Prenatal diagnosis of variant late infantile neuronal ceroid lipofuscinosis (vLINCL[Finnish]; CLN5).

The first prenatal diagnosis of variant late infantile neuronal ceroid lipofuscinosis (vLINCL[Finnish]; CLN5) is reported. The disease belongs to the group of progressive encephalopathies in children with psycho-motor deterioration, visual failure and premature death. Neurons and several extraneural cells harbour lysosomal inclusions showing accumulation of material with histochemical characteristics of ceroid and lipofuscin. A Finnish woman with a daughter with vLINCL came for genetic counselling for her current pregnancy. Electron microscopy of a chorionic villus sample (CVS) at the 11th week of gestation did not reveal inclusions characteristic for NCL. DNA analysis showed that the fetus had inherited the major mutation, a 2 bp deletion of the CLN5 gene from the mother, and the same paternal (and maternal) haplotypes for COLAC1 and AC224 as the affected daughter. The pregnancy was terminated. Electron microscopy of the CVS of the aborted fetus at the 14th week of pregnancy showed lysosomal electron dense inclusions with straight and curved lamellar profiles consistent with vLINCL. Prenatal diagnosis of NCL-disorders (CLN1, CLN2, CLN3) can be made from CVS by demonstrating the mutations of the affected genes or by haplotype analysis using the closely linked markers in most cases. In various clinical settings the DNA diagnostics may not be possible. Demonstration of the characteristic inclusions of the placenta and fetal tissues remains a helpful adjunct in such cases.

Characterisation of epitopes on human p53 using phage-displayed peptide libraries: insights into antibody-peptide interactions.

We previously described the use of a phage-displayed library of random hexapeptides to define and localise the epitope on the human tumor suppressor protein p53 recognised by the monoclonal antibody PAb240. Here we have extended these results to a further eight anti-p53 monoclonal antibodies and to two further libraries, which display 12-mer and 20-mer peptides, respectively. First, we showed that selection of PAb240 binding clones from the 12-mer and 20-mer libraries gives essentially identical results to those obtained by screening the 6-mer library. Second, we used the 6-mer and 12-mer libraries to define the detailed specificity profiles of six antibodies (DO-1, DO-2, DO-7, Bp53-11, Bp53-12 and Bp53-19), which recognise the same short, highly immunogenic N-terminal segment of p53. Finally, we employed all three libraries to reveal the distinct mechanisms by which PAb421 and PAb122, two monoclonal antibodies that allosterically activate sequence-specific DNA binding by p53, react specifically with the same positively-charged C-terminal segment. In each case the epitope locations inferred from the selected sequences were confirmed by probing an array of overlapping synthetic peptides representing the primary sequence of p53. The results emphasise the consequences for epitope mapping of screening random, as opposed to antigen-derived, peptide libraries; specifically (1) that comparison of selected sequences reveals the contribution of individual residues to binding energy and specificity; (2) that heteroclitic reactions can lead to definition of a consensus that is related to but distinct from the immunising epitope and (3) that isolation of non-immunogen-homologous "mimotope" sequences reveals discrete, alternative ligand structures. The results with PAb421 and PAb122 provide examples where, while selection from the 12-mer and 20-mer libraries leads to isolation of immunogen-homologous sequences, selection from the 6-mer library results in the isolation either of no binding clones (PAb122) or solely of "mimotope" sequences with no discernible homology to the original antigen (PAb421). In addition the results with PAb421 reveal that linear epitopes can be longer than previously thought and can be formally discontinuous, consisting of independent contact motifs, which show promiscuous relative positioning.

Paternity after bone marrow transplantation following conditioning with total body irradiation.

A 28-year-old man with chronic myeloid leukaemia received an allogeneic bone marrow transplant after conditioning with daunorubicin, cyclophosphamide and fractionated total body irradiation (TBI). Four years later his wife gave birth to a healthy child. Although the patient was azospermic serologic HLA testing suggested that the patient was the father of the child. DNA fingerprinting as well as analysis of three variable number of tandem repeats (VNTR) loci D1S80 (MCT118), D17S30 (YNZ22) and the apolipoprotein B hypervariable region (apo B 3') gave unequivocal results showing that the patient was the father. Fathering a child after TBI-containing regimen has been very rare and this is the first case where the paternity has been proven with DNA methodology.

