Klinefelter syndrome and fertility-Impact of X-chromosomal inheritance on spermatogenesis.

With the use of testicular sperm extraction (TESE), spermatozoa can be retrieved in about 30%-50% of men with Klinefelter syndrome (KS). The reason for the absence or presence of spermatozoa in half of the men with KS remains unknown. Therefore, the search for an objective marker for a positive prediction in finding spermatozoa is of significant clinical value to avoid unnecessary testicular biopsies in males with (mostly) low testicular volume and impaired testosterone. The objective of this study was to determine whether paternal or maternal inheritance of the additional X-chromosome can predict the absence or presence of spermatogenesis in men with KS. Men with KS who have had a testicular biopsy for diagnostic fertility workup TESE were eligible for inclusion. Buccal swabs from nine KS patients and parents (trios) were taken to compare X-chromosomal inheritance to determine the parental origin of both X-chromosomes in the males with KS. Spermatozoa were found in TESE biopsies 8 of 35 (23%) patients after performing a unilateral or bilateral TESE. Different levels of spermatogenesis (from the only presence of spermatogonia, up to maturation arrest or hypospermatogenesis) appeared to be present in 19 of 35 (54%) men, meaning that the presence of spermatogenesis not always yields mature spermatozoa. From the nine KS-trios that were genetically analysed for X-chromosomal inheritance origin, no evidence of a correlation between the maternal or paternal origin of the additional X-chromosome and the presence of spermatogenesis was found. In conclusion, the maternal or paternal origin of the additional X-chromosome in men with KS does not predict the presence or absence of spermatogenesis.

Klinefelter syndrome and fertility: sperm preservation should not be offered to children with Klinefelter syndrome.

STUDY QUESTION: Should fertility preservation be offered to children with Klinefelter syndrome (KS)? SUMMARY ANSWER: Current evidence shows that fertility preservation should not be offered to adolescents with KS younger than 16 years because of lower retrieval rates for germ cells by testicular sperm extraction (TESE) compared with retrieval rates for adolescents and adults between 16 and 30 years. WHAT IS KNOWN ALREADY: KS, the most common chromosomal disorder in men leading to non-obstructive azoospermia, is caused by the presence of at least one additional X chromosome. The onset of puberty in adolescents with KS leads to progressive degeneration of the testicular environment. The impact of the subsequent tissue degeneration on fertility potential of patients with KS is unknown, but in previous literature it has been suggested that fertility preservation should be started in adolescents as early as possible. However spermatozoa can be found by TESE in about 50% of adults with KS despite severe testicular degeneration. This review discusses the current evidence for fertility preservation in children and adolescents and possible prognostic markers for fertility treatment in KS. STUDY DESIGN, SIZE, DURATION: An extensive literature search was conducted, searching Pubmed, Embase, Cinahl and Web of Science from origin until April 2016 for 'Klinefelter syndrome' and 'fertility' and various synonyms. Titles and abstracts have been scanned manually by the authors for eligibility. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total 76 studies were found to be eligible for inclusion in this review. Information from the papers was extracted separately by two authors. MAIN RESULTS AND THE ROLE OF CHANCE: Various studies have shown that pre-pubertal children with KS already have a reduced number of germ cells despite a normal hormonal profile during childhood. The presence of spermatozoa in the ejaculate of adolescents with KS is extremely rare. Using TESE, the retrieval rates of spermatozoa for adolescents younger than 16 years old are much lower (0-20%) compared with those for adolescents and young adults between 16 and 30 years old (40-70%). Although spermatogonia can be found by TESE in about half of the peri-pubertal adolescents, there are currently no clinically functional techniques for their future use. Children and adolescents need to be informed that early fertility preservation before the age of 16 cannot guarantee fertility later in life and may even reduce the chances for offspring by removing functional immature germ cells which may possibly develop into spermatozoa after puberty. Furthermore, except for the age of patients with KS, there are no identified factors that can reliably be used as a predictive marker for fertility preservation. LIMITATIONS, REASONS FOR CAUTION: Most of the evidence presented in this review is based on studies including a small number of adolescents with KS. Therefore, the studies may have been underpowered to detect clinically significant differences for their various outcomes, especially for potential predictive factors for fertility preservation, such as hormone levels. Furthermore, the population of patients with KS diagnosed during childhood might be different from the adult population with KS where the diagnosis is based on infertility. Results based on comparisons between the two groups must be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: Despite the limitations, this review summarizes the current evidence for managing fertility preservation in patients with KS to provide optimal health care. STUDY FUNDING/COMPETING INTERESTS: There was no funding for this study. S.F., Y.H., K.D., W.L.M.N., D.S., H.L.C.-v.d.G. and L.R. declare to have no conflicts of interests. D.D.M.B. reports grants from Merck Serono, grants from Ferring and grants from MSD, outside the submitted work. K.F. reports personal fees from MSD (commercial sponsor), personal fees from Ferring (commercial sponsor), grants from Merck-Serono (commercial sponsor), grants from Ferring (commercial sponsor) and grants from MSD (commercial sponsor), outside the submitted work.

