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Background Small supernumerary marker chromosomes (sSMC) are additional centric chromosome fragments too small to be identified by banding cytogenetics alone. A sSMC can originate from any chromosome and it is estimated that 70% of sSMC are de novo, while 30% are inherited. Cases of sSMC derived from chromosome 5 (sSMC5) are rare, accounting for1.4% of all reported sSMC cases. In these patients, the most common reported features are macrocephaly, dysmorphic facial features, heart defects, growth retardation, hypotonia, and intellectual disability. Also sSMC derived from chromosome 8 are very rare and the phenotype of patients with sSMC8 is very variable. Common clinical features of the patients include developmental delay, mental retardation, intellectual disability, hypotonia, hypospadias, attention deficit hyperactivity disorders (ADHD), skeletal anomalies, dysmorphic facial features, and renal dysplasia. To the best of our knowledge, in literature there are no cases with coexistence of sSMC5 and sSMC8, so we reviewed the literature to compare cases with SMC5 and those with SMC8 separately. This study is aimed to highlight the unique findings of a patient with the coexistence of sSMC5 and sSMC8. Case presentation We describe a female patient with two supernumerary markers derived from chromosome 5 (SMC5) and chromosome 8 (SMC8). The patient was born prematurely at 30 weeks with respiratory distress and bronchodysplasia. On physical examination she presented dysmorphic features, respiratory issues, congenital heart defect, developmental delay, and intellectual disability. The G-banded chromosome analysis on cultured lymphocytes revealed in all the analyzed cells a female karyotype with the presence of two supernumerary chromosomal markers and the array-CGH highlighted the region and the size of these two duplications. We also used the fluorescent in situ hybridization analysis (FISH) using painting of chromosomes 5 and 8 to confirm the origin of the two sSMC. So, the karyotype of the patient was: 48, XX, +mar1, +mar2. Conclusions This is the first case with two markers: one from chromosome 5 and one from chromosome 8. Based on the data reported, we can affirm that the phenotype of our patient is probably caused mainly by the presence of the sSMC.

Background Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome (Online Mendelian Inheritance in Man [OMIM] #277000) is a congenital condition characterized by the total or partial agenesis of vagina and uterus. Agenesis can be isolated (MRKH 1) or associated with other renal, vertebral or cardiac defects (MRKH 2). Case presentation In this paper, we report a case of a Caucasian patient showing the clinical signs associated with MRKH. Array-based comparative genomic hybridization (a-CGH) analysis revealed a microduplication of approximately 3.01 megabases (Mb) located on the long arm of chromosome 22 (22q11.21). Microduplications affecting the 22q11.21 region have been shown to be associated with MRKH syndrome and Müllerian aplasia. The phenotype of patients with 22q11.2 duplication (OMIM #608363) appears extremely variable, ranging from apparently normal to mild learning difficulties or with multiple defects, sharing features with DiGeorge/velocardiofacial (DGS/VCFS) syndrome. Conclusions The altered gene expression together with other genetic, nongenetic, epigenetic or environmental factors can cause the extremely variable phenotype in patients carrying such duplication. Therefore, we can consider MRKH syndrome to be one of the clinical features of DGS/VCFS syndrome.

Interstitial duplications of the short arm of the X chromosome have been rarely described, especially in males. Usually boys present mental retardation, multiple congenital abnormalities and short stature. We describe two sons one with a 2q37.3 deletion and a Xp11.4 duplication and the other with Xp11.4 duplication only, identified by array-CGH. They both presented a phenotype characterized by poor growth, mild facial dysmorphisms, autism and developmental delay. The 2q37.3 identified chromosomal anomaly was inherited from the healthy father and included approximately 8 known genes, while the Xp11.4 duplication resulted inherited from the healthy mother and involved 13 known genes. Of these TSPAN7 and CASK, localized on Xp11.4, genes are of special interest. The alteration on the X chromosome could be more related to the clinical feature presented by the two brothers, while the anomaly on the chromosome 2 is more likely a polymorphism or might influence the phenotype correlated to the Xp11.4 duplication. The healthy phenotype of the mother could be explained by X chromosome inactivation (XCI) phenomenon.

Hsa-miR-155-5p (miR-155) is overexpressed in most solid and hematological malignancies. It promotes loss of genomic integrity in cancer cells by targeting genes involved in microsatellite instability and DNA repair; however, the link between miR-155 and aneuploidy has been scarcely investigated. Here we describe a novel mechanism by which miR-155 causes chromosomal instability. Using osteosarcoma cells (U2OS) and normal human dermal fibroblast (HDF), two well-established models for the study of chromosome congression, we demonstrate that miR-155 targets the spindle checkpoint proteins BUB1, CENP-F, and ZW10, thus compromising chromosome alignment at the metaphase plate. In U2OS cells, exogenous miR-155 expression reduced the recruitment of BUB1, CENP-F, and ZW10 to the kinetochores which resulted in defective chromosome congression. In contrast, during in vitro transformation of HDF by enforced expression of SV40 Large T antigen and human telomerase (HDFLT/hTERT), inhibition of miR-155 reduced chromosome congression errors and aneuploidy at early passages. Using live-cell imaging we observed that miR-155 delays progression through mitosis, indicating an activated mitotic spindle checkpoint, which likely fails to reduce aneuploidy. Overall, this study provides insight into a mechanism that generates aneuploidy at early stages of cellular transformation, pointing to a role for miR-155 in chromosomal instability at tumor onset.

