The human gene for plasminogen activator inhibitor 2 (PAI2) exhibits an EcoRI RFLP.
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Chromosome Mapping↗
Biomedical subjects
E Bakker
Publications and source records attributed to E Bakker.
Infectivity of blood seropositive for hepatitis C virus antibodies.
Stored serum samples from 5150 blood product transfusions and 383 recipients were tested for antibodies to hepatitis C virus (anti-HCV) by a recombinant enzyme-linked immunosorbent assay (ELISA) as part of a prospective study on post-transfusion non-A, non-B hepatitis (NANBH). Donor cofactors associated with HCV infectivity of anti-HCV-positive blood products were raised alanine aminotransferase concentrations (6 of 9 infective vs 1 of 26 not infective); a mean ELISA optical density/cut-off ratio greater than or equal to 2 (7 of 9 vs 9 of 26); both preceding factors (together in 6 blood products, all of which transmitted infection); and persistent donor anti-HCV seropositivity. Use of anti-HCV screening to prevent post-transfusion NANBH was compared with measurement of alanine aminotransferase concentrations: a corrected efficacy of 63% and 65%, a specificity of 93% and 64%, and a positive predictive value of 16.2% and 3.6% were found, respectively; 0.7% or 3.8% of blood donations, respectively, would be discarded. Blood donor screening for anti-HCV is recommended to reduce the incidence of post-transfusion NANBH.
Alanine Transaminase↗
An unusual variant of Becker muscular dystrophy.
We report on 5 brothers with slowly progressive limbgirdle weakness. Calf hypertrophy was absent. The levels of creatine kinase, electromyography, and findings from a muscle biopsy specimen were compatible with muscular dystrophy. The propositus's biopsy specimen also showed numerous rimmed vacuoles. DNA analysis revealed a deletion in the dystrophin gene, establishing a diagnosis of Becker muscular dystrophy. Both the absence of calf hypertrophy and the presence of rimmed vacuoles are unusual features in this disorder.
Adult↗
Detection of truncated dystrophin in fetal DMD myotubes.
An immunohistochemical study was carried out on a twelve-week old fetus, aborted for high risk of Duchenne muscular dystrophy. Southern and FIGE analysis showed an intragenic duplication in the DMD gene, which had previously resulted in a severe Duchenne phenotype in three relatives. Polyclonal antibodies directed against the NH2-terminal half of dystrophin showed a positive reaction an a similar distribution of dystrophin in the skeletal myotubes of a twelve-week old normal fetus and the affected fetus. In contrast, a polyclonal antibody directed against the COOH-terminus of dystrophin, i.e., distal to the mutation in this family, did only react with the myotubes of the normal fetus and not with those of the affected fetus. This indicates the presence of a truncated dystrophin in the affected fetus. Apparently at this stage, before binding of dystrophin to the sarcolemma, no distinction is made yet between normal and abnormal dystrophins. This implies that the potential to bind to the sarcolemma could be a major point of discrimination between normal and defective dystrophins. The truncated dystrophin will probably be degraded in a later stage during fetal development. So it appears that the use of dystrophin immunostaining to confirm high Duchenne risk abortions requires great caution. To prevent false-positive results, the combined use of NH2- and COOH-terminal antibodies is mandatory.
Dystrophin↗
New polymorphic DNA marker close to the fragile site FRAXA.
DNA from a human-hamster hybrid cell line, 908-K1B17, containing a small terminal portion of the long arm of the human X chromosome as well as the pericentric region of 19q was used as starting material for the isolation of an X-chromosome-specific DNA segment, RN1 (DXS369), which identifies a XmnI RFLP. Linkage analysis in fragile X families resulted in a maximum lod score of 15.3 at a recombination fraction of 0.05 between RN1 and fra(X). Analysis of recombinations around the fra(X) and distal to DXS105. Analysis of the marker content of hybrid cell line 908K1B17 suggests the localization of RN1 between DXS98 and fra(X). Heterozygosity of DXS369 is approximately 50%, which extends the diagnostic potential of RFLP analysis in fragile X families significantly.
Animals↗
An infrequent DNA polymorphism associated with severe von Willebrand's disease.
Genomic DNA of six unrelated Dutch patients with severe von Willebrand's disease (vWD) was submitted to restriction fragment length polymorphism analysis. We observed a strong association between a 36 kb allele detected by a partial complementary DNA probe (pvWF 1100) and the restriction enzyme XbaI with severe von Willebrand's disease. This 36 kb allele is rare (allele frequency of 7%) both in the general population and in patients with autosomal dominant types of von Willebrand's disease. Three of our six patients were found to be homozygous for this allele while two others were heterozygous. The association of this rare XbaI allele with severe vWD enables carrier detection and prenatal diagnosis in these families. The high frequency (67%) of the 36 kb allele observed in this patient group raises the possibility that a subgroup of patients with severe vWD has a genetic defect with a common origin.
Alleles↗
Two step procedure for early diagnosis of polycystic kidney disease with polymorphic DNA markers on both sides of the gene.
