Cross-border reprogenetic services.

The purpose of this review is to synthesize the current knowledge on the international movement of patients and biopsied embryo cells for pre-implantation genetic diagnosis and its different applications. Thus far, few attempts have been made to identify the specific nature of this phenomenon called 'cross-border reprogenetic services'. There is scattered evidence, both empirical and speculative, suggesting that these services raise major issues in terms of service provision, risks for patients and the children-to-come, the legal liabilities of physicians, as well as social justice. To compile this evidence, this review uses the narrative overview protocol combined with thematic analysis. Five major themes have emerged from the literature at the conjunction of cross-border treatments and reprogenetics: 'scope', 'scale', 'motivations', 'concerns', and 'governance'. Similar themes have already been observed in the case of other medical tourism activities, but this review highlights their singularity with reprogenetic services. It emphasizes the diagnostic and autologous feature of reprogenetics, the constant risk of misdiagnosis, the restriction on certain tests for medically controversial conditions, and the uncertain accessibility of genetic counseling in cross-border settings.

Quantitative measurement of FMRP in blood platelets as a new screening test for fragile X syndrome.

The fragile X syndrome usually results from CGG repeats expansion and methylation of the FMR1 gene leading to the absence of expression of its encoded protein, fragile X mental retardation protein (FMRP). Therefore, its diagnosis is traditionally based on the detection of these molecular alterations. As an alternative, FMRP-based screening methods have been proposed over the years. Most of them are based on immunohistochemistry analyses applied to a restricted number of lymphocytes (100) or hair roots (10-20) with limited diagnosis potential. In this study, we describe a truly quantitative approach using a new model, the blood platelet, which can be recovered easily with very high purity (99.9%). FMRP levels in platelets were first measured in a control population (n = 124) and reference values were established. FMRP measurements were also performed in confirmed fragile X subjects. Receiver operating characteristic curve analysis has shown that our test can easily discriminate fragile X males and females from controls (area under curve, AUC = 0.948). Cognitive functions were also assessed in these individuals using age-specific Wechsler Intelligence Scales for Children and the Vineland Adaptive Behavior Scales. A proportional relationship between FMRP levels, intelligence quotient and adaptive behavior was observed among fragile X individuals, suggesting that our test would be able to detect fragile X cases and may predict cognitive functions.

DNA repair rates mapped along the human PGK1 gene at nucleotide resolution.

The repair of cyclobutane pyrimidine dimers (CPDs), DNA lesions induced by ultraviolet light, was studied at nucleotide resolution. Human fibroblasts were irradiated with ultraviolet light and allowed to repair. The DNA was enzymatically cleaved at the CPDs, and the induced breaks along the promoter and exon 1 of the PGK1 gene were mapped by ligation-mediated polymerase chain reaction. Repair rates within the nontranscribed strand varied as much as 15-fold, depending on nucleotide position. Preferential repair of the transcribed strand began just downstream of the transcription start site but was most pronounced beginning at nucleotide +140 in exon 1. The promoter contained two slowly repaired regions that coincided with two transcription factor binding sites.

Phenotypic variability among patients with hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome homozygous for the delF188 mutation in SLC25A15.

BACKGROUND: Hyperornithinaemia-hyperammonaemia-homocitrullinuria (HHH) syndrome (OMIM 238970) is caused by impaired ornithine transport across the inner mitochondrial membrane due to mutations in SLC25A15. To date, 22 different mutations of the SLC25A15 gene have been described in 49 patients belonging to 31 unrelated families. OBJECTIVE: To further delineate the phenotypic spectrum of HHH syndrome from a description of a genetically homogeneous cohort of patients and identify prognostic factors based on long-term follow-up. METHODS: Sixteen French-Canadian patients were retrospectively and prospectively clinically assessed. RESULTS: Owing to a founder effect, 15 of the 16 patients were homozygous for the F188del mutation in the SLC25A15 gene. The main clinical features at presentation were liver dysfunction (6/16) and neurological disease (9/16), including chronic neurological symptoms (6/9) and acute encephalopathy (3/9). Hyperammonaemia was not constant and usually mild and uncommon after start of treatment. Long-term follow-up showed that variable intellectual impairment and lower limb spasticity often occur, together or separately, with no obvious relationship to age at diagnosis and compliance with treatment. CONCLUSION: We report the largest known cohort to date of patients with HHH syndrome. A similar range of severity occurred in the clinical course and outcome of patients homozygous for delF188 and in the 33 other reported patients compiled from the literature. The poor clinical outcome of some patients with HHH syndrome despite early treatment and repeatedly normal plasma ammonia levels emphasises the need to better understand the pathophysiology and to reconsider the therapeutic goals for HHH.

