Analysis of the Fragile X Trinucleotide Repeat in Basques: Association of Premutation and Intermediate Sizes, Anchoring AGGs and Linked Microsatellites with Unstable Alleles.

Fragile X Syndrome (FXS) is associated with an unstable CGG repeat sequence in the 5' untranslated region in the first exon of the FMR1 gene which resides at chromosome position Xq27.3 and is coincident with the fragile site FRAXA. The CGG sequence is polymorphic with respect to size and purity of the repeat. Interpopulation variation in the polymorphism of the FMR1 gene and consequently, in the predisposition to FXS due to the prevalence of certain unstable alleles has been observed. Spanish Basque population is distributed among narrow valleys in northeastern Spain with little migration between them until recently. This characteristic may have had an effect on allelic frequency distributions. We had previously reported preliminary data on the existence of FMR1 allele differences between two Basque valleys (Markina and Arratia). In the present work we extended the study to Uribe, Gernika, Durango, Goierri and Larraun, another five isolated valleys enclosing the whole area within the Spanish Basque region. We analyzed the prevalence of FMR1 premutated and intermediate/grey zone alleles. With the aim to complete the previous investigation about the stability of the Fragile X CGG repeat in Basque valleys, we also analyzed the existence of potentially unstable alleles, not only in relation with size and purity of CGG repeat but also in relation with DXS548 and FRAXAC1 haplotypes implicated in repeat instability. The data show that differences in allele frequencies as well as in the distribution of the mutational pathways previously identified are present among Basques. The data also suggest that compared with the analyzed Basque valleys, Gernika had increased frequency of susceptibility to instability alleles, although the prevalence of premutation and intermediate/grey zone alleles in all the analyzed valleys was lower than that reported in Caucasian populations.

Fragile X gene stability in Basque Valleys: prevalence of premutation and intermediate alleles.

Fragile X syndrome is the most common form of inherited mental retardation. The molecular basis is usually the unstable expansion of a CGG repeat in the FMR1 gene. We previously analyzed a sample of two Basque valleys. In the present work we extend the study to another five isolated valleys. The results show that differences in factors implicated in CGG repeat instability--CGG repeat size, XS548/FRAXAC1 haplotypes, and AGG interspersion pattern-are present in the Basque populations analyzed.

Detection of survivin mRNA in urine of patients with superficial urothelial cell carcinomas.

INTRODUCTION: The aim of the present study was to assess if the presence of survivin mRNA in exfoliated cells present in urine samples can be a reliable marker of the presence of bladder tumour and recurrence. MATERIALS AND METHODS: Urine samples from 30 patients with superficial urothelial cell carcinomas (UCC) were collected prior to transurethral resection (TUR) of the tumour and in the first routine follow-up, three months after TUR. Detection of survivin mRNA was performed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: No correlation was observed between survivin detection and the clinicopathological variables analysed, nevertheless, when patients were grouped into low-grade (G1) and high-grade (G2+G3) tumours, statistically significant differences were found between both groups (p=0.04). When we analysed the results of survivin detection and urinary cytology together, we observed that informative cases rose from 27.8% to 44.4%. Also, Kaplan-Meier curves for patients with negative cytology in the first followup, categorised according to survivin detection, revealed that survivin mRNA positive cases recurred earlier than negative ones. CONCLUSIONS: From our results we can conclude that detection of survivin expression can be a reliable tumour marker, but more studies are needed to clarify the potential of survivin to predict recurrences. These results showed that survivin detection in combination with conventional urinary cytology can be a useful tool to increase the sensitivity in detecting the presence of a recurrence after TUR.