Polymorphic DNA markers can now be used for presymptomatic and prenatal diagnosis of the autosomal dominant form of polycystic kidney disease (PKD). A detailed map is known for the chromosomal region around the PKD1 gene on the short arm of chromosome 16. We present here a simple, two step procedure for diagnosis of PKD1 by family studies. Using this approach, at least 92% of random subjects are informative for polymorphic DNA markers bracketing the PKD1 gene. The recombination rate between these flanking markers is on average 10%. In non-recombinants (90% of family members), the accuracy of diagnosis using DNA markers is greater than 99%. We conclude that sufficient well defined DNA markers are now available for routine diagnosis of PKD1. We recommend, however, that prenatal diagnosis of PKD by chorionic villi sampling should be attempted only after the linkage phase of the DNA markers has been established by haplotyping the index family. Since autosomal dominant PKD has been found to be genetically heterogeneous, families should be of sufficient size to rule out the rare form of PKD not caused by a mutation on the short arm of chromosome 16.
Chromosomes, Human, Pair 16↗
Immunological study of dystrophin in Duchenne fetus.
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Dystrophin↗
High resolution deletion breakpoint mapping in the DMD gene by whole cosmid hybridization.
The locus DXS269 (P20) defines a deletion hotspot in the distal part of the Duchenne Muscular Dystrophy gene. We have cloned over 90 kilobase-pairs of genomic DNA from this region in overlapping cosmids. The use of whole cosmids as probes in a competitive DNA hybridization analysis proves a fast and convenient method for identifying rearrangements in this region. A rapid survey of P20-deletion patients is carried out to elucidate the nature of the propensity to deletions in this region. Using this technique, deletion breakpoints are pinpointed to individual restriction fragments in patient DNAs without the need for tedious isolation of single copy sequences. Simultaneously, the deletion data yield a consistent restriction map of the region and permit detection of several RFLPs. A 176 bp exon was identified within the cloned DNA, located 3' of an intron exceeding 150 Kb in length. Its deletion causes a frameshift in the dystrophin reading frame and produces the DMD phenotype. This exon is one of the most frequently deleted exons in BMD/DMD patients and its sequence is applied in a pilot study for diagnostic deletion screening using Polymerase Chain Reaction amplification.
Base Sequence↗
Lack of immune potentiation by complexing HBsAg in a heat-inactivated hepatitis B vaccine with antibody in hepatitis B immunoglobulin.
In a randomized, dose-response study among 305 health care workers, we examined whether the immunogenicity of a heat-inactivated hepatitis B vaccine could be enhanced when HBsAg was complexed by anti-HBs contained in hepatitis B immunoglobulin either at equivalent proportions or at 10-fold antigen excess. The dose of HBsAg in the control vaccine as well as in the two complexed vaccine preparations could be reduced from the standard value (3 micrograms) to 0.6 micrograms per injection without affecting the antibody response in the vaccinees. Still lower dosages of HBsAg in the three vaccine preparations induced significantly lower but comparable anti-HBs responses. These results indicate that, in man, using a heat-inactivated plasma vaccine, addition of anti-HBs contained in hepatitis B immunoglobulin does not potentiate the immunogenicity of HBsAg.
Antibody Formation↗
DNA restriction fragment length polymorphisms in differential diagnosis of genetic disease: application in neuromuscular diseases.
Three families, in which several male individuals suffer from a hereditary neuromuscular disease, were examined by analysis of naturally occurring restriction fragment length polymorphisms (RFLPs) and by screening for deletions. Originally, differential diagnosis included spinal muscular atrophy (two families) and limb girdle syndrome (one family) or Becker muscular dystrophy. Since deletions were not detectable, an X-chromosomal segment, carrying DNA markers for the dystrophin gene and its flanking regions was reconstructed; this demonstrated Becker muscular dystrophy is the most probable primary cause of illness in these families. Furthermore, the possible carriership of female members of these families could be determined accurately.
Chromosome Deletion↗
Prenatal diagnosis of Duchenne muscular dystrophy: a three-year experience in a rapidly evolving field.
Application of molecular genetic techniques has greatly increased diagnostic possibilities of hereditary disorders. In 1983 the first linkage of Duchenne muscular dystrophy with flanking DNA probes was described, which made carrier detection possible in a limited number of cases. The first published prenatal diagnosis for Duchenne muscular dystrophy dates from 1985. DNA-analysis for Duchenne muscular dystrophy and Becker muscular dystrophy has become increasingly informative, firstly by the development of more flanking markers, followed by intragenic probes detecting deletions and, more recently, by the use of cDNA probes detecting a deletion or duplication mutation in over 60% of the Duchenne and Becker muscular dystrophy patients. Although these developments allow a highly reliable (greater than 99%) carrier detection and prenatal diagnosis in over 90% of cases, the continuing introduction of new probes and/or technologies has necessitated constant reappraisal of many families to derive maximum information. During the past 3 years we applied prenatal diagnosis for Duchenne and Becker muscular dystrophies with DNA-analysis on 53 male fetuses in 47 families. Twenty-two healthy male babies were born, after being diagnosed to have a low Duchenne muscular dystrophy risk. Two pregnancies also diagnosed as low risk have not yet come to term. In the other cases a high risk for Duchenne muscular dystrophy was found and the parents chose abortion. Our studies also revealed a number of important diagnostic pitfalls, such as non-paternity, karyotypic anomalies and gonadal mosaicism.