Over the past two decades, there has been a rapid evolution in prenatal screening for fetal chromosome abnormalities. Initially, testing was focused on the identification of affected pregnancies in either the first, or, the second trimester (e.g. the Combined test or the triple test). This was replaced by sequential modalities (e.g. contingent screening) that have enhanced detection while reducing the need for invasive testing. More recently, the introduction of technologies based on cell-free DNA (cfDNA) in maternal plasma and enrichment of fetal cells in maternal circulation have further refined the concept of sequential screening. In this review, we document our experience with serum and ultrasound-based contingent screening where we were able to achieve a detection rate of 96.8%, a false-positive rate of 2.8% and an odds of being affected given a positive result of 1:11. We also describe our initial experience with a novel sequential protocol that includes the analysis of fetal cells in maternal blood.Methods for enrichment for fetal cells cfDNA and cfDNA technologies offer the possibility of greater sensitivity and specificity as well as expansion in the scope of genetic disorders detectable. As costs decline, these technologies will become increasingly used as primary screening tools. In the meantime, sequential use offers a practical approach to maximizing the benefits of prenatal testing. Keywords: Maternal serum, Ultrasound, Screening, Chromosome abnormality, Fetal cells, Cell-free DNA

Aneuploidy and overexpression of hsa-miR-155-5p (miR-155) characterize most solid and hematological malignancies. We recently demonstrated that miR-155 sustains aneuploidy at early stages of in vitro cellular transformation. During in vitro transformation of normal human fibroblast, upregulation of miR-155 downregulates spindle checkpoint proteins as the mitotic checkpoint serine/threonine kinase budding uninhibited by benzimidazoles 1 (BUB1), the centromere protein F (CENPF) and the zw10 kinetochore protein (ZW10), compromising the chromosome alignment at the metaphase plate and leading to aneuploidy in daughter cells. Here we show that the heterogeneous nuclear ribonucleoprotein L (HNRNPL) binds to the polymorphic marker D2S1888 at the 3&prime, UTR of BUB1 gene, impairs the miR-155 targeting, and restores BUB1 expression in chronic lymphocytic leukemia. This mechanism occurs at advanced passages of cell transformation and allows the expansion of more favorable clones. Our findings have revealed, at least in part, the molecular mechanisms behind the chromosomal stabilization of cell lines and the concept that, to survive, tumor cells cannot continuously change their genetic heritage but need to stabilize the most suitable karyotype.

Background A long sought goal in medical genetics has been the replacement of invasive procedures for the detection of chromosomal aneuploidies by isolating and analyzing fetal cells or free fetal DNA from maternal blood, avoiding risk to the fetus. However, a rapid, simple, consistent, and low-cost procedure suitable for routine clinical practice has not yet been achieved. The purpose of this study was to assess the feasibility of predicting fetal aneuploidy by applying our recently established dual-probe FISH protocol to fetal cells isolated and enriched from maternal blood. Methods A total of 172 pregnant women underwent prospective testing for fetal aneuploidy by FISH analysis of fetal cells isolated from maternal blood. Results were compared with the karyotype determined through invasive procedures or at birth. Results Seven of the samples exhibited fetal aneuploidy, which was confirmed by invasive prenatal diagnosis procedures. After enrichment for fetal cells, the frequency of trisomic cells was at least double in samples from aneuploid pregnancies (range 0.38–0.90%) compared to samples from normal pregnancies (≤0.18%). One false negative result was also obtained. Conclusions Noninvasive prenatal aneuploidy screening using fetal cells isolated from maternal blood is feasible and could substantially reduce the need for invasive procedures.

Interstitial 16p13.3 microduplication, encompassing the CREBBP gene, is now considered a well recognizable syndrome. To date, 28 patients have been reported with a 16p13.3 microduplication. The majority of the patients share a similar phenotype which is mainly characterized by typical facial dysmorphisms and variable intellectual disability. Other features include microcephaly, growth retardation, limb anomalies and defects of the brain, heart, genitalia, palate and eyes. We report on a de novo microduplication of chromosome 16p13.3 revealed using array-comparative genomic hybridization (array-CGH) technology in a patient presenting with variable congenital anomalies and typical facial dysmorphisms, but with no evidence of developmental delay. This case highlights the importance of an accurate clinical examination and the utility of array-CGH in pediatric patients with a characteristic phenotype but without intellectual disability.

A prenatal case of a de novo interstitial deletion distal to 8q24 was reported. Ultrasound examination and postmortem evaluation demonstrated no apparent phenotypic alterations. Array CGH showed an 11.4-Mb loss in chromosome 8 ranging from 8q24.13 to 8q24.23. This case partially overlaps the 2 cases previously described in the literature.

