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The use of new technologies in the routine diagnosis of constitutional abnormalities, such as high-resolution chromosomal microarray and next-generation sequencing, has unmasked new mechanisms for generating structural variation of the human genome. For example, complex chromosome rearrangements can originate by a chromosome catastrophe phenomenon in which numerous genomic rearrangements are apparently acquired in a single catastrophic event. This phenomenon is named chromoanagenesis (from the Greek “chromo” for chromosome and “anagenesis” for rebirth). Herein, we report 2 cases of genomic chaos detected at prenatal diagnosis. The terms “chromothripsis” and “chromoanasynthesis” and the challenge of genetic counseling are discussed.

MECP2 duplication syndrome (MDS) is an X-linked neurodevelopmental disorder characterized by a severe to profound intellectual disability, early onset hypotonia and diverse psycho-motor and behavioural features. To date, fewer than 200 cases have been published. We report the clinical and molecular characterization of a Spanish MDS cohort that included 19 boys and 2 girls. Clinical suspicions were confirmed by array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA). Using, a custom in-house MLPA assay, we performed a thorough study of the minimal duplicated region, from which we concluded a complete duplication of both MECP2 and IRAK1 was necessary for a correct MDS diagnosis, as patients with partial MECP2 duplications lacked some typical clinical traits present in other MDS patients. In addition, the duplication location may be related to phenotypic severity. This observation may provide a new approach for genotype-phenotype correlations, and thus more personalized genetic counselling.

1q44 deletion is a rare syndrome associated with facial dysmorphism and developmental delay, in particular related with expressive speech, seizures, and hypotonia (ORPHA:238769). Until today, the distinct genetic causes for the different symptoms remain not entirely clear. We present a patient with a 2.3-Mb 1q44 deletion, including AKT3, ZBTB18, and HNRNPU, who shows microcephaly, developmental delay, abnormal corpus callosum, and seizures. The genetic findings in this case and a review of the literature spotlight a region between 243 Mb and 245 Mb on chromosome 1q related to the genesis of the typical symptoms of 1q44 deletion.

Copy number variants (CNVs) of the Williams-Beuren syndrome (WBS) 7q11.23 region are responsible for neurodevelopmental disorders with multisystem involvement and variable expressivity. We found 2 patients with a deletion and 1 patient with a duplication in this region sharing a common breakpoint located between the LIMK1 and EIF4H(WBSCR1) genes. One patient had a WBS phenotype, although testing with a commercially available FISH assay was negative for the deletion. A further test using array CGH showed an atypical WBS region deletion. The second patient showed global developmental delay, speech delay and poor motor skills with a deletion outside the WBS region. The third patient had manifestations compatible with an autism spectrum disorder showing a duplication in the WBS region. Our findings point to the existence of a previously unrecognized recurrent breakpoint responsible for rearrangements in the WBS region. Given that most commercial FISH assays include probes flanking this novel breakpoint, further testing with array CGH should be performed in patients with WBS and negative FISH results.

Small supernumerary marker chromosomes (sSMC) originating from chromosome 10 are rare and usually found in mosaic form. We present a de novo apparently non-mosaic sSMC(10) prenatally diagnosed in amniotic fluid and postnatally confirmed in peripheral blood. Characterization by array-CGH showed a pericentromeric duplication of 7.1 Mb of chromosome 10. The fetus did not show ultrasound abnormalities, and a normal female phenotype was observed during a 3-year postnatal follow-up. The absence of phenotypic abnormalities in the present case provides evidence of a non-critical pericentromeric region in 10p11.21q11.1 (hg19 35,355,570-42,448,569) associated with a duplication.

Resumen Introduccion Se conocen numerosas reestructuraciones cromosomicas compatibles con un fenotipo normal, principalmente algunos cromosomas marcadores sin contenido geneticamente relevante y heteromorfismos cromosomicos. Material y metodos Estudio retrospectivo de 20.098 casos prenatales. Resultados Se han detectado 24/17.784 casos (0,13%) de pequenos cromosomas marcadores (SMC) en liquido amniotico, 8/2.223 (0,36%) en vellosidad corial y 31/20.007 (0,15%) reestructuraciones estructurales clasificadas como heteromorfismos. Conclusiones Se proponen guias de actuacion basandose en nuestra experiencia y la bibliografia existente.