Detection of cross-sex chimerism in the common marmoset monkey (Callithrix jacchus) in interphase cells using fluorescence in situ hybridisation probes specific for the marmoset X and Y chromosomes.

Chimerism associated with placental sharing in marmosets has been traditionally analysed using conventional chromosome staining on metaphase spreads or polymerase chain reaction. However, the former technique requires the presence of proliferating cells, whereas the latter may be associated with possible blood cell contamination. Therefore, we aimed to develop a single-cell analysis technique for sexing marmoset cells. We applied fluorescent in situ hybridisation (FISH) to cell nuclei using differentially labelled X and Y chromosome-specific probes. Herein we present the validation of this method in metaphase cells from a marmoset lymphoblastoid cell line, as well as application of the method for evaluation of cross-sex chimerism in interphase blood lymphocytes and haematopoietic bone marrow cells from marmosets of same- and mixed-sex litters. The results show conclusively that haematopoietic cells of bone marrow and leucocytes from blood are cross-sex chimeric when the litter is mixed sex. In addition, single samples of liver and spleen cell suspensions from one individual were tested. Cross-sex chimerism was observed in the spleen but not in liver cells. We conclude that FISH is the method of choice to identify cross-sex chimerism, especially when combined with morphological identification of nuclei of different cell types, which will allow a targeted tissue-specific analysis.

Long-term effects of oxandrolone treatment in childhood on neurocognition, quality of life and social-emotional functioning in young adults with Turner syndrome.

Turner syndrome (TS) is the result of (partial) absence of one X-chromosome. Besides short stature, gonadal dysgenesis and other physical aspects, TS women have typical psychological features. Since psychological effects of androgen exposure in childhood probably are long-lasting, we explored long-term psychological functioning after oxandrolone (Ox) therapy during childhood in adults with TS in terms of neurocognition, quality of life and social-emotional functioning. During the initial study, girls were treated with growth hormone (GH) combined with placebo (Pl), Ox 0.03 mg/kg/day, or Ox 0.06 mg/kg/day from the age of eight, and estrogen from the age of twelve. Sixty-eight women participated in the current double-blinded follow-up study (mean age 24.0 years, mean time since stopping GH/Ox 8.7 years). We found no effects on neurocognition. Concerning quality of life women treated with Ox had higher anxiety levels (STAI 37.4 ± 8.4 vs 31.8 ± 5.0, p=0.002) and higher scores on the depression subscale of the SCL-90-R (25.7 ± 10.7 vs 20.5 ± 4.7, p=0.01). Regarding social-emotional functioning, emotion perception for fearful faces was lower in the Ox-treated patients, without effect on interpersonal behavior. Our exploratory study is the first to suggest that androgen treatment in adolescence possibly has long-term effects on adult quality of life and social-emotional functioning. However, differences are small and clinical implications of our results seem limited. Therefore we would not recommend against the use of Ox in light of psychological consequences.

The rs1800716 variant in CYP2D6 is associated with an increased double endometrial thickness in postmenopausal women on tamoxifen.