We describe a newborn female with a de novo duplication of chromosomes 2q31.2 and 2q37.3, and a de novo monosomy 9p24.3. The clinical findings of this patient include congenital heart defects, dysmorphic facial features, hypotonia, feeding difficulties and microcephaly. Ultrasonographic prenatal findings were negative for foetal malformations. Only a mild pyelectasis was reported. This is the first report of molecular cytogenetic characterization of a partial trisomy 2q31.2–37.3 with monosomy 9p24.3.

From January 1st 2013 to August 31st 2016, 24408 pregnant women received the first trimester Combined test and contingently offered second trimester maternal serum screening to identify those women who would most benefit from invasive prenatal diagnosis (IPD). The screening was based on first trimester cut-offs of ≥1:30 (IPD indicated), 1:31 to 1:899 (second trimester screening indicated) and ≤1:900 (no further action), and a second trimester cut-off of ≥1:250. From January 2014, analysis of fetal cells from peripheral maternal blood was also offered to women with positive screening results. For fetal Down syndrome, the overall detection rate was 96.8% for a false-positive rate of 2.8% resulting in an odds of being affected given a positive result (OAPR) of 1:11, equivalent to a positive predictive value (PPV) of 8.1%. Additional chromosome abnormalities were also identified resulting in an OAPR for any chromosome abnormality of 1:6.6 (PPV 11.9%). For a sub-set of cases with positive contingent test results, FISH analysis of circulating fetal cells in maternal circulation identified 7 abnormal and 39 as normal cases with 100% specificity and 100% sensitivity. We conclude that contingent screening using conventional Combined and second trimester screening tests is effective but can potentially be considerably enhanced through the addition of fetal cell analysis.

Study design A population of 10 156 pregnant women with singleton pregnancies were screened by the integrated test. Risks were retrospectively recalculated for contingent test strategies with first step intermediate risk groups defined by first trimester upper cut-offs of 1 : 10, 1 : 30, 1 : 50, and 1 : 70 and lower cut-offs 1 : 1500, 1 : 1200, 1 : 1100, and 1 : 900. The second trimester high risk group was based on a single cut-off of 1 : 250. Results In the first trimester, the detection rate (DR) ranged from 21% (6/29) to 52% (15/29) as the high risk first trimester cut-off was changed from 1 : 10 to 1 : 70. The corresponding first trimester false positive rate (FPR) increased from 0.2% to 1.4%. In the second trimester, an additional 21/29 (72%) to 12/29 (41%) affected pregnancies could be detected with an additional 1.6% to 2.7% false positives when lower first trimester cut-offs of 1 : 900 to 1 : 1500 were used. The best results were obtained with the upper first trimester cut-off of 1 : 30 and lower first trimester cut-off of 1 : 900, which yielded a rate of women requiring a second trimester test of only 12%, with overall DR and FPR of 93% and 2.8%, respectively. Conclusions Although the study population was relatively small, the results confirm the advantage of using contingent screening and suggest optimal first trimester cut-offs of 1 : 30 (lower cut-off) and 1 : 900 (upper cut-off). © 2012 John Wiley & Sons, Ltd.

We report on a 10-year-old patient with childhood apraxia of speech (CAS) and mild dysmorphic features. Although multiple karyotypes were reported as normal, a bacterial artificial chromosome array comparative genomic hybridization revealed the presence of a de novo 14.8-Mb mosaic deletion of chromosome 7q31. The deleted region involved several genes, including FOXP2, which has been associated with CAS. Interestingly, the deletion reported here was observed in about 50% of cells, which is the first case of mosaicism in a 7q31 deletion. Despite the presence of the deletion in only 50% of cells, the phenotype of the patient was not milder than other published cases. To date, 6 cases with a deletion of 9.1-20 Mb involving the FOXP2 gene have been reported, suggesting a new contiguous gene deletion syndrome characterized mainly by CAS caused by haploinsufficiency of the genes encompassed in the 7q critical region. This report suggests that children found with a deletion involving the FOXP2 region should be evaluated for CAS and that analysis of the FOXP2 gene including array comparative genomic hybridization should be considered in selected patients with CAS. Mosaic deletions in this area may also be considered as causative of CAS.