Detection of survivin mRNA in urine of patients with superficial urothelial cell carcinomas

INTRODUCTION: The aim of the present study was to assess if the presence of survivin mRNA in exfoliated cells present in urine samples can be a reliable marker of the presence of bladder tumour and recurrence. MATERIALS AND METHODS: Urine samples from 30 patients with superficial urothelial cell carcinomas (UCC) were collected prior to transurethral resection (TUR) of the tumour and in the first routine follow-up, three months after TUR. Detection of survivin mRNA was performed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: No correlation was observed between survivin detection and the clinicopathological variables analysed, nevertheless, when patients were grouped into low-grade (G1) and high-grade (G2+G3) tumours, statistically significant differences were found between both groups (p=0.04). When we analysed the results of survivin detection and urinary cytology together, we observed that informative cases rose from 27.8% to 44.4%. Also, Kaplan-Meier curves for patients with negative cytology in the first followup, categorised according to survivin detection, revealed that survivin mRNA positive cases recurred earlier than negative ones. CONCLUSIONS: From our results we can conclude that detection of survivin expression can be a reliable tumour marker, but more studies are needed to clarify the potential of survivin to predict recurrences. These results showed that survivin detection in combination with conventional urinary cytology can be a useful tool to increase the sensitivity in detecting the presence of a recurrence after TUR (AU)

Near-infrared reflectance spectroscopy as a fast and non-destructive tool to predict foliar organic constituents of several woody species.

Near-infrared reflectance spectroscopy (NIRS) was used to estimate N, neutral detergent fibre (NDF), acid detergent fibre (ADF), lignin and cellulose contents in leaves of a heterogeneous group of 17 woody species from the Central Western region of the Iberian Peninsula. The sample set consisted of 182 samples of leaves of deciduous and evergreen species, showing a wide range of concentrations determined by reference methods: 6.60-35.2 g kg-1 (N), 15.5-66.0% (NDF), 10.2-57.3% (ADF), 3.45-27.4% (lignin) and 5.79-31.3% (cellulose). Reflectance spectra, obtained for samples of dried and ground leaves, were recorded as log1/R (R=reflectance) from 1,100 to 2,500 nm. NIRS calibrations were developed using multiple linear (MLR) and partial least-squares (PLSR) regressions, and tested by external validation. Spectral data were transformed to the first and second derivative (1D, 2D). The PLSR method and derivative transformations provided the best statistics and showed lower standard errors of calibration (SEC) and higher coefficients of multiple determination (R2). In the external validation the standard errors of prediction (SEP) were 0.76 g kg-1 (N), 2.11% (NDF), 1.47% (ADF), 0.85% (lignin) and 0.86% (cellulose). The results obtained show that NIRS is very effective for the estimation of these organic constituents in leaf tissue of woody species. This technique can be used in ecological or ecophysiological studies as an alternative to the more time-consuming standard methods.

Use of near-infrared reflectance spectroscopy in predicting nitrogen, phosphorus and calcium contents in heterogeneous woody plant species.

Near-infrared reflectance spectroscopy was applied to determine nitrogen (N), phosphorus (P) and calcium (Ca) content in leaf samples of 18 woody species. A total of 183 samples from mountain, riparian and dry areas from the Central-Western Iberian Peninsula were collected for this purpose. The wide intervals of variation observed in nutrient concentrations (6.6-45.0 g kg(-1) for N, 0.24-2.97 g kg(-1) for P, and 1.00-20.06 g kg(-1) for Ca) were due to the great heterogeneity of the samples. To develop calibration equations, multiple linear regression, and partial least-squares regression (PLSR) were used. In both cases, three mathematical transformations of the data were applied: log1/R and first and second derivatives. The best calibration statistics were obtained using PLSR and derivative transformations (second derivative for N and first derivative for P and Ca). The following coefficients of multiple determination (R2) and standard errors of cross validation were obtained: 0.99 and 0.93 for N, 0.94 and 0.15 for P, and 0.95 and 0.88 for Ca. In the external validation the standard errors of prediction obtained were 0.76 (N), 0.11 (P) and 0.60 (Ca).

The FMR1 CGG repeat and linked microsatellite markers in two Basque valleys.

