Microsatellite genome screening: rapid non-denaturing, non-isotopic dinucleotide repeat analysis.

The study of reverse genetics has made it possible for scientists to isolate and identify the genes responsible for such human diseases as cystic fibrosis and Duchenne- and Becker-type muscular dystrophy. The expensive and time-consuming process of detecting restriction fragment length polymorphisms by Southern blotting was the only method available to localize these genes. With the discovery of polymorphic microsatellite sequences in the human genome, the speed and ease of which a genome screening can be performed has increased dramatically. This paper reports on advanced methods for detection of microsatellite-repeated sequences making the task of mapping human genes simpler, safer and more economical.

Carrier detection in X-linked retinitis pigmentosa.

X-linked retinitis pigmentosa (XLRP) is manifested in affected males in their first decade and results in blindness by the third or fourth decade. Carrier detection is difficult since most carrier females show no or only equivocal signs well into or beyond their reproductive years. The genes, or the mutations causing RP have not been identified but at least two have been localised to the short arm of the X chromosome provisionally named RP2 and RP3. Identifying inheritance of one or other of these genes must be done by linkage in families using close, informative DNA markers. Here we report the localisation of a highly informative polymerase chain reaction (PCR) detectable microsatellite marker DXS538 using a previously studied family with X-linked RP3 in which recombination had occurred in the region of importance. The DXS538 dinucleotide repeat locus was previously localised to Xp21.1-p11.21 to study RP3 in one XLRP family. Using published RFLP data we narrowed the localisation of DXS538 to the region Xp21.1-p11.23. Thus DXS538 is now a convenient diagnostic tool, aiding carrier detection of XLRP in females, as shown in the family presented here.

The rapid analysis of dystrophin gene deletions shows variable electrophoretic mobility.

The introduction of PCR technology to the molecular diagnosis of genetic diseases has increased the speed and range of DNA tests available. Previous analyses of dystrophin gene mutations were time consuming, taking weeks to complete, and used radioisotopic methods. Further developments in DNA amplification and post-amplification techniques have made conventional tube PCR redundant. The rapid methodologies described enable the efficient screening of large populations for genetic disorders, although precautions must be taken when analysing the PCR products.

Detection of polymorphisms using thermal cycling with a single oligonucleotide on a DNA sequencing gel.

A method is described for the detection of restriction fragment length polymorphisms (RFLPs) in single copy genes in mammalian cells using one 5'-labelled oligonucleotide. This linear amplification (LA) method employs a single oligonucleotide as primer, which is extended by Taq DNA polymerase up to a restriction enzyme cleavage site. The products are arithmetically amplified by thermal cycling. The size of the products are determined by the sequence of the oligonucleotide and the position of the restriction enzyme cleavage site. Hence, an RFLP can be observed by measuring the size of the products. Polymorphisms which differ in size by a small number of base pairs, as are found in (CA)n repeats, are especially suitable for analysis by the LA procedure since the products are run on DNA sequencing gels. A number of genes were examined by the procedure and all produced a satisfactory signal including GC-rich template. It is proposed that the LA method would be suitable for large-scale genetic linkage analysis. The LA procedure has many advantages including the ability to multiplex signals under the same conditions, and lower cost since only one primer is needed.

Direct PCR sequencing of dystrophin polymorphic CACA alleles after purification to remove shadow bands.

A method is described that allows the sequencing of polymerase chain reaction (PCR) products containing CACA repeats. The method was tested using a DNA polymorphism that exists at the 3' end of the dystrophin gene. This polymorphism consists of a variation in the length of a CACA dinucleotide repeat. Four alleles from a total of 16 individuals were sequenced at this locus after the DNA sequence had been amplified by the PCR. Five examples of each of the common alleles were sequenced. For each allele all five sequences were the same. The only example of a rare allele was also sequenced. The PCR products of DNA sequences containing dinucleotide repeats consist of a number of bands differing by 2 bp below the most intense main band. Previously, direct sequencing of the PCR products lead to ambiguities and smearing at and above the CACA repeat. In this paper, the main PCR band was cut out of a sequencing gel and directly sequenced to give a clear DNA sequence. Our results indicate that for a particular allele, all individuals had exactly the same DNA sequence. This implies that with the appropriate choice of oligonucleotide primers, polymorphisms could be detected without electrophoresis.

