X-autosome and X-Y Translocations in Female Carriers: X-chromosome Inactivation Easily Detectable by 5-ethynyl-2-deoxyuridine (EdU).

Here we report one new case each of an X-autosome translocation (maternally derived), and an X-Y-chromosome translocation. Besides characterizing the involved breakpoints and/or imbalances in detail by molecular cyto-genetics, also skewed X-chromosome inactivation was determined on single cell level using 5-ethynyl-2-deoxyuridine (EdU). Thus, we confirmed that the recently suggested EdU approach can be simply adapted for routine diagnostic use. The latter is important, as only by knowing the real pattern of the skewed X-chromosome inactivation, correct interpretation of obtained results and subsequent reliable genetic counseling, can be done.

Mouse white adipose tissue-derived mesenchymal stem cells gain pericentral and periportal hepatocyte features after differentiation in vitro, which are preserved in vivo after hepatic transplantation.

AIM: Mesenchymal stem cells may differentiate into hepatocyte-like cells in vitro and in vivo. Therefore, they are considered a novel cell resource for the treatment of various liver diseases. Here, the aim was to demonstrate that mesenchymal stem cells may adopt both perivenous and periportal hepatocyte-specific functions in vitro and in vivo. METHODS: Adipose tissue-derived mesenchymal stem cells were isolated from immunodeficient C57BL/6 (B6.129S6-Rag2(tm1Fwa) Prf1(tm1Clrk) ) mice and differentiated into the hepatocytic phenotype by applying a simple protocol. Their physiological and metabolic functions were analysed in vitro and after hepatic transplantation in vivo. RESULTS: Mesenchymal stem cells changed their morphology from a fibroblastoid into shapes of osteocytes, chondrocytes, adipocytes and hepatocytes. Typical for mesenchymal stem cells, hematopoietic marker genes were not expressed. CD90, which is not expressed on mature hepatocytes, decreased significantly after hepatocytic differentiation. Markers indicative for liver development like hepatic nuclear factor 4 alpha, or for perivenous hepatocyte specification like cytochrome P450 subtype 3a11, and CD26 were significantly elevated. Periportal hepatocyte-specific markers like carbamoylphosphate synthetase 1, the entry enzyme of the urea cycle, were up-regulated. Consequently, cytochrome P450 enzyme activity and urea synthesis increased significantly to values comparable to cultured primary hepatocytes. Both perivenous and periportal qualities were preserved after hepatic transplantation and integration into the host parenchyma. CONCLUSIONS: Adult mesenchymal stem cells from adipose tissue differentiated into hepatocyte-like cells featuring both periportal and perivenous functions. Hence, they are promising candidates for the treatment of region-specific liver cell damage and may support organ regeneration in acute and chronic liver diseases.

Atypical polycystic ovary syndrome--a genetic analysis.

BACKGROUND/AIMS: Although polycystic ovary syndrome (PCOS) is a common endocrinopathy the pathogenesis is not entirely understood. Typically, high androgen levels are associated with increased virilization. We report 2 rare groups of patients with either unexpectedly high testosterone levels despite low virilization as well as patients with low testosterone levels despite high grade of virilization. One possibility for the atypical PCOS may be based on an altered androgen receptor (AR) signaling. METHODS: 6 patients and when available the parents were included in this study. Alterations of the metaphase chromosomes by GTG staining, the length of both the trinucleotide CAG- and GGC-repeats of the androgen receptor (AR) gene was determined by PCR, further the entire AR gene was sequenced and analyzed. RESULTS: The GTG banding revealed no chromosomal alterations and the range of CAG- and GGC-repeat lengths are within the normal range. Interestingly, by sequencing of the entire AR gene few genetic mutations were identified. CONCLUSION: The detected mutations do not alter the AR protein sequence but they change the codon usage towards less frequent codons that potentially may alter AR protein levels and androgen signaling. In addition to this, we postulate also other causes for manifestation of atypical PCOS, which may include AR-coregulators or epigenetic alterations. To our knowledge this is the first report of combining chromosomal analysis of PCOS patients with full sequencing of the human AR gene and linking codon usage to PCOS.

Clinical impact of somatic mosaicism in cases with small supernumerary marker chromosomes.