Fragile X syndrome is associated with an unstable CGG repeat sequence in the 5' untranslated region of the first exon of the FMR1 gene. The present study involved the evaluation of factors implicated in CGG repeat stability in a normal sample from two Basque valleys (Markina and Arratia), to discover whether the Basque population shows allelic diversity and to identify factors involved, by using the data in conjunction with previous findings. The study was based on a sample of 204 and 58 X chromosomes from the Markina and Arratia valleys, respectively. The CGG repeat, the AGG interspersion and two flanking microsatellite markers, FRAXAC1 and DXS548, were examined. In the Markina valley, gray zone alleles (> or =35 CGG repeats) were associated with anchoring AGGs, with the longest 3' pure CGG repeats of the valley (=15), with the 5' instability structure 9+n and with one principal fragile X FRAXAC1-DXS548 haplotype 42-50. In the Arratia valley, gray zone alleles (> or =35 CGG repeats) showed the highest frequency among the Basque samples analyzed, and were associated with anchoring AGGs, with the longest 3' pure repeats (> or =20), with the 5' instability structure 9+n and with one "normal" FRAXAC1-DXS548 haplotype 38-40 (these data from Arratia suggest the existence of a "protective" haplotype). The results showed, on the one hand, differences between Markina and Arratia in factors implicated in CGG repeat instability and, on the other hand, a great similarity between the general Basque sample from Biscay and the Markina valley.

Expression of cell-cycle regulatory proteins and their prognostic value in superficial low-grade urothelial cell carcinoma of the bladder.

AIMS: Cell-cycle regulatory proteins are important indicators in determining progression trough the cell-cycle and progression to invasive cancer in patients presenting with superficial bladder cancer. We performed an immunohistochemical study in order to evaluate the prognostic value of the expression of p16, p27, pRb, p53 and Ki-67 in superficial grade I and II papillary urothelial cell carcinoma of the bladder. METHODS: p16, p27, p53, pRb and Ki-67 immunoexpression was studied in 14 pTa, 35 pT1a and 7 pT1b bladder tumours at presentation and at recurrence of their tumours. The recurrence-free survival and the progression-free survival were analysed according to these regulatory cell-cycle proteins expression. RESULTS: For survival in univariate analysis a high Ki-67 labelling index was a poor prognostic factor for recurrence-free and progression-free survival (P=0.0014 and P=0.012, respectively). Ki-67 labelling index was also an independent recurrence-free survival prognostic factor (P=0.0005). The p16, p27, p53 and pRb immunoreactivity was not significantly associated with recurrence or progression rate in this group of bladder carcinomas. CONCLUSIONS: These data suggest that the Ki-67 labelling index can be a reliable marker in predicting recurrence and/or progression in superficial low-grade bladder carcinomas and may be relevant in planning adjuvant therapy.

First Report of Choke Disease Caused by Epichloë baconii in the Grass Agrostis castellana.

Agrostis castellana is common in semiarid natural grasslands of the province of Salamanca, Spain. In this area, plants showing fungal stromata in their stems were observed in July of 2001. These symptoms are typical of choke disease, caused by Epichloë species in several grasses (3). In this disease, external fungal stromata develop around the leaf sheath of the flag leaf during the reproductive cycle of the plant host. As a result, the inflorescence does not emerge. In natural populations of A. castellana, less than 1% of plants showed disease symptoms, and all the stems of infected plants were sterilized by stromata. Intercellular endophytic mycelium was observed by microscopy in stem pith of diseased plants, but not on samples of 30 apparently healthy plants (1). Ergovaline, a fungal alkaloid, was not detected in lyophilized samples of infected plant tissue (2). In a fungal culture obtained from surface-disinfected leaf sheaths of a diseased plant (1), reniform conidia and conidiophores characteristic of the genus Epichloë were observed (4). To determine the fungal species, the nucleotide sequence of the ITS1-5.8SrRNA-ITS2 region and the three first introns of the beta-tubulin gene were obtained (EMBL Accession Nos. AJ490938 and AJ490939). When compared to those of other Epichloë species, these sequences identified the fungus from A. castellana as E. baconii (3). This fungus has been previously described as a pathogenic fungal endophyte in other Agrostis and Calamagrostis species (3,4). The fact that all stems of infected plants were diseased, infection incidence was low, and no alkaloids were detected in plants suggests that this grass-endophyte interaction is pathogenic and not mixed or mutualistic. References: (1) E. Clark et al. J. Microbiol. Methods 1:149, 1983. (2) N. Hill et al. Crop Sci. 33:331, 1993. (3) A. Leuchtmann et al. Mycol. Res. 102:1169, 1998. (4) J. White Jr. Mycologia 85:444, 1993.