Objective To compare the efficacy of combined, stepwise sequential, and contingent screening versus the integrated test in detecting fetal aneuploidies. Study design First trimester combined test, sequential second trimester, and contingent risks were retrospectively calculated for 7292 unselected pregnant women with singleton pregnancies who had received integrated screening. The first trimester testing was based on nuchal translucency, pregnancy-associated plasma protein-A, and free-beta-human chorionic gonadotrophin (free β-hCG) and the second trimester tests were alpha-fetoprotein, hCG, and unconjugated estriol. A second trimester risk of 1:250 defined a positive result for all protocols with the contingent protocol based on additional second trimester testing for those with risks between 1:30 and 1:1200. Results Among the population submitted for the integrated test, the detection rate was 19/21 (90%) for Down syndrome (DS) and 6/6 (100%) for Edwards syndrome (ES) and the DS false-positive rate (FPR) was 247/7271 (3.4%). Provision of the first trimester combined test alone would have resulted in a 17/21 (81%) detection rate for DS, that of 4/6 (67%) for ES and a DS FPR of 292/7271 (4.0%). The sequential and contingent approaches had the same final detection rates as the integrated test but potentially allowed a high proportion of the affected pregnancies to be detected in the first trimester. The lowest net DS FPR was seen with the contingent approach (2.6%) and using this protocol only 12.7% of women would have required second trimester testing. Conclusions Integrated, sequential, and contingent screenings are all more efficacious than the combined test. Overall, the contingent approach was the most efficient with a high-detection rate, the lowest FPR, and the least amount of testing. Copyright © 2011 John Wiley & Sons, Ltd.

A fetus with rhombencephalosynapsis and prenatally diagnosed tetrasomy 9p is reported. Chromosomal analysis from amniocyte culture revealed non-mosaic supernumerary chromosome identified as isochromosome 9p (9p24--q13::q13--p24). Ultrasound scan revealed intrauterine growth retardation, renal anomalies, cardiac anomalies, ventriculomegaly, and agenesis of cerebellar vermis with fusion of the cerebellar hemispheres.Although most cases of cerebellar vermis agenesis in tetrasomy 9p are described with cystic malformation such as Dandy-Walker anomaly, our case indicates that this chromosomal disorder should be taken into account in fetuses with the development of cystic and non-cystic malformations of cerebellar vermis and posterior fossa.

Mutations in the X-linked androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS), resulting in an impaired embryonic sex differentiation in 46,XY genetic men. Complete androgen insensitivity (CAIS) produces a female external phenotype, whereas cases with partial androgen insensitivity (PAIS) have various ambiguities of the genitalia. Mild androgen insensitivity (MAIS) is characterized by undermasculinization and gynecomastia. Here we describe a 2-month-old 46,XY female patient, with all of the characteristics of CAIS. Defects in testosterone (T) and dihydrotestosterone (DHT) synthesis were excluded. Sequencing of the AR gene showed the presence in exon 6 of a T to C transition in the second base of codon 790, nucleotide position 2369, causing a novel missense Leu790Pro mutation in the ligand-binding domain of the AR protein. The identification of a novel AR mutation in a girl with CAIS provides significant information due to the importance of missense mutations in the ligand-binding domain of the AR, which are able to induce functional abnormalities in the androgen binding capability, stabilization of active conformation, or interaction with coactivators.

Supernumerary invdup(15) chromosomes, now also reported as sSMC(15), containing two additional copies of Prader-Willi/Angelman critical region (PWACR) have been associated with distinct clinical phenotype that includes hypotonia, dysmorphisms, developmental delay/mental retardation, autistic behaviour, and epilepsy. We report on a healthy adult male carrying an sSMC(15) with two copies of PWACR in 20-50% of cells from different tissues. Molecular analyses showed the sSMC(15) as resulting from a PWACR-duplicated region spanning 8Mb which is larger than those in the only two other healthy PWACR-duplicated sSMC(15) carriers previously reported. Mosaicism level and mosaic cell line rate variation among different tissues observed in our case support mosaicism in critical tissues as of relevance for sSMC(15) phenotype-genotype correlations.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused in the majority of cases by deletions of the DMD gene and are readily detectable in affected males by multiplex polymerase chain reaction (PCR). However, different approaches must be used for the identification of female carriers, in which deletions are not detectable by PCR, because of the presence of a normal X chromosome. In this study, we used the multiple ligation probe amplification (MLPA) tool for the identification of female carriers of DMD deletions or duplications in 12 families with a single affected male, 10 of which were previously diagnosed as carriers of a DMD rearrangement, and the remaining two as having an unknown disease-causing mutation. In all the investigated affected males, MLPA analysis confirmed the presence of a DMD rearrangement, and in six of them allowed the refinement of the breakpoints. In 12 female relatives of the affected patients, MLPA analysis showed a DMD deletion or duplication, confirming their carrier status. Two of these were the mother and the sister of a patient whose disease-causing mutation was not known. MLPA analysis thus proved to be an useful tool for the analysis of both affected males and females carriers of DMD rearrangements in cases in which the disease-causing mutation in the affected male was not known, providing useful information for the genetic counselling of the family.

Extra structurally abnormal chromosomes (ESACs) and cryptic rearrangements are often associated with mental retardation and phenotypic abnormalities. In some cases their characterisation, using standard cytogenetic techniques and fluorescence in situ hybridization (FISH), is difficult and time consuming, where a fast and accurate identification is essential, especially when such chromosomal aberrations are found in prenatal diagnosis. A recent molecular technique, spectral karyotyping (SKY), based on the spectral signature of 24 chromosome-specific painting probes labelled with different combinations of five fluorochromes, allows the simultaneous visualisation of all human chromosomes in different colours. We used SKY analysis on 14 cases with rare ESACs or cryptic unbalanced rearrangements found at pre- or postnatal diagnosis. SKY analysis permitted the classification of chromosome rearrangements in all 14 cases analysed in combination with FISH analysis.