Impaired plasma nonesterified fatty acid tolerance is an early defect in the natural history of type 2 diabetes.

CONTEXT: Abnormal plasma nonesterified fatty acid (NEFA) metabolism may play a role in the development of type 2 diabetes. OBJECTIVES: Our objectives were to demonstrate whether there is a defect in insulin-mediated suppression of plasma NEFA appearance (RaNEFA) and oxidation (OxNEFA) during enhanced intravascular triacylglycerol lipolysis early in the natural history of type 2 diabetes, and if so, to determine whether other mechanisms than reduced insulin-mediated suppression of intracellular lipolysis are involved. DESIGN: These are cross-sectional studies. SETTING: The studies were performed at an academic clinical research center. PARTICIPANTS: Nine healthy subjects with both parents with type 2 diabetes (FH+) and nine healthy subjects with no first-degree relatives with type 2 diabetes (FH-) with similar anthropometric features were included in the studies. INTERVENTIONS: Pancreatic clamps and iv infusion of stable isotopic tracers ([1,1,2,3,3-(2)H5]-glycerol and [U-(13)C]-palmitate or [1,2-(13)C]-acetate) were performed while intravascular triacylglycerol lipolysis was simultaneously clamped by iv infusion of heparin plus Intralipid at low (fasting) and high insulin levels. Oral nicotinic acid (NA) was used to inhibit intracellular lipolysis. MAIN OUTCOME MEASURES: RaNEFA and OxNEFA were determined. RESULTS: During heparin plus Intralipid infusion at high plasma insulin levels, and despite similar intravascular lipolytic rates, FH+ had higher RaNEFA and OxNEFA than FH- (RaNEFA: 17.4+/-6.3 vs. 9.2+/-4.2; OxNEFA: 4.5+/-1.8 vs. 2.3+/-1.5 micromol/kg lean body mass/min), independent of NA intake, gender, age, and body composition. In the presence of NA, insulin-mediated suppression of RaNEFA was still observed in FH-, but not in FH+. CONCLUSIONS: Increased RaNEFA and OxNEFA during intravascular lipolysis at high insulin levels occur early in the natural history of type 2 diabetes.

Quebec neonatal mass urinary screening programme: from micromolecules to macromolecules.

The Quebec Mass Urinary Screening Programme, initiated in 1971, has resulted in the screening of more than 2,500,000 newborns in the province of Quebec for 25 inherited Mendelian disorders divided into two groups. The first group concerns urea cycle disorders (citrullinaemia, hyperargininaemia, argininosuccinic aciduria), ketotic hyperglycinaemia, and organic acidurias (methylmalonic aciduria, glutaric aciduria type I, etc.); the second group relates to disorders of amino acid metabolism (cystathioninuria, prolidase deficiency, etc.) and transport (Fanconi syndrome, cystinurias, Hartnup syndrome, etc.). The main goal of the Programme is to detect and prevent these genetic diseases, some detectable only in urine, before the onset of clinical symptoms. A multiplex thin-layer chromatography methodology was developed, in which metabolites in urine are resolved and visualized by the sequential application of four different reagents to detect aminoacidopathies and organic acidurias. The technique is simple, reproducible, inexpensive and rapid, allowing the analysis of 500 samples daily by a single technician. The voluntary compliance of the parents is excellent, averaging 90% per year. Over the years, we have established a dynamic process, developing techniques or new reagents to detect as many treatable disorders as possible, now evaluating macromolecules associated with lysosomal storage disorders, mainly globotriaosylceramide (Gb3) for Fabry disease. We present here the methodology, infrastructure in place, results and recent statistics of the well-established Quebec Mass Urinary Screening Programme. We also report a study by tandem mass spectrometric analysis of urinary Gb3 in Fabry disease for the follow-up and monitoring of Fabry patients, as well as for its possible application to mass and high-risk screening programmes.

