affected boys for deletions is considerably less, and provides an accurate genetic marker for subsequent prenatal diagnosis in the family and prospective counseling for female relatives. It seems likely that, once the entire gene (cDNA) is available for screening, most DMD boys will show deletions.

Alzheimer's disease is a devastating neurological disorder and the leading cause of dementia among the elderly. Recent studies have localized the gene for familial Alzheimer's disease to chromosome 21 in a series of early onset AD families (mean age of onset less than 60). Familial late onset AD (mean age of onset greater than 60) is a more common clinical form of the disorder. Thirteen families with multiply affected Alzheimer's disease family members were identified and sampled. Ten of these families were of the late onset Alzheimer's disease type. Simulation studies were used to evaluate the usefulness of these pedigrees in linkage studies in familial Alzheimer's disease. Linkage studies undertaken to test the localization of both early onset and late onset Alzheimer's disease families to chromosome 21 failed to establish linkage and excluded linkage from a large portion of the region where the early onset Alzheimer's disease gene was localized. These findings suggest that more than one etiology may exist for familial Alzheimer's disease and indicate the need for continued screening of the genome in familial Alzheimer's disease families.

The cDNA and genomic probes for apolipoprotein C2 detect two restriction fragment length polymorphisms on chromosome 19. The combined estimated percentage of heterozygosity, assuming equilibrium, is approximately 75%, ie, apolipoprotein C2 is informative in 75% of matings. We have analyzed over 350 individuals in large multigenerational families for linkage of apolipoprotein C2 to myotonic muscular dystrophy. The maximum lod score was 16.29 with the maximum recombination fraction (theta) of 0.02, with 95% confidence limits for theta of 0.001 to 0.065. Thus, apolipoprotein C2 is useful in carrier detection and prenatal diagnosis with an accuracy of about 98%.