Genomic chondrocyte culture profiling by array-CGH, interphase-FISH and RT-PCR.

OBJECTIVE: In vitro expansion is an important step to acquire sufficient cells in human tissue engineering technologies. The high number of chondrocytes needed for human articular cartilage implants requires in vitro expansion of the primary cells, bearing a theoretical risk of in vitro induced changes in the genomes. To gain more insights into this situation, model cultures were prepared and analyzed. DESIGN: 25 chondrocyte cell DNA samples from nine donors were analyzed by array comparative genomic hybridization (aCGH) on whole genome level and 28 chondrocyte cell samples from 16 individuals were analyzed by fluorescence in situ hybridization (FISH) on single cell level. The expanded cells were further characterized upon the chondrocytic mRNA phenotype by reverse-transciptase polymerase chain reaction (RT-PCR). RESULTS: The molecular karyotyping results revealed autosomal stability, but all male samples analyzed by aCGH displayed a variable loss of the Y-chromosome. These data were confirmed by FISH-experiments and suggest an age dependant effect toward the loss of the Y-chromosome in cultured chondrocytes. RT-PCR data for the mRNAs from collagen types I, II, and aggrecan and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) did not reveal any correlation of transcriptional activity in cultures with Y-chromosome losses, nor were there statistically significant differences between cells from female and male donors. CONCLUSIONS: While cells of male origin may suffer from an age-related loss of the Y-chromosome, there was no indication of a functional impairment. The data suggest some caution toward applying proliferative steps when considering chondrocytes from elderly male patients for tissue engineering approaches.

Dystocia and fetotomy associated with cerebral aplasia in a greater one-horned rhinoceros (Rhinoceros unicornis).

The captive greater one-horned rhinoceros population consists of 176 animals. Since 1971, a total of 226 calves were born into this captive population. However, 24% of the offspring born were either stillborn or did not survive the first 3 months. The causes for this high rate of stillbirth and neonate mortality have not yet been documented. Here, we report on the veterinary management of a dystocia and foetotomy resulting from a malpositioned greater one-horned rhinoceros foetus. The dead foetus presented with a forelimb flexed at the shoulder joint, with all other joints extended. The foetus was dissected into five parts and extracted during two anaesthesias on two consecutive days. The dam recovered fully and came into oestrous 31 days after surgery. Post-mortem and CT examination of the malformed foetal head revealed cranioschisis with cerebral aplasia and cerebellar hypoplasia. The cerebral aplasia presented here and in other recent cases suggests that neural tube defects and cranial malformations may be associated with more captive rhinoceros stillbirths than previously considered. Epidemiologic studies of these phenomena and possible nutritional deficiencies or hereditary defects are warranted.

The impact of first trimester screening and early fetal anomaly scan on invasive testing rates in women with advanced maternal age.

PURPOSE: To evaluate the acceptance of noninvasive screening for trisomy 13, 18, 21 and the impact on invasive testing rates in women at an age≥35 years. MATERIALS AND METHODS: In a retrospective analysis from 2003-2006 including 13 268 women≥35 years old with singleton pregnancies and 3133 invasive procedures, we evaluated the prenatal detection rate of aneuploidies in two cohorts. Group 1: advanced maternal age as sole indication, group 2: additional abnormalities and/or suspicious maternal serum parameters. In an additional analysis from 1998-2006 including 31,076 patients≥35 years, we investigated the shift in time of sonography at 11+0-13+6, 14+0-17+6 and 18+0-22+6 gestational weeks (gw). RESULTS: Among 13,268 women, 3133 invasive tests were performed with a significant decrease over time (-17%). 9% of women chose invasive testing after a normal ultrasound (group 1, n=1,267) and 14% in the case of additional markers (group 2, n=1,866). 102 cases of aneuploidy were disclosed. The proportion of detected aneuploidies was 0.86% in group 1 and 4.9% in group 2. No change in the overall detection rate (90-93%) was observed. The number of patients≥40 years increased significantly (+2.8%). There was an increase in examinations at 11+0-13+6 gw (+8%), a decrease at 14+0-17+6 gw (-10.3%) and no significant change at 18+0-22+6 gw over time. CONCLUSION: Increasing numbers of women≥35 years of age rely on the individually adjusted risk figure to make a decision about invasive testing. The application of these selective procedures can reduce the rates of invasive testing with fewer losses of normal fetuses and led to an earlier diagnosis of aneuploidies.

A mixture model of nuchal translucency thickness in screening for chromosomal defects: validation of a single operator dataset.

