Heterogeneity of a Constitutional Complex Chromosomal Rearrangement in 2q.

Complex chromosome rearrangements (CCRs) are unusual structural chromosome alterations found in humans, and to date only a few have been characterized molecularly. New mechanisms, such as chromothripsis, have been proposed to explain the presence of the CCRs in cancer cells and in patients with congenital disorders and/or mental retardation. The aim of the present study was the molecular characterization of a constitutional CCR in a girl with multiple congenital disorders and intellectual disability in order to determine the genotype-phenotype relation and to clarify whether the CCR could have been caused by chromosomal catastrophic events. The present CCR was characterized by G-banding, high-resolution CGH, multiplex ligation-dependent probe amplification and subtelomeric 2q-FISH analyses. Preliminary results indicate that the de novo CCR is unbalanced showing a 2q37.3 deletion and 2q34q37.2 partial trisomy. Our patient shows some of the typical traits and intellectual disability described in patients with 2q37 deletion and also in carriers of 2q34q37.2 partial trisomy; thus, the clinical disorders could be explained by additional effects of both chromosome alterations (deletions and duplications). A posterior, sequential FISH study using BAC probes revealed the unexpected presence of at least 17 different reorganizations affecting 2q34q37.2, suggesting the existence of chromosome instability in this region. The present CCR is the first case described in the literature of heterogeneity of unbalanced CCRs affecting a small region of 2q, indicating that the mechanisms involved in constitutional chromosome rearrangement may be more complex than previously thought.

Development and validation of multiplex real-time PCR assay for noninvasive prenatal assessment of fetal RhD status and fetal sex in maternal plasma.

OBJECTIVE: Noninvasive prenatal detection of RhD status and fetal sex is becoming part of daily practice in clinical laboratories. We evaluated a high throughput procedure for automated DNA extraction and developed a multiplex real-time PCR (rt-PCR) for the simultaneous detection of three fetal loci in a single reaction to assess fetal sex and RhD status in maternal plasma. METHODS: An automated DNA extraction method was evaluated together with a new multiplex rt-PCR assay for the simultaneous detection of exons 5 and 7 of the RHD gene together with the Y chromosome marker DYS14 in maternal plasma. The test was evaluated on 60 samples of known fetal genotype obtained from RhD-negative pregnant women before being applied prospectively on 158 consecutive clinical cases. Results were compared with newborn phenotypes. RESULTS: Automated DNA extraction allowed successful analysis of all samples. DYS14 was detected in 118 cases (male fetuses) and both RHD exon 5 and 7 were detected in 148 samples. In 70 samples neither RHD exon 5 nor RHD exon 7 were detected (RhD-negative fetuses). Absence of all three sequences (female RhD-negative fetuses) was assessed in 33 samples. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. CONCLUSION: The automated DNA extraction procedure coupled with a novel multiplex rt-PCR assay proved accurate, efficient and reliable allowing rapid and high throughput noninvasive determination of fetal sex and RhD status in clinical samples.

Persistent respiratory symptoms in clean-up workers 5 years after the Prestige oil spill.

OBJECTIVES: Fishermen who had participated in clean-up activities of the Prestige oil spill showed an excess risk of respiratory symptoms 1-2 years later, but the long-term persistence of these health effects is unclear. The aim of this study was to evaluate the persistence of these respiratory symptoms 5 years after clean-up work. METHODS: Subgroups of 501 fishermen who had been exposed to clean-up work and 177 non-exposed individuals were re-interviewed by telephone in 2008, including the same symptom questions as in the initial survey. Associations between participation in clean-up work and respiratory symptoms were assessed using log-binomial and multinomial regression analyses adjusting for sex, age and smoking. RESULTS: Information from 466 exposed (93%) and 156 non-exposed (88%) fishermen was obtained. The prevalence of lower respiratory tract symptoms (including wheeze, shortness of breath, cough and phlegm) had slightly decreased in both groups, but remained higher among the exposed (RR 1.4, 95% CI 1.1 to 1.9). The risk of having persistent respiratory symptoms (reported both at baseline and at follow-up) increased with the degree of exposure: RR ratio 1.7 (95% CI 0.9 to 3.1) and 3.3 (95% CI 1.8 to 6.2) for moderately and highly exposed, respectively, when compared with those without any symptoms. Findings for nasal symptoms and for respiratory medication usage were similar. CONCLUSIONS: Participation in clean-up activities of oil spills may result in respiratory symptoms that persist up to 5 years after exposure. Guidelines for preventive measures and a continued surveillance of clean-up workers of oil spills are necessary.

