Altered expression of key cell cycle regulators in renal cell carcinoma associated with Xp11.2 translocation.

Renal cell carcinoma (RCC) is a rare tumor in the pediatric population. Recently, a phenotypically and genetically distinct kidney carcinoma, mainly prevalent in children and associated with an Xp11.2 translocation or TFE3 gene fusion, has been described. It has been advanced that in this subtype of RCC, there is an accumulation of cyclin D1, cyclin D3, and p21 ((wafl/cip1)). The aim of the present study was to figure out in two pediatric RCC recently diagnosed in our department (one clear cell-type RCC and one TFE3-positive RCC) whether those features are indeed specific of the latter tumor or occur in pediatric RCC irrespective of the tumor type. The following immunostains were performed in both cases: Ki67, p16(ink4a), p21 ((wafl/cip1)), p27(kip1), p53, p63, mdm2, cyclin D1, cyclin D3, TFE3, CD10, vimentin, E-cadherin, and RCC-antigen. We observed in the TFE3-positive carcinoma an intense immunoreaction for p21 ((wafl/cip1)), cyclin D1, and cyclin D3, without expression for p53, p16, p27(kip1), and mdm2, whereas the immunoexpression profile observed in the classic RCC was similar to that of clear cell, adult-type RCC. Our study confirms that TFE3-positive RCC exhibits a deregulation of the cell cycle apparently unrelated to the young age of the patients.

MSH6 germline mutations in early-onset colorectal cancer patients without family history of the disease.

Germline MLH1 and MSH2 mutations are scarce in young colorectal cancer patients with negative family history of the disease. To evaluate the contribution of germline MSH6 mutations to early-onset colorectal cancer, we have analysed peripheral blood of 38 patients diagnosed with this disease before 45 years of age and who presented no family history of hereditary nonpolyposis colorectal cancer-related cancers. Blood samples from 108 healthy volunteers were analysed for those genetic alterations suspected to affect the function of MSH6. Of the seven (18.4%) MSH6 alterations found, we have identified three novel germline mutations, one 8 bp deletion leading to a truncated protein and two missense mutations resulting in the substitution of amino acids belonging to different polarity groups. High-frequency microsatellite instability was found in the patient with the MSH6 deletion, but not in the other 27 carcinomas analysed. No MLH1 promoter methylation was detected in tumour tissue. Our findings suggest that germline MSH6 mutations contribute to a subset of early-onset colorectal cancer. Further studies are warranted to understand the genetic and environmental factors responsible for the variable penetration of MSH6 germline mutations, as well as to identify other causes of early-onset colorectal cancer.

Cleft lip/palate and CDH1/E-cadherin mutations in families with hereditary diffuse gastric cancer.

We report the association of CDH1/E-cadherin mutations with cleft lip, with or without cleft palate (CLP), in two families with hereditary diffuse gastric cancer (HDGC). In each family, the CDH1 mutation was a splicing mutation generating aberrant transcripts with an in-frame deletion, removing the extracellular cadherin repeat domains involved in cell-cell adhesion. Such transcripts might encode mutant proteins with trans-dominant negative effects. We found that CDH1 is highly expressed at 4 and 5 weeks in the frontonasal prominence, and at 6 weeks in the lateral and medial nasal prominences of human embryos, and is therefore expressed during the critical stages of lip and palate development. These findings suggest that alteration of the E-cadherin pathway can contribute to human clefting.

Pathological exon skipping in an HNPCC proband with MLH1 splice acceptor site mutation.

One of the most commonly mutated mismatch repair genes in human nonpolyposis colorectal cancer (HNPCC) is MLH1. We identified a splice site mutation in MLH1 in a colorectal cancer proband (T-to-A at position -11 of intron 1 splice acceptor) and investigated its functional consequences by RT-PCR, using lymphocyte mRNA from the proband, two noncarrying siblings, and one unrelated individual. Subcloning of PCR products followed by sequencing of individual clones revealed increased transcript heterogeneity in the mutation carrier, attributable to the presence of a variety of mRNA forms lacking exon 2, or combinations of exons 2, 4, 6, 9, and 10. The full-length transcript subcloned from the mutation carrier was detected with a much reduced frequency, suggesting that only the wild-type allele produced functional MLH1 mRNA. The three noncarriers expressed some previously described transcripts and several novel variants, but none that lacked exon 2. The results are consistent with the hypothesis that this splice site mutation causes skipping of MLH1 exon 2 in a large proportion of mRNA transcripts derived from the mutated allele. Such an observation strengthens the case for identifying the mutation as pathogenic in this HNPCC family, which is of interest given the rarity of exon skipping defects resulting from splice acceptor site mutations outside the invariant AG dinucleotide.

Eleven novel APC mutations identified in Portuguese FAP families.

Germline mutations of the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to colorectal cancer. In the present study we screened all of the exons of the APC gene in individuals belonging to 85 Portuguese FAP families. We here report eleven novel mutations which are predominantly frameshifts or single base substitutions, resulting in premature stop codons. Hum Mutat 16:178, 2000.