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.

Tetrasomy of short arm of chromosome 9 constitutes a clinically recognizable chromosomal syndrome. Isochromosome 9p shows a strong propensity to tissue-limited mosaicism. It occurs predominantly in peripheral blood cultures, often at a lower frequency or even absent in skin, amniotic fluid or chorionic villous cell cultures. Tissue-limited nature of mosaicism may render prenatal detection of this condition very difficult. Herein, we report two new cases of mosaic tetrasomy 9p. Conventional cytogenetics (CC) and FISH studies demonstrated a differential expression of the mosaicism in several tissues. We review the literature and discuss the implications of these findings in cytogenetic prenatal diagnosis.

We report a new dic(17;18)(p11.2;p11.2) in a 61-year-old male patient diagnosed with atypical B-cell chronic lymphocytic leukemia. The dic(17;18)(p11.2;p11.2) was detected in 90%, 10%, and 100% of metaphases in the peripheral blood, bone marrow, and lymph node, respectively. Fluorescence in situ hybridization studies with chromosome 17 and 18 centromeric probes revealed the presence of two normal centromeres of both chromosomes 17 and 18. The centromere of one chromosome 17 was found together with the centromere of one chromosome 18, confirming the dicentric nature of the rearrangement. In addition, with the use of a 17p13.1 region probe, monosomy of the 17p13 region, where the Tp53 gene is located, was observed.

We have studied 13 cases of histologically confirmed mantle cell lymphomas (MCL) combining cytological-immunological features with conventional cytogenetics and in situ hybridization (ISH) techniques. Peripheral blood smears and lymph node biopsies expressed the typical mantle zone pattern with a B-cell phenotype. Most of the cases (11 of 13) had lymphomatous cells in the peripheral blood. Chromosome analysis was carried out on lymphoid cells from peripheral blood and/or lymph node biopsies. Phytohemagglutinin (PHA) and phorbol 12-myristate 13 acetate (TPA) were used as mitogens. Biotin-labeled libraries of whole chromosomes implicated in complex karyotypes were used to improve their interpretation. Clonal chromosome abnormalities were found in 10 of 13 patients (77%); 7 of these had a complex abnormality. The most frequent recurrent structural abnormalities were: t(11;14)(q13;q32), involvement of chromosome 1 (der[1], del[1], dup[1]), chromosome 2 (del[2], der[2]), chromosome 9 (der[9], −9), chromosome 13 (add[13], t[13q]), and chromosome 17 (add[17], der[17], t[17q]). The most frequent numerical abnormalities were monosomy 21 and loss of the Y chromosome.

We present a clinical and molecular cytogenetic characterization of two new patients with a complex supernumerary marker consisting of the entire short arm of chromosome 18 with a chromosome 13/21 centromere. One patient is a girl with a nonsyndromic intellectual disability and the second is a prenatally diagnosed fetus. To our knowledge, these are the fourth and fifth such cases to be described in the literature, suggesting the existence of a possible recurring constitutional structural chromosome abnormality. © 2013 Wiley Periodicals, Inc.

Characterization of marker chromosomes before the introduction of array CGH (aCGH) assays was only based on their banding patterns (G, C, and NOR staining) and fluorescent in situ hybridization techniques. The use of aCGH greatly improves the identification of marker chromosomes in some cases. We describe an atypical case of Pallister-Killian syndrome (PKS) detected at prenatal diagnosis with a very unusual cytogenetic presentation: a supernumerary ring chromosome including two copies of 12p. A similar anomaly described in a postnatal patient suggests ring chromosome as a possible cause of PKS. Extra ring chromosomes might be a more common etiology for PKS than previously thought, given the difficulty in their characterization before the advent of aCGH.

Keywords: cytogenetics; in situ hybridization ; splenic marginal zone; B-cell lymphoma; splenic lymphoma; villous lymphocytes

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.

