One-cell biopsy significantly improves the outcome of preimplantation genetic diagnosis (PGD) treatment: retrospective analysis of 569 PGD cycles at the Stockholm PGD centre.

STUDY QUESTION: What are the significant factors that influence the outcome of a PGD treatment? SUMMARY ANSWER: The age of the woman and the number of biopsied cells per embryo are of significant importance for a successful PGD treatment. WHAT IS KNOWN ALREADY: Younger women are more likely to succeed with an IVF treatment. STUDY DESIGN, SIZE, DURATION: Cohort study, retrospective analysis of 569 PGD cycles, 1996-2009. PARTICIPANTS, SETTING, METHODS: 256 couples and 569 PGD treatments at 'Stockholm PGD centre'. At this centre after 2003, a 1-cell policy was applied, when possible, with respect to the reliability of the diagnostic test and since 2009, 1-cell biopsy policy was also applied for monogenic disorders. MAIN RESULTS AND THE ROLE OF CHANCE: The women under 36 years of age were three times more likely to get pregnant after PGD treatment, P = 0.003 and odds ratio 3.1 [95% confidence interval (CI) 1.5-6.5]. The 1-cell biopsy cycles were twice as likely to result in a pregnancy in comparison with cycles were 2 cells were removed from the embryo, P = 0.0013 and odds ratio 2.55 (95% CI 1.44-4.52). No other factors were found to be significant for the outcome. LIMITATIONS, REASONS FOR CAUTION: Retrospective analysis with 1- and 2-cell biopsies at different times. WIDER IMPLICATIONS OF THE FINDINGS: The results will have an impact on the implementation of PGD in general, thereby making it possible to significantly improve the treatment outcome.

Improved structural characterization of chromosomal breakpoints using high resolution custom array-CGH.

Array-CGH is a powerful tool for the rapid detection of genomic imbalances. By customizing the array it is possible to increase the resolution in a targeted genomic region of interest and determine the structure of the breakpoints with high accuracy, as well as to detect very small imbalances. We have used targeted custom arrays to zoom in on 38 chromosomal breakpoints from 12 different patients carrying both balanced and unbalanced rearrangements. We show that it is possible to characterize unbalanced breakpoints within 17-20,000 bp, depending on the structure of the genome. All of the deletion and duplication breakpoints were further refined and potential underlying molecular mechanisms of formation are discussed. In one of seven carriers of apparently balanced reciprocal translocations we detected a small deletion of 200 bp within the previously FISH-defined breakpoint, and in another patient, a large deletion of 11 Mb was identified on a chromosome not involved in the translocation. Targeted custom oligonucleotide arrays make it possible to perform fine mapping of breakpoints with a resolution within the breakpoint region much higher compared to commercially available array platforms. In addition, identification of small deletions or duplications in apparently balanced rearrangements may contribute to the identification of new disease causing genes.

Detailed molecular and clinical characterization of three patients with 21q deletions.

We have investigated three patients with 21q deletions, two with developmental delay, dysmorphic features and internal organ malformations, and one with cognitive function within the normal range but with some deficits in gross and fine motor development. All aberrations were characterized by array-comparative genomic hybridization (array-CGH). In addition, extensive fluorescence in situ hybridization (FISH) mapping on metaphase chromosomes and mechanically stretched chromosomes was performed on patient 1 who had an extremely complex intrachromosomal rearrangement with 16 breakpoints, four deletions and four duplications. Patients 2 and 3 had interstitial deletions comprising 21q21.1-21q22.11 and 21q11.2-21q21.3, respectively. Partial deletions of 21q are rare and these patients display a highly variable phenotype depending on the size and position of the deletion. A review of the literature identified 38 cases with pure 21q deletions. Twenty-three of these had reliable mapping data. The combined information of present and previous cases suggests that the ITSN1 gene is involved in severe mental retardation in patients with 21q deletion. In addition, a critical region of 0.56 Mb containing four genes, KCNE1, DSCR1, CLIC6 and RUNX1, is associated with severe congenital heart defects, and deletions of the most proximal 15-17 Mb of 21q is associated with mild or no cognitive impairment, but may lead to problems with balance and motor function.

Concurrent microdeletion and duplication of 22q11.2.