Somatic mosaicism is present in slightly more than 50% of small supernumerary marker chromosome (sSMC) carriers. Interestingly, non-acrocentric derived sSMC show mosaicism much more frequently than acrocentric ones. sSMC can be present in different mosaic rates, which may go below 5% of the studied cells. Also cryptic mosaicism can be present and mosaics may be differently expressed in different tissues of the body. Even though in the overwhelming majority of the cases somatic sSMC mosaicism has no direct clinical effect, there are also cases with altered clinical outcomes due to mosaicism. Also clinically important is the fact that a de novo sSMC, even present in mosaic, may be a hint of uniparental disomy (UPD). As it is under discussion to possibly replace standard karyotyping by methods like array-CGH, the impracticality of the latter to detect low-level sSMC mosaics and/or UPD has to be considered as well. Overall, sSMC mosaicism has to be studied carefully in each individual case, as it can be extremely informative and of importance, especially for prenatal genetic counseling.

Back-gated spray-deposited carbon nanotube thin film transistors operated in electrolytic solutions: an assessment towards future biosensing applications.

We report on back-gated carbon nanotube (CNT) thin-film transistors (CNTFETs) and their performance in electrolytic solutions to assess their suitability for future application as biosensors. Spray-deposited CNT networks were used as the sensitive active layer which offers the opportunity for integration on flexible sensing platforms at low-cost. We characterized the transistors' behavior in electrolytes by analyzing the response to different KCl solutions and buffers over a wide pH range. We observed a linear response of the drain current upon changing the pH in low molarity buffers and obtained an exponential dependence on the salt concentration of the electrolyte. These responses can be attributed to electrostatic gating effects that go along with shifts in the threshold voltage. Even though a lot of effort has been put into understanding the biosensing mechanism a detailed theory is still missing. Back-gated CNTFETs operated in electrolytic solutions can be a further tool to investigate and clarify the existing unsolved phenomena.

Five novel locations of Neocentromeres in human: 18q22.1, Xq27.1∼27.2, Acro p13, Acro p12, and heterochromatin of unknown origin.

Since the first report in 1993, an ectopic centromere, i.e. neocentromere formation, has been reported in more than 100 small supernumerary marker chromosomes (sSMC), in 7 instances of centromere repositioning, and in about a dozen cases with more complex chromosomal rearrangements. Here we report 2 new cases with centromere repositioning and 3 neocentric sSMC consisting exclusively of heterochromatic material. Yet, no centromere formation was reported for the regions 18q22.1 and Xq27.1∼27.2 as it was observed in the 2 cases with centromere repositioning here; in both cases, cytogenetically an inversion was suggested. Two of the 3 neocentric sSMC were derived from a short arm of an acrocentric chromosome. The remainder neocentric sSMC case was previously reported and was stainable only by material derived from itself.

Clinical impact of proximal autosomal imbalances.

Centromere-near gain of copy number can be induced by intra- or inter-chromosomal rearrangements or by the presence of a small supernumerary marker chromosome (sSMC). Interestingly, partial trisomy to hexasomy of euchromatic material may be present in clinically healthy or affected individuals, depending on origin and size of chromosomal material involved. Here we report the known minimal sizes of all centromere-near, i.e., proximal auto-somal regions in humans, which are tolerated; over 100 Mb of coding DNA are comprised in these regions. Additionally, we have summarized the typical symptoms for nine proximal autosomal regions including genes obviously sensitive to copy numbers. Overall, studying the carriers of specific chromosomal imbalances using genomics-based medicine, combined with single cell analysis can provide the genotype-phenotype correlations and can also give hints where copy-number-sensitive genes are located in the human genome.

[Medium-term results of the Mayo™ short-stem hip prosthesis after avascular necrosis of the femoral head]. / Mittelfristige Ergebnisse der Mayo™-Kurzschaftprothese bei Hüftkopfnekrose.