Objective To present a case of premature ovarian failure (POF) and a complex chromosomal rearrangement involving band Xq21. Design Case report. Setting Department of cytogenetics, general analysis laboratory. Patient(s) A woman with POF and a complex translocation involving chromosomes X and 2 and a nucleolus organizer region (NOR) structure inserted in the critical region Xq21. Intervention(s) Chromosomal analysis, NOR banding, hysteroscopy. Main Outcome Measure(s) Fluorescence in situ hybridization, comparative genome hybridization, human androgen receptor gene technique. Result(s) Four mechanisms may explain a causal relationship between the phenotype of the patient and her chromosome constitution. The presence of a NOR structure at the breakpoint of chromosome X suggests a complex reorganization and is of interest per se. Conclusion(s) Cytogenetic analysis is essential in women with unexplained POF and may provide valuable information on the chromosome location of critical regions implicated in ovarian function. However, in very complex reorganizations such as that described herein, classical cytogenetic techniques must be combined with molecular techniques to achieve a more complete characterization of the anomaly.

We have found a high correlation of non-random bending of human metaphase chromosome 12 with the intranuclear arrangement deduced by Nogami et al. (Chromosoma 108 (2000) 514), providing further evidence of the relation of non-random bending and the interphase organization of the nucleus.

Tetrasomy of short arm of chromosome 9 constitutes a clinically recognizable chromosomal syndrome. Isochromosome 9p shows a strong propensity to tissue-limited mosaicism. It occurs predominantly in peripheral blood cultures, often at a lower frequency or even absent in skin, amniotic fluid or chorionic villous cell cultures. Tissue-limited nature of mosaicism may render prenatal detection of this condition very difficult. Herein, we report two new cases of mosaic tetrasomy 9p. Conventional cytogenetics (CC) and FISH studies demonstrated a differential expression of the mosaicism in several tissues. We review the literature and discuss the implications of these findings in cytogenetic prenatal diagnosis.

We report cytogenetic results in a series of 60 patients affected with multiple myeloma (MM) and plasma cell leukemia (PCL) and compare the results with those previously reported. In our series, a total of 41% of MM patients and 71% of PCL patients displayed chromosome abnormalities. To evaluate the clinical value of monosomy 18, we obtained fluorescence in situ hybridization results (using centromeric probe for chromosome 18) of 22 MM patients who displayed a normal karyotype. Monosomy 18 was present in 3 of 22 patients (14%). Using conventional cytogenetics, we detected monosomy 18 in one patient affected with PCL. Two of four cases with monosomy 18 followed an aggressive course, with overall survival of 1 and 9 months. The remaining two are in follow-up and remain stable. The association of monosomy 18 with IgA subtype predominance and poor prognosis was not observed in this series of MMs and PCLs. Although these results do not confirm our previous hypothesis, further observations of this group of patients (with monosomy 18) regarding malignant transformation is warranted.

Rearrangements involving long arm of chromosome 12 are rare events. To our knowledge, we present the first case of an interstitial deletion of the long arm of chromosome 12 in a prenatal diagnosis. A review of the literature is included in our report.

We report the prenatal detection of an inherited paracentric inversion 16(q11.2q13).

Autologous stem cell transplantation is a therapeutic option for chronic myeloid leukemia (CML) patients who are not candidates for allogeneic transplant. To reduce the risk of post-autografting disease recurrence, different strategies of stem cell selection have been attempted. The results of using recombinant human granulocyte colony-stimulating factor (rHuG-CSF) for harvesting hematopoietic progenitors in CML patients treated with interferon-a (IFN) are reported.Twenty-one CML patients who received IFN for a median of 21 (8-68) months were mobilized with rHuG-CSF (10 mg/kg/day). Twelve were in complete (CCR) or major (MCR) cytogenetic response. Complete success was considered a sufficient harvest (1 x 10(6)/kg CD34(+) cells/kg) without Philadelphia (Ph)+ metaphases in at least one apheresis; a partial success was a sufficient harvest with 1-35% Ph(+) cells.A total of 78 aphereses were performed. No patient had major side-effects. The median number (range) of mononuclear and CD34(+) cells obtained was, respectively, 8.6 x 10(8)/kg (0.9-22.6) and 3.3 x 10(6)/kg (0.4-26.3) per patient. A sufficient cell yield was collected in all but three patients. A complete/partial success was achieved in seven CCR/MCR patients (63%) and in three (33%) with other responses. Four patients underwent successful autografting using the stem cells obtained after rHuG-CSF mobilization.Mobilization of IFN-treated patients using rHuG-CSF is safe and provides a significant proportion of Ph-negative progenitors in CML patients in complete or major cytogenetic response.