Detection of prognostic significant translocations in childhood acute lymphoblastic leukaemia by one-step multiplex reverse transcription polymerase chain reaction.

The search for chromosomal translocations in de novo cases of childhood acute lymphoblastic leukaemia (ALL) is crucial for the selection of the appropriate therapeutic protocol. In this work, we describe a new method - one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) - to screen for prognostic significant translocations in childhood ALL. Our approach involves a single PCR reaction for the simultaneous detection of the molecular rearrangements resulting from the t(9;22), t(12;21), t(4;11) and t(1;19), with a turnaround time of less than 24 h. This assay proved to be highly sensitive, specific, reproducible and easy to implement in a routine genetics laboratory.

Molecular cytogenetic characterization of a small, familial supernumerary ring chromosome 7 associated with mental retardation and an abnormal phenotype.

A family is described in which a mother and two of her children were mosaic for a small supernumerary ring chromosome. As the origin of the ring chromosome could not be determined by routine cytogenetic studies, fluorescent in situ hybridization was performed, which indicated that the ring chromosome was derived from the pericentromeric region of chromosome 7. Further characterization with a YAC-probe showed the involvement of the proximal q-arm of chromosome 7. Both sibs had speech difficulties and were mildly mentally retarded whereas the mother's intelligence was at the lower end of the normal range. They all had an unusual face, characterized by a flat profile, short forehead, downslant of the palpebral fissures, high and broad nasal bridge, simply formed ears, and prognathia. This is the second report of a small supernumerary ring chromosome derived from the pericentromeric region of chromosome 7, and the described clinical phenotype differs from that delineated in the previous report.

Four novel MSH2 / MLH1 gene mutations in portuguese HNPCC families.

Hereditary non-polyposis colorectal cancer (HNPCC) is considered to be determined by germline mutations in the mismatch repair (MMR) genes, especially MSH2 and MLH1. While screening for mutations in these two genes in HNPCC portuguese families, 3 previously unreported MSH2 and 1 MLH1 mutations have been identified in families meeting strict Amsterdam criteria. Hum Mutat 15:116, 2000.

Significance of trisomy 7 and 12 in thyroid lesions with follicular differentiation: a cytogenetic and in situ hybridization study.

In a group of benign and malignant follicular thyroid lesions, previously analyzed by conventional cytogenetics, single- and double-target fluorescence in situ hybridization studies with pericentromeric probes specific for chromosomes 7 and 12 were performed, by using isolated nuclei from paraffin-embedded specimens of: 11 goiters, 21 adenomas, 9 follicular carcinomas, and adjacent normal thyroid tissue. Nonisotopic in situ hybridization with the same probes was used in 4-microm histologic sections of four follicular carcinomas. By fluorescence in situ hybridization analysis, the percentage of goiters, adenomas, and follicular carcinomas with gains of No. 7 was 18.2%, 52.4%, and 66.0%, respectively, and with gains of No. 12 was 9.0%, 42.9%, and 66.0%, respectively. The percentage of the same lesions (goiters, adenomas, carcinomas) exhibiting polysomies of No. 7 and No. 12, as assessed by cytogenetic analysis, was 5.0% and 0.0%, 20.0% and 20.0%, and 15.8% and 10.5%, respectively. Numerical alterations of these chromosomes were not observed in normal tissue. These findings reveal that gain of chromosomes 7 and 12 is a characteristic of the morphologically altered thyroid tissue; polysomies of chromosomes 7 and 12 are more frequent in thyroid lesions than it can be detected by conventional cytogenetic studies; the increasing frequency of polysomies of chromosomes 7 and 12 from hyperplastic lesions to benign and malignant tumors seem to substantiate the existence of a multistep pathway, ie, normal thyroid --> goiter --> adenoma --> follicular carcinoma in the pathogenesis of some thyroid neoplasms.

Analysis of FMR1 and flanking microsatellite markers in normal and fragile X chromosomes in Portugal: evidence for a "protector" haplotype.

In order to look for linkage disequilibrium between the fragile X locus and its flanking markers, we analysed the FRAXAC1 and DXS548 microsatellites in normal and fragile X individuals of Portuguese origin. We observed differences in allele and haplotype frequencies between these two samples. Four haplotypes (A-2, C-2, C-5 and D-6) accounted for 76% of all fragile X chromosomes, whereas a single haplotype (C-7) accounted for 70% of the normal population and less than 3% of the fragile X chromosomes. Among the four observed high-risk haplotypes, A-2 and D-6 had been previously reported in other studies, but C-2 and C-5 seem characteristic of Portuguese patients, as suggested by the high frequency (38%) in fragile X chromosomes and virtual absence in controls. In accordance with previous studies, a greater heterozygosity of the fragile X sample was noted when compared to that of controls. The high frequency of C-7 haplotype in the normal population and its virtual absence in the fragile X sample may reflect the existence of linkage disequilibrium between the two loci and/or selective advantage (protector effect) of this haplotype.

Three cases of mosaicism for balanced reciprocal translocations.

