A fluorescence in situ hybridization (FISH) procedure to assist in differentiating benign from malignant melanocytic lesions.

OBJECTIVE: Misdiagnosis of melanocytic lesions can result in unnecessary psychological distress to patients, under- or overtreatment, inaccurate prognosis and improper follow-up and family member surveillance. It is well recognized that, despite many attempts to 1) put forth a set of histologic criteria that can accurately and reproducibly be used to diagnose melanocytic lesions, and 2) identify reliable markers of malignancy as an adjunct to routine histopathology, misdiagnoses still occur in a significant number of cases. METHOD: A multi-color FISH probe mixture has been devised to assist pathologists in differential diagnosis of difficult melanocytic lesions. The mixture includes a centromeric probe for chromosome 6 and unique sequence probes for three other chromosomal regions that have most frequently shown amplifications or deletions in melanoma. We have carried out a preliminary evaluation of this new probe set in 25 cases of benign and malignant pigmented lesions. RESULTS: The tool reliably identified all nevi and ordinary melanomas, and only failed to identify a pigmented epithelioid melanocytoma and two malignant lesions that, by morphology and behavior, have distinct features from common invasive melanomas, i.e., a desmoplastic melanoma and a nevoid melanoma. Considering this, 100% specificity and 75% sensitivity was achieved. CONCLUSION: The FISH tool used in this study was able to separate accurately benign nevi from ordinary melanoma. Failure to identify uncommon melanocytic lesions adds to its advantage and calls for further studies to unveil the molecular profile of these rare entities.

Chromosomal abnormalities in miscarriages after different assisted reproduction procedures.

About 10-15% of all recognised pregnancies end in spontaneous abortion and around 60% of these show a chromosomal abnormality. The finding of an abnormal karyotype allows one to avoid unnecessary and controversial testing and treatment, providing accurate reproductive and genetic counselling to the couple. Consequently, chromosome study of products of conception (POC) is routinely performed in our Institute, starting from 2002. Cytogenetic analysis on spontaneous metaphases, obtained using direct method, was successfully performed on a total of 277 cases: 133 from patients who underwent assisted reproductive technology (ART) and 144 samples from infertile couples that had natural conceptions (NC). An abnormal karyotype was observed in 84 (63.2%) cases after ART and in 103 (71.5%) after NC demonstrating that the ART group doesn't seem subjected to a higher cytogenetic risk due to the application of technical procedures and higher than the expected risk in the fertile population. Moreover we did not observe a significant difference in the incidence of chromosome anomalies between intracytoplasmic sperm injection (ICSI) (61.5%) and in vitro fertilisation (IVF) (54.5%). However, in the ICSI with testicular sperm extraction (ICSI-TESE) group, 80% of the cases were abnormal with 50% showing triploid/tetraploid karyotypes. Chromosomal abnormalities were present in 54.5% of miscarriages which occurred after ICSI with cryo-preserved oocytes (cryo-ICSI) and in 85.7% after intra-uterine insemination (IUI). To our knowledge this study analysed the largest number of POCs after ART and NC in an infertile population. Moreover, for the first time the cytogenetic results of POC from different ART procedures were included.

Clinical and molecular cytogenetic studies in three infertile patients with mosaic rearranged Y chromosomes.

Isodicentrics (idic) are structural anomalies of the Y chromosome associated with a 45,X cell line and a broad spectrum of phenotypes. We characterized the rearranged Y chromosomes from three azoospermic males by fluorescence in-situ hybridization (FISH) and PCR. Chromosome study was performed on lymphocytes and testicular biopsy. FISH analysis and PCR established the degree of mosaicism and analysed specific Y regions. Two patients showed a 45,X/46,X,?idic(Y) karyotype with varying degrees of mosaicism. FISH demonstrated the presence of two centromeres and two SRY regions. In the lymphocytes of the third patient, the presence of a small Y-derived marker was also observed. An additional cell line with two idic(Y) was present in the testicular biopsy of the same patient. PCR showed the breakpoint between SY182 (KALY) and SY121 in Yq11.221-q11.222 region in all the cases. For the evaluation of the mosaicism, different tissues must be investigated. The phenotypical sex depends more on the number of copies of the SRY gene rather than on the percentage of 45,X cells, at least in the gonads. The combined use of classical and molecular cytogenetics is necessary for delineating the chromosome regions involved allowing a better genotype-phenotype correlation.