Monitoring of adult B-cell lineage acute lymphoblastic leukemia: validation of a simple method for detecting immunoglobulin heavy chain gene clonality.

Most approaches to demonstrating immunoglobulin heavy chain gene rearrangements are relatively laborious for routine follow-up of acute lymphoblastic leukemia (ALL). Here the use of a simple polymerase chain reaction (PCR) approach to monitor ALL disease activity has been validated. In the dilution experiments the method revealed a detection sensitivity 0.5% clonal cells in a background of 99.5% normal cells. To validate the immunoglobulin heavy chain gene PCH (IgH-PCR) in practice, we monitored the disease activity of 26 adult ALL patients showing a B-cell lineage component in immunophenotyping at the diagnosis of the disease. In 18 of those 26 patients, an IgH-PCR product could be demonstrated in the samples taken either at diagnosis or in relapse. These 18 patients were followed with a total of 158 consecutive samples by IgH-PCR. The mean follow-up time for the IgH-PCR-positive patients was 13.6 months (range 4 to 26 months). Eleven of these patients underwent altogether 18 relapses. In nine patients (81.8%), ten relapses (55.6%) could be predicted using the IgH-PCR approach. The mean time of IgH-PCR clonality detection, preceding a cytologic relapse, was 9.1 weeks (range 1.0 to 30.7 weeks). It seems that in three patients the predictive value of the IgH-PCR was remarkable, showing a repetitive positivity in spite of a cytologic remission, even one year prior to the relapse. We find that IgH-PCR provides a straightforward additional tool for monitoring B-cell lineage ALL. Due to the straightforward technical performance the method has low running costs and it is thus suitable for a routine service laboratory. Even if a negative finding in IgH-PCR does not rule out a forthcoming relapse in the patient, a positive finding is a definitive warning signal. All of the patients that showed an IgH-PCR clonality in the follow-up samples relapsed sooner or later.

Amplification of three hypervariable DNA regions by polymerase chain reaction for paternity determinations: comparison with conventional methods and DNA fingerprinting.

The present study evaluates the usefulness of a PCR-based method for routine paternity testing in 35 paternity cases. This identification method which is based on amplification of three hypervariable genetic loci, apoB, D1S80 and HLA-DQ alpha, is compared, with regard to reliability and technical feasibility, to the conventional identification methods based on protein polymorphisms and to Southern blot hybridizations with multi- and single locus probes. Data obtained by PCR-amplification of these three loci resulted in paternity indices (56.1, geometric mean value) which are at the same level as the corresponding values derived from standard genetic blood group markers (42.7). The geometric mean value of the paternity indices obtained by Southern blot hybridization using three single locus probes (190.6) was more informative, and the most informative analysis proved to be Southern blot hybridization with multilocus probes. The technical feasibility and the reproducibility of the PCR-based analysis is, however, overwhelming, and if several highly polymorphic loci are amplified, the resolving power of PCR-analysis is similar to that obtained using multilocus probes.

Does chemotherapy of hematological malignancies affect DNA fingerprint pattern?

We analyzed DNA fingerprints of lymphoma patients to find out whether DNA damage caused by irradiation and chemotherapy can result in DNA fingerprint changes, and whether the differences found previously in leukemia patients could be partially due to the treatment. In this study we did not find any post-treatment DNA fingerprint differences in 33 lymphoma patients, concluding, that the therapy of hematological malignancies does not affect DNA fingerprint patterns. Further, the variations of methylation do not either explain the detected differences in leukemic patients.

Proving paternity of children with deceased fathers.