A patient with refractory anaemia with excess of blasts (RAEB) had a complex karyotype with multiple markers. Spectral karyotyping (SKY) showed rearrangements including three different der(11), containing a very high number of MLL gene copies, shown by fluorescence in situ hybridization (FISH) analysis. Fibre-FISH experiments disclosed the presence of chromatin fibres with multiple MLL copies with a head-to-tail pattern. Apparently, no other region flanking the MLL site was present in the three der(11). MLL amplification was confirmed by the reverse transcription polymerase chain reaction (RT-PCR). The patient died 6 months after diagnosis, supporting the severe prognosis of sole MLL amplification.

Deletions in distal Yq interval 6 represent the cause of 10–15% of idiopathic severe male infertility and map to a region defined AZFc (azoospermia factor c). The testis-specific gene DAZ is considered a major AZFc candidate, and its deletion has been associated with a severe disruption in spermatogenesis. However, DAZ is actually a multicopy gene family consisting of seven clustered copies spanning about 1 megabase. Only deletions removing the entire DAZ gene cluster together with other genes have been reported in infertile males. Because no case of spermatogenic failure has been traced to intragenic deletions, point mutations, or even deletions not involving all the DAZ copies, the definitive proof for a requirement of DAZ for spermatogenesis is still debatable. Here we report the first case of a partial deletion of the DAZ cluster removing all but one of the copies. This deletion is present in a patient affected with severe oligozoospermia who had a testicular phenotype characterized by a great quantitative reduction of germ cells (severe hypospermatogenesis). The absence of this deletion in the fertile brother of the patient suggests that this de novo mutation indeed caused the spermatogenic failure.

Pallister-Killian syndrome (PKS) is a sporadic chromosomal anomaly, caused by a tissue-specific mosaic distribution of an additional isochromosome 12p. About 60 cases of prenatal diagnosis of PKS have been reported. Only 1 case of PKS is described on the basis of prenatal screening, presenting increased nuchal translucency. An abnormal fetal facial profile is described prenatally as sonographic evidence of PKS. We report a case of prenatal diagnosis in a fetus undergoing second-level scan due to positive triple screen with ultrasound features of PKS.

A longitudinal investigation using fluorescence in situ hybridization (FISH) analysis, PCR-SSCP, and in situ detection of apoptosis by the terminal deoxynucleotidyl Transferase (TdT) method was carried out on 13 chronic myelogenous leukemia (CML) patients to study the p53 gene behavior and the apoptotic process during the course of the disease. At diagnosis, FISH showed no loss of the p53 gene on interphase nuclei, and no point mutation was detected by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and sequencing. During the disease course, FISH analysis showed a significative loss of allele (LOA) rate for the p53 gene in eight patients that in seven cases was associated with a suppression of apoptotic process and the progressive expansion of the p53+/p53- clone. DNA sequencing showed in two of these eight patients a point mutation on the other allele, consisting in the formation of a stop codon in one case, and in a frameshift mutation in the other. Six patients had a myeloid blastic crisis (BC), five a lymphoid BC, and the other two an erythroid and an undifferentiated BC, respectively. All patients with myeloid BC and the one with undifferentiated BC disclosed a progressive expansion of the clone with p53 loss that was associated with a significant reduction in apoptosis. On the contrary in the 5 patients with lymphoid BC no significant p53 LOA rate was observed during the course of the disease. In these patients apoptotic process also persisted in the acute phase although in a lower rate as compared to CP.

We report a case of a two year-old girl with persistent thrombocytopenia, syndactyly, and mild psychomotor delay with speech delay. Proband chromosomal analysis from peripheral blood detected a mosaicism with two cell lines: the first with a ring chromosome 21 46,XX,r (21) (88%), and the second 45,XX,-21 (12%). In uncultured cells the level of mosaicism was 15% for the 46,XX,r(21) cell line and 85% for the 45,XX,-21 cell line. A different level of mosaicism was detected on buccal smear cells with a r(21) in 94%, and monosomy 21 in 6% of cells. The findings in the different cell lines are consistent with loss of the ring chromosome in the blood line.

Three patients carrying an isodicentric (idic) Y chromosome associated with a mosaic 45,X cell line were studied using molecular techniques. Genotype-phenotype correlations suggested an effect of the 45,X cell line on sexual differentiation. A relationship was established between instability of the idic(Y) chromosome and localization of the breakpoint on Yq, and between azoospermia and deletion of interval 6 on Yq.