Microduplication of 22q11.2 has been reported in fewer than 40 cases, all of them including the DiGeorge critical region (DGCR). We here present the characterization of a new duplication that does not include the DGCR. The duplication was initially found by multiplex ligation-dependent probe amplification analysis of 22q11.2 in a young girl with a concurrent deletion of the DGCR in 70% of her peripheral blood lymphocytes. Her phenotype included many of the features of the velocardiofacial syndrome, with velopharyngeal insufficiency, recurrent infections, learning and concentration problems as well as difficulties in social interactions. However, there were no congenital malformations, and her facial appearance was not typical for the syndrome. Further investigations included array comparative genomic hybridization (CGH) to size map the deletion/duplication and interphase fluorescent in situ hybridization to investigate mosaicism and the structure of the rearrangement. An identical duplication of this part of 22q11.2 has not been reported before, and the duplication itself seems to be associated with very mild or no symptoms. This study contributes to the growing knowledge regarding new deletions and duplications of 22q11.2, most of them mediated by the pre-disposing high number of low-copy repeats in the region.

Frequent amplification of the telomerase reverse transcriptase gene in human tumors.

Activation of telomerase is a crucial step during cellular immortalization and malignant transformation of human cells and requires the induction of the catalytic component, human telomerase reverse transcriptase (hTERT), encoded by the hTERT gene. It is poorly understood how the hTERT gene is activated in human cancer cells. In the present study, we examined the hTERT gene copy number in human cancer cell lines and in primary tumor tissues. Amplification of the hTERT gene was observed in 8 of 26 (31%) tumor cell lines and 17 of 58 (30%) primary tumors examined (8 of 21 lung tumors, 3 of 10 cervical tumors, 5 of 19 breast carcinomas, and 1 of 8 neuroblastomas). In addition, 13 of 26 (50%) cell lines and 13 of 58 (22%) primary tumors displayed gain of hTERT gene copies with 3-4 copies/cell. The present findings imply that the hTERT locus may be a frequent target for amplification during tumorigenesis and that this genetic event probably contributes to the dysregulation of telomerase activity occurring in human tumors.

Inv dup(22), del(22)(q11) and r(22) in the father of a child with DiGeorge syndrome.

We here report a unique inherited case of DiGeorge syndrome. The asymptomatic father had a mosaic karyotype with a 21q11 deletion in three different cell lines. In two of the cell lines there was an additional supernumerary inv dup(22) or an r(22), respectively. In the third cell line the del(22) was the sole anomaly. FISH analysis showed that both the inv dup(22) and the r(22) included the DGS region. We hypothesize that an inter-chromosomal recombination between inverted repeats, together with a recombination between sister chromatids during meiosis I, gave rise to a deletion of 22q11 as well as an inv dup(22) containing the DGS region. The inv dup(22) was later rearranged into a ring chromosome during mitosis which was subsequently lost during cell division, thereby resulting in three different cell lines. This is the first case reported with an inv dup(22) and a del(22)(q11) in the same cell line. Our findings support a related mechanism in the formation of these two rearrangements mediated by low-copy repeats.

Tetrasomy 18p de novo: parental origin and different mechanisms of formation.

We have used eight PCR-based DNA polymorphisms to determine the parental origin and mechanisms of formation in 9 patients with de novo nonmosaic tetrasomy 18p. The 9 patients, 4 girls and 5 boys, had clinical features characteristic of i(18p) syndrome. The supernumerary marker chromosome was identified by fluorescence in situ hybridization (FISH) analysis using centromeric probes and a flow-sorted 18p-specific library. The isochromosome was of maternal origin in all 9 cases. The formation of tetrasomy 18p cannot be explained by a single model. In 6 cases, meiosis II nondisjunction, followed by subsequent postzygotic misdivsion, and in 1 case postzygotic nondisjunction and postzygotic misdivision were the most likely mechanisms of formation. Alternative mechanisms are suggested in the remaining 2 cases.

Origin of nondisjunction in trisomy 8 and trisomy 8 mosaicism.

Causes of chromosomal nondisjunction is one of the remaining unanswered questions in human genetics. In order to increase our understanding of the mechanisms underlying nondisjunction we have performed a molecular study on trisomy 8 and trisomy 8 mosaicism. We report the results on analyses of 26 probands (and parents) using 19 microsatellite DNA markers mapping along the length of chromosome 8. The 26 cases represented 20 live births, four spontaneous abortions, and two prenatal diagnoses (CVS). The results of the nondisjunction studies show that 20 cases (13 maternal, 7 paternal) were probably due to mitotic (postzygotic) duplication as reduction to homozygosity of all informative markers was observed and as no third allele was ever detected. Only two cases from spontaneous abortions were due to maternal meiotic nondisjunction. In four cases we were not able to detect the extra chromosome due to a low level of mosaicism. These results are in contrast to the common autosomal trisomies (including mosaics), where the majority of cases are due to errors in maternal meiosis.

