Wolfram Henn

Genotyping in 46 patients with tentative diagnosis of Treacher Collins syndrome revealed unexpected phenotypic variation

To define the range of phenotypic expression in Treacher Collins syndrome (TCS; Franceschetti–Klein syndrome), we performed mutation analysis in the TCOF1 gene in 46 patients with tentative diagnosis of TCS and evaluated the clinical data, including a scoring system. A total of 27 coding exons of TCOF1 and adjacent splice junctions were analysed by direct sequencing. In 36 patients with a clinically unequivocal diagnosis of TCS, we detected 28 pathogenic mutations, including 25 novel alterations. No mutation was identified in the remaining eight patients with unequivocal diagnosis of TCS and 10 further patients, in whom the referring diagnosis of TCS was clinically doubtful. There is no overt genotype–phenotype correlation except that conductive deafness is significantly less frequent in patients with mutations in the 3′ part of the open reading frame. Inter- and intrafamilial variation is wide. Some mutation carriers, parents of typically affected patients, are so mildly affected that the diagnosis might be overlooked clinically. This suggests that modifying factors are important for phenotypic expression. Based on these findings, minimal diagnostic criteria were defined: downward slanting palpebral fissures and hypoplasia of the zygomatic arch. The difficulties in genetic counselling, especially diagnosis of family members with a mild phenotype, are described.

Polysomy of chromosome 7 is correlated with overexpression of the erbB oncogene in human glioblastoma cell lines

SummaryChromosome analysis in a series of human glioblastoma cell lines (HeRo, HeRo-SV1, A172, T406, T508, T705) has indicated characteristic changes in the karyotype, the most striking and consistent of which is a significant increase in the copy number of chromosome 7, with up to 8 copies per metaphase. As determined by Spurr et al., chromosome 7 represents the genomic locus for the oncogene erbB (7pter-q22). Therefore, we have compared the number of chromosomes 7 to the levels of expression of the erbB oncogene. Interestingly, in all of them erbB-specific mRNA was found to be increased at levels even higher than expected from the number of chromosomes 7 found. In contrast, in an astrocytoma of slightly lower grade of malignancy (cell line T567), neither polysomy 7 nor significant expression of the erbB oncogene was noted.

Homologous sequences at human chromosome 9 bands p12 and q13-21.1 are involved in different patterns of pericentric rearrangements

A thorough study of the heterochromatin organisation in the pericentromeric region and the proximal long (q) and short (p) arms of human chromsome 9 (HSA 9) revealed homology between 9p12 and 9q13-21.1, two regions that are usually not distinguishable by molecular cytogenetic techniques. Furthermore, the chromosomal regions 9p12 and 9q13-21.1 showed some level of homology with the short arms of the human acrocentric chromosomes. We studied five normal controls and 51 clinical cases: 48 with chromosome 9 heteromorphisms, one with an exceptionally large inversion and two with an additional derivative chromosome 9. Using fluorescence in situ hybridisation (FISH) with three differentially labelled chromosome 9-specific probes we were able to distinguish 12 heteromorphic patterns in addition to the most frequent pattern (defined as normal). In addition, we studied one inversion 9 case with the recently described multicolour banding (MCB) technique. Our results, and previously published findings, suggest several hotspots for recombination in the pericentromeric heterochromatin of HSA 9. They also demonstrate that constitutional inversions affecting the pericentromeric region of chromosome 9 carry breakpoints located preferentially in 9p12 or 9q13-21.1 and less frequently in 9q12.

Homozygous PMS2 germline mutations in two families with early-onset haematological malignancy, brain tumours, HNPCC-associated tumours, and signs of neurofibromatosis type 1

Heterozygous germline mutations in mismatch repair (MMR) genes MLH1, PMS2, MSH2, and MSH6 cause Lynch syndrome. New studies have indicated that biallelic mutations lead to a distinctive syndrome, childhood cancer syndrome (CCS), with haematological malignancies and tumours of brain and bowel early in childhood, often associated with signs of neurofibromatosis type 1. We provide further evidence for CCS reporting on six children from two consanguineous families carrying homozygous PMS2 germline mutations. In family 1, all four children had the homozygous p.I590Xfs mutation. Two had a glioblastoma at the age of 6 years and one of them had three additional Lynch-syndrome associated tumours at 15. Another sibling suffered from a glioblastoma at age 9, and the fourth sibling had infantile myofibromatosis at 1. In family 2, two of four siblings were homozygous for the p.G271V mutation. One had two colorectal cancers diagnosed at ages 13 and 14, the other had a Non-Hodgkins lymphoma and a colorectal cancer at ages 10 and 11, respectively. All children with malignancies had multiple café-au-lait spots. After reviewing published cases of biallelic MMR gene mutations, we provide a concise description of CCS, revealing similarities in age distribution with carriers of heterozygous MMR gene mutations.

