Iskra Petković

Expand Long PCR for fragile X mutation detection.

Fragile X mutation detection by DNA analysis enables accurate diagnosis of the fragile X syndrome. The mutation involves the expansion of CGG repeats in the FMR1 gene and has been primarily detected by the Southern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutated and full‐mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by ethidium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. The amplified premutations and full mutations were identified by hybridization with a digoxigenin‐labeled 5′‐(CGG)5–3′ probe, followed by chemiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this paper we propose a new strategy for molecular diagnosis of the fragile X syndrome in which our Expand Long PCR assay is used as the first screening test for fragile X mutation detection.

Interstitial deletion 4q and Rieger syndrome

In a 9‐year‐old girl, the diagnosis of the Rieger syndrome, an autosomal dominant disorder of variable expressivity, was established on the basis of characteristic congenital ocular and dental anomalies. Cytogenetic analysis revealed a de novo interstitial deletion of 4q.

Constitutive heterochromatin of chromosomes No. 1, 9, and 16 in 90 patients with malignant disease and 91 controls

The variability of constitutive heterochromatin in chromosomes #1, #9, and #16 was investigated in 90 patients with malignant tumors (neoplasms of the head, neck, breast, and genital organs) and in 91 controls. The investigation was carried out on chromosome preparations from peripheral blood cultures. An analysis showed no significant difference in the frequency of localization variants, that is, inversions, in the patients and in the normal controls. Analysis of the variability of the C-segment size was made by measuring the length of heterochromatin blocks and showed that a correlation with the process of malignant transformation can be sought in the consequences of an uneven distribution (chromosome #1) and in quantitative changes in the constitutive heterochromatin (chromosomes #1 and #9).

Cytogenetic, cytomorphologic, and immunologic analysis in 55 children with acute lymphoblastic leukemia

This paper presents the results of cytogenetic analysis in 55 children with acute lymphatic leukemia (ALL). Acquired chromosome aberrations were identified in 35 (63.6%) children. Difference in frequency of clonal aberrations depending on age of patients was observed. Thus, acquired aberrations were detected in all three children up to 6 months of age, in 57.8% in the group from 1-10 years, and in six (85.7%) of seven children older than 10 years of age at diagnosis. The analysis revealed chromosome aberrations specific for ALL. Hyperdiploidy >50 was identified in 20% of children, and the association with CALLA+ early pre-B and L2-ALL was observed. del(6) and t(1;19) were identified in 7.3% and 5.5% of children, respectively. No association with a specific morphology or specific immunophenotype for both structural aberrations has been established. The association of structural aberrations involving regions 14q11-12 and T-cell ALL, however, has been observed, as an aberration was identified in two (3.6%) children, i.e., in 25% of our T-cell leukemias. Interstitial deletion of the long arm of chromosome 13, a rare chromosomal aberration in ALL, was identified in addition to del(9)(q31) in a 17-month-old girl with constitutional trisomy of chromosome 21 and B-cell ALL-L2. Interesting is the finding of hyperdiploidy with 52 chromosomes and structural aberrations of chromosome 1 in a 1-month-old girl with morphologically unclassified CALLA+ pre-T acute leukemia. To our knowledge this is the first case of hyperdiploidy >50 in a neonatal leukemia.

Cytogenetic analysis of hepatoblastoma

A cytogenetic investigation was performed in a case of hepatoblastoma; the analyses revealed a pseudodiploid karyotype.

Cytogenetic analysis in ataxia telangiectasia with malignant lymphoma

We present the results of cytogenetic analysis in a brother and sister with ataxia telangiectasia (AT), one of whom had malignant T-cell lymphoma. In both children, cytogenetic analysis of phytohemagglutinin (PHA)-stimulated lymphocytes showed chromosomal instability and inv(7) in 10% of the cells examined. The malignant lymphoma was analyzed cytogenetically on slides obtained from short-term culture of the lymph node cells; 64 cells were analyzed. A heterogeneous cell population was noted. Fourteen cells (21.9%) had a normal male karyotype; t(7;14)(p14;q12) and inv(7)(p14q35) were observed in 6.3% and 3.1% of metaphases. Owing to low frequency, these cells are probably a characteristic of the basic disease and have no features of malignant cells. Forty cells (62.5%) had a pseudodiploid karyotype 46,XY,dup(1)(p22p36),del(5)(q33),del(12)(p11), without cytogenetically evident aberrations of chromosomes 7 and 14. The results of these investigations suggest that the cells with rearrangements of chromosomes 1, 5, and 12 are malignant cells and did not originate by transformation of cells with inv(7) and t(7;14).