BACKGROUND: Tamoxifen remains important in the treatment and prevention of estrogen receptor-positive breast cancer. In postmenopausal women, it can lead to endometrial changes such as cystic appearances, hyperplasia, polyps and endometrial cancer. Tamoxifen is metabolized by cytochrome P450 (CYP450) enzymes to the more active metabolite endoxifen. Several genetic variants in the CYP450 enzymes reduce tamoxifen metabolism, leading to reduced endoxifen levels. We hypothesize that carriers of these variants, which are established poor metabolizers of tamoxifen, do not have the typical tamoxifen-induced increase in endometrial thickness. We test the association between genetic variability in CYP450 enzymes and the increase in double endometrial thickness (DET) as measured through transvaginal ultrasound (TVU). PATIENTS AND METHODS: We carried out a retrospective study on postmenopausal tamoxifen users for which germline DNA was available and at least one DET measurement was made between January 2000 and October 2011. Genotyping of 33 single nucleotide polymorphisms in CYP450 genes involved in tamoxifen metabolism was carried out using Sequenom MassARRAY. The association between these variants and TVU outcome (DET ≥5 mm) was assessed by proportional hazards regression. RESULTS: Data were available for 184 women: 47 with a DET of <5 mm on all ultrasounds and 137 with a DET of ≥5 mm on at least one ultrasound. The rs1800716 variant in CYP2D6 showed a statistically significant association with DET. In particular, mutant carriers of rs1800716 had an increased chance of having a DET of ≥5 mm (P = 0.0022, false discovery rate 0.0179). None of the other variants were associated with DET. CONCLUSION: Although mutant carriers of rs1800716 are characterized by reduced CYP2D6 enzyme activity and by low levels of endoxifen, we observed that mutant alleles of rs1800716 were associated with an increased chance of having a DET of ≥5 mm in postmenopausal women on tamoxifen. We conclude that the increase in endometrial thickness seen under tamoxifen cannot be used as a marker for favorable genotypes. CLINICAL TRIAL NUMBER: B32220084284.

Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer.

BACKGROUND: Molecular screening programs use next-generation sequencing (NGS) of cancer gene panels to analyze metastatic biopsies. We interrogated whether plasma could be used as an alternative to metastatic biopsies. PATIENTS AND METHODS: The Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze 69 tumor (primary/metastases) and 31 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tumor DNA was ×1000 and for plasma cell-free DNA ×25 000. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used to confirm observed mutations. RESULTS: Evaluable NGS results were obtained for 60 tumor and 31 plasma samples from 17 patients. When tumor samples were analyzed, 12 of 17 (71%, 95% confidence interval (CI) 44% to 90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma samples were analyzed, 12 of 17 (71%, 95% CI: 44-90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All mutations were confirmed. When we focused on tumor and plasma samples collected at the same time-point, we observed that, in four patients, no mutation was identified in either tumor or plasma; in nine patients, the same mutations was identified in tumor and plasma; in two patients, a mutation was identified in tumor but not in plasma; in two patients, a mutation was identified in plasma but not in tumor. Thus, in 13 of 17 (76%, 95% CI 50% to 93%) patients, tumor and plasma provided concordant results whereas in 4 of 17 (24%, 95% CI 7% to 50%) patients, the results were discordant, providing complementary information. CONCLUSION: Plasma can be prospectively tested as an alternative to metastatic biopsies in molecular screening programs.

Positional cloning of the gene for Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS), also known as ataxia-telangiectasia (AT) variant, is an autosomal recessive disorder characterized by microcephaly, growth retardation, severe combined immunodeficiency and a high incidence of lymphoid cancers. Cells from NBS patients display chromosome instability, hypersensitivity to ionizing radiation and abnormal cell-cycle regulation after irradiation, all of which are characteristics shared with AT. Recently, the NBS locus was mapped at 8q21 by two independent approaches, complementation studies and linkage analysis. Here, we report the positional cloning of the NBS gene, NBS1, from an 800-kb candidate region. The gene comprises 50 kb and encodes a protein of 754 amino acids. The amino-terminal region of the protein shows weak homology to the yeast XRS2, MEK1, CDS1 and SPK1 proteins. The gene is expressed at high levels in the testes, suggesting that it might be involved in meiotic recombination. We detected the same 5-bp deletion in 13 individuals, and conclude that it is likely to be a founder mutation.