FISH characterization of two supernumerary r(1) associated with distinct clinical phenotypes.

Only a few reports on supernumerary r(1) chromosomes associated with a clinical phenotype have been published. We describe two unrelated patients with congenital malformations and developmental delay who were found to have a de novo supernumerary r(1) in 50% (Case 1) and 80% (Case 2) of the examined cells. Conventional cytogenetic techniques (QFQ, CBG, and DA-DAPI), complemented by fluorescence in situ hybridization studies using alpha satellite probes, showed that both small marker chromosomes (SMCs) primarily consisted of the centromere and heterochromatin of chromosome 1, a conclusion that was also supported by chromosome 1 painting. In an attempt to establish phenotype-genotype correlations, a further investigation was performed using YACs mapped to the chromosome 1 pericentromeric region. A fluorescent signal was evident after hybridization with Y934G9 (1q21) in Case 1 and Y959C4 (1p11.1-12) in Case 2. Partial trisomy of unique sequences flanking pericentromeric sequences is shown to underlie the clinical phenotype in both patients. This evidence should be taken into account when SMCs are ascertained, particularly in prenatal diagnosis.

Reduced growth hormone (GH) responsiveness to combined GH-releasing hormone and pyridostigmine administration in the Prader-Willi syndrome.

OBJECTIVE: It is unclear whether the blunted GH secretion in Prader-Willi Syndrome (PWS) is a true deficiency, or merely secondary to obesity. We have investigated the role of obesity in the blunted GH secretion in PWS. DESIGN: We studied the GH response to a combined administration of GHRH (1 microgram/kg i.v. at 0 min) and pyridostigmine (PD) (60 and 120 mg by mouth for children and adults, respectively, at time -60 min), as well as the baseline IGF-I levels, in a group of patients with PWS. Two different control groups were studied with GHRH + PD using the same doses and methods as above: prepubertal and pubertal obese subjects, and prepubertal short normal children. Moreover, in 14 patients with PWS and in the group of short normals the GH response to at least two stimulation tests (insulin tolerance test, clonidine, L-dopa, arginine) had been previously determined. PATIENTS: Twenty-two PWS patients (10 males and 12 females), 21 with essential obesity (11 males and 10 females), and eight short normal children (4 males and 4 females) were studied after obtaining informed consent. MEASUREMENTS: Blood samples were taken at -60, -30 and 0 min and then 15, 30, 45, 60, 90 and 120 min after GHRH administration. Serum GH was measured in duplicate by IRMA, and IGF-I by RIA after acid ethanol extraction. Statistical analysis was performed by t-test for unpaired data, and analysis of variance for parametric or nonparametric data, where appropriate. RESULTS: The GH response to GHRH + PD was significantly lower in PWS patients (AUC: mean +/- SE: 599 +/- 99 micrograms/l/h) if compared with either short normal children (3294 +/- 461 micrograms/l/h: P < 0.0001) or obese subjects (1445 +/- 210 micrograms/l/h: P < 0.005). Low IGF-I concentrations were found in all PWS patients, so that PWS group had mean IGF-I levels significantly lower than the other groups. CONCLUSIONS: Our results showed that subjects with PWS had a reduced GH responsiveness to GHRH + PD associated with subnormal IGF-I levels. These findings suggested that short stature in PWS may be at least partially correlated to the presence of GH deficiency, and that impaired GH secretion is not secondary to obesity.

Cytogenetic study of pituitary adenomas.