Mosaicism for a balanced reciprocal translocation (BRTM) is rare. As far as we know only 26 cases of BRTM, demonstrated in lymphocyte cultures, have been described, five of which had an abnormal phenotype. Prenatally three confirmed cases with a normal phenotypic outcome have been described. Here we present three further cases of BRTM in lymphocyte cultures. The first was detected during a family study, the second after an abnormal karyotype in chorionic villus sampling, and the third because of a history of stillborn children. All three carriers have normal phenotypes. An inventory of the BRTM cases reported so far is made.

Cytogenetic and fluorescence in situ hybridization studies in a case of anaplastic thyroid carcinoma.

Cytogenetic analysis of a case of anaplastic thyroid carcinoma revealed multiple numerical and structural chromosomal changes, including a der(9) add(9)(p22)hsr(9)(p?). Fluorescence in situ hybridization (FISH) studies performed to identify the genetic nature of this derivative chromosome showed that both the additional material and the hsr region were composed of only chromosome 9 sequences and that the C-ABL oncogene was not one of the genes harbored at the hsr region. We suggest that amplification of gene(s) located at chromosome 9, other than the C-ABL, may play a significant role in anaplastic evolution of thyroid carcinomas.

Follicular thyroid carcinoma: chromosome analysis of 19 cases.

Short-term cultures of 19 follicular thyroid carcinomas were examined cytogenetically. Clonal chromosomal changes were detected in 12 tumors. Two follicular carcinomas had only numerical alterations: one with a hyperdiploid karyotype with trisomies/polysomies of chromosomes 7 and 12, similar to the karyotypes previously identified in a sub-group of benign thyroid lesions, and the other with monosomy 20. In the remaining ten cases several structural chromosome anomalies were found. Loss of the short arm of chromosome 3 was observed in one tumor. In two widely invasive and metastasizing follicular carcinomas there was a t(7;8)(p15;q24) as the sole abnormality in one case and a der(8)t(7;8)(p15;q24) together with other cytogenetic alterations in the other case. This finding suggests that t(7;8)(p15;q24) may be related to an aggressive behavior of follicular thyroid carcinomas.

Cytogenetic investigations of 340 thyroid hyperplasias and adenomas revealing correlations between cytogenetic findings and histology.

Cytogenetic analyses were performed on 340 follicular thyroid adenomas and goiters after short-term culture. Clonal chromosomal changes were found in 67 cases. Trisomy 7 as the sole abnormality or along with other trisomies was the most frequent type of aberration (19 cases). Other recurrent numerical changes were loss of chromosome 22 (4 cases) and the second X or the Y chromosome (5 cases). Translocations involving 19q13 (12 cases) were frequent structural chromosomal changes. Dicentric chromosomes or telomeric associations were frequent in goiters (12 cases). After a histopathologic classification of all cases, we have correlated the cytogenetic findings with the histology of the tumors. Only 8.4% of the goiters showed clonal abnormalities, whereas 44.9% of the adenomas revealed clonal abnormalities. Furthermore, simple clonal changes were predominantly found in goiters and complex changes in adenomas. The most impressive correlation was found in the group of lesions with trisomy 7. Although all but one lesion with one or two additional trisomies were goiters, those having three or more additional trisomies were all adenomas or adenomatous goiters.

Increased fetal nuchal translucency: possible involvement of early cardiac failure.

The ultrasonographic measurement of nuchal translucency thickness at 10-13 weeks of gestation is accepted as an efficient method of screening for chromosomal abnormalities. However, the underlying mechanism producing increased nuchal translucency thickness is still poorly understood. The purpose of this study was to investigate the possible contribution of impaired cardiac function to such an increase, by studying the venous return in the ductus venosus, using Doppler ultrasound. In a total of 65 fetuses, nuchal translucency thickness was measured at 10-13 weeks of gestation by means of a transvaginal probe. Color-coded and pulsed Doppler ultrasound were also used to evaluate different hemodynamic parameters in the ductus venosus: maximum systolic and diastolic velocities, pulsatility index, lowest forward velocity during atrial contraction and fetal heart rate. Fetal nuchal translucency thickness of > or = 3 mm was found in 17 cases; in five of them there were chromosomal anomalies: four trisomy 21 and one trisomy 18. Of interest is the finding that in the five chromosomally abnormal fetuses with increased nuchal translucency thickness, the forward velocity during atrial contraction was consistently less than 2 cm/s (p < 0.001). This impairment of atrial contraction may well implicate cardiac failure and/or heart defects in the pathogenesis of increased nuchal translucency thickness in the first trimester of pregnancy. Furthermore, nuchal translucency may prove to be a sensitive marker for the early identification of fetal cardiac anomalies.

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.

Probability tables for exclusion of mosaicism in prenatal diagnosis.

The decision concerning the number of metaphases that need to be analysed to detect mosaicism of a certain degree depends mainly, for the same confidence levels, on the culture method used (in situ or flask methods). Several probability tables, designed for either the in situ or the flask method, have been reported and can be used to assist laboratories in making the decisions referred to above. However, there are instances where part of the analysis is done using the in situ and flask methods. In such situations, the previously published tables are of limited use. We have generated a new table that can be used in such situations, as well as in cases where only the flask method is used.