Development of a filter paper method potentially applicable to mass and high-risk urinary screenings for Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting from a deficiency of the enzyme alpha-galactosidase A, and leading to the progressive accumulation of one biomarker, globotriaosylceramide (Gb(3)), predominantly elevated in the urine of these patients. We have developed a technique for the analysis of total Gb(3) in urine samples collected on filter paper, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a triple quadrupole instrument. Existing Gb(3) techniques being both time- and labour-intensive, this filter paper method eliminates lipid extraction, glycolipid isolation, centrifugation and evaporation steps, while maintaining sensitivity and efficiency. The stability of Gb(3) on filter paper was good for a 7-week period under different temperature conditions. Normal control values were established and the technique was tested with anonymized samples from Fabry hemizygotes and heterozygotes. The levels of total Gb(3) in all classical hemizygotes were well above the control values and all heterozygotes, except two nonexcretors, were above the reference level. The proposed novel filter paper method favours the collection, storage and shipment of samples. It is simple and efficient for a feasibility study, potentially applicable to the determination of total urinary Gb(3) in the newborn population as part of a screening programme, and could also be used in high-risk screening laboratories. Since the incidence of Fabry disease is hard to establish, owing to the heterogeneous clinical expression of the visible phenotype, this feasibility study could help determine its actual incidence in the Quebec population.

Binding of transcription factors creates hot spots for UV photoproducts in vivo.

Cyclobutane dipyrimidines and less than mean value of 6-4 dipyrimidines are the two major classes of mutagenic DNA photoproducts produced by UV irradiation of cells. We developed a method to map cyclobutane dipyrimidines at the DNA sequence level in mammalian cells. The frequency of this class of photoproducts was determined at every dipyrimidine along the human phosphoglycerate kinase-1 (PGK1) promoter sequence and was compared to the UV-induced frequency distribution of mean value of 6-4 dipyrimidines. After irradiation of living cells containing active or inactive PGK1 genes, enzymatic or chemical cleavage at UV photoproducts, and amplification by ligation-mediated polymerase chain reaction, photofootprints were seen in all regions which bind transcription factors and appear as DNase I footprints. Photoproduct frequency within transcription factor binding sites was suppressed or enhanced relative to inactive genes or naked DNA with enhancements of up to 30-fold. Since photoproducts are mutagenic, this indicates that photoproduct (mutation?) hot spots may be tissue specific in mammals.

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols.

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.

Ablation of p21waf1cip1 expression enhances the capacity of p53-deficient human tumor cells to repair UVB-induced DNA damage.

During periods of genotoxic stress, the cyclin-dependent kinase inhibitor p21waf1cip1 (hereafter referred to as p21) is transcriptionally up-regulated by the p53 tumor suppressor and subsequently plays a key role in cellular growth arrest. Investigations have also indicated that p21 may regulate nucleotide excision repair, a critical pathway that removes carcinogenic DNA damage induced by UV light and other mutagens. In this study, we examined whether low levels of endogenous p21 expression can modulate nucleotide excision repair in p53-deficient human tumor cells after UVB exposure. For this purpose, we used the well-characterized p53-/-p21+/+ adenocarcinoma cell strain DLD1 and its isogenic counterpart carrying a homozygous knockout for p21 (p53-/-p21-/- DLD1). Because p53-/-p21+/+ DLD1 expresses very low levels of endogenous p21 protein that are not up-regulated after mutagen exposure, this strain has been considered functionally p21-deficient in the cellular response to DNA damage. Nonetheless, the ligation-mediated PCR technology was used here to demonstrate, at nucleotide resolution, that p53-/-p21+/+ DLD1 excises UVB-induced cyclobutane pyrimidine dimers from the c-jun proto-oncogene at a significantly lower rate than the isogenic p53-/-p21-/- derivative. The higher efficiency of DNA repair in UVB-exposed p53-/-p21-/- DLD1 cells is accompanied by increased clonogenic survival and reduced levels of apoptosis, relative to the p53-/-p21+/+ counterpart. Our results show that ablation of p21 expression can significantly enhance the capacity of p53-deficient human tumor cells to repair UVB-induced DNA damage.

The multilayered organization of engineered human skin does not influence the formation of sunlight-induced cyclobutane pyrimidine dimers in cellular DNA.