Amyloid plaque core protein in Alzheimer disease and Down syndrome.

We have purified and characterized the cerebral amyloid protein that forms the plaque core in Alzheimer disease and in aged individuals with Down syndrome. The protein consists of multimeric aggregates of a polypeptide of about 40 residues (4 kDa). The amino acid composition, molecular mass, and NH2-terminal sequence of this amyloid protein are almost identical to those described for the amyloid deposited in the congophilic angiopathy of Alzheimer disease and Down syndrome, but the plaque core proteins have ragged NH2 termini. The shared 4-kDa subunit indicates a common origin for the amyloids of the plaque core and of the congophilic angiopathy. There are superficial resemblances between the solubility characteristics of the plaque core and some of the properties of scrapie infectivity, but there are no similarities in amino acid sequences between the plaque core and scrapie polypeptides.

Loop arrays in mouse brain demonstrated with antisera to cytokeratins and monoclonal antibodies to several classes of intermediate filaments: strain differences and developmental expression.

Some monoclonal antibodies raised against mouse brain antigens display a novel loop array apparently localized within the cytoplasm of neurons in fresh frozen sections of adult mouse brain. By indirect immunofluorescence, these loops are detectable in the cerebral cortex, thalamus, brainstem, and are particularly striking in association with pyramidal neurons of the hippocampus. The loops are also seen with polyclonal antibodies to the cytokeratin class of intermediate filaments. The antibodies which react with these loops also react with ependymal cells. Western blot analysis of crude insoluble cytoskeletal components of mouse brain with antibodies of cytokeratins confirm the presence of reactive bands in the range of 40-60 kdalton, appropriate in molecular weight for this class of cytoskeletal filaments. This evidence suggests that the loops share antigenic determinants with non-neural cytokeratins. During development, immunoreactive structures are first seen as small punctate or curvilinear profiles, which change into a loop array at approximately 14 days postnatal age in several mouse strains. However, in 8 of 15 different mouse strains, these immature punctate profiles remain without morphological alteration to loops throughout adult age. The F1 crosses between strains with and without the loops develop loops, but on average they are of smaller size than in the positive parent.

Alpha-Lactalbumin and lactose concentrations in rat milk during lactation.

Homogeneous rat alpha-lactalbumin was prepared from whey by chromatography on DEAE-Sephadex A-50 and Ultrogel AcA 44. Two biologically active forms of alpha-lactalbumin were apparent after ion-exchange chromatography, but on gel filtration the combined forms were eluted as a single peak with a molecular weight of approx. 33000. The molecular weight when determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was 15100. Antiserum to alpha-lactalbumin was prepared from rabbits, and single radial immunodiffusion was used to measure the concentration of alpha-lactalbumin in milk expressed from rats during lactation and for 2 days after the cessation of lactation. A significant positive correlation (r = + 0.89) between the concentrations of alpha-lactalbumin and lactose was obtained for the first 20 days of lactation. This is consistent with the suggestion that alpha-lactalbumin may control the concentration of lactose in milk. However, a significant negative correlation (r = -0.91) between the concentration of alpha-lactalbumin and lactose was obtained for 2 days after the cessation of lactation on day 20.

Myasthenia gravis and D-penicillamine.

There has been some uncertainty as to whether the apparent association between myasthenia gravis (MG) and D-penicillamine (D-P)-treated rheumatoid arthritis (RA) could be due to chance or whether the drug is responsible. In the absence of D-P, RA is found in association with MG, but this may simply reflect the high prevalence of RA. Although MG may be more common than expected after D-P treatment of RA, it probably occurs in only approximately 1% of such patients. In these circumstances, it is difficult to prove that D-P can induce MG, but compelling evidence in support for this possibility comes from the finding of differences between autoantibodies when spontaneous and D-P-associated MG are compared. These serologic differences could be explained in terms of an effect of D-P on antigen presentation and/or immunoregulation.

Immunobiology of D-penicillamine.

D-penicillamine holds the key to a better understanding of autoimmunization and the pathogenesis of autoimmune disease. Analysis of its mode of action is complicated by its multiplicity of effects. In respect to anti-acetylcholine receptors and myasthenia gravis, the major effect may be at the level of immunoregulation and/or immunogenicity. Anti-striated muscle antibody is much more common and is influenced by the HLA antigen of the patient. Thus, HLA-linked immune response genes may be involved.