Genetic structure of natural populations of the grass endophyte Epichloë festucae in semiarid grasslands.

Plants of red fescue (Festuca rubra), a commercially important turf grass, are infected by the fungal endophyte Epichloë festucae in semiarid natural grasslands, known as dehesas, in western Spain. We used amplified fragment length polymorphism (AFLP) markers to analyse the genetic polymorphism existing in two natural populations of Epichloë festucae. Linkage disequilibrium and the presence of clonal lineages indicated that nonrecombinant asexual reproduction predominates in both populations. However, most genetic variation detected was found to occur within populations, with only a moderate amount of genetic differentiation between populations (F(ST): 0.197). Overall, the study suggests that dehesa grasslands are useful reservoirs of Epichloë festucae endophytes, and provides information on population structure which is relevant to design sampling strategies.

Identification of the Fungal Endophyte Epichloe festucae in the Fine Fescue Festuca ampla.

Festuca ampla is native to the Iberian Peninsula (4). Endophytic mycelium was observed by microscopy (2) in stem pith samples of two of eight asymptomatic plants of F. ampla collected in one population from a natural grassland in Salamanca, Spain. The fungus could be isolated (2) only from these two plants, and conidiophores and reniform conidia typical of Epichloe species (3) were observed in pure cultures. The two infected plants maintained in pots outside, developed ectostromata in some reproductive stems (choke disease) the year after the field sampling. Six seeds collected from an infected plant were germinated, and all six seedlings were found to be infected based on microscopy (2), implying seed transmission of the endophyte. These observations suggest that this is a pleiotropic symbiont, having both mutualistic and pathogenic states in its host. An ergovaline concentration of 120 ng/g dry weight was detected in a sample of leaves and leaf sheaths of an infected plant. All of the above characteristics are typical of the genus Epichloe, and in particular of the fine fescue endophyte E. festucae (1,3). To determine the species, internal transcribed spacer and 5.8-rDNA sequences as well as a partial sequence of the ß-tubulin gene were obtained. These two sequences (EMBL Accession Nos. AJ488497 and AJ488498) showed 100% sequence homology to the corresponding sequences in E. festucae. To our knowledge, this is the first report of this endophyte species in the grass F. ampla. References: (1) L. P. Bush et al. Plant Physiol. 114:1, 1997. (2) E. M. Clark et al. J. Microbiol. Methods 1:149, 1983. (3) A. Leuchtmann et al. Mycologia 86:802, 1994. (4) I. Markgraff-Dannenberg. Festuca. Pages 125-153 in: Flora Europaea, Vol 5. Cambridge University Press, Cambridge, 1980.

Evaluation of the cytogenetic damage induced by the antihypertensive drug nimodipine in human lymphocytes.

The aim of this work was a study of the genotoxic potential of chronic long-term therapy with the antihypertensive drug nimodipine by measures of sister chromatid exchanges (SCE) and micronuclei (MN) in peripheral human lymphocytes of patients with long-term exposure to this drug. Peripheral human lymphocytes of control individuals exposed in vitro to nimodipine were also studied to assess the effect of the drug itself. Fluorescence in situ hybridization (FISH) with a centromeric probe was performed to determine the origin of the induced MN. The in vivo study was carried out on five patients under antihypertensive treatment with nimodipine. The in vitro study was performed on five control individuals by adding the drug to the culture medium at a final concentration similar to the levels found in plasma (controls/medium). The in vivo study showed no genotoxic effects of long-term therapy with nimodipine because the frequencies of SCE and MN in exposed patients did not show significant differences as compared with control individuals. A statistically significant increase in the frequency of MN was detected in controls/medium as compared with control individuals without the drug. FISH analysis revealed statistically significant differences with respect to the frequency of centromeric signals in nimodipine-induced MN in vitro. With regard to the in vivo results, chronic long-term therapy with nimodipine is not associated with increased genotoxicity. The differing results in vivo and in vitro could be due to extensive metabolism of nimodipine, indicating that the cytogenetic effect observed was due to the drug itself rather than its metabolites or to an adaptive response to nimodipine in vivo.