Maternal isodisomy of chromosome 9 with no impact on the phenotype in a woman with two isochromosomes: i(9p) and i(9q).

We describe a 34-year-old healthy woman with isochromosomes for the short and long arm of chromosome 9 who was ascertained because of repeated spontaneous abortions. Molecular analysis demonstrated maternal uniparental isodisomy for the whole chromosome 9, thus the origin of the isochromosomes was maternal. Because the patient had a normal phenotype, the maternal isodisomy supports the previous assumption that there are no maternally imprinted genes on chromosome 9.

Trisomy 5q12-->q13.3 in a patient with add(13q): characterization of an interchromosomal insertion by forward and reverse chromosome painting.

We report on a patient with azoospermia, mild mental retardation, and minor physical anomalies. Chromosome analysis demonstrated the presence of additional material on the long arm of one chromosome 13. Forward chromosome painting using chromosome-specific libraries showed an insertion of material from chromosome 5. Further characterization with flow sorting of the aberrant chromosome and amplification by DOP-PCR followed by reverse chromosome painting showed specific trisomy of 5q12-->q13.3.

Monosomy 1p36.31-33-->pter due to a paternal reciprocal translocation: prognostic significance of FISH analysis.

A rare monosomy 1p36.31-33-->pter was found in a child with physical anomalies, psycho-motor retardation, and seizures. Cytogenetic investigation suggested an unbalanced translocation between 1p and an acrocentric chromosome, but the rearrangement was difficult to assess accurately using conventional chromosome banding techniques. The half-cryptic translocation was further characterized using fluorescence in situ hybridization, and the aberrant chromosome 1 was shown to be a derivate of a paternal reciprocal translocation t(1;15) (p36.31-33;p11.2-12). The breakpoints on chromosome 1 and 15 were defined in detail using locus specific probes. The rearrangement did not include the region on chromosome 1p which previously has been suggested to predispose to the development of neuroblastoma in a case with a constitutional translocation. At 3 6/12 years, the patient has no clinical signs of this disease, which illustrates the prognostic significance of this investigation.

Molecular cytogenetic characterization and origin of two de novo duplication 9p cases.

We report on two additional cases with duplication of 9p, minor with facial anomalies and developmental delay. Using fluorescence in situ hybridization and single-copy probes, we showed that the first case was a direct duplication, whereas the second case was inverted. The extent of the direct duplication was defined as 9p12 --> p24 by microdissection and microcloning of the aberrant chromosome and subsequent chromosome-specific comparative genomic hybridization. DNA polymorphism analysis with eight microsatellite markers revealed that the origin of the dup(9p) was maternal in the first case, whereas it was paternal in the second.

Fifty probands with extra structurally abnormal chromosomes characterized by fluorescence in situ hybridization.

Extra structurally abnormal chromosomes (ESACs) are small supernumerary chromosomes often associated with developmental abnormalities and malformations. We present 50 probands with ESACs characterized by fluorescence in situ hybridization using centromere-specific probes and chromosome-specific libraries. ESAC-specific libraries were constructed by flow sorting and subsequent amplification by DOP-PCR. Using such ESAC-specific libraries we were able to outline the chromosome regions involved. Twenty-three of the 50 ESACs were inverted duplications of chromosome 15 [inv dup(15)], including patients with normal phenotypes and others with similar clinical symptoms. These 2 groups differed in size and shape of the inv dup(15). Patients with a large inv dup(15), which included the Prader-Willi region, had a high risk of abnormality, whereas patients with a small inv dup(15), not including the Prader-Willi region, were normal. ESACs derived from chromosomes 13 or 21 appeared to have a low risk of abnormality, while one out of 3 patients with an ESAC derived from chromosome 14 had discrete symptoms. One out of 3 patients with an ESAC derived from chromosome 22 had severe anomalies, corresponding to some of the manifestations of the cat eye syndrome. Small extra ring chromosomes of autosomal origin and ESACs identified as i(12p) or i(18p) were all associated with a high risk of abnormality.