AIM: Short-stemmed prostheses are increasingly regarded as implants of first choice in coxarthrosis, especially in young patients. Despite promising short-term results, long-term follow-up studies are still lacking. Short-stemmed femoral implants are characterised by a metaphyseal osseointegration and strain distribution. Therefore a reduced stress shielding of the proximal femur is hypothesized and in some studies already proven. There is histological evidence that osteonecrosis (ON) of the femoral head may involve not only the intracapital region but also the femoral neck and metaphyseal area. This could lead to a higher rate of aseptic loosening of short-stemmed implants. The aim of this retrospective study was to analyze the midterm results of the Mayo™ short-stem prosthesis after ON with particular attention on osseointegration. METHOD: From 2002-2004, in 21 patients (2 females, 19 males; mean age 45 years; mean BMI = 27) with secondary coxarthrosis after ON implantation of 26 Mayo™ Conservative Hips was performed. Postoperatively, all patients were mobilised with full weight-bearing. Using the specially developed Wristing® software, longitudinal stem migration and varus-valgus femoral stem alignment were examined digitally in anteroposterior X-rays taken immediately after surgery and in standing AP radiographs after 8.2 months and on average after 7.9 years (16 patients). The incidence of periprosthetic radiolucent lines was captured in the anteroposterior X-rays and assigned to the Gruen zones and a DEXA scan was performed. The X-rays of a matched control group with implantation of a Mayo™ short-stem prosthesis in primary coxarthrosis were analyzed by the same method. In all patients the Harris hip score (HHS) was obtained pre- and postoperatively. RESULTS: There was no significant migration or valgus tilt of the Mayo™ prosthesis in the study and control groups during postoperative follow-up (paired t-test, p = 0.13 and 0.69, respectively). In six of 26 Mayo™-Stems 12 radiolucent lines (RL) of the Mayo™ prosthesis were observed. The control group showed at ten of 30 Mayo™ stems 17 radiolucent lines. The difference between the groups was not statistically different (chi-square test for the total number of RL: χ² = 0.001, p = 1.0 and χ² = 0.06, p = 0.79 for the number of Mayo™ stems with RL). The DEXA scan showed a slightly higher bone mineral density (BMD) in Gruen zones 3 and 5 compared with a control group: study group. In the study group the postoperative HHS was 93.5 (SD 5.6) compared to 94.2 (SD 6.9) in the control group (t-test, p = 0.63). CONCLUSION: In the mid-term course no increased migration or tilt could be proven for Mayo™ short-stem THA in patients with osteonecrosis of the femoral head. Due to the absence of differences in the occurrence of radiolucent lines and the same results in the DEXA scan an unimpaired osseointegration of the Mayo™ stem is assumed. Therefore it is concluded that the Mayo™ Conservative Hip can be regarded as an alternative for operative treatment of ON of the femoral head.

Is there a yet unreported unbalanced chromosomal abnormality without phenotypic consequences in proximal 4p?

Unbalanced chromosomal abnormalities (UBCA) are reported for >50 euchromatic regions of almost all human autosomes. UBCA are comprised of a few megabases of DNA, and carriers are in many cases clinically healthy. Here we report on a partial trisomy of chromosome 4 of the centromere-near region of the short arm of chromosome 4 present as a small supernumerary marker chromosome (sSMC). The sSMC was present in >70% of amnion cells and in 60% of placenta. Further delineation of the size of the duplicated region was done by molecular cytogenetics and array comparative genomic hybridization. Even though the sSMC lead to a partial trisomy of ~9 megabase pairs, a healthy child was born, developing normally at 1 year of age. No comparable cases are available in the literature. Thus, we discuss here the possibility of having found a yet unrecognized chromosomal region subject to UBCA.

Evidence for correlation of fragile sites and chromosomal breakpoints in carriers of constitutional balanced chromosomal rearrangements.

A molecular cytogenetic study of 251 cases with balanced chromosomal rearrangements detected due to infertility of unclear origin or in prenatal diagnostics with a later normal outcome was done. Balanced translocations (127 cases), inversions (105 cases), insertions (three cases), balanced complex rearrangements (four cases), or derivative chromosomes leading to no imbalance (12 cases), were studied by multicolor banding (MCB) and/or subcentromeric multicolor fluorescence in situ hybridization (subcenM-FISH). Five-hundred and twenty-nine break-events were characterized by molecular cytogenetics. Only 150 of these were unique breakpoints, the remainder were observed between two and 10 times. According to the results obtained, there was cytogenetic co-localization of fragile site (FS) in ~71% of the studied 529 break-events. Nine selected cases with evidence for breakpoints within FS were further analyzed by FS-specific bacterial artificial chromosome (BAC) probes; only one did not show a co-localization. Further detailed molecular analysis will be necessary to characterize the mechanisms and genetic basis for this phenomenon.

Prevalence of CYP450 gene variations in patients with type 2 diabetes.