Cytogenetic analysis in children with acute nonlymphocytic leukemia

In this work we present the results of cytogenetic analysis of the malignant cells in 27 children with acute nonlymphocytic leukemia (ANLL). The aim of our investigations was to determine the frequency and types of chromosome aberrations in our population of children with ANLL. Successful cytogenetic analysis was carried out in 24 (89%) patients. Aberrant karyotypes of malignant cells were established in 58% of the cases. The most frequent chromosomal abnormality was t(8;21), identified in 5 (20.8%) patients, i.e., 4 of 10 M2-ANLL. Aberration frequency of chromosome 11 was 16.6% and was identified in 3 of 8 M5-ANLL. Trisomy 8 and monosomy 7 were identified in one patient each with M3 and M2-ANLL, respectively. del(13), a rare chromosome aberration in hemoblastoses, was found in a child with M1,t(8;21) and the loss of chromosome Y. Translocation t(1;11;21) with a break in regions 1q23, 11q23, and 21q22, is unusual and was identified in a boy with M2-ANLL; it can be considered as a variant form of the t(8;21).

Unusual segregation of t(11;22) resulting from crossing-over followed by 3:1 disjunction at meiosis I

Reciprocal translocation t(11;22)(q23;q11) is of particular interest because the unbalanced offspring of the translocation carriers usually present with a supernumerary derivative chromosome 22. This common unbalanced karyotype is the result of 3:1 chromosome segregation during meiosis. We report the third case of a rare segregation pattern of a paternal 11; 22 translocation. The probands karyotype revealed the presence of a der(11) and two copies of a der(22), i.e. 47, XX, t(11; 22)(q23;q11), +der(22) t(11;22)pat. The karyotype is the result of paternal 3:1 segregation after crossing‐over involving the derived and the normal chromosome 22, as revealed by chromosome polymorphism analysis. Contrary to the preferential maternal transmission of this common unbalanced translocation, the data from the literature, including our case, may suggest preferential paternal transmission of this rare type of unbalanced translocation.

Variability of chromosomes 1, 9, and 16 in children with malignant diseases

Heterochromatic segments of chromosomes #1, #9, and #16 were analyzed in 38 children with malignant disease and 42 healthy persons. The analysis was carried out on C-banded metaphases obtained by peripheral blood culture. Using a quantitative method of analysis, an association was established between C-segment length of chromosome #9 and malignant disease in children. A disturbed quantitative relation of C-heterochromatin of chromosomes #1, #9, and #16 was also found in the group of children with malignant disease.

Paternal origin of der(X)t(X;6) in a girl with trisomy 6p and unbalanced t(6;10) mosaicism in her mother

We present a case of trisomy for the whole short arm of chromosome 6 in a 3‐year‐old girl with moderate mental retardation, mild facial dysmorphism, short stature, failure to thrive, and no abnormalities of the visceral organs. Cytogenetic and fluorescence in situ hybridization (FISH) analysis revealed a 46, X, der(X)t(X;6)(q22; p11.1) karyotype. The derived X was late replicating with variable spreading of X chromosome inactivation onto the translocated 6p. A normal karyotype was observed in the father, while the mother presented 46,XX/46,XX, der(10)t(6;10)(p11;p11). The mother is a mosaic with unbalanced t(6;10) in 4.7% of cells. To the best of our knowledge, this unusual mosaicism has not yet been reported. In this family the short arm of chromosome 6 was involved in an unbalanced rearrangement with chromosome X in the proband and with chromosome 10 in the mother. In order to study the mechanism of the formation of t(X;6) in the girl we performed DNA polymorphism analysis. These investigations revealed that chromosomes X and 6 involved in the rearrangement are of paternal origin. Our patient exhibits only discrete facial features characteristic of partial trisomy 6p. We suggest that mild phenotypic expression be probably due to X chromosome inactivation spreading onto the translocated 6p. This report show that combined cytogenetic, FISH, and molecular analysis of chromosomal aberrations are necessary for the understanding of the mechanism of formation, parental origin, and genetic counseling.