OBJECTIVE: (1) To validate the mixture model in a single operator dataset and (2) to compare the detection rates for fetal chromosomal defects obtained from the mixture model with those obtained from either the delta nuchal translucency (NT) or log multiple of the median (MoM) approach. METHODS: Database query, viable singletons [crown-rump length (CRL) 45-84 mm corresponding to 11-13(+6) weeks], December 1997 to November 2006, examined by Adam Gasiorek-Wiens, the statistical mixture model was applied. RESULTS: Seventy-four of 4171 were lost to follow-up (1.8%), 4097 singleton pregnancies included trisomy 21 (n = 34, 0.8%), trisomy 18 (n = 20, 0.5%), trisomy 13 (n = 8, 0.2%), Turner syndrome (n = 9, 0.2%) and other chromosomal abnormalities (n = 14, 0.3%). The main findings are that (1) the log-transformed NT measurements follow a mixture of two Gaussian distributions and (2) the criteria to apply either the delta-NT or log MoM models are not met. In the normal group, the majority of NT measurements were dependent on the CRL, a small group showed a median independent of the CRL. In the abnormal group it was the opposite. For a 5% false-positive rate (FPR), the trisomy 21 detection rate was 83%. CONCLUSIONS: The use of the mixture model in a single operator dataset produces results compatible with the original study. The mixture model has thus been validated.

First-trimester prenatal diagnosis of Okihiro syndrome.

A case of Okihiro syndrome (OS) detected by 2- and confirmed by 3-dimensional ultrasound at 13+2 gestational weeks is reported. While the pregnant woman affected by the OS presented with limb anomalies, the fetus showed severe thoracoabdominal and skeletal anomalies. Termination of pregnancy was performed at 14+1 gestational weeks and confirmed the sonographically detected symptoms. The diagnosis was confirmed by autoptic, radiologic and molecular genetic analysis. To our knowledge, this is the first case of prenatal diagnosis of OS.

10p11.2 to 10q11.2 is a yet unreported region leading to unbalanced chromosomal abnormalities without phenotypic consequences.

Directly transmitted unbalanced chromosomal abnormalities (UBCA) or euchromatic variants (EV) were recently reported for >50 euchromatic regions of almost all human autosomes. UBCA and EV are comprised of a few megabases of DNA, and carriers are in many cases clinically healthy. Here we report on partial trisomies of chromosome 10 within the pericentromeric region which were detected by standard G banding. Those were referred for further delineation of the size of these duplicated regions for molecular cytogenetics and/or array-CGH. Partial trisomies of chromosome 10 in the pericentromeric region were identified prenatally in seven cases. A maximum of three copies of the region from 10p12.1 to 10q11.22 was observed in all cases without apparent clinical abnormalities. The imbalances were either caused by a direct duplication in one familial case or by de novo small supernumerary marker chromosomes (sSMC). Thus, we report a yet unrecognized chromosomal region subject to UBCA detected in seven unrelated cases. To the best of our knowledge, this is the first report of a UBCA in the pericentromeric region of chromosome 10 that is not correlated with any clinical consequences.

Interphase M-FISH applications using commercial probes in prenatal and PGD diagnostics.

Early, rapid and reliable diagnosis is of first priority in prenatal medicine. The combination of specific sonographic markers (e.g. nuchal translucency) and biochemical parameters in maternal serum (e.g. free beta-human chorionic gonadotropin, pregnancy-associated plasma protein A), has already dramatically improved the sensitivity of non-invasive first trimester risk screening in pregnancy. In invasive prenatal diagnosis, in addition to well-established chorionic villi short-term culture, interphase multi-colour-fluorescence in situ hybridisation (M-FISH) on uncultured amnion cells has become a reliable tool for the rapid detection of fetal aneuploidies. Interphase M-FISH applications have enabled the diagnosis of selected chromosomal abnormalities in single cells and, therefore, have also become an important diagnostic tool for preimplantation diagnosis (PGD). The development of commercially available probe sets, in particular, has led to a broad use of interphase M-FISH in prenatal and PGD diagnosis.

Characterization of cell cycle checkpoint responses after ionizing radiation in Nijmegen breakage syndrome cells.