The TERT-CLPTM1L locus for lung cancer predisposes to bronchial obstruction and emphysema.

Clinical studies suggest that bronchial obstruction and emphysema increase susceptibility to lung cancer. We assessed the possibility of a common genetic origin and investigated whether the lung cancer susceptibility locus on chromosome 5p15.33 increases the risk for bronchial obstruction and emphysema. Three variants in the 5p15.33 locus encompassing the TERT and CLPTM1L genes were genotyped in 777 heavy smokers and 212 lung cancer patients. Participants underwent pulmonary function tests and computed tomography of the chest, and completed questionnaires assessing smoking behaviour. The rs31489 C-allele correlated with reduced forced expiratory volume in 1 s (p=0.006). Homozygous carriers of the rs31489 C-allele exhibited increased susceptibility to bronchial obstruction (OR 1.82, 95% CI 1.24-2.69; p=0.002). A similar association was observed for diffusing capacity of the lung for carbon monoxide (p=0.004). Consistent with this, CC-carriers had an increased risk of emphysema (OR 2.04, 95% CI 1.41-2.94; p=1.73 × 10(-4)) and displayed greater alveolar destruction. Finally, CC-carriers also had an increased risk for lung cancer (OR 1.90, 95% CI 1.21-2.99; p=0.005), and were more susceptible to developing both lung cancer and bronchial obstruction than lung cancer alone (OR 2.11, 95% CI 1.04-4.26; p=0.038). The rs31489 variant on 5p15.33 is associated with bronchial obstruction, presence and severity of emphysema, and lung cancer.

SNP array analysis in constitutional and cancer genome diagnostics--copy number variants, genotyping and quality control.

Array-based comparative genomic hybridization analysis of genomic DNA was first applied in postnatal diagnosis for patients with intellectual disability (ID) and/or congenital anomalies (CA). Genome-wide single-nucleotide polymorphism (SNP) array analysis was subsequently implemented as the first line diagnostic test for ID/CA patients in our laboratory in 2009, because its diagnostic yield is significantly higher than that of routine cytogenetic analysis. In addition to the detection of copy number variations, the genotype information obtained with SNP array analysis enables the detection of stretches of homozygosity and thereby the possible identification of recessive disease genes, mosaic aneuploidy, or uniparental disomy. Patient-parent (trio) information analysis is used to screen for the presence of any form of uniparental disomy in the patient and can determine the parental origin of a de novo copy number variation. Moreover, the outcome of a genotype analysis is used as a final quality control by ruling out potential sample mismatches due to non-paternity or sample mix-up. SNP array analysis is now also used in our laboratory for patients with disorders for which locus heterogeneity is known (homozygosity pre-screening), in prenatal diagnosis in case of structural ultrasound anomalies, and for patients with leukemia. In this report, we summarize our array findings and experiences in the various diagnostic applications and demonstrate the power of a SNP-based array platform for molecular karyotyping, because it not only significantly improves the diagnostic yield in both constitutional and cancer genome diagnostics, but it also enhances the quality of the diagnostic laboratory workflow.

Changes in yearly birth prevalence rates of children with Down syndrome in the period 1986-2007 in The Netherlands.

BACKGROUND: The Netherlands are lacking reliable national empirical data in relation to the development of birth prevalence of Down syndrome. Our study aims at assessing valid national live birth prevalence rates for the period 1986-2007. METHOD: On the basis of the annual child/adult ratio of Down syndrome diagnoses in five out of the eight Dutch cytogenetic centres, the national annual figures of the National Cytogenetic Network on total numbers of postnatal Down syndrome diagnoses were transformed into national figures on total numbers of postnatal Down syndrome diagnoses in newborn children only. In combination with the national annual data of the Working Group for Prenatal Diagnostics and Therapeutics on numbers of Down syndrome pregnancies not aborted after diagnosis, national figures on birth prevalence were constructed. RESULTS: For the period 1986-2007, results based on the data of the cytogenetic centres are almost similar to the theory-based model data of de Graaf et al., with a small discrepancy of approximately 4%. Down syndrome birth prevalence in the Netherlands shows an upward trend from around 11 per 10,000 births in the early 1990s to around 14 per 10,000 births nowadays. CONCLUSION: In spite of expansion of antenatal screening in the Netherlands, Down syndrome live birth prevalence has risen in the last two decades as a result of rising maternal age. This increase in Down syndrome birth prevalence is in contrast to studies from other European countries.