Health changes in fishermen 2 years after clean-up of the Prestige oil spill.

BACKGROUND: In 2002, the oil tanker Prestige spilled more than 67,000 tons of bunker oil, heavily contaminating the coast of northwestern Spain. OBJECTIVE: To assess respiratory effects and chromosomal damage in clean-up workers of the oil spill 2 years after the exposure. DESIGN: Cross-sectional study. SETTING: Fishermen cooperatives in coastal villages. PARTICIPANTS: Local fishermen who were highly exposed (n = 501) or not exposed (n = 177) to oil 2 years after the spill. MEASUREMENTS: Respiratory symptoms; forced spirometry; methacholine challenge; markers of oxidative stress (8-isoprostane), airway inflammation (interleukins, tumor necrosis factor-α, and interferon-γ), and growth factor activity in exhaled breath condensate; and chromosomal lesions and structural alterations in circulating lymphocytes. RESULTS: Compared with nonexposed participants, persons exposed to oil were at increased risk for lower respiratory tract symptoms (risk difference, 8.0 [95% CI, 1.1 to 14.8]). Lung function did not significantly differ between the groups. Among nonsmoking participants, exposed individuals had higher exhaled 8-isoprostane levels than nonexposed individuals (geometric mean ratio, 2.5 [CI, 1.7 to 3.7]), and exposed individuals with lower respiratory tract symptoms had higher 8-isoprostane levels than those of exposed individuals without symptoms. Exposed nonsmoking participants also had higher levels of exhaled vascular endothelial growth factor (risk difference, 44.8 [CI, 27.9 to 61.6]) and basic fibroblast growth factor (risk difference, 16.0 [CI, 3.5 to 28.6]). A higher proportion of exposed participants had structural chromosomal alterations (risk difference, 27.4 [CI, 10.0 to 44.8]), predominantly unbalanced alterations. The risk for elevated levels of exhaled 8-isoprostane, vascular endothelial growth factor, and basic fibroblast growth factor and structural chromosomal alterations seemed to increase with intensity of exposure to clean-up work. LIMITATIONS: The clinical significance of exhaled biomarkers and chromosomal findings are uncertain. The association between oil exposure and the observed changes may not be causal. The findings may not apply to spills involving other types of oil or to different populations of oil spill workers. CONCLUSION: Participation in clean-up of a major oil spill was associated with persistent respiratory symptoms, elevated markers of airway injury in breath condensate, and chromosomal damage.

Characterization of a de novo complex chromosomal rearrangement in a patient with cri-du-chat and trisomy 5p syndromes.

Two syndromes with abnormalities of the short arm of chromosome 5 have been described: cri-du-chat (resulting from 5p deletion) and trisomy 5p. We report for the first time a patient with both syndromes, resulting from a complex chromosomal rearrangement with an inverted duplication of 5p13.1-p14.2, a deletion of 5p14.2-pter, and a duplication of 5p12, characterized by array-CGH and BAC clones. The patient showed phenotypic characteristics of both syndromes and died at 3 months of age as a result of cardiorespiratory failure, probably associated with the clinical severity of the trisomy 5p syndrome. We propose a potential causative mechanism for this rearrangement.

Intrachromosomal partial triplication of chromosome 13 secondary to a paternal duplication with mild phenotypic effect.

