Quality assessment of genetic markers used for therapy stratification.

PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

Preimplantation genetic diagnosis (PGD): the Gothenburg experience.

BACKGROUND: A program for preimplantation genetic diagnosis of pre-embryos from patients with hereditary disorders was set up in our unit at Sahlgrenska University Hospital in 1994. The majority of the patients were carriers of X-chromosome linked disorders; a few patients were translocation carriers. In this paper we describe our experiences of our first 36 cycles, 30 gender determinations and six analyses of embryos with possible translocations. METHODS: Conventional hormone replacement treatment with intracytoplasmic sperm injection to fertilize the eggs followed by blastomere biopsy and fluorescent in situ hybridization at the eight cell stage was used for sexing as well as detection of translocations. RESULTS: Out of the 30 cycles in 13 patients for gender determination, blastomere biopsies could be carried out in 25 cycles. Transfer of normal female embryos (XX) was performed in 18 cycles, resulting in five pregnancies (pregnancy rate 27.8%) and an implantation rate of 20% per transfer. Three girls have been born. Hence the take home baby rate was 16.7% per transfer and 10% per started cycle. Six cycles (three patients) for detection of translocations in embryos were performed. Diagnosis was possible in four cycles. Transfer of normal embryos was carried out in one cycle. No pregnancy was achieved. CONCLUSION: Successful PGD in its clinical application demands close collaboration between a large group of specialists. Even so, the success rate is considerably lower than after conventional IVF or ICSI procedures. Taking into account the stress caused to the parents facing late interruption of pregnancy following conventional prenatal diagnosis we are convinced that this technique is well worthwhile continuing and refining.

Congenital variant Rett syndrome in a girl with terminal deletion of chromosome 3p.

A girl fulfilling four/five of six inclusion criteria and eight/nine of 11 supportive criteria for atypical Rett syndrome had a cytogenetic deletion of chromosome 3p, del(3)(pter-->3p25.1 approximately 25.2). The deletion was situated on the maternally derived chromosome and by molecular analysis the deletion breakpoint was shown to be between DNA markers D3S3589 and D3S1263.

The use of fine-needle aspiration cytology in the molecular characterization of neuroblastoma in children.

BACKGROUND: The utility of fine-needle aspiration (FNA) material in the molecular characterization of a group of neuroblastic tumors (NT) in children is presented. METHODS: A recent study showed a high accuracy rate for FNA cytology (FNAC) in combination with immunostaining as a diagnostic method in 26 children with NT. In the current study FNA smears from 18 children were analyzed, either at the time of diagnosis or retrospectively, on available stored smears for cellular DNA content (DNA ploidy) by means of image cytometry (ICM) and 1p deletion and N-myc amplification by interphase fluorescent in situ hybridization (FISH). RESULTS: A total 62 analyses (DNA ploidy, 20 analyses; chromosome 1 or 2 number, 11 analyses; 1p deletion, 16 analyses; and N-myc, 15 analyses) resulted in clear information in 60 cases, whereas 2 tests for N-myc amplification failed. The results were compared with each other and with cytometric DNA analyses on tumor touch imprints (n = 12) and N-myc analyses on material from surgical specimens (Southern blot analysis, n = 12). The results were concordant in all but seven analyses (all with clear informative results) in six children. These discrepancies may be explained as an effect of either chemotherapy or tumor heterogeneity. CONCLUSIONS: FNA is a fast and noninvasive diagnostic technique that yields sufficient material for the molecular characterization of neuroblastic tumors by means of FISH and ICM. Such analyses are of prognostic significance because they predict tumor behavior and response to therapy according to International Neuroblastoma Staging System/International Neuroblastoma Risk Groups criteria. In the majority of cases it also is possible to obtain material for storage and future investigations.

The two-exon gene of the human forkhead transcription factor FREAC-2 (FKHL6) is located at 6p25.3.

The gene for the human transcription factor forkhead related activator 2 (FREAC-2; HGMW-approved symbol FKHL6) has been characterized and found to consist of two exons separated by an intron of 3.6 kb. The first exon encodes the forkhead DNA-binding domain and one of the transcriptional activation domains, AD2. The second exon contains the coding sequence corresponding to the C-terminal activation domain AD1. The full-length FREAC-2 protein is predicted to be 444 amino acids, which adds 39 amino acids to the previously published partial cDNA sequence. A 2-kb CG island is centered around the 5' end of the FREAC-2 gene. Fluorescence in situ hybridization was used to localize the human FREAC-2 gene to chromosomal position 6p24-p25, and the localization was further refined by radiation hybrid mapping to 6p25.3.