Artificial neural networks applied to the analysis of synchrotron nuclear resonant scattering data.

The capabilities of artificial neural networks (ANNs) have been investigated for the analysis of nuclear resonant scattering (NRS) data obtained at a synchrotron source. The major advantage of ANNs over conventional analysis methods is that, after an initial training phase, the analysis is fully automatic and practically instantaneous, which allows for a direct intervention of the experimentalist on-site. This is particularly interesting for NRS experiments, where large amounts of data are obtained in very short time intervals and where the conventional analysis method may become quite time-consuming and complicated. To test the capability of ANNs for the automation of the NRS data analysis, a neural network was trained and applied to the specific case of an Fe/Cr multilayer. It was shown how the hyperfine field parameters of the system could be extracted from the experimental NRS spectra. The reliability and accuracy of the ANN was verified by comparing the output of the network with the results obtained by conventional data analysis.

Study of reorientation processes in L10-ordered FePt thin films.

We report on the development of structural and magnetic order in epitaxially grown L10 FePt thin films. Upon annealing, the easy axis of magnetization changes from the out-of-plain into the in-plain direction. We found that the overall fraction of reoriented domains first increases but after certain time decreases before achieving a saturated state. The results are based on conversion electron Mössbauer spectroscopy studies and confirm Monte Carlo simulations in nano-layered FePt. We present a modified version of the Johnson-Mehl-Avrami (JMA) model adequately describing the experimental findings. Two dynamical processes, the first being a 2D-growth, dominate the initial state of sample annealing and the second being a 3D-growth, dominate the late stage close to saturation. From an Arrhenius plots of JMA coefficients for both processes we extracted the activation energies of the underlying dynamics which are 1.5(1) eV for disordering and 0.8(2) eV for ordering.

Iron silicide nanostructure formation on Au induced superstructures on Si(111).

Using scanning tunnelling microscopy we have investigated the formation of low dimensional Fe-Si structures on Au covered Si(111) surfaces. The ultrathin Au layer induces a variety of surface reconstructions, depending on the coverage and temperature: Si(111)-5 x 2, [alpha-squareroot 3 x squareroot 3,beta-squareroot 3 x squareroot 3], and 6 x 6-Au. The subsequent deposition of 0.28 ML (monolayers) of Fe at 400 degrees C results in the formation of Fe-Si nanostructures whose morphological properties critically depend on the underlying surface. All Au induced reconstructions give rise to an increase in diffusion length as compared to the bare Si(111)-7 x 7 surface, thereby allowing the growth of well-separated nanostructures at considerably lower temperatures. Ultimately, the decoupling of surface diffusion and temperature, induced by the Au layer, can be exploited to tailor the island dimensions and density. With an appropriate choice of substrate, passivating layer and deposited material, nanostructures with the desired properties can be grown in a controlled way.

Protein profiling of non-malignant and malignant ascites by SELDI-TOF MS: proof of principle.

Ascites is a common clinical symptom in liver cirrhosis, inflammatory disorders of the abdomen and a major late manifestation of metastatic malignancies. Standard cytopathological techniques and immunocytochemistry have specificities and sensitivities of approximately 95 and 60%, respectively for the presence of tumor cells. Development of faster and more accurate screening methods would be of great clinical utility. In this work we examined differential analysis of the unbound proteins in the supernatant of ascites fluid by Protein-Chip SELDI mass spectrometry. There were 21 tumor cell-positive and 34 tumor cell-negative samples. We used principal component analysis coupled with linear regression applied to the mass spectra of the samples to distinguish between the sample groups. Two sample sets for statistical analysis were created after randomization, a training set with 37 samples and a validation set with 18 samples resulting in a specificity of 93% and a sensitivity of 83% on the training set. The validation set yielded a specificity and sensitivity of 75%. This study suggests that SELDI-TOF mass spectrometry appears to have great potential as a surrogate diagnostic tool to evaluate effusion specimens.