In vitro and in vivo evaluation of the antihypertensive drug atenolol in cultured human lymphocytes: effects of long-term therapy.

The genotoxicity of atenolol, a beta-blocker antihypertensive drug, both in vitro and in vivo, was cytogenetically tested for its ability to induce sister chromatid exchange (SCE) and micronuclei (MN) in cultured peripheral lymphocytes. Also, fluorescence in situ hybridization (FISH) with a centromeric probe was performed to determine the origin of the induced MN. The in vivo study was carried out, on the one hand, on four patients under antihypertensive treatment with atenolol and, on the other hand, on four matched control individuals taking an oral dose of atenolol. The in vitro study was performed on the control individuals by adding the drug to the culture medium at a final concentration similar to the levels found in plasma. When a comparison was made, the frequency of SCE did not show significant differences in any case. A statistically significant increase in the frequency of MN was detected in patients but not in control individuals either in vitro or in vivo. FISH analysis revealed statistically significant differences between patients and control individuals without the drug with respect to the frequency of centromeric signals in MN. Taking all these observations together, our data suggest that chronic exposure to atenolol resulted mainly in the induction of chromosome loss, so an aneugenic activity could be predicted. Different sensitivity to the compound was observed among control individuals. Nevertheless, all of them responded to the presence of atenolol in the same way in both assays. Interindividual variability was also reported. The intervariability seen in patients suggested an adaptive response to the chemical after long-term therapy.

Stability of the FMR1 CGG repeat in a Basque sample.

The fragile X syndrome is an X-chromosome-linked dominant disorder with reduced penetrance. It is the most common inherited form of mental retardation. The molecular basis is usually the unstable expansion of a CGG trinucleotide repeat in the 5' untranslated region of the first exon of the FMR1 gene, which resides at chromosome position Xq27.3 and is coincident with the cytogenetic fragile site FRAXA, which characterizes the syndrome. In the Biscay province of the Basque Country the prevalence of FRAXA in a mentally retarded sample of non-Basque origin is in the range of other analyzed Spanish populations. In the sample of Basque origin we have not found FRAXA site expression and the repeat size is in the normal range. Based on this, we have examined FMR1 gene stability in normal individuals of Basque origin from the Biscay province. This study is based on a sample of 242 X chromosomes. The results from the CGG repeat region of FMR1 indicate that a prevalence of predisposing normal alleles toward repeat instability in the Basque population is 0.00% or near to it. This could be 1 of the explanations of the apparently low fragile X syndrome incidence found in the Basque mentally retarded sample analyzed by us. This low incidence does not seem to be associated with the flanking microsatellite markers.

A survey of fragile X syndrome in a sample from Spanish Basque country.

Fragile X syndrome is the most common inherited form of mental retardation. The syndrome is associated with a CGG repeat expansion in the 5'UTR of the first exon of the FMR1 gene. This gene maps to Xq27.3 and coincides with the cytogenetic fragile site (FRAXA). The present study deals with the prevalence of fragile X syndrome among individuals with mental retardation of unknown cause from institutions and special schools from the Spanish Basque Country. Results of cytogenetic and molecular studies, performed in a group of 134 unrelated individuals (92 males and 42 females) are presented. The cytogenetic marker at Xq27.3 was identified in 12 patients. Other chromosomal abnormalities were found in two cases that this and previous studies confirmed as Angelman and Prader-Willi syndromes. Two males, in whom the cytogenetic marker was identified, were found negative for FRAXA and FRAXE expansion at the molecular level. The present study shows that the frequency of the FRAXA full mutation in individuals of Spanish non-Basque origin is in the range of other Spanish populations. In the sample of Spanish Basque origin we have not found cytogenetic FRAXA site expression, and the CGG repeat size of FMR1 gene is in the normal range. The significance of these results are discussed.

Prenatal diagnosis of partial trisomy 2q. Case report.