Detailed characterization of 12 supernumerary ring chromosomes using micro-FISH and search for uniparental disomy.

Twelve patients with varying degrees of mosaicism for a supernumerary ring chromosome were studied. The ring chromosomes were characterized using microdissection in combination with degenerate nucleotide-primed polymerase chain reaction (PCR) and reverse painting (micro-FISH). This method made it possible to determine the chromosomal origin of the ring chromosomes in detail, and thus to compare the phenotypes of similar cases. Eleven of the marker chromosomes were derived from the most proximal part of 1p, 3p, 3q, 5p, 7q, 8p, 8q, 9p, 10p and 20p. One marker chromosome had a complex origin, including the proximal and the most distal part of 20q. Eight of the families were also investigated for uniparental disomy (UPD) using microsatellite analysis. One case with maternal UPD 9 was found in a child with a ring chromosome derived from chromosome 9, r(9)(p10p12).

Detailed characterization of a complex karyotype in a patient with primary plasma cell leukaemia using multicolour spectral karyotyping and micro-FISH.

INTRODUCTION: Plasma cell leukaemia is a rare disorder that usually carries an aggressive course with a rapidly fatal outcome. A variety of chromosomal abnormalities have been reported in plasma cell leukaemia but the clinical significance of an abnormal karyotype is still unclear. MATERIALS AND METHODS: We have applied the molecular cytogenetic techniques multicolour spectral karyotyping and microdissection in combination with fluorescence in situ hybridization on metaphases from a patient with primary plasma cell leukaemia and a fatal outcome. RESULTS AND CONCLUSION: The chromosome analysis showed severe hypodiploidy and 12 marker chromosomes. Identification of the structural rearrangements was not possible using routine cytogenetic methods. Utilizing the methods above, all marker chromosomes could be identified in detail and the karyotype was shown to be very complex. Forty-three breakpoints were found, and 25 could be identified at the band level, among others 14q32 where the immunoglobulin heavy chain locus is situated. Thus, these techniques provide the opportunity to resolve very complex chromosomal changes in a way that has not been previously possible and will consequently be of great importance in the search for hot spots that may harbour new cancer genes.

Physical state of HPV16 and chromosomal mapping of the integrated form in cervical carcinomas.

Using a procedure based on restriction enzyme cleavage, self-ligation, and inverse polymerase chain reaction (rliPCR), the authors investigated 18 cervical intraepithelial neoplasia III (CIN III) cases and 37 invasive squamous carcinomas for integration of human papillomavirus type 16 (HPV16). All eighteen CIN III cases (severe dysplasia or high-grade squamous intraepithelial lesion) were found to harbor episomal HPV, but one of the samples contained mixed episomal and integrated forms. Seventeen of 37 invasive cervical carcinoma samples were identified previously as containing the completely integrated HPV16 genome by using PCR covering the entire E1/E2 gene, and this was confirmed by rliPCR in 16 cases. One case, however, showed a low level of episomal deoxyribonucleic acid in addition to the predominant integrated form. Of the remaining 20 carcinoma samples showing episomal forms in the previous analysis, 14 were found to contain integrated forms using rliPCR, and four contained multimeric episomal forms. Thus, in total, 31 of 37 of the carcinomas (84%) showed the integrated HPV16 genome. The rliPCR product from five carcinoma cases was cloned into a plasmid vector and used as a template for "primer walking" deoxyribonucleic acid sequencing to deduce human sequences flanking the integrated HPV genome. Based on this information, bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones were obtained and used as probes in fluorescent in situ hybridization experiments on human metaphase chromosomes. The results of the fluorescent in situ hybridization experiments showed evidence for HPV16 integration in chromosome regions 1q25, 3q28, 6p25, 11p13, and 18q22. Sixteen carcinoma samples, containing episomal HPV16, were sequenced in the long control region. Evidence for changes in E2 binding or silencer YY1 sequences was found in only two samples.

A case of acute lymphoblastic leukemia, near-triploidy, and poor outcome: characterization by fluorescence in situ hybridization using chromosome-specific libraries from all human chromosomes.