We report the results of cytogenetic studies on 23 pituitary adenoma specimens, using both the direct and short-term tissue culture methods. The direct method was applied to all of the specimens and allowed a karyotype to be identified in 15 of the processed samples (65%). Four tumors were shown to have a hypotriploid chromosomal constitution, two of which also presented structural clonal rearrangements: an isochromosome 1q,i(1)(q10) and a der(1)t(1;3)(p22;q21) were observed in two PRL-secreting adenomas, one of which also had a telomeric association involving the short arms of chromosomes 14 and 19. Telomeric associations of the long arms of chromosomes 11, 19, and 22 were observed in a near-diploid, non-secreting tumor showing monosomy 13. One other adenoma showed trisomies 8 and 12, a finding that was confirmed by means of the FISH analysis of chromosome 8 and 12 centromeric probes in the more than 300 scored nuclei. An apparently normal chromosome constitution was observed in the remaining nine cases. Short-term cultures were set up in 21 of the 23 samples, allowing us to obtain a karyotype in 18 specimens (85%). The six tumors that could not be analyzed using the direct method showed a normal karyotype. A diploid chromosome constitution was observed in the four tumors shown to be hypotriploid by the direct method as well as in the tumor with monosomy 13. The trisomies 8 and 12 identified by the direct method in one tumor were still observed, but a clone with a normal karyotype was also found. To the best of our knowledge, this is the only report of the results of cytogenetic studies on pituitary adenomas performed using both direct preparation and short-term culture.

FISH characterization of small supernumerary marker chromosomes in two Prader-Willi patients.

A small supernumerary chromosome was observed in two Prader-Willi syndrome (PWS) patients. The clinical diagnosis of PWS was confirmed by the ascertainment of the deletion of region 15q11-13 in one case and uniparental disomy (UPD) of the same region in the other. The markers were negative for dystamycinA/DAPI banding, did not contain NOR-positive satellites, and had an appearance consistent with a very small ring chromosome. Fluorescent in situ hybridization (FISH) analysis with the "all human centromere" probe indicated the presence of centromeric sequences in both markers. Chromosomal in situ suppression hybridization with chromosome specific libraries demonstrated that the small markers in the deleted and UPD patient originated from chromosome 15 and X, respectively. To the best of our knowledge these are the only PWS patients reported with a supernumerary marker chromosome other than inv dup(15) characterized by FISH.

Isochromosome 15q of maternal origin in a Prader-Willi patient with pituitary adenoma.

We report on a Prader-Willi syndrome (PWS) patient carrier of a balanced 15q15q translocation and affected by a prolactin-secreting pituitary adenoma. Evidence provided by molecular studies indicates that the structural rearrangement is an isochromosome of maternal origin. According to the identification of isodisomy as the basis of the association of rare disorders and the recent report on chromosome 15 monosomy and nullisomy in pituitary adenoma, we suggest that in our case PWS and pituitary adenoma might be related.

Seizure and EEG patterns in Angelman's syndrome.

We studied the seizure and polygraphic patterns of 18 patients with Angelman's syndrome. All patients showed movement problems. Eleven patients were also reported to have long-lasting periods of jerky movements. The polygraphic recording showed a myoclonic status epilepticus in nine of them. Seven patients had partial seizures with eye deviation and vomiting, similar to those of childhood occipital epilepsies. These seizures and electroencephalographic patterns suggest that Angelman's syndrome occurs in most of the patients as a nonprogressive, age-dependent myoclonic encephalopathy with a prominent occipital involvement. These findings indicate that, whereas ataxia is a constant symptom in Angelman's syndrome, the occurrence of a transient myoclonic status epilepticus may account for the recurrence of different abnormal movements, namely the jerky ones.

FISH analysis in Prader-Willi and Angelman syndrome patients.

We report on a combined high resolution cytogenetic and fluorescent in situ hybridization study (FISH) on 15 Prader-Willi syndrome (PWS) and 14 Angelman syndrome (AS) patients. High resolution banding showed a microdeletion in the 15q11-q13 region in 7 out of 15 PWS patients, and FISH analysis of the D15S11 and SNRPN cosmids demonstrated absence of the critical region in three additional cases. Likewise 8 out of 14 AS patients were found to be deleted with FISH, using the GABRB3 specific cosmid, whereas only 4 of them had a cytogenetically detectable deletion.

Familial translocation (X;3) (p22.3;p23): chromosomal in situ suppression (CISS) hybridization and inactivation pattern study.