We report on nine patients submitted to BMT with sex-matched donors and investigated by means of PCR amplification of the VNTRs ApoB, D1S80, DXS52, and D17S5. In all cases it was possible to detect a polymorphism able to distinguish between donor and patient cells, thus allowing us to recognize the presence of complete or mixed chimerism. In eight patients PCR analysis showed a complete chimerism during the entire follow-up. Only one of these patients relapsed, while the others are alive and without any sign of relapse 56.2 months (mean) after BMT. Mixed chimerism was detected in only one patient, who relapsed 3 months after this finding. These results confirm the usefulness of the study of PCR-amplified VNTRs in the assessment of marrow engraftment after BMT, mostly in sex-matched transplants where, in the absence of specific chromosome rearrangements, cytogenetic or FISH analysis cannot be used.

A population of 10,156 pregnant women with singleton pregnancies were screened by the integrated test. Risks were retrospectively recalculated for contingent test strategies with first step intermediate risk groups defined by first trimester upper cut-offs of 1 : 10, 1 : 30, 1 : 50, and 1 : 70 and lower cut-offs 1 : 1500, 1 : 1200, 1 : 1100, and 1 : 900. The second trimester high risk group was based on a single cut-off of 1 : 250.In the first trimester, the detection rate (DR) ranged from 21% (6/29) to 52% (15/29) as the high risk first trimester cut-off was changed from 1 : 10 to 1 : 70. The corresponding first trimester false positive rate (FPR) increased from 0.2% to 1.4%. In the second trimester, an additional 21/29 (72%) to 12/29 (41%) affected pregnancies could be detected with an additional 1.6% to 2.7% false positives when lower first trimester cut-offs of 1 : 900 to 1 : 1500 were used. The best results were obtained with the upper first trimester cut-off of 1 : 30 and lower first trimester cut-off of 1 : 900, which yielded a rate of women requiring a second trimester test of only 12%, with overall DR and FPR of 93% and 2.8%, respectively.Although the study population was relatively small, the results confirm the advantage of using contingent screening and suggest optimal first trimester cut-offs of 1 : 30 (lower cut-off) and 1 : 900 (upper cut-off).

The authors report on 13 patients with chronic myeloid leukemia (CML) studied by serial karyotyping and fluorescence in situ hybridization (FISH) of their bone marrow cells. Ten patients had complex translocations of the Ph chromosome while the remaining three were Ph negative. FISH analysis revealed in all 13 patients the translocation of the ABL protooncogene into chromosome 22 at band q11. Moreover, in all complex translocations but one, FISH with a chromosome 22 painting probe demonstrated on one chromosome 9 at band q34 the presence of material from chromosome 22, in addition to signals on the third chromosome involved in complex changes. Therefore, in this study complex translocations appeared as secondary changes resulting from two consecutive translocations with a total of at least four breaks. The first translocation gave rise to the standard t(9;22)(q34;q11). The second one included a break distal to the original breakpoint at band 9q34 and another one on a third chromosome. Furthermore FISH using S1 and S15 probes, mapped at band 22q11.2 or 22q12, gave evidence that in complex translocations the secondary breakpoint on der(9) was in the translocated segment 22q11-qter between bands q11 and q12. FISH analysis also disclosed the presence of material from chromosome 22 on one chromosome 9 in the three patients with Ph negative CML, demonstrating that in these cases a retranslocation between chromosomes 9q+ and 22q- had occurred. Consequently, the four-break mechanism could also be invoked for the three Ph negative CML patients.

To compare the efficacy of combined, stepwise sequential, and contingent screening versus the integrated test in detecting fetal aneuploidies.First trimester combined test, sequential second trimester, and contingent risks were retrospectively calculated for 7292 unselected pregnant women with singleton pregnancies who had received integrated screening. The first trimester testing was based on nuchal translucency, pregnancy-associated plasma protein-A, and free-beta-human chorionic gonadotrophin (free β-hCG) and the second trimester tests were alpha-fetoprotein, hCG, and unconjugated estriol. A second trimester risk of 1:250 defined a positive result for all protocols with the contingent protocol based on additional second trimester testing for those with risks between 1:30 and 1:1200.Among the population submitted for the integrated test, the detection rate was 19/21 (90%) for Down syndrome (DS) and 6/6 (100%) for Edwards syndrome (ES) and the DS false-positive rate (FPR) was 247/7271 (3.4%). Provision of the first trimester combined test alone would have resulted in a 17/21 (81%) detection rate for DS, that of 4/6 (67%) for ES and a DS FPR of 292/7271 (4.0%). The sequential and contingent approaches had the same final detection rates as the integrated test but potentially allowed a high proportion of the affected pregnancies to be detected in the first trimester. The lowest net DS FPR was seen with the contingent approach (2.6%) and using this protocol only 12.7% of women would have required second trimester testing.Integrated, sequential, and contingent screenings are all more efficacious than the combined test. Overall, the contingent approach was the most efficient with a high-detection rate, the lowest FPR, and the least amount of testing.