We have applied fluorescence in situ hybridization (FISH) using chromosome-specific libraries from all 24 chromosomes on metaphase spreads from bone marrow cells, in order to resolve the chromosomal changes in leukemic cells from a 10-year-old boy with acute lymphoblastic leukemia (ALL), near-triploidy, and a subsequent poor outcome. The FISH analysis revealed a pattern of chromosome gains and losses that differed from all cases previously described. Most of the affected chromosomes were present in three copies (trisomy for chromosomes 1, 2, 5, 6, 7, 11, 12, 13, 14, 16, 17, 18, 19, 20, and 22), but the patient had four copies of chromosomes 8 and 21, two copies of chromosomes 3, 4, 9, 10, 15, and X, and one Y chromosome. No structural abnormalities could be detected. Thus, the karyotype of the malignant clone was 66,XXY-3,-4,+8,-9,-10,-15,+21.

DNA image analysis in childhood acute lymphoblastic leukemia.

DNA index (DI) and percentages of cells in S and G2/M phase were determined in Feulgen stained nuclei of blasts from 31 cases of childhood ALL at diagnosis. In 6 cases the results of DNA analysis and cytogenetics were concordant showing hyperdiploidy. Two other cases with normal karyotype were revealed as DNA aneuploid with image analysis. Cases with cytogenetic abnormalities like translocation, deletion or presence of single or double supernumerary chromosomes had DI within normal ranges. Nine ALL cases (29%) were found to be DNA aneuploid--8 hyperdiploid and 1 hypodiploid. The percentages of cells in S and G2/M phase for blasts from bone marrow (mean 17.6%) were significantly higher than those estimated in the peripheral blood (mean 1.57%). We conclude that analysis by image cytometry can detect aneuploid DNA content even in cases, which showed a normal karyotype and provides new information concerning the biological aspects of leukemic blasts.

New chromosomal breakpoints in non-Hodgkin's lymphomas revealed by spectral karyotyping and G-banding.

Chromosomal rearrangements in short term cultures from nine cases of non-Hodgkin's lymphomas (NHL) were characterized by G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). Eight of the nine cases showed complex karyotypes with chromosomal aberrations which, in most cases, could not be fully characterized by traditional G-banding analysis alone. Karyotypic abnormalities of special interest were marker chromosomes and chromosomes with added unidentified chromosomal material, as previously non-identified chromosomal translocations were hidden behind these aberrations. SKY and FISH analysis, as a complement to banding analysis, significantly improved the karyotypes in seven of the nine cases and unveiled 21 previously unidentified rearrangements with novel translocation breakpoints. Traditional G-banding alone revealed seven new rearrangements, which were all confirmed by SKY. None of these new aberrations occurred as single clonal rearrangements but as parts of complex karyotypes. Nevertheless, the chromosomal break-point regions identified should be considered as potential hot spots for genes involved in the tumorigenesis of the malignancy.

Generation of dendritic cells from peripheral blood of patients at different stages of chronic myeloid leukemia.

We report a method to generate dendritic cells (DC) from frozen leukapheresis products of patients with chronic myeloid leukemia (CML), using sterile culture bags and serum-free culture medium, ie conditions feasible for re-infusion into the patient as part of immunotherapeutic protocols. Leukapheresis products were stored from harvests performed either at diagnosis (13 patients) or after chemotherapy with subsequent granulocyte colony stimulating factor (G-CSF) administration (9 patients), for Peripheral Blood Stem Cell (PBSC) collections. In the presence of optimal concentrations of GM-CSF (50 ng/ml) and IL-4 (40 ng/ml) CML progenitors differentiated on day 7 and 14 of culture to DC, expressing CD1a,HLA-DR and CD86 surface antigens. Mature DCs exhibited on average 12-fold higher allo-stimulatory capacity for CD4+ and CD8+ cells compared to non-cultured PBMC in mixed lymphocyte reaction (MLR). Only DCs obtained from CML patients at diagnosis exhibited bcr/abl fusion gene when tested by fluorescent in situ hybridization (FISH). CD34-selection on leukapheresis products from diagnosis (7 patients) resulted in later maturation of DCs (after 14-15 d), compared to the nonselected PBMC. CD34-selection significantly increased the DC growth, and improved the allo-stimulatory capacity in MLR (on average on day 14, 3.5- and 2.3-fold, respectively). Large differences were observed between individual patients and different leukapheresis products from the same patient. Our report demonstrates the possibility to generate ex vivo autologous functionally active DC in CML in a way that allows their clinical application as immunotherapeutic agents.