DNA↗
The DMD gene analysed by field inversion gel electrophoresis.
Genomic and cDNA probes were used to construct a physical map of the DMD region including the 2.3 Mb DMD gene. FIGE-analysis allows rapid screening of the complete region using only a few probes, detecting deletions or duplications in over 60% of the patients. The technique is especially powerful in the analysis of carrier females. We have found two mutational hotspots; a minor hotspot located proximally and a major hotspot within a large, centrally located intron. Deletions involving this latter intron were studied using 100 kb of cloned DNA sequences. Although breakpoints are spread over the entire region, 5 are clustered within 3 kb. Analysis of over 250 BMB/DMD families has underscored the importance of germinal mosaicism as a major diagnostic pitfall. At least 14% of new mutation cases involve germinal mosaicism and this still is a lower estimate, due to small family sizes. Hence, relatives of apparent new mutation patients should be considered to have high risk, and require appropriate counselling.
Electrophoresis↗
Germinal mosaicism increases the recurrence risk for 'new' Duchenne muscular dystrophy mutations.
In 288 Dutch and Belgian Duchenne and Becker muscular dystrophy families, the parental origin of 41 new deletion or duplication mutations was determined. Twenty seven of the new mutations occurred in the maternal X chromosome and nine in the grandmaternal and five in the grandpaternal X chromosome. The grandparental data are compatible with equal mutation rates for DMD in male and female X chromosomes. New mutations were defined by their presence in one or more progeny and absence in the lymphocytes of the mother or the grandparents. In one family a fraction of the maternal lymphocytes was found to carry the mutation, suggesting somatic mosaicism. In six cases out of 41, the mutation was transmitted more than once by a parent in whom the mutation was absent in lymphocytes, suggesting gonadal mosaicism as the explanation for the multiple transmission. Using our data for the recurrence of the mutations among the total of at risk haplotypes transmitted, we arrive at a recurrence risk of 14% for the at risk haplotype. The observation of this high risk of germinal mosaicism is crucially important for all physicians counselling females in DMD families. Recently, germinal mosaicism has been observed also in a number of other X linked and autosomal disorders. The implications and appropriate diagnostic precautions are discussed.
DNA Probes↗
Assignment of the gene coding for human peroxisomal 3-oxoacyl-CoA thiolase (ACAA) to chromosome region 3p22----p23.
The chromosomal location of the human gene coding for peroxisomal 3-oxoacyl-CoA thiolase (ACAA) was determined with the aid of cDNA and genomic probes by screening of rodent x human somatic cell hybrids and in situ hybridization. The results localize the gene to chromosome region 3p22----p23.
Acetyl-CoA C-Acyltransferase↗
Mapping of recombinants near the Huntington disease locus by using G8 (D4S10) and newly isolated markers in the D4S10 region.
Genetic linkage between the marker G8 (D4S10) and Huntington disease (HD) was studied in six Dutch pedigrees. The informativeness of the D4S10 locus was increased by isolation of a cosmid, C5.5, with a G8 subclone used as probe. We present a restriction map of 70 kb in the D4S10 region. Two subclones of C5.5, H5.52 and F5.53, detect MspI and SinI RFLPs, respectively. These probes increase the informativeness of D4S10 in the Dutch HD population from 55% to 95%. Seven recombinations were found in 124 informative meioses in which multipoint segregation of D4S10 haplotypes and the HD locus was studied. Two of the recombinations occurred within the D4S10 region. The other five recombinations are highly valuable for the mapping of present and future markers relative to each other and to the HD gene. In addition, several recombinations between markers in meioses from unaffected parents were noted, which will also be useful in ordering new markers. On the basis of our three-point recombination data, the orientation of the D4S10 region relative to HD is HD-H5.52-G8-F5.53, which independently confirms the previously derived polarity for D4S10.
Chromosome Mapping↗
Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications.
We have studied 34 Becker and 160 Duchenne muscular dystrophy (DMD) patients with the dystrophin cDNA, using conventional blots and FIGE analysis. One hundred twenty-eight mutations (65%) were found, 115 deletions and 13 duplications, of which 106 deletions and 11 duplications could be precisely mapped in relation to both the mRNA and the major and minor mutation hot spots. Junction fragments, ideal markers for carrier detection, were found in 23 (17%) of the 128 cases. We identified eight new cDNA RFLPs within the DMD gene. With the use of cDNA probes we have completed the long-range map of the DMD gene, by the identification of a 680-kb SfiI fragment containing the gene's 3' end. The size of the DMD gene is now determined to be about 2.3 million basepairs. The combination of cDNA hybridizations with long-range analysis of deletion and duplication patients yields a global picture of the exon spacing within the dystrophin gene. The gene shows a large variability of intron size, ranging from only a few kilobases to 160-180 kb for the P20 intron.
Chromosome Deletion↗
Carrier detection and prenatal diagnosis of Duchenne/Becker muscular dystrophy (D/BMD) by DNA-analysis.
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DNA↗