Thirteen male patients affected by different hematologic diseases who underwent bone marrow transplantation (BMT) with female donors were investigated by cytogenetic analysis and polymerase chain reaction (PCR) amplification of a DNA sequence specific for the Y chromosome. In six of these patients, PCR showed the presence of the Y chromosome-related sequence; in only three of these did cytogenetic analysis confirm the presence of mixed chimerism. In the remaining three patients, the results of the PCR were confirmed by in situ hybridization on cell nuclei with a probe for the alpha-satellite of the Y chromosome. We compare results obtained with the two methods and discuss the meaning of the minimal residual disease detected by PCR in patients submitted to BMT.

Mutations in the X-linked androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS), resulting in an impaired embryonic sex differentiation in 46,XY genetic men. Complete androgen insensitivity (CAIS) produces a female external phenotype, whereas cases with partial androgen insensitivity (PAIS) have various ambiguities of the genitalia. Mild androgen insensitivity (MAIS) is characterized by undermasculinization and gynecomastia. Here we describe a 2-month-old 46,XY female patient, with all of the characteristics of CAIS. Defects in testosterone (T) and dihydrotestosterone (DHT) synthesis were excluded. Sequencing of the AR gene showed the presence in exon 6 of a T to C transition in the second base of codon 790, nucleotide position 2369, causing a novel missense Leu790Pro mutation in the ligand-binding domain of the AR protein. The identification of a novel AR mutation in a girl with CAIS provides significant information due to the importance of missense mutations in the ligand-binding domain of the AR, which are able to induce functional abnormalities in the androgen binding capability, stabilization of active conformation, or interaction with coactivators.

Objective: To investigate the relationship between maternal serum screening markers and pregnancy outcome in fetuses with cystic hygroma at 15–18 weeks of gestation. Study design: We retrospectively reviewed case-notes of 34 consecutive singleton fetuses with cystic hygroma referred at 15–18 weeks of gestation. All cases had maternal blood sampled for triple screening at the time of the ultrasound scan. Results In total, 62% of fetuses with cystic hygroma had abnormal chromosome complements and 80% had a poor outcome. Six fetuses presenting normal values of human chorionic gonadotropin (0.5–2.5 MoM [multiples of the median]), serum alpha-fetoprotein (0.5–2.5 MoM) and unconjugated estriol (>0.5 MoM), normal karyotype and absence of associated structural anomalies had an uneventful outcome. Conclusions Our data demonstrated that cystic hygroma at 15–18 weeks has a strong association with chromosomal abnormalities. In euploid fetuses, maternal serum screening results may have a role in the diagnostic work-up of the pregnancy.

BCR/ABL-positive acute myeloid leukemia (AML) is a rare disease, characterized by a poor prognosis, with resistance to induction chemotherapy and frequent relapses in responsive patients. Here we report a case of BCR/ABL-positive AML-M6 who, after relapse, was treated with Imatinib Mesylate (600 mg/die) and within 4 months achieved a cytogenetic and molecular complete response. After more than 4 years of continuous Imatinib therapy, nested RT-PCR for BCR/ABL is persistently negative. The case reported shows that the response obtained with Imatinib Mesylate in BCR/ABL-positive AML may be long lasting, offering a chance of successful treatment for this poor prognosis group of patients.

Objective Isolated elevations in midtrimester maternal serum human chorionic gonadotrophin concentrations (MShCG) have been reported to be associated with a substantially increased likelihood of fetal congenital malformations. The reported malformations included a wide range of organ systems, originating at different embryologic developmental stages. The purpose of our study was to determine the significance of an isolated elevated MShCG (>2.5 MoM) in midtrimester for the detection of fetal structural anomalies in a large population. Methods Among 10 144 women who underwent a biochemical triple screen at 15 to 18 weeks' gestation, 463 patients, who had an elevated MShCG, but normal α-fetoprotein (AFP) and unconjugated estriol (uE3) levels, were identified. Patients with an integrated calculated Down syndrome risk above 1:250 were excluded. Only nonsmokers, at ages

BACKGROUND: This study analyses the prevalence of karyotype changes and Yq11 microdeletions among couples referred for assisted reproduction techniques. METHODS: Prior to receiving either IVF or ICSI treatment, each partner of 2078 infertile couples was screened for karyotype changes by GTG-banding technique on peripheral lymphocytes. No subject presented with obvious phenotype of chromosomal rearrangement. All the oligo/azoospermic men with normal karyotype were further investigated by PCR for Yq11 microdeletions. RESULTS: Eighty-two out of 2078 couples (3.95%) had one partner carrying a chromosomal change, and 10 out of 202 (4.95%) men showed Yq11 microdeletions. The chromosomal rearrangements were 44 (2.1%) translocations, 23 (1.1%) gonosomal mosaics, six (0.3%) 47,XXY, five (0.24%) marker chromosomes, three (0.14%) inversions and one (0.05%) duplication. Frequency of anomalies in men and women were similar: 42 and 40 cases respectively. CONCLUSIONS: Partners of infertile couples requiring IVF or ICSI treatment appear to be affected by higher frequency of chromosomal rearrangements than the general population. Categories with greater risk were represented by men with sperm cell count < 20 3 10 6 sperm/ml, and women with history of pregnancy loss.