Solar UVB initiates skin cancer mainly by generating highly premutagenic cyclobutane pyrimidine dimers (CPDs) and subsequent mutations in critical growth control genes. It is universally presumed that the upper epidermis in human skin blocks a significant portion of the incident UVB, thereby protecting the cancer-prone basal layer from CPD formation. Using two sensitive techniques for measuring CPD in cellular DNA, we confirmed that the multilayered organization of engineered human skin efficiently shields the basal layer against 254-nm UVC (which is not present in terrestrial sunlight) but, very unexpectedly, provides virtually no protection against environmentally relevant UVB. This underscores the importance of regular sunscreen use, which, in light of our data, may constitute a considerably more important first line of defense against photocarcinogenesis than previously believed.

Alkylating agent and chromatin structure determine sequence context-dependent formation of alkylpurines.

We determined the adduct maps of S(N)1 and S(N)2 alkylating agents in cultured human cells (in vivo) and in vitro to probe DNA-protein interactions along sequences of the promoter and exon 1 of the Fragile-X mental retardation 1 (FMR1) gene. Using ligation-mediated polymerase chain reaction (LMPCR), we compared the piperidine-sensitive alkylpurines sites generated by treating cultured cells (in vivo) and naked DNA (in vitro) with S(N)1 (N-methyl-N-nitrosourea, N-nitroso(acetoxymethyl)methylamine and 1-methyl-3-nitro-1-nitrosoguanidine) and S(N)2 alkylating agents (dimethyl sulfate (DMS), methane sulfonic acid methyl ester, iodo methane, diethyl sulfate, methane sulfonic acid ethyl ester and iodo ethane). The FMR1 promoter has four sites where DNA-protein interactions are observed. In these regions, the S(N)1 methylating agent reactions produced only hypo-reactive sites. In contrast, iodoalkane S(N)2 alkylating agents (MeI and EtI) reactions generated only hyper-reactive sites. Although there are hyper-reactive sites for the other S(N)2 reagents, the hyper-reactive site at +14 on the FMR1 map is more pronounced for the sulfate and sulfonate-derived alkylating agents than for the iodoalkanes. However, DMS modification in the presence of methyl sulfone, a compound that does not alkylate DNA, eliminates the hyper-reactive site observed at +14. This suggests that the electron-rich oxygen atoms of the sulfate and sulfonate-derived S(N)2 alkylating agent structure position the alkylating moiety to the neighboring N-7-guanine position to favor alkyl transfer to the guanine. Using KMnO(4) to probe for single-strand DNA, an unpaired cytosine base was detected at the 5'-side of the hyper- reactive guanine base at position +14, consistent with the formation of a local DNA single-strand bulge. In conclusion, we show that the sequence context-dependent formation of alkylpurines is determined by the chemical nature of the alkylating agent, the DNA sequence context, chromatin structure, and the presence of other non-reactive molecules that can inhibit alkylation.

Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA.

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify. Therefore, we use the NNK analogue 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentration-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA. Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine. In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation. Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248. We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV. These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA. The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks. These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds.

The efficacy of a broad-spectrum sunscreen to protect engineered human skin from tissue and DNA damage induced by solar ultraviolet exposure.

Sunscreens are known to protect against sunlight-induced erythema and sunburn, but their efficiency at protecting against skin cancer is still a matter of debate. Specifically, the capacity of sunscreens to prevent or reduce tissue and DNA damage has not been thoroughly investigated. The present study was undertaken to assess the ability of a chemical broad-spectrum sunscreen to protect human skin against tissue and DNA damage after solar UV radiation. Engineered human skin was generated and either treated or not with a broad-spectrum SPF 30 sunscreen and exposed to increasing doses of simulated sunlight (SSL). Immediately after irradiation, histological, immunohistochemical, and molecular quantitative analyses were performed. The unprotected irradiated engineered human skin showed significant epidermal disorganization accompanied by a complete absence of laminin deposition. The sunscreen prevented SSL-induced epidermal damage at low doses and allowed laminin deposition at almost all SSL doses tested. The frequencies of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts, and photooxidative lesions measured by alkaline gel electrophoresis and radioimmunoassay were significantly reduced by the sunscreen. Thus, tissue and DNA damage may provide excellent quantitative end points for assessing the photoprotective efficacy of sunscreens.

Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications.

The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2. The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.

Electron microscopy of gold-labeled human and equine chromosomes.

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.

Ultrastructural, immunohistochemical, and cytogenetic study of a malignant peripheral neuroectodermal tumor in a patient seropositive for human immunodeficiency virus.