Monoclonal gammopathies of undetermined significance (MGUS) are characterized by the presence of a monoclonal protein in serum in quite asymptomatic patients. Ten to 33% of MGUS patients eventually will develop overt multiple myeloma, but no single laboratory test exists that can predict changes toward a malignant evolution. The aim of the present study was to apply conventional cytogenetics, the MAC (morphology, antibody, chromosome) method and fluorescence in situ hybridization (FISH) techniques in a series of 50 MGUS patients and 4 "smoldering" multiple myeloma patients to test the usefulness of their approaches as predictive methodologies. All patients studied by conventional cytogenetics presented a normal karyotype independent of the culture conditions used. The MAC method revealed that all mitotic cells showing a normal karyotype were positive for anti-MOP7 or anti-CD3 in 12 patients studied. In addition, two of them presented a numerical abnormality detected by FISH. Using a FISH technique with direct labeled centromeric probes for chromosomes 3, 7, 11, and 18 we showed a numerical abnormality in eight of 35 patients (23%) with a normal karyotype. The common occurrence of MGUS and the fact that they may evolve toward lymphoproliferative disorders displays the importance of being able to identify laboratory results that are capable of predicting the evolution of these patients. In the literature, patients who presented an IgA peak of immunoglobulin type have been associated with a higher risk of evolving to a malignant condition. Our study shows the correlation of MGUS patients who presented monosomy 18 with the presence of an immunoglobulin peak of the IgA type. Prospective follow-up is needed to evaluate the clinical value of monosomy 18 as a predictive factor for defining a high risk of malignant transformation in MGUS patients.

We present a cytogenetic and fluorescence in situ hybridization (FISH) study, using centromeric probes for chromosomes 3, 7, 11, and 18, TP53 gene (17p13), and RB-1 locus (13q14) DNA probes, in four cases of plasma cell leukemia (PCL). Among the four cases, three presented monosomy of the RB-1 locus and one monoallelic deletion of the TP53 gene. The present report shows the usefulness of the FISH technique to detect abnormalities not previously observed by conventional cytogenetics.

We studied five cases of hairy cell leukemia (HCL) using conventional cytogenetics. All patients were diagnosed with typical HCL. Chromosome analysis was carried out on a 72-hour culture of peripheral blood. Phytohemagglutinin (PHA) mitogen was used. Clonal chromosome abnormalities were found in 2/5 patients (40%), a complex abnormality being identified in one of them. The chromosomes involved were: 1, 6, 7, 8, and 17. No patient showed trisomy 12 or a 14q+. An interesting result was the finding of del(7)(q32) as a sole anomaly.

We present the cytological features, conventional cytogenetics, and in situ hybridization (ISH) findings of three cases of B-cell prolymphocytic leukemia (B-PLL). The diagnosis was made according to the French-American-British (FAB) criteria. We considered a diagnosis of B-PLL when a predominance (> 50%) of lymphoid cells with coarse chromatin but prominent central nucleoli and more abundant cytoplasm than typical chronic lymphocytic leukemia (CLL) cells were present. B-PLL express strong SIg, B-cell antigens, and reactivity with the monoclonal antibody FMC7. Chromosome analysis was carried out on lymphoid cells from peripheral blood and, in one patient, from lymph node. The phytohemagglutinin (PHA) mitogen was used. ISH was performed with two types of probes: the biotin-labeled chromosome 12-specific alpha satellite DNA probe to detect trisomy 12, and biotin-labeled libraries of whole chromosomes 1, 7, and 14. Clonal chromosome abnormalities were found in all three patients; in one, a complex karyotype was observed. The most frequent recurrent abnormality was trisomy 12. Our results suggest that PLL usually presents with cytogenetic abnormalities. The finding of translocation (11;14) is noteworthy; chromosomes 1 and 3 are also involved.

A 62-year-old woman with phenotypic stigmata of Turner syndrome and a mosaic cytogenetic pattern, 45,X/45,XX, developed multiple myeloma. The affected cells had a number of karyotypic changes in addition to the loss of the X chromosome.