BACKGROUND: Drug degradation in the human organism is driven by detoxification mechanisms that can be affected in their efficiency by genetic mutations. The purpose of this pilot investigation was to investigate whether Type 2 diabetes is associated with mutations in prominent members of the CYP 450 isoenzyme family. METHODS: Genomic DNA was isolated from EDTA blood samples of 203 Caucasian subjects (101 patients with Type 2 diabetes and 102 non-diabetic subjects, age (mean +/- STD): 49 +/- 16 years) was analyzed. Genomic DNA was isolated from EDTA blood. Mutation analysis for CYP2C8 (*2/*3/*4), CYP2C9 (*2/*3), CYP2C19 (*2/*3), CYP2D6 (*3/*4/*5/*6) and PPARgamma (P12A) was performed by means of real-time PCR methods (Light-Cycler, Roche Diagnostics, Indianapolis, IN, USA). RESULTS: The genotyping revealed the following allele frequency distributions for the two investigated groups: CYP2C8: *2 (type 2 diabetes 3% vs. 1%, n.s.), *3 (16% vs. 3%, n.s.), *4 (15% vs. 2%, p < 0.05), CYP2C9: *2 (20% vs. 24%, n.s.), *3 (22% vs. 21%, n.s.), CYP2C19: *2 (23% s. 33%, n.s.), *3 (0% vs. 0%, n.s.), CYP2D6: *3 (3% vs. 4%, n.s.), *4 (40% vs. 37%, n.s.), *5 (3% vs. 2%, n.s.), *6 (0% vs. 0%, n.s.), PPARgamma P12A (15% vs. 21%, n.s.), i.e. all but one mutation (CYP2C8*4) were found with equal prevalence in the two cohorts. CONCLUSIONS: In this pilot investigation, we found an increased prevalence of the CYP2C8*4 mutation in the Type 2 diabetic patient group. This may result in a modification of drug degradation and drug efficacy in these patients and may have an influence, e.g. on the choice of anti-diabetic drugs. However, further trials are necessary in order to confirm our findings.

An HDAC1-binding domain within FATS bridges p21 turnover to radiation-induced tumorigenesis.

There is a gap between the initial formation of cells carrying radiation-induced genetic damage and their contribution to cancer development. Herein, we reveal a previously uncharacterized gene FATS through a genome-wide approach and demonstrate its essential role in regulating the abundance of p21 in surveillance of genome integrity. A large exon coding the NH2-terminal domain of FATS, deleted in spontaneous mouse lymphomas, is much more frequently deleted in radiation-induced mouse lymphomas. Its human counterpart is a fragile site gene at a previously identified loss of heterozygosity site. FATS is essential for maintaining steady-state level of p21 protein and sustaining DNA damage checkpoint. Furthermore, the NH2-terminal FATS physically interacts with histone deacetylase 1 (HDAC1) to enhance the acetylation of endogenous p21, leading to the stabilization of p21. Our results reveal a molecular linkage between p21 abundance and radiation-induced carcinogenesis.

Characterization of 23 small supernumerary marker chromosomes detected at pre-natal diagnosis: The value of fluorescence in situ hybridization.

Small supernumerary marker chromosomes (sSMCs) cannot be identified or characterized unambiguously by conventional cytogenetic banding techniques. Until recently, the large variety of marker chromosomes, as well as the limitations in their identification, have presented a diagnostic problem. In order to determine the origin of sSMCs, we used a variety of fluorescence in situ hybridization (FISH) methods, including centromere-specific multicolor FISH, acrocentric specific multicolor FISH, subcentromere-specific multicolor FISH and multicolor FISH with whole chromosome paint probes. Moreover, uniparental disomy testing was in all cases attempted. From a total of 28,000 pre-natal samples from four diagnostic genetics laboratories in Greece, 23 (0.082%) supernumerary marker chromosomes were detected. The mean maternal age was 36.2 years (range 27-43) and the mean gestational age at which amniocentesis was performed was 18.5 weeks (range 16-23). Eighteen markers were de novo and 5 markers were inherited. Molecular cytogenetic methods were applied to determine the chromosomal origin and composition of the sSMC. In total, 17 markers were derived from acrocentric chromosomes (14, 15, 21 and 22) and 6 markers were non-acrocentric, derived from chromosomes 9, 16, 18, 20 and Y. Uniparental disomy was not detected in any of the cases studied. With regard to pregnancy outcome, 13 pregnancies resulted in normal healthy neonates, while 10 pregnancies were terminated due to ultrasound abnormalities. A total of 23 marker chromosomes from 28,000 pre-natal samples (0.082%) were identified. Molecular cytogenetic techniques provided valuable information on the chromosomal origin and composition of all the sSMCs. Especially in cases with normal ultrasound, the FISH results rendered genetic counseling possible in a category of cases previously considered a diagnostic problem. Abnormal outcome was observed in 10 cases (43,5%), 7 of which showed abnormal ultrasound findings. New technologies, such as array-comparative genomic hybridization, should be used in future genotype-phenotype correlation studies, although the high mosaicism rate poses a problem.