Intrachromosomal triplications are rare and can be mistaken for duplications. The majority of triplications reported are de novo, mostly involving chromosome 15q, and have a middle inverted repeat. We report on the clinical, cytogenetic, and molecular analyses of a patient with a novel triplication 13q21.1-q21.33 secondary to a familial duplication 13q21.1-q21.33 with mild phenotypic effect in three generations. The propositus was an 8-year-old boy referred because of language delay and mild mental retardation. His weight, height and OFC were above the 97th centile. He had delayed tooth eruption and subtle dysmorphic features. Chromosome analysis (550 band stage) showed extra material in 13q21. Family history was unremarkable except for adult-onset sensorineural hearing loss in the father and paternal grandfather. Their karyotypes and those of both brothers of the propositus also showed an abnormal chromosome 13 but with less extra genetic material. FISH analysis with several BAC clones showed a triplication in the propositus between 204N9 and 184B18 (which mapped to 13q21.1 and 13q21.33, respectively) and a direct duplication for the same fragment (around 12 Mb) in the rest of the family members with the abnormal chromosome 13. The FISH signals did not show a middle inverted repeat. We describe the first intrachromosomal triplication 13q21.1-q21.33 derived from a paternal duplication. Meiotic instability in the transmission of a duplication has not been previously observed. Phenotypic variability may be explained by chromosomal non-penetrance or dosage critical loci located in the triplicate/duplicate segment.

Thirty-two new cases with small supernumerary marker chromosomes detected in connection with fertility problems: detailed molecular cytogenetic characterization and review of the literature.

Thirty-two patients with fertility problems were identified as carriers of small supernumerary marker chromosomes (sSMC). Molecular cytogenetic techniques were used to characterize their chromosomal origin. Together with the other cases available in the literature 111 sSMC cases have now been detected in connection with fertility problems in otherwise clinically healthy persons and characterized for their genetic content. According to this study, in 60% of the cases the sSMC originated from chromosomes 14 or 15. Euchromatic imbalances were caused by the sSMC presence in 30% of the cases. Notably, in 53% of infertile sSMC carriers, the sSMC was parentally transmitted. As we found indications of an as yet unknown mechanism for the elimination of sSMC from the human gene pool, sSMC could also play a role in elucidating the process of chromosome gain and loss during evolution. Nonetheless, further detailed molecular analysis will be necessary in the future to characterize the mechanisms and genetic basis for this phenomenon.

Rapid prenatal diagnosis by QF-PCR: evaluation of 30,000 consecutive clinical samples and future applications.

Rapid prenatal diagnoses of major chromosome abnormalities can be performed on a large scale using highly polymorphic short tandem repeats (STRs) amplified by the quantitative fluorescent polymerase chain reaction (QF-PCR). The assay was introduced as a preliminary investigation to remove the anxiety of the parents waiting for the results by conventional cytogenetic analysis using amniotic fluid or chorionic cells. However, recent studies, on the basis of the analyses of several thousand samples, have shown that this rapid approach has a very high rate of success and could reduce the need for cytogenetic investigations. Its high efficiency, for example, allows early interruption of affected fetuses without the need of waiting for completion of fetal karyotype. The main advantages of the QF-PCR are its accuracy, speed, automation, and low cost that allows very large number of samples to be analyzed by few operators. Here, we report the results of using QF-PCR in a large series of consecutive clinical cases and discuss the possibility that, in a near future, it may even replace conventional cytogenetic analyses on selected samples.

Persistence of Breakage in Specific Chromosome Bands 6 Years after Acute Exposure to Oil.