Nijmegen breakage syndrome (NBS), which in the past also has been classified as a variant of ataxia telangiectasia (AT), is characterized by cancer proneness and extreme sensitivity to ionizing radiation. We investigated the DNA damage responses of four independent primary NBS fibroblast cell lines. Following a low dose of ionizing radiation, p53 is mostly induced with slower kinetics and shows more transient induction in NBS fibroblasts. Nonetheless, this damage-induced protein appears biologically functional: unsynchronized and synchronized NBS cells show a G1 arrest after ionizing radiation as determined by bivariate flow cytometry. Neither an AT cell line nor a NBS cell line transformed with human papillomavirus genes E6 and E7 shows a G1 arrest. Furthermore, NBS cells show a normal G2 block, unlike that shown for AT cells. These data provide a cellular distinction between NBS and AT, thereby clearly separating the NBS from the AT syndrome.

Prenatal diagnosis in Germany.

Prenatal diagnosis (PND) in Germany is well established. A wide spectrum of sonographic, cytogenetic, molecular and biochemical investigations can be chosen by pregnant women. While sonographic examinations are offered to all pregnant women, the methods requiring invasive procedures are performed predominantly when there is a higher risk than in the general population. However, PND is also performed on request by the mother in the absence of an increased risk. Pretest genetic counselling is recommended before invasive techniques. PND is performed in public as well as in private settings. Guidelines are in effect for specialisation of gynaecologists and human geneticists. Quality assessment for cytogenetic and molecular laboratories is performed on a voluntary basis. Since October 1995, termination of pregnancies is regulated by a new law.

Localisation of a Fanconi anaemia gene to chromosome 9p.

Using homozygosity mapping in a large consanguineous family, we have localised to chromosome 9p a further gene for the autosomal recessive, genetically heterogeneous disease Fanconi anaemia (FA). This is the fourth of at least eight FA genes to be localised to a discrete chromosomal region. Previously localised genes are FAA, FAC and FAD. By analysis of assigned families we show that the gene localised to chromosome 9p is FAF, FAG or FAH, or a new FA gene, and refine the localisation to the 21 cM region between markers D9S1678 and D9S175.

Partial trisomy 1q, an uncommon chromosomal aberration in erythroleukemia.

A 2 years 4 months old boy with erythroleukemia (FAB-M6) and Down's syndrome is described. Chromosome analysis of bone marrow leukemic blasts revealed apart from the trisomy 21 a partial trisomy of the long arm of chromosome 1:1q23-->1qter in all cells and trisomy 8 mosaicism. To the best of our knowledge this is the first time that a partial trisomy of chromosome 1 in association with erythroleukemia has been described. Previous reports of other hematologic malignancies with aberrations of chromosome 1 indicate that the breakpoint 1q23 is nonrandom and that trisomies of chromosome 1 plays a crucial role in the course of development of hematologic malignancies. This could probably also be true for erythroleukemias.

Genetic haplotyping of ataxia-telangiectasia families localizes the major gene to an approximately 850 kb region on chromosome 11q23.1.

The genotyping data given localize the major A-T gene to an approximately 850 kb region. They also localize the group A A-T gene (ATA) to a region that contains the approximately 850 kb region. They are compatible with linking A-TFresno to 11q22-23. NBS-V2 does not link to this region. Four non-linking families contain only single affecteds, suggesting that these may be spontaneous mutations rather than evidence for an A-T gene outside the 11q22-23 region. Finally, two other non-linking families contain recombinant haplotypes that are compatible with a second A-T gene at 11q22-23, slightly distal to the approximately 850 kb region. However, convincing evidence for a second gene is still lacking.

Frequency of trisomy 21 in Germany before and after the Chernobyl accident.

For Berlin (West) the rate of trisomy 21 among newborn and all prenatally diagnosed cases can be almost completely recorded, including the maternal age distribution. During the 9-year-period from 1980 and 1988 the average number of trisomy 21 per month was about 2, following a Poisson distribution. A significant increase (P less than 0.01) was observed in January 1987, exactly 9 months after the Chernobyl accident. In a supraregional study based on greater than 30,000 prenatal diagnoses performed in 1986, no significant effect could be observed. However, the highest rates of trisomy 21 were observed in the more heavily contaminated, southern part of Germany. The majority of these fetuses were conceived during the period of greatest radioactive exposure. The data are discussed with respect to the effect of low-dose radiation around the time of conception on the induction of non-disjunction in man.

Investigation into the possible genetic effects of diagnostic ultrasound.