The case of an infertile male with an uncommon reciprocal X-autosomal translocation: how does this affect male fertility?

Infertility is defined as the inability to conceive after one year of regular unprotected intercourse. Constitutional numerical and/or structural chromosomal aberrations like sex-chromosome aberrations are one of the possible factors involved in fertility problems. Reciprocal translocations between an X-chromosome and an autosome are rarely seen in men. Male carriers of an X-autosome translocation are invariably sterile, regardless of the position of the breakpoint in the X-chromosome. Breakpoints in autosomal chromosomes could also be involved in male infertility. In this paper, we describe a 31-year-old male with azoospermia. GTG banding with high resolution multicolor-banding (MCB) techniques revealed a karyotype 46,Y,t(X;1)(p22.3;q25), and we discuss how the breakpoint of this translocation could affect male infertility. As a conclusion, cytogenetic evaluation of infertile subjects with azoospermia should be considered in the first place before in vitro fertilisation procedures are planned.

First case of a neocentromere formation in an otherwise normal chromosome 7.

According to cytogenetic and molecular cytogenetic characterization, an otherwise not-altered chromosome 7 formed a neocentromere in band 7q32.1 in a clinically normal female. The alpha satellite sequence D7Z1 remained in its place but was not used for formation of the primary chromosomal incision. Similar observations of centromere repositioning have been made for chromosomes 3 (2x), 4, 8 and Y (2x). Even though data is available for some neocentromeres whose positions are correlated with evolutionary new centromeres for 7q32.1, no correlation could be found for an ancestral inactivated centromere in any of the presently living primates. Overall, we report a new case of centromere repositioning at a position not known to harbor an ancestral inactivated centromere.

A neocentric isochromosome Yp present as additional small supernumerary marker chromosome--evidence against U-type exchange mechanism?

Here we report the first case of an inverted duplicated neocentric small supernumerary marker chromosome present in a karyotype 47,XX,+mar(Y). As expected a partial disomy of Ypter to Yp11.2 did not lead to any major malformations. However, the formation of an inverted duplicated chromosome from a Y chromosome is not possible by a U-type exchange, as has been suggested for such kind of neocentric marker chromosomes. Thus, some evidence is here provided that this concept might not always be true.

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.

Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo.

OBJECTIVE: The hepatic integration of human adipose tissue derived mesenchymal stem cells (hAT-MSCs) in vivo with or without prior differentiation to hepatocyte-like cells in vitro was investigated. METHODS AND RESULTS: Cells, isolated either from peritoneal or subcutaneous adipose tissue, expressed mesenchymal stem cell surface markers and featured multiple lineage differentiation. Under conditions favouring hepatocyte differentiation, hAT-MSCs gained hepatocytic functions in vitro including urea formation, glycogen synthesis, cytochrome P450 enzyme activity, and expression of hepatocyte-specific transcripts of carbamoylphosphate synthetase, albumin and cytochrome P450 type 3A4 (CYP3A4). Transgenic expression of green fluorescent protein emerged upon hepatocyte differentiation when driven by the hepatocyte-specific promoter of the cytosolic phosphoenolpyruvate carboxykinase gene but was constitutive from the ubiquitin gene promoter. Human AT-MSCs were transplanted into livers of immunodeficient Pfp/Rag2-/- mice with or without prior hepatocyte differentiation in vitro. Donor-derived human cells engrafted in the mouse host liver predominantly in the periportal region of the liver lobule. They expressed HepPar1 and albumin, typical features of differentiated human hepatocytes, in the otherwise negative mouse liver background. Engraftment was significantly more efficient using hAT-MSCs pre-differentiated to hepatocyte-like cells in vitro as compared with undifferentiated cells. CONCLUSIONS: Pre-differentiation of human MSCs from adipose tissue into hepatocyte-like cells in vitro facilitates long term functional hepatic integration in vivo.

A new unbalanced chromosomal abnormality in 1q31.1 to 1q32 without phenotypic consequences.

A familial duplication in the long arm of one chromosome 1 was detected due to recurrent abortions in a couple. The duplication was present in the male partner of the couple and in his mother, both clinically healthy. By reverse FISH, the duplication was determined to be located in 1q31. Multicolor banding (MCB) and application of locus-specific probes narrowed down the breakpoints to 1q31.1 and 1q32. The duplication spans a region of 13.9 Mb. None of the two breakpoints was colocalized with a known fragile site in 1q31.2, which, however, was duplicated. To the best of our knowledge, this is the first report of an unbalanced chromosome abnormality in this region that is not correlated with any clinical consequences.