Clinical and molecular characteristics of 1qter microdeletion syndrome: delineating a critical region for corpus callosum agenesis/hypogenesis.

BACKGROUND: Patients with a microscopically visible deletion of the distal part of the long arm of chromosome 1 have a recognisable phenotype, including mental retardation, microcephaly, growth retardation, a distinct facial appearance and various midline defects including corpus callosum abnormalities, cardiac, gastro-oesophageal and urogenital defects, as well as various central nervous system anomalies. Patients with a submicroscopic, subtelomeric 1qter deletion have a similar phenotype, suggesting that the main phenotype of these patients is caused by haploinsufficiency of genes in this region. OBJECTIVE: To describe the clinical presentation of 13 new patients with a submicroscopic deletion of 1q43q44, of which nine were interstitial, and to report on the molecular characterisation of the deletion size. RESULTS AND CONCLUSIONS: The clinical presentation of these patients has clear similarities with previously reported cases with a terminal 1q deletion. Corpus callosum abnormalities were present in 10 of our patients. The AKT3 gene has been reported as an important candidate gene causing this abnormality. However, through detailed molecular analysis of the deletion sizes in our patient cohort, we were able to delineate the critical region for corpus callosum abnormalities to a 360 kb genomic segment which contains four possible candidate genes, but excluding the AKT3 gene.

A novel duplication of chromosome (13)(q14.1q21.3) in a patient with mental retardation and microcephaly.

We report on a mentally retarded female with behavioural problems, microcephaly, mild facial dysmorphisms, short stature and small hands with thin fingers due to a de novo partial duplication within the long arm of chromosome 13(q14.1q21.3). She was primarily referred to the outpatient department of neuropsychiatry because of short lasting psychotic episodes. No formal psychiatric diagnosis was made and the behavioural problems appeared the result of anxieties provoked by novel situations, enhanced by the intellectual disability. To the author's knowledge, this duplication has not been published previously and it is considered causative of the phenotype.

Nijmegen breakage syndrome cells fail to induce the p53-mediated DNA damage response following exposure to ionizing radiation.

The functionality of the p53-mediated pathway, activated in response to DNA damage, has been assessed in primary fibroblast cell cultures and Epstein-Barr virus-transformed lymphoblastoid cell lines derived from Nijmegen breakage syndrome (NBS) patients. This autosomal recessive disease is characterized by microcephaly, growth and mental retardation, chromosomal instability, radiosensitivity, and high cancer incidence. The recent mapping of the NBS gene to chromosome 8q21 demonstrates that NBS is genetically distinct from ataxia telangiectasia (AT). Changes in p53 protein levels were significantly reduced and delayed in all the NBS fibroblast cell cultures and lymphoblastoid cell lines examined compared to normal cultures over a 4-h period postirradiation (5 Gy). The transcriptional activation of p21(WAF1/CIP1) mRNA was also lower in 12 NBS fibroblast cultures examined. In agreement with an abrogated p53 function, NBS cells exposed to ionizing radiation show an abnormal cell cycle arrest at G1-S and a prolonged accumulation of cells in the G2 phase. In contrast, exposure to the alkylating agent methyl methanesulfonate results in similar increases of p53 and p21(WAF1/CIP1) mRNA in both cell types. The ATM gene transcript was found to be expressed at similar levels in NBS and normal cells, whereas it was strongly reduced in the AT homozygote cells examined. These results suggest that the ATM gene product cannot substitute for that of the NBS gene in the signaling of cellular damage produced by ionizing radiation and that both are involved in the activation of p53. The suboptimal p53-mediated response could contribute to the high cancer risk and radiosensitivity seen in NBS patients.

Cryptic duplication of the distal segment of 22q due to a translocation (21;22): three case reports and a review of the literature.