A case of malignant peripheral neuroectodermal tumor occurring during the course of a human immunodeficiency virus (HIV) infection is reported. The patient was a male homosexual who presented with a rapidly enlarging tumor of the posterior lower thoracic wall. By light microscopic examination the tumor was a small cell tumor showing occasional structures suggestive of Homer-Wright rosettes. The strong positivity for neuron-specific enolase and the neurosecretory granules indicated the neural differentiation of the tumor. Its precise nature was shown cytogenetically by the presence of the t(11;22) translocation, which distinguished it from the classical neuroblastoma.

Tetrasomy 8 is associated with a major cellular proliferative advantage and a poor prognosis. two cases of myeloid hematologic disorders and review of the literature.

We report two cases of acute myeloid leukemia (AML) with tetrasomy 8 detected in patients' bone marrow samples using chromosome GTG-banding, fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) techniques. Case 1 was a myelodysplastic syndrome (MDS) in transition to AML-M4 and case 2 was an AML-M2. In case 1, the tetrasomy 8 was found in 40% of metaphase cells and constituted the only chromosome abnormality. In case 2, it was accompanied by a double Ph, trisomy 18 and disomy Y and was found in 68% of metaphase cells. However, FISH and PRINS techniques revealed the coexistence of tetrasomy 8 and trisomy 8 in interphase nuclei of both cases. When the proportion of cells with tetrasomy 8 was compared between metaphases and interphase nuclei, it showed a much higher percentage of cells with tetrasomy 8 in metaphases than in interphase nuclei. Moreover, in case 2, although multi-PRINS and FISH-PRINS techniques showed other populations of interphase nuclei with different combinations of chromosome anomalies with respect to the copy numbers for chromosomes 8, 18, Y and Ph, only cells that contained either a single Ph or tetrasomy 8 plus trisomy 18, disomy Y, and double Ph could be seen in metaphases. This strongly suggests that tetrasomy 8 confers a higher proliferative advantage to cells. Our cases also show that the tetrasomy 8 is associated with a poor prognosis.

Trisomy 8 and monosomy 7 detected in bone marrow using primed in situ labeling, fluorescence in situ hybridization, and conventional cytogenetic analyses. A study of 54 cases with hematological disorders.

Trisomy 8 and monosomy 7 are the two most frequent aneuploidies found in hematological disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, primed in situ labeling (PRINS), fluorescence in situ hybridization (FISH) and conventional cytogenetic approaches were used to investigate 54 cases of hematopoietic disorders. Of these cases, there were 22 cases of trisomy 8, 2 cases of tetrasomy 8, 14 cases of monosomy 7, and 16 cases with two copies of both chromosomes 7 and 8. PRINS was carried out in interphase nuclei of bone marrow samples using primers that can specifically detect alpha-satellite DNA sequences of chromosomes 7 and 8. In parallel, using the alpha-satellite probes for chromosomes 7 and 8, FISH was performed for all the cases. PRINS and FISH techniques showed similar specificity and sensitivity. In comparison to FISH, PRINS is more advantageous since it is a faster, easier, and more cost-effective technique. Additionally, for most of the cases, a higher proportion of aneuploidy was detected in metaphases using conventional cytogenetics, as compared to the one found in interphase nuclei identified with PRINS and FISH techniques. In other words, for these cases, the cells with trisomy 8 or monosomy 7, had a distinct proliferative advantage compared to the disomic cell population. Therefore, to better quantify the proportion of aneuploid cells in bone marrow, we recommend to investigate the frequency of aneuploidy in nuclei using PRINS, rather than studying only the dividing cells as detected by conventional cytogenetic techniques.

Detection by electron microscopy of a small subband 13q14.11 deletion in an hereditary retinoblastoma.

High-resolution banding, specific for electron microscopy, was applied to chromosomes of synchronized blood lymphocytes obtained from a child with bilateral retinoblastoma. Ultrastructural analysis of the subbands in region q14.1, after synchronization and immunochemical banding, showed that the deletion in the abnormal chromosome 13 corresponds to subband 14.11, thus evidencing that the retinoblastoma gene is located within subband q14.11. This first application to a diagnostic problem of immunochemical banding suggests that, coupled with electron microscopy, this banding provides a higher resolution than that obtained with light microscopy and should be useful to pinpoint important localizations.