High-resolution chromosome banding and chromosomal in situ suppression hybridization were used to identify a derivative X in a 10-month-old female patient with congenital heart defect and slight dysmorphism. The unbalanced karyotype was monosomic for Xp22.3-pter and trisomic for 3p23-pter regions. The derivative X was inherited from the mother carrier of a balanced translocation (X;3) (p22.3;p23). Replication study of the patient showed the abnormal X,t(X;3) to be late replicating, except for the translocated segment. This patient demonstrated only epicanthus and congenital heart defect, despite her partial trisomy 3. The clinical phenotype may be less severe when the X-chromosome is involved in an unbalanced translocation.

A de novo 6q11-q15 duplication investigated by chromosome painting.

A de novo interstitial duplication of the 6q11-q15 chromosome region, confirmed by the application of a chromosome 6 painting probe, was observed in a patient with craniofacial dysmorphism, psychomotor retardation, cryptorchidism and hypospadias. Despite the publication of several cases showing partial trisomy 6q, to our knowledge the duplication of the proximal region q11-q15 has not previously been reported.

Cytogenetic abnormalities detected by direct analysis in a case of choriocarcinoma.

Malignant trophoblastic cells from a case of choriocarcinoma were cytogenetically investigated by direct analysis of fresh tissue from the tumor. To our knowledge, previous cytogenetic studies have been performed only on established cell lines. In this study, 54 metaphases were observed, of which 41 were fully karyotyped. Three chromosomally abnormal lines were identified. In all of them, trisomy 3 and 10, a supernumerary isochromosome 1q,i(1)(q10), an i(8)(q10) replacing one chromosome 8, and a marker chromosome were observed. In addition, involvement of chromosome 12 was observed in two of the three lines, trisomic in one and an i(12)(q10) in the other.

12q13 fragility in a family with recurrent spontaneous abortions: expression of the fragile site under different culture conditions.

A fragile site at the 12q13 band was found in metaphases from lymphocyte cultures of three members of a family. A comparison of the frequency and expression of the fragile site was carried out on cells cultured in RPM-I 1640 with and without BrdU and in 199 media. The fragile site was not typically folate-sensitive, being expressed in standard medium as well as in cultures after exposure to BrdU.

Seven cases of trisomy 3 mosaicism in chorionic villi.

This paper describes seven cases of confined chorionic mosaicism with trisomy 3. The chromosomally abnormal cell line in chorionic villi was revealed in three cases at diagnostic CVS and in four cases at the evacuation of the uterine cavity after a missed abortion had been diagnosed by ultrasound. In two of these cases, the abortion occurred after apparently normal development of the fetus during the second trimester of pregnancy. An evaluation of the effect of confined chorionic mosaicism with trisomy 3 on the viability of the conceptus has been attempted.

Prevalence and distribution of chromosome abnormalities in a sample of first trimester internal abortions.

Cytogenetic analysis was performed directly on villus material from 202 samples obtained at the evacuation of the uterine cavity in cases of retained abortion in the first trimester, identified as such by ultrasound examination. A precise delineation of the karyotype was obtained in 94% of the cases, while the efficiency of karyotype analysis in samples of spontaneous abortion was not higher than 50%. An abnormal chromosome constitution was found in 145 fetuses (76.7%) of which 117, including mosaics, were aneuploid (70%), 16 polyploid (8.5%) and 12 had structural abnormalities (6.3%). The relative proportion of chromosome abnormalities in this material is higher than that found in spontaneous abortion for trisomies and double trisomies, but lower for 45,X and polyploidy. The method was found to be efficient in obtaining fetal karyotypes also in those cases in which the villous material was scarce (1 mg), and thus it seems appropriate for routine cytogenetic studies in the first trimester abortions.

Orphenadrine serum levels in a poisoned patient.

A patient ingested about 5 g of orphenadrine hydrochloride. He had gastric lavage and oral administration of activated charcoal. The main symptoms were neuropsychiatric in nature. Possible relation between serum levels of the drug and time course of the toxic effects are described.