BACKGROUND: The identification of breakpoints involved in chromosomal damage could help to detect genes involved in genetic disorders, most notably cancer. Until now, only one published study, carried out by our group, has identified chromosome bands affected by exposure to oil from an oil spill. In that study, which was performed two years after the initial oil exposure in individuals who had participated in clean-up tasks following the wreck of the Prestige, three chromosomal bands (2q21, 3q27, 5q31) were found to be especially prone to breakage. A recent follow-up study, performed on the same individuals, revealed that the genotoxic damage had persisted six years after oil exposure. OBJECTIVES: To determine whether there exist chromosome bands which are especially prone to breakages and to know if there is some correlation with those detected in the previous study. In addition, to investigate if the DNA repair problems detected previously persist in the present study. DESIGN: Follow-up study performed six years after the Prestige oil spill. SETTING: Fishermen cooperatives in coastal villages. PARTICIPANTS: Fishermen highly exposed to oil spill who participated in previous genotoxic study six years after the oil. MEASUREMENTS: Chromosome damage in peripheral lymphocytes. For accurate identification of the breakpoints involved in chromosome damage of circulating lymphocytes, a sequential stain/G-banding technique was employed. To determine the most break-prone chromosome bands, two statistical methods, the Fragile Site Multinomial and the chi-square tests (where the bands were corrected by their length) were used. To compare the chromosome lesions, structural chromosome alterations and gaps/breaks between two groups of individuals we used the GEE test which takes into account a possible within-individual correlation. Dysfunctions in DNA repair mechanisms, expressed as chromosome damage, were assessed in cultures with aphidicolin by the GEE test. RESULTS: Cytogenetic analyses were performed in 47 exposed individuals. A total of 251 breakpoints in exposed individuals) were identified, showing a non-uniform distribution in the human ideogram. Ten chromosome bands were found to be especially prone to breakage through both statistical methods. By comparing these bands with those observed in certain exposed individuals who had already participated the previous study, it was found in both studies that four bands (2q21, 3q27, 5q31 and 17p11.2) are particularly sensitive to breakage. Additionally, the dysfunction in DNA repair mechanisms was not significantly higher in oil-exposed individuals than in non-exposed individuals. LIMITATIONS: The sample size and the possibility of some kind of selection bias should be considered. Genotoxic results cannot be extrapolated to the high number of individuals who participated occasionally in clean-up tasks. CONCLUSION: Our findings show the existence of at least four target bands (2q21, 3q27, 5q31 and 17p11.2) with a greater propensity to break over time after an acute exposure to oil. The breaks in these bands, which are commonly involved in hematological cancer, may explain the increase of cancer risk reported in chronically benzene-exposed individuals. In addition, a more efficiency of the DNA repair mechanisms has been detected six years after in fishermen who were highly exposed to the oil spill. To date, only this study, performed by our group on the previous and present genotoxic effects, has analyzed the chromosomal regions affected by breakage after an acute oil exposure.

Chromosomal instability in amniocytes from fetuses of mothers who smoke.

CONTEXT: Tobacco increases the risk of systemic diseases, and it has adverse effects on pregnancy. However, only indirect data have been published on a possible genotoxic effect on pregnancy in humans. OBJECTIVES: To determine whether maternal smoking has a genotoxic effect on amniotic cells, expressed as an increased chromosomal instability, and to analyze whether any chromosomal regions are especially affected by exposure to tobacco. DESIGN, SETTING, AND PATIENTS: In this prospective study, amniocytes were obtained by routine amniocentesis for prenatal diagnosis from 25 controls and 25 women who smoke (> or =10 cigarettes/d for > or =10 years), who were asked to fill out a smoking questionnaire concerning their smoking habits. Chromosomal instability was analyzed in blinded fashion by 2 independent observers in routine chromosome spreads. Breakpoints implicated in chromosomal abnormalities were identified by G-banding. MAIN OUTCOME MEASURES: Association between maternal smoking and increased chromosomal instability in amniotic fluid cells, expressed as chromosomal lesions (gaps and breaks) and structural chromosomal abnormalities. RESULTS: Comparison of cytogenetic data between smokers and nonsmokers (controls) showed important differences for the proportion of structural chromosomal abnormalities (smokers: 12.1% [96/793]; controls: 3.5% [26/752]; P = .002) and to a lesser degree for the proportion of metaphases with chromosomal instability (smokers: 10.5% [262/2492]; controls: 8.0% [210/2637]; P = .04), and for the proportion of chromosomal lesions (smokers: 15.7% [391/2492]; controls: 10.1% [267/2637]; P = .045). Statistical analysis of the 689 breakpoints detected showed that band 11q23, which is a band commonly implicated in hematopoietic malignancies, was the chromosomal region most affected by tobacco. CONCLUSIONS: Our findings show that smoking 10 or more cigarettes per day for at least 10 years and during pregnancy is associated with increased chromosomal instability in amniocytes. Band 11q23, known to be involved in leukemogenesis, seems especially sensitive to genotoxic compounds contained in tobacco.

Follow-Up Genotoxic Study: Chromosome Damage Two and Six Years after Exposure to the Prestige Oil Spill.