X-linked myotubular myopathy: a linkage study.

Two families with the congenital X-linked infantile form of myotubular myopathy have been investigated by linkage analysis using markers from the X-chromosome. Linkage was found at the locus Xq28 (with DXS52). The analysis gave a peak lod score of 2.41 at the recombination fraction zero. Free recombinations (theta = 0.50) were seen using the markers DXS84, DXS14 and DXS146 from the p arm of the X-chromosome. Since the disorder is very rare, it is important to add cumulative linkage data from the few families that do exist.

X-linked myotubular myopathy: clinical and pathological findings in a family.

A five-generation family with recessively inherited X-linked myotubular myopathy was investigated. Two of the affected boys, who were siblings and were verified by muscle biopsy to have the disease, died 3 days and 3 months, respectively, after birth. They showed marked hypotonus from birth, general muscle weakness and asphyxia. Three other boys, who were probably affected by the disease, had severe asphyxia and died shortly after birth. In three of the five cases there was polyhydramnios. The muscle biopsies of the two siblings revealed predominance of small fibres with central nuclei and accumulation of mitochondria in the central parts of the fibres. In one of the boys mainly the type 1 fibres were hypotrophic. The postmortem examination revealed variation in the involvement of different muscles, the anterior tibial muscle being the most severely affected. Intrafusal muscle fibres and myocardium were apparently unaffected. There was no involvement of the spinal cord. The clinical examination of two obligate carriers in the family revealed no muscle weakness but the muscle biopsy showed pathological changes including greatly increased variability of fibre size, and many fibres with central nuclei. The findings indicate that muscle biopsy is of value in genetic counselling to detect carriers although the observed changes were unspecific.

XbaI restriction fragment length polymorphism of apolipoprotein B in Swedish myocardial infarction patients.

Apolipoprotein B (apoB) is a major importance to the metabolism of lipoproteins, and there is also evidence which suggests that apoB plays a central role in atherogenesis. In order to study whether there is a link between one of the mutations of the apoB gene and premature coronary heart disease, the frequency of the XbaI RFLP for the apoB gene was analysed in 52 male myocardial infarction patients. These were compared with a control group matched for age and sex (n = 52), and a random population sample of middle-aged men (n = 106). Two alleles were identified by the presence (X2) or the absence (X1) of an XbaI cleavage site. A somewhat higher frequency of the X2 allele was seen among the patients, however there was no significant difference between patients and controls regarding the genotypes or allele frequencies. This observation does not confirm one earlier report where a higher frequency of the X1 allele was seen in myocardial infarction patients. Differences between the studied populations or epidemiological designs of the studies might explain the diverging results. Further studies are evidently needed to fully resolve the relation between the XbaI RFLP and risk of atherosclerotic disease or lipoprotein metabolism.

Lack of correlation between the apolipoprotein B XbaI polymorphism and blood lipid levels in a Swedish population.

The possible connections between the apolipoprotein B (apo B) XbaI polymorphism and the serum levels of total cholesterol, triglycerides, LDL, HDL and apo B have been investigated among 187 randomly selected subjects from Gothenburg, Sweden. The interferences of age and sex on the serum lipoproteins and apo B concentrations were considered. Using multiple regression analysis, we compared the different lipid levels and the levels of apo B with the genotypes X1X1, X1X2 and X2X2 (X1 = without the XbaI restriction site, X2 = with the site), with age and with sex and with those factors combined with each other. A significantly higher concentration of serum cholesterol and LDL among men than among women was found and total serum cholesterol, LDL and apo B were positively correlated with age. The allele frequency of the XbaI polymorphism in the sample was 0.45 for the allele without the XbaI restriction site. No correlation was found between the apo B genotypes and the levels of serum lipoproteins or apo B.

Apolipoprotein B gene variants are involved in the determination of serum cholesterol levels: a study in normo- and hyperlipidaemic individuals.

We have investigated the frequencies of 3 restriction fragment length polymorphisms (RFLPs) of the apolipoprotein B (apo B) gene in normo- and hyperlipidaemic individuals. In individuals with type III hyperlipidaemia, the allele frequency for the RFLP detected with XbaI was significantly different from the allele frequency in normolipidaemic individuals and in those with other types of hyperlipidaemia. No significant difference in allele frequency was found among these groups for the RFLPs detected with MspI or EcoRI. Within a sample of 62 normolipidaemic individuals, homozygotes for the X2 allele (cutting site) of the XbaI RFLP had a significantly higher serum cholesterol level than homozygotes for the XI allele, with individuals of the genotype X1X2 having an intermediate value (X2X2 mean 5.71 mmol/l, X1X1 mean 4.81 mmol/l, X1X2 mean 5.30 mmol/l). There were also significant differences in serum triglyceride levels in individuals with different XbaI genotypes. In these normolipidaemic individuals there was no correlation between the EcoRI and MspI RFLP genotypes and levels of any serum lipid variable. Information from the XbaI and EcoRI RFLPs was used in conjunction to define apo B haplotypes. These haplotypes are a more precise measure of the genotypic variation, and they explain a greater fraction of the serum cholesterol and triglyceride levels than the single-site polymorphisms considered separately. This study suggests that variations in the gene for apo B are associated with the determination of serum cholesterol and triglyceride levels both in patients with type III hyperlipidaemia and in the normal population.