The mutagenic effect of DUS (diagnostic ultrasound) was examined using two generators emitting continuous waves and pulsed waves, respectively. Three different test systems for mutagenic activity were employed in this study. 1. The frequency of sister chromatid exchanges (SCEs) was determined in metaphase chromosomes of human lymphocytes and of Chinese hamster ovary (CHO) cells sonicated at different stages of the cell cycle. 2. The induction of DNA single strand breaks was tested in CHO cells treated with continuous wave ultrasound. Following sonication an endonuclease was introduced into the cells converting single strand breaks to chromosomal aberrations scorable in the following metaphase. 3. The influence of DUS on the number of point mutations was evaluated in the Ames test. A tester strain of Salmonella typhimurium indicating base substitutions was sonicated. At the present stage, all results proved to be completely negative and could thus not support the view of any mutagenic activity of diagnostic ultrasound.

[Mixed gonadal dysgenesis--a rare cause of primary infertility. Report of 2 cases and review of the literature]. / Die gemischte Gonadendysgenesie--eine seltene Ursache der primären Infertilität. Bericht über 2 Fälle und Literaturüberblick.

We report on two cases of primary male infertility attributable to mixed gonadal dysgenesis. In one case the diagnosis was made when the patient was 30 years old; in the other, the 44-year-old patient had been evaluated 4 years before for erectile dysfunction, which had been treated with a penile prosthesis without the diagnosis being made. These two cases are life following exceptional in that the diagnosis was made in adult-life following an uncomplicated male adolescence.

Significant increase in trisomy 21 in Berlin nine months after the Chernobyl reactor accident: temporal correlation or causal relation?

OBJECTIVE: To assess whether the increased prevalence of trisomy 21 in West Berlin in January 1987 might have been causally related to exposure to ionising radiation as a result of the Chernobyl reactor accident or was merely a chance event. DESIGN: Analysis of monthly prevalence of trisomy 21 in West Berlin from January 1980 to December 1989. SETTING: Confines of West Berlin. RESULTS: Owing to the former "island" situation of West Berlin and its well organised health services, ascertainment of trisomy 21 was thought to be almost complete. A cluster of 12 cases occurred in January 1987 as compared with two or three expected. After exclusion of factors that might have explained the increase, including maternal age distribution, only exposure to radiation as a result of the Chernobyl reactor accident remained. In six of seven cases that could be studied cytogenetically the extra chromosome was of maternal origin, confirming that nondisjunction had occurred at about the time of conception. CONCLUSION: On the basis of two assumptions--(a) that maternal meiosis is an error prone process susceptible to exogenous factors at the time of conception; (b) that owing to the high prevalence of iodine deficiency in Berlin a large amount of iodine-131 would have been accumulated over a short period--it is concluded that the increased prevalence of trisomy 21 in West Berlin in January 1987 was causally related to a short period of exposure to ionising radiation as a result of the Chernobyl reactor accident.

Detailed screening for fetal anomalies and cardiac defects at the 11-13-week scan.

OBJECTIVE: To assess the diagnostic efficacy of the first-trimester anomaly scan including first-trimester fetal echocardiography as a screening procedure in a 'medium-risk' population. METHODS: In a prospective study, we evaluated 3094 consecutive fetuses with a crown-rump length (CRL) of 45-84 mm and gestational age between 11 + 0 and 13 + 6 weeks, using transabdominal and transvaginal ultrasonography. The majority of patients were referred without prior abnormal scan or increased nuchal translucency (NT) thickness, the median maternal age was, however, 35 (range, 15-46) years, and 53.8% of the mothers (1580/2936) were 35 years or older. This was therefore a self-selected population reflecting an increased percentage of older mothers opting for prenatal diagnosis. The follow-up rate was 92.7% (3117/3363). RESULTS: The prevalence of major abnormalities in 3094 fetuses was 2.8% (86/3094). The detection rate of major anomalies at the 11 + 0 to 13 + 6-week scan was 83.7% (72/86), 51.9% (14/27) for NT < 2.5 mm and 98.3% (58/59) for NT >or= 2.5 mm. The prevalence of major congenital heart defects (CHD) was 1.2% (38/3094). The detection rate of major CHD at the 11 to 13 + 6-week scan was 84.2% (32/38), 37.5% (3/8) for NT < 2.5 mm and 96.7% (29/30) for NT >or= 2.5 mm. CONCLUSION: The overall detection rate of fetal anomalies including fetal cardiac defects following a specialist scan at 11 + 0 to 13 + 6 weeks' gestation is about 84% and is increased when NT >or= 2.5 mm. This extends the possibilities of a first-trimester scan beyond risk assessment for fetal chromosomal defects. In experienced hands with adequate equipment, the majority of severe malformations as well as major CHD may be detected at the end of the first trimester, which offers parents the option of deciding early in pregnancy how to deal with fetuses affected by genetic or structural abnormalities without pressure of time.