BACKGROUND: The north-west coast of Spain was heavily contaminated by the Prestige oil spill, in 2002. Individuals who participated in the clean-up tasks showed increased chromosome damage two years after exposure. Long-term clinical implications of chromosome damage are still unknown. OBJECTIVE: To realize a follow-up genotoxic study to detect whether the chromosome damage persisted six years after exposure to the oil. DESIGN: Follow-up study. SETTING: Fishermen cooperatives in coastal villages. PARTICIPANTS: Local fishermen who were highly exposed (n = 52) and non-exposed (n = 23) to oil seven years after the spill. MEASUREMENTS: Chromosome damage in circulating lymphocytes. RESULTS: Chromosome damage in exposed individuals persists six years after oil exposure, with a similar incidence than those previously detected four years before. A surprising increase in chromosome damage in non-exposed individual was found six years after Prestige spill vs. those detected two years after the exposure. LIMITATIONS: The sample size and the possibility of some kind of selection bias should be considered. Genotoxic results cannot be extrapolated to the approximately 300,000 individuals who participated occasionally in clean-up tasks. CONCLUSION: The persistence of chromosome damage detected in exposed individuals six years after oil exposure seems to indicate that the cells of the bone marrow are affected. A surprising increase in chromosome damage in non-exposed individuals detected in the follow-up study suggests an indirect exposition of these individuals to some oil compounds or to other toxic agents during the last four years. More long-term studies are needed to confirm the presence of chromosome damage in exposed and non-exposed fishermen due to the association between increased chromosomal damage and increased risk of cancer. Understanding and detecting chromosome damage is important for detecting cancer in its early stages. The present work is the first follow-up cytogenetic study carried out in lymphocytes to determine genotoxic damage evolution between two and six years after oil exposure in same individuals.

Analysis of kidney tumors by comparative genomic hybridization and conventional cytogenetics.

Comparative genomic hybridization (CGH) and conventional cytogenetic karyotyping were used to screen for losses and gains of DNA sequences along chromosomes in ten renal tumors (RCC) of different histologic types (clear-cell RCC, papillary RCC, and one oncocytoma). Loss of 3p was the most common change in clear-cell RCC. All papillary tumors, either adenomas or carcinomas revealed gains of chromosomes 7 and 17q without limitation to size and grade. Homozygotic loss of the pseudoautosomal Xp or Yp region was detected in three RCC tumors. A dicentric (Y;14) was present as the sole chromosome abnormality in the oncocytoma. Both techniques showed concordant results in tumors with homogeneous karyotype. However, in tumors with several composite clones some discrepancies were observed, especially in cases of clear-cell RCC where chromosomal abnormalities present in a low number of metaphases could not be detected by CGH.

A comparative genomic hybridization study in a 46,XX male.

OBJECTIVE: To identify Y chromosome material in an azoospermic male with an XX karyotype. DESIGN: Case report. SETTING: Faculty of medicine and Centro de Patologia Celular (CPC) medical center. PATIENT(S): A 33-year-old man with infertility. INTERVENTION(S): G-banding, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and comparative genomic hybridization (CGH). MAIN OUTCOME MEASURE(S): FISH for X and Y chromosomes, PCR for the SRYgene and amelogenin gene in the Xp (AMGX) and (AMGY), and losses or gains with CGH. RESULT(S): FISH analysis using X and Y chromosome-specific probes showed an X chromosome containing Y chromosome sequences on the top of the short arm; this Y chromosome region was not visible by conventional cytogenetic analysis. PCR amplification of DNA showed the presence of the sex-determining region of the Y chromosome (SRY) and the amelogenin gene in the pseudoautosomal boundary of the X chromosome (AMGX). CGH confirmed the presence of the chromosome region Yp11.2-pter and detected the presence of the two otherwise normal X chromosomes. CONCLUSION(S): The two Xpter (XPAR1) pseudoautosomal regions present in this XX male suggest the need to reevaluate XX males using CGH and PCR to characterize the clinical variability in XX males due to genes other than those located on the Y chromosome.

Primary amenorrhea in a woman with a cryptic complex chromosome rearrangement involving the critical regions Xp11.2 and Xq24.