Duplications of the proximal segment of chromosome 22q are not uncommon, like Cat-eye syndrome and duplications due to familial (11;22) translocations. However, duplications of the distal long arm of chromosome 22 (22qter) seem to be exceedingly rare. So far, duplications of 22q12 or 22q13 to 22qter have been described in 21 patients, of whom 13 had a pure duplication 22qter. Here we report on three new cases with a pure duplication of the distal part of 22q. The first patient carries a duplication of terminal 22q due to a de novo unbalanced translocation, 46,XX,der(21)t(21;22) (p13;q13.2), detected by NOR-staining, while the other patients have a familial cryptic duplication of terminal 22q due to an unbalanced translocation, 46,XY,der(21)t(21;22)(p10;q13.3). The last two patients were initially thought to have a polymorphic variant of 21p, but additional subtelomeric screening using FISH showed the extra material was derived from chromosome 22. Terminal duplications of 22qter may be more common than generally assumed, but due to its small size, especially when located on an acrocentric chromosome and/or possibly relatively mild phenotype remain undetected thus far.

Interphase cytogenetics of hematological cancer: comparison of classical karyotyping and in situ hybridization using a panel of eleven chromosome specific DNA probes.

Numerical chromosome aberrations were detected in hematological cancers by nonradioactive in situ hybridization (ISH) procedures, using centromere specific probes for chromosomes 1, 7, 8, 9, 10, 11, 16, 17, 18, X, and Y. All 15 cases could be evaluated by ISH for these 11 probes. Our experiments show that in seven of these randomly selected leukemia bone marrow cell suspensions numerical aberrations for one or two chromosomes could be detected by this method. The results of ISH on interphase nuclei and in some cases on metaphase preparations were compared with karyotyping data. Seven cases of chromosomal aberrations observed with ISH (three for monosomy and four for trisomy) were confirmed by this classical cytogenetic technique, whereas in five instances an aberration was found only with ISH (twice for monosomy, twice for trisomy, and one disomy for the Y-probe). One case of a trisomy for chromosome 1 observed by ISH on interphase nuclei could be explained by a marker chromosome, a finding that was further substantiated by ISH on metaphase spreads. In this case double-target ISH on interphase cells with the probes for chromosomes 1 and 16 strongly suggested a translocation between these chromosomes. Also, in one case a marker chromosome could be characterized as a translocation between chromosomes 7 and 17. In this latter case the cytogenetic examinations revealed only monosomy for chromosomes 7 and 17 in addition to noncharacterized marker chromosomes. Our results indicate that the nonradioactive ISH procedure in combination with chromosome specific repetitive centromeric probes is a powerful tool for studying both numerical and structural chromosomal aberrations in interphase nuclei of leukemias. It may therefore become a valuable and routine diagnostic tool in addition to the existing karyotyping procedures.

Prospective screening for subtelomeric rearrangements in children with mental retardation of unknown aetiology: the Amsterdam experience.

OBJECTIVE: The frequency of subtelomeric rearrangements in patients with unexplained mental retardation (MR) is uncertain, as most studies have been retrospective and case retrieval may have been biased towards cases more likely to have a chromosome anomaly. To ascertain the frequency of cytogenetic anomalies, including subtelomeric rearrangements, we prospectively screened a consecutive cohort of cases with unexplained MR in an academic tertiary centre. METHODS: Inclusion criteria were: age <18 years at referral, IQ<85, no aetiological diagnosis after complete examination, which included karyotyping with high resolution banding (HRB). RESULTS: In 266 karyotyped children, anomalies were detected in 20 (7.5%, seven numerical, 13 structural); 39 cases were analysed by FISH for specific interstitial microdeletions, and anomalies were found in nine (23%). FISH analyses for subtelomeric microdeletions were performed in 184 children (44% moderate-profound MR, 51% familial MR), and one rearrangement (0.5%) was identified in a non-familial MR female with mild MR (de novo deletion 12q24.33-qter). The number of probable polymorphisms was considerable: 2qter (n=7), Xpter (n=3), and Ypter (n=1). A significantly higher total number of malformations and minor anomalies was present in the cytogenetic anomaly group compared to the group without cytogenetic anomalies. CONCLUSIONS: The total frequency of cytogenetic anomalies in this prospective study was high (1:10), but the frequency of subtelomeric rearrangements was low. The most likely explanations are the high quality of HRB cytogenetic studies and the lack of clinical selection bias. Conventional cytogenetic analyses, combined with targeted microdeletion testing, remain the single most effective way of additional investigation in mentally retarded children, also in a tertiary centre.