Extra structurally abnormal chromosomes (ESACs) and cryptic rearrangements are often associated with mental retardation and phenotypic abnormalities. In some cases their characterisation, using standard cytogenetic techniques and fluorescence in situ hybridization (FISH), is difficult and time consuming, where a fast and accurate identification is essential, especially when such chromosomal aberrations are found in prenatal diagnosis. A recent molecular technique, spectral karyotyping (SKY), based on the spectral signature of 24 chromosome-specific painting probes labelled with different combinations of five fluorochromes, allows the simultaneous visualisation of all human chromosomes in different colours. We used SKY analysis on 14 cases with rare ESACs or cryptic unbalanced rearrangements found at pre- or postnatal diagnosis. SKY analysis permitted the classification of chromosome rearrangements in all 14 cases analysed in combination with FISH analysis.

Myelodysplastic syndrome (MDS) is an adult hematological disease that evolves into acute myeloid leukemia (AML) in about 30% of the cases. The availability of a highly specific probe moved us to perform in patients affected with MDS/AML, associated with normal karyotype, painting and fluorescence in situ hybridization (FISH) analysis aimed to check the inositide-specific phospholipase C (PI-PLC) beta1 gene, a player in the control of some checkpoints of the cell cycle. Here we present a preliminary observation in which FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PI-PLCbeta1 gene. On the contrary, PI-PLC beta4, another gene coding for a signaling molecule, located on 20p12.3 at a distance as far as less than 1Mb from PI-PLCbeta1, is unaffected in MDS patients with the deletion of PI-PLC beta1 gene, hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML. The data suggest the possible involvement of PI-PLCbeta1 in the progression of the disease and pave the way for a larger investigation aimed at identifying a possible high-risk group among MDS patients with a normal karyotype.

A newborn infant is reported who had aganglionic megacolon, renal hypoplasia, severe growth retardation, generalised hypotonia, and various dysmorphic features. Chromosome analysis of lymphocytes and fibroblasts showed a ring chromosome 10 with breakpoints at p13-15 and q26. AluI digestion showed that the ring chromosome was monocentric. FISH with an alpha satellite probe specific for chromosome 10 showed one signal only in about 20% of interphase nuclei. It is suggested that aganglionic megacolon could result from dynamic somatic mosaicism owing to loss of the ring chromosome.

The authors describe three patients with myelodysplastic syndrome without a history of exposure to chemical agents and who showed many chromosome rearrangements not previously reported in this hematologic disorder, and a rapid outcome of the disease. The authors discuss the significance of the chromosome changes, suggesting, in agreement with others, that patients with complex rearrangements have a poor prognosis.

The authors report on a sequential cytogenetic study carried out on 31 patients with acute leukemia (20 with acute lymphoblastic leukemia and 11 with acute non-lymphocytic leukemia) who underwent bone marrow transplantation (BMT). Engraftment was documented in all patients with sex-mismatched donors and with donor constitutional aberrations. During the follow-up, ranging from 6 to 110 months, clinical and hematologic relapse was observed in 11 patients (35.5%). Five of these cases showed a normal karyotype, 3 were of undefined relapse origin, 2 were aneuploid karyotypes, and one was donor (male) metaphases. Cytogenetic and immunologic data in the latter patient were suggestive of relapse in donor cells.

A patient with acute non-lymphoblastic leukemia, FAB type M2, showed a complex karyotype involving chromosomes 1 and 11. The breakpoints could not be exactly identified by GTG and QFQ banding. A subsequent analysis with Alul and HaeIII restriction enzyme staining allowed the detection of a translocation of the heterochromatic region of chromosome 1 to 11q21.

The restriction endonucleases HpaII and MspI both cleave the nucleotide sequence CCGG, but the action of HpaII is inhibited if the internal cytosine is methylated. HpaII and MspI were used on fixed chromosomes from bone marrow cells of individuals suffering from chronic myelogenous leukemia and healthy individuals. We found that MspI acts with the same efficiency on all chromosome samples, whereas HpaII extracts more DNA from the chromosomes of leukemic individuals than from the chromosomes of nonleukemic individuals. We postulate that demethylation of cytosine in the CpG dinucleotide of leukemic cell DNA accounts for our findings.

Cytogenetic data are reported from 16 patients with Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML) treated with bone marrow transplantation (BMT). The usefulness of cytogenetic investigations for the assessment of marrow engraftment is stressed. The significance of persistence or reappearance of Ph after BMT, possibly due to a defective leukemic clone eradication by the conditioning regimen, is also discussed. Generally, Ph-positive cells are damaged and disappear within the first year of BMT. Sometimes, however, the cells may repair the damage and proliferate again, resulting in disease relapse. Rarely, clinical and hematologic relapse does not follow Ph-positive clone expansion although leukemic cells represent more than 50% of marrow metaphases examined. Finally, the effect of interferon on Ph-positive clones after BMT and random chromosome changes, that appear transiently after BMT and are of uncertain significance, are discussed.