OBJECTIVE: To characterize a complex chromosome rearrangement (CCR) previously detected by G-banding in peripheral blood lymphocytes, as 46,X,-2,-11,-22,-X,+mar 1+mar2+mar3+mar4 in a patient with primary amenorrhea. DESIGN: Case report. SETTING: University faculty of Medicine and hospital. PATIENT(S): A 36-year-old woman with primary amenorrhea. INTERVENTION(S): Fluorescence in situ hybridization (FISH). MAIN OUTCOME MEASURE(S): Use of commercially available M-FISH probe (24 colors simultaneously) and whole chromosome painting probes for chromosomes 2, 11, 22, and X to characterize the CCR. RESULT(S): The use of conventional and multiple FISH allowed the redefinition of the CCR, showing a cryptic insertion of chromosome 11 in marker 3 previously suspected by M-FISH. The combination of G-banding and FISH data revealed that four chromosomes and seven breakpoints, including 2q21, 2q31, 11q22.1, 11q22.3, 22q13.3, Xp11.21, and Xq24, were implicated in this CCR. CONCLUSION(S): This report confirms the importance of a combination of G-banding and FISH (M-FISH and conventional FISH) techniques to characterize the de novo CCR. These techniques also were useful in defining two possible critical chromosome regions, Xp11.21 and Xq24, in which genes of potential interest for a primary amenorrhea could be located.

Fast-proliferating adipose tissue mesenchymal-stromal-like cells for therapy.

Human mesenchymal stromal cells, whether from the bone marrow or adipose tissue (hASCs), are promising cell therapy agents. However, generation of abundant cells for therapy remains to be a challenge, due to the need of lengthy expansion and the risk of accumulating genomic defects during the process. We show that hASCs can be easily induced to a reversible fast-proliferating phenotype (FP-ASCs) that allows rapid generation of a clinically useful quantity of cells in <2 weeks of culture. Expanded FP-ASCs retain their finite expansion capacity and pluripotent properties. Despite the high proliferation rate, FP-ASCs show genomic stability by array-comparative genomic hybridization, and did not generate tumors when implanted for a long time in an SCID mouse model. Comparative analysis of gene expression patterns revealed a set of genes that can be used to characterize FP-ASCs and distinguish them from hASCs. As potential candidate therapeutic agents, FP-ASCs displayed high vasculogenic capacity in Matrigel assays. Moreover, application of hASCs and FP-ASCs in a fibrin scaffold over a myocardium infarct model in SCID mice showed that both cell types can differentiate to endothelial and myocardium lineages, although FP-ASCs were more potent angiogenesis inducers than hASCs, at promoting myocardium revascularization.

Evaluation of the persistence of functional and biological respiratory health effects in clean-up workers 6 years after the prestige oil spill.

Fishermen who had participated in clean-up activities of the Prestige oil spill showed increased bronchial responsiveness and higher levels of respiratory biomarkers 2 years later. We aimed to evaluate the persistence of these functional and biological respiratory health effects 6 years after clean-up work. In 2008/2009 a follow-up study was done in 230 never-smoking fishermen who had been exposed to clean-up work in 2002/2003 and 87 non-exposed fishermen. Lung function and bronchial responsiveness testing and the determination of respiratory biomarkers in exhaled breath condensate were done identically as in the baseline survey in 2004/2005. Associations between participation in clean-up work and respiratory health parameters were assessed using linear and logistic regression analyses adjusting for sex and age. Information from 158 exposed (69%) and 57 non-exposed (66%) fishermen was obtained. Loss to follow-up in the non-exposed was characterised by less respiratory symptoms at baseline. During the 4-year follow-up period lung function, bronchial hyperresponsiveness and the levels of respiratory biomarkers of oxidative stress and growth factors had deteriorated notably more among non-exposed than among exposed. At follow-up, respiratory health indices were similar or better in clean-up workers than in non-exposed. No clear differences between highly exposed and moderately exposed clean-up workers were found. In conclusion, we could not detect long-term respiratory health effects in clean-up workers 6 years after the Prestige oil spill. Methodological issues that need to be considered in this type of studies include the choice of a non-exposed control group and limitation of follow-up to subgroups such as never smokers.

Evaluation of sample stability and automated DNA extraction for fetal sex determination using cell-free fetal DNA in maternal plasma.

OBJECTIVE: The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. METHODS: A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. RESULTS: Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. CONCLUSION: We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

Chromosomal bands affected by acute oil exposure and DNA repair errors.