A case of partial trisomy 2(q21q33) detected by cordocentesis at 27 weeks' gestation in a polymalformed fetus is described. This is the second case of a prenatally detected de novo duplication of 2q and the first involving the region referred to above.

Angelman syndrome caused by loss of a marker chromosome: cytogenetic and fluorescence in situ hybridization analysis.

We report a cytogenetic and fluorescence in situ hybridization study of a family in which a female child showed all the main characteristics of Angelman syndrome. Her karyotype revealed a translocation between chromosomes 5 and 15 with a partial deletion from 15pter to the Angelman region. Several members of her family appeared to be carriers of the same translocation, but showed no symptoms. The karyotypes showed a marker chromosome, that was not present in the female with Angelman syndrome. Fluorescence in situ hybridization revealed that the marker chromosome corresponded to material from chromosome 15. The present study is in agreement with the suggestion that genomic imprinting is one of the mechanisms involved in Angelman syndrome.

Cytogenetics of autosomal fragile sites: A Basque population study.

Population cytogenetic data on autosomal fragile sites show differences among different ethnic groups. The Basques are an ancient population; their origin is not exactly known and many studies using several traits have shown peculiarities in the Basques. This is the first study about the incidence of autosomal fragile sites in a healthy Basque sample. The results show interindividual variability, no sex differences at a global level, but differences for some fragile sites. Compared with other populations, a higher incidence of rare autosomal fragile sites has been demonstrated (8%). © 1996 Wiley-Liss, Inc.

Detection of numerical alterations for chromosomes 7 and 12 in benign thyroid lesions by in situ hybridization. Histological implications.

Polysomies of chromosomes 7 and 12 have been frequently observed by conventional cytogenetics in a subgroup of thyroid follicular adenomas and in some cases of thyroid goiters. To further study possible cytogenetic similarities between these two types of thyroid lesions, we have used fluorescence in situ hybridization (FISH) to detect polysomies of chromosomes 7 and/or 12 in isolated nuclei from frozen and paraffin-embedded material of goiters and thyroid follicular adenomas and compared results with previous ones obtained by flow cytometry and conventional cytogenetics. With a set of two alpha-satellite DNA probes specific for the centromeric regions of chromosomes 7 and 12, used either separately (single-target fluorescence in situ hybridization) or simultaneously (double-target fluorescence in situ hybridization), we detected polysomies of chromosome 7 in 35.7% of the thyroid follicular adenomas and in 10.7% of the goiters. Polysomies of chromosome 12 were detected in 29.6% of the thyroid follicular adenomas and 6.7% of the goiters. The significantly higher frequency of adenomas with numerical alterations for chromosomes 7 and/or 12 supports the idea of a biological continuum and karyotypic evolution between both lesions. It is also noteworthy that polysomies of chromosomes 7 and/or 12 were observed only in lesions with an exclusive (or predominant) microfollicular histological component, as detected by enzymatic in situ hybridization on frozen sections.

Increasing levels of MYC and MET co-amplification during tumor progression of a case of gastric cancer.

The cytogenetic study of a nodal metastasis from a gastric carcinoma, after two passages in nude mice, revealed a large number of double minutes. Comparative genomic in situ hybridization (CGH) analysis using DNA extracted from this xenograft revealed the existence of three clear amplification units that originated from the chromosomal subregions 6q24-25, 7q31-32, and 8q24 in the xenograft DNA. Similar, though less prominent, CGH results were found with DNAs extracted from the primary tumor and its metastasis, implying that the same amplicons were also present, albeit less abundantly, in the DNAs of these neoplastic tissues. Southern analysis of the second-passage xenograft detected 18- and 10-fold amplification of MET (located at 7q31) and MYC (located at 8q24), respectively. The retrospective study of the first passage of the xenograft, as well as of the metastatic and primary tumors before xenografting, showed amplification levels of MET of, respectively, 12-, 9-, and 5-fold and MYC of, respectively, 8-, 7-, and 5-fold. Our results suggest that increased levels of co-amplification of MYC and MET correlate with enhanced growth potential in this case of gastric carcinoma.