The isolation of genomic recombinants for the human apolipoprotein B gene and the mapping of three common DNA polymorphisms of the gene--a useful marker for human chromosome 2.

We have used four independently isolated cDNA probes for human apolipoprotein B (apo B), to isolate overlapping genomic recombinants for the 3' portion of the apo B gene. The cDNA clones and a unique fragment from the genomic recombinant have been used to identify the human apo B gene in DNA from a series of rodent X human somatic cell hybrids. Our results provide evidence for the assignment of this gene to the short arm of human chromosome 2 (p23-pter). We have used the cDNA probes to identify three common DNA polymorphisms. The first, detected with the restriction enzyme XbaI and our probe pAB4, has a rare allele frequency of 0.48. The other two polymorphisms are detected with the probe pAB3. The enzyme MspI detects at least three alleles, with frequencies of 0.67, 0.16 and 0.15, while that detected with the enzyme EcoRI has a rare allele frequency of 0.12. The relative position of these polymorphisms has been mapped using the genomic recombinants. Investigation of a small number of haplotypes indicates that there is linkage equilibrium between the polymorphisms, which have a total polymorphism information content (PIC) value of more than 0.8. These polymorphisms will provide useful markers for genetic studies on chromosome 2 and for the analysis of the involvement of variants of the apo B gene in the development of hyperlipidaemia.

Subcellular localisation of the middle and large T-antigens of polyoma virus.

The distribution of two of the polyoma virus early proteins (the large and middle T-antigens) in lytically infected mouse cells and transformed rat cells has been investigated by indirect immunofluorescence and immuno-electron microscopy using well-characterised monoclonal antibodies. By these techniques, the viral large T-antigen was found almost exclusively in the nucleus, sometimes in association with nuclear pores, but never in the nucleolus. In lytically infected, but not transformed cells, fluorescence was detected in discrete areas ('hot spots') within the nucleus and, in a minor population of lytically infected cells, cytoplasmic immunoreactive material was observed. The viral middle T-antigen was found in association with most cytoplasmic membranes and in the majority of cells mainly in the endoplasmic reticulum. Only a fraction of the staining was observed in the plasma membrane and no staining in the nucleoplasm was observed. The data suggest that the site of action of the major transforming activity of polyoma virus need not be at the plasma membrane. Functions associated with the viral antigens are discussed in terms of their subcellular distributions within cells.

Analysis of the human apolipoprotein B gene; complete structure of the B-74 region.

In this paper we describe the nucleotide sequence of the B-74 region of human apolipoprotein B-100 mRNA. This region comprises the 3'-proximal three-quarters of the mRNA and contains 10,089 nucleotides (nt), 9786 of which are coding. Combining our data with the published sequence of the 5'-proximal one-quarter (i.e., the B-26 region [Protter et al., Proc. Natl. Acad. Sci. USA 83 (1986) 5678-5682] assigns 14,059 nt to the apoB-100 mRNA. The coding sequence spans 13,548 nt or 4516 amino acids (leader peptide excluded). The B-74 part of the apoB gene is built up of five exons separated by small introns, and is dominated by an unusually large exon of 7.5 kb. The derivation of two (EcoRI and XbaI) restriction fragment length polymorphisms occurring in the coding region is discussed.

Molecular cloning of human apolipoprotein B cDNA.

In this paper we describe the isolation of cDNA clones which code for parts of apolipoprotein B (apoB). The clones were obtained by immunoscreening of an expression library (lambda gt 11) derived from a human hepatoma cell line (Hep G2). The relationship between positive clones and apoB was established with immunochemical techniques using polyclonal as well as monoclonal antibodies. Recombinants, expressing nonoverlapping regions of apoB are described, all hybridizing with a very large mRNA (approximately 20,000 bases long). The nucleotide sequence obtained predicts a primary protein structure with a composition suitable for the formation of stretches of an amphipatic alpha-helix.