BACKGROUND: In a previous study, we showed that individuals who had participated in oil clean-up tasks after the wreckage of the Prestige presented an increase of structural chromosomal alterations two years after the acute exposure had occurred. Other studies have also reported the presence of DNA damage during acute oil exposure, but little is known about the long term persistence of chromosomal alterations, which can be considered as a marker of cancer risk. OBJECTIVES: We analyzed whether the breakpoints involved in chromosomal damage can help to assess the risk of cancer as well as to investigate their possible association with DNA repair efficiency. METHODS: Cytogenetic analyses were carried out on the same individuals of our previous study and DNA repair errors were assessed in cultures with aphidicolin. RESULTS: Three chromosomal bands, 2q21, 3q27 and 5q31, were most affected by acute oil exposure. The dysfunction in DNA repair mechanisms, expressed as chromosomal damage, was significantly higher in exposed-oil participants than in those not exposed (p= 0.016). CONCLUSION: The present study shows that breaks in 2q21, 3q27 and 5q31 chromosomal bands, which are commonly involved in hematological cancer, could be considered useful genotoxic oil biomarkers. Moreover, breakages in these bands could induce chromosomal instability, which can explain the increased risk of cancer (leukemia and lymphomas) reported in chronically benzene-exposed individuals. In addition, it has been determined that the individuals who participated in clean-up of the oil spill presented an alteration of their DNA repair mechanisms two years after exposure.

Phylloid hypomelanosis and mosaic partial trisomy 13: two cases that provide further evidence of a distinct clinicogenetic entity.

BACKGROUND: Phylloid hypomelanosis is a rare neurocutaneous syndrome characterized by a pattern of hypopigmentation consisting of leaflike or oblong macules reminiscent of floral ornaments. Associated extracutaneous anomalies include cerebral, ocular, and skeletal defects. Recently it has been suggested that this phenotype originates from mosaic partial or complete trisomy 13. We report clinical and cytogenetic data for 2 cases. OBSERVATIONS: A bizarre pattern of multiple leaflike macules was noted in 2 girls with mental deficiency. In patient 1, additional anomalies included syndactyly, clinodactyly, trichomegaly of the eyelashes, low frontal hairline, and several pale pink telangiectatic macules. In patient 2, epileptic seizures, dental malposition, oligodontia, preauricular fistulas, scoliosis, tethered cord, and syringomyelia were noted. A diagnosis of phylloid hypomelanosis was made in both patients. In both patients, blood lymphocytes showed a normal karyotype 46,XX; however, fibroblasts derived from lesional skin demonstrated tetrasomy of chromosome 13q21-qter in patient 1 and trisomy of 13q22-qter in patient 2. CONCLUSIONS: These 2 cases lend further support to the concept that phylloid hypomelanosis is a distinct clinicogenetic entity that should no longer be confused with pigmentary mosaicism of the Ito type. From a comparison of our cytogenetic findings with those documented in previous articles, we infer that phylloid hypomelanosis is most likely related to the 13q region.

Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR, results of 9 years of clinical experience.

BACKGROUND: Despite being deliberately targeted to common chromosome aneuploidies, the rapid quantitative fluorescent polymerase chain reaction (QF-PCR) tests can detect the majority of chromosome abnormalities in prenatal diagnosis. The main advantages of this assay are low cost, speed and automation allowing large-scale application. METHODS: We developed a QF-PCR test that was applied on 43 000 clinical samples reporting results in 24 h. Most common indications were biochemical risk (32%) and advanced maternal age (30%). Samples were also tested by cytogenetic analysis and the results compared. RESULTS: Aneuploidies involving chromosomes 21, 18, 13, X and Y were detected with 100% specificity. Several cases of partial trisomies and mosaicism were also identified. Overall 95% of clinically relevant abnormalities were readily detected and termination of affected pregnancies could be performed without waiting for the cytogenetic results. CONCLUSIONS: Our study supports the possibility of reducing the load of prenatal cytogenetic tests if the pregnancies are carefully monitored by non-invasive screening. In case of abnormal QF-PCR results, medical action can be taken within few hours from sampling. In cases of negative QF-PCR results, cytogenetic analyses might only be performed for fetuses with ultrasound abnormalities. In countries where large-scale cytogenetic tests are not available, QF-PCR may be used as the only prenatal diagnostic procedure.