Determination of paternity was attempted in the case of three children whose putative fathers are dead using DNA samples of the paternal grandparents. The DNA analyses were performed with both multilocus and single-locus probes which resolve highly polymorphic areas of human genome. The results were conclusive with both types of probes and facilitated, for example, the exclusion of the brother of the putative father. The evidence for true paternity obtained with DNA analyses can be considered reliable in this type of "indirect" paternity in which tests based on protein polymorphism are inconclusive.

DNA-fingerprint changes compared to karyotypes in acute leukemia.

Chromosomal analysis is routinely used in follow-up studies of acute leukemia, but it cannot be used in patients who do not have karyotypic abnormalities in blastic phase (10-46% of all cases). Recently we have demonstrated that unspecific DNA-fingerprint (DNA-F) changes can be detected in blastic phase of leukemia by DNA-F analysis. These changes can be used as molecular markers of the disease and in the follow-up studies of acute leukemia karyotyping and DNA-F analysis are complementary. The comparative analyses of leukemia samples with both these methods could help to localize minisatellite loci in chromosomes and reveal new DNA areas related to leukemia. New, more specific probes targeted to specific chromosomes could consequently be developed. We compared the DNA-F to the karyotypes of 50 acute leukemia patients. In blastic phase, 19 patients had DNA-F alterations and 31 patients abnormal karyotypes, 12 patients had both DNA-F alterations and karyotypic abnormalities. DNA-F changes were detected in six of the 16 patients with normal karyotypes and 19 patients with normal DNA-F had abnormal karyotypes at the time of diagnosis. Eleven patients showed neither DNA-F changes nor abnormal karyotypes in blastic phase. Three acute myeloid leukemia (AML) patients with DNA-F changes in blastic phase had trisomy eight as a sole abnormality or combined with balanced translocation, which suggests that there might be AML associated minisatellite locus in chromosome 8. In other cases the karyotypes were complicated and no clear evidence of the relationship of DNA-F changes with other chromosomes could be found.

New molecular marker in AML: DNA-fingerprint differences between leukemic phase and remission in acute myeloid leukemia.

DNA-fingerprint (DNA-F) analysis, based on the polymorphism of the tandem repeats of minisatellite areas in human genome, has a capacity to reveal minor changes in dispersed areas of human genome. In this study we have applied DNA-F analysis to the detection of differences between leukemic phase and remission in acute myeloid leukemia (AML). In order to identify normal and leukemic cell populations we used two molecular probes: Jeffreys' minisatellite probes 33.6 + 33.15 and M13 wild-type phage probe. Comparison of varying minisatellite fragments between remission and diagnosis/relapse was performed by Southern blot hybridization in 21 patients with AML. The results demonstrate that Southern hybridization with minisatellite probes can detect differences in DNA-fingerprints between leukemic phase and remission in 44% of AML patients. Thus differences in DNA-fingerprinting provides a new molecular marker, which can be useful in the detection of residual disease as well as in the study of the pathogenesis of AML.

Application of DNA "fingerprints" to paternity determinations.

26 cases of disputed paternity were tested by the methods routinely used in Scandinavian countries and by the DNA "fingerprinting" technique. In all the studied cases the results of DNA analyses were similar to those obtained with the routine examinations based on protein polymorphisms; and, in the cases where even HLA typing did not distinguish between tentative fathers, the results obtained with DNA analyses were unambiguous.

Differences in DNA-fingerprints between remission and relapse in childhood acute lymphoblastic leukemia.

DNA "fingerprint" (DNA-F) analysis, based on the polymorphism caused by numeric variations in the tandem repeats of minisatellite areas of the human genome, has a potential capacity to reveal even minor genomic changes. In this study we have applied DNA-F to the detection of residual disease in leukemia. In order to identify normal and leukemic cell populations, we used two molecular probes: Jeffrey's minisatellite probes and M13 wild type phage probe, which detect different sets of polymorphic fragments in the human genome. Comparison of varying minisatellite fragments between remission and relapse was performed by Southern blot hybridization in seven patients with acute lymphoblastic leukemia (ALL). The results suggest that Southern hybridization with DNA "fingerprint" probes can prove to be a sensitive method in the detection of minimal residual disease in ALL.