Myosin autoantibodies reacting with selective muscle fiber types.

Myosin in striated muscle exists in at least two immunologically distinct forms. Human autoantibodies specific for either form show selective muscle fiber reactivity. One group of antibodies reacts with type 2b (white) muscle fibers, the other with type 2a (red) and type 1 (intermediate) fibers. All of these antibodies react with the A band of glycerinated myofibrils. Both groups of antibodies react with cardiac muscle. Muscle-fiber-specific antibodies are detected in approximately 1% of sera submitted for diagnostic screening. Sera from eight patients were studied further. The two patients with antibodies against type 2b (white) muscle myosin had rheumatoid arthritis. The six patients with antibodies against type 2a (red) and type 1 (intermediate) myosin had clinical features which included various autoimmune and infectious disorders.

Characterization of human heterophile antibodies apparently induced by alloimmunization.

Human heterophile antibodies reactive with various tissues of different species may be detected by indirect immunofluorescence. Results of screening sera obtained after rhesus immunization and before and after blood transfusion and renal transplantation suggest that such antibodies can appear following alloimmunization of group A and O individuals. Eleven positive sera selected for detailed study were found to have anti-B isohaemagglutinins together with rat erythrocyte agglutinins. Absorption studies suggested that there may be some relationship between the group B antigen and the rat antigen concerned but the precise explanation for alloimmunization remains to be determined.

Characterisation of immunofluorescent heterophile antibodies which may be confused with autoantibodies.

Approximately 6% of sera submitted for routine immunofluorescent autoantibody screeening produced characteristic reaction patterns against a variety of animal and human tissues. It is suggested that these non-tissue-specific patterns represent a complex family of heterophile antibodies which could be confused with certain autoantibodies. It is further suggested that these heterophile antibodies bear a relationship to IgG isohaemagglutinins.

Evidence for immunological cross-reactivity between smooth and skeletal muscle.

In 1965 Johnson, Holborow & Glynn showed that some patients with chronic liver disease have antibodies which react with smooth muscle by indirect immunofluorescence. This work was rapidly confirmed and it was soon showed that the same serum may also react with renal glomeruli and other tissue components. Reactions with skeletal muscle were not demonstrated. In 1960 Strauss and co-workers deomonstrated that patients with myasthenia gravis may have antibodies which react with skeletal muscle. Subsequent work has suggested that these antibodies are specific for skeletal as opposed to smooth muscle. More recently smooth muscles antibodies (SMA) have been shown to react with a wide range of tissues and cells including neoplastic cells (Gabbiani et al,, 1973; Holborow et al., 1975). Throughout this work it has been assumed that such reactivity is due to the presence of smooth muscle contractile proteins. This conclusion was further supported by the demonstration that animals immunized with smooth muscle contractile proteins developed antibodies which produce similar patterns to those found with the naturally occurring antismooth muscle antibodies (Trenchev, Sneyd & Holborow, 1974). From these studies it appeared that there was very little if any cross reactivity between smooth and skeletal muscle, although Bray (1974) pointed to the need to re-examine this conclusion. In the course of screening human sera for autoantibodies we have been impressed by the occasional occurrence of sera which gave both smooth and skeletal muscle staining patterns. In this report we describe selected sera which react with antigens which appear specific for either smooth or striated muscle, and additional sera which react with antigens which appear common to both smooth and striated muscle. Immunization of rabbits with skeletal muscle contractile protein has provided further evidence for such cross reactivity,

The reactivity of the antistriational antibodies associated with thymoma and myasthenia gravis.

Using immunofluorescence forty-nine sera giving striational staining of skeletal and cardiac muscle have been tested on glycerinated myofibrils. By comparing immunofluorescence and phase contrast it has been found that these antibodies are rather heterogeneous but at least three different staining patterns can be identified. Some patterns may be associated with the presence of a thymoma or may be induced by penicillamine treatment of patients with rheumatoid arthritis. Routine screening for antistriational antibodies may lead to the recognition of undiagnosed myasthenia gravis and thymoma. Antistriational autoantibodies may be useful in the further study of the structure and function of the sarcomere.