Loss of Alkaline Phosphatase Activity in Meningiomas: A Rapid Histochemical Technique Indicating Progression-associated Deletion of a Putative Tumor Suppressor Gene on the Distal Part of the Short Arm of Chromosome 1

Apart from defined histomorphologic features, increased Ki-67 indices and various numeric and structural chromosome aberrations, meningiomas of the intermediate (WHO grade II, atypical meningioma) and anaplastic type (WHO grade III) are cytogenetically distinguished from common-type meningiomas (WHO grade I) by frequent loss of the distal part of the short arm of one chromosome 1 (1p-), which formerly proved to be an independent predictor of shorter recurrence-free intervals. Histochemically, loss of alkaline phosphatase activity (ALPL, liver/bone/kidney type, EC 3.1.3.1) was another frequent, specific finding in meningiomas with signs of dedifferentiation. In a prospective study including 66 meningiomas, all common-type meningiomas except one case (18/19) were reactive for ALPL, whereas 75% (30/39) of intermediate type and all anaplastic meningiomas (8/8) showed loss of enzyme activity in large areas of the tumor. Exclusively, the ALPL negative phenotype was associated with 1p loss (15/19). Our data suggest that ALPL, which is coded as a single copy gene on chromosome 1p36.1-p34, is a useful marker enzyme for the loss of a putative regulatory (tumor suppressor) gene on chromosome 1p, or that ALPL itself represents a new tumor suppressor gene homozygously inactivated in meningiomas.

Mosaicism in Turner's syndrome

Skuse et al. report that females with Turners syndrome who have retained the paternal X chromosome (Xp) tend to achieve better cognition and social adjustment scores than those with the maternal X (Xm). As sex-chromosome mosaicism is frequent in Turners syndrome, we argue that the presence of residual Y chromosomal sequences in the brain, which is exclusively possible in Xm Turner patients, might be a realistic alternative to the hypothesis of an imprinted X-linked locus as a reason for the behavioural differences between Xm and Xp Turner patients.

An offer you can't refuse? Ethical implications of non-invasive prenatal diagnosis.

aneuploidies has been an integral part of prenatal medicine for over 30 years. It is usually performed as a combination of initial non-invasive risk screening (NIRS) strategies, and suspicious results are followed by invasive diagnostic procedures such as amniocentesis. Recently, however, several breakthroughs in non-invasive prenatal diagnosis (NIPD) indicate the upcoming clinical applicability of this procedure. In 2007, after years of largely fruitless efforts to enrich fetal cells in maternal plasma, Lo et al. reported the ability to correctly diagnose trisomy 21 in the fetus using quantitative analysis of cell-free fetal RNA in maternal blood. And in 2008, Fan et al. used shotgun sequencing of cell-free DNA in maternal blood to non-invasively diagnose fetal aneuploidy. Recently, at least two companies, using different technical approaches, have announced plans to introduce NIPD into health care: in February 2009, Lenetix, Inc. reported the launch of an Institutional Review Board-approved screening study on NIPD for Down’s syndrome, and Sequenom, Inc. has revealed that NIPD will be available for clinical use in the fourth quarter of 2009 (ref. 4). With regard to the clinical implementation of NIPD, three scenarios are possible: NIPD might replace current prenatal screening tests or be added to them; NIPD might be interposed between NIRS and invasive PND; or NIPD might replace invasive PND. Which of these options is followed will depend primarily on the technical accuracy of the NIPD strategies (in terms of sensitivity and specificity) but the third option seems to be the most attractive, especially in the long term. What will the effects be of this third option, which sees invasive methods no longer being required for routine PND of aneuploidies? Depending on the NIPD method used three effects can be anticipated, which, especially in combination, might pose serious threats to the autonomous decision making of the pregnant woman. First, prenatal (cyto)genetic diagnosis will be achieved much earlier in pregnancy. From a medical perspective this is a positive development as termination-related risks for the mother are minimized at an earlier gestational age. But it is debatable whether such a shift would be ethically desirable, because early diagnosis could increase the proportion of pregnant women opting for termination, including for non-medical reasons (such as for ‘unwanted’ fetal sex). Second, uptake rates of NIPD might become as high as they are for NIRS strategies now, which in Germany, for example, could mean up to 85%. Providing adequate pre-test counselling for such a high percentage of pregnant women would be challenging. Finally, the two-step approach of prenatal screening potentially followed by invasive diagnosis will be transformed into a one-step procedure. Although the abolishment of invasive PND has some positive aspects, as women who undergo prenatal testing would no longer have to risk fetal loss or their own health, the one-step diagnostic procedure might worsen current tendencies to neglect the reproductive autonomy of pregnant women. There will be only one contact between the pregnant woman and the physician to discuss the pros and cons of NIPD, which — if NIPD is implemented analogously to NIRS — might take place in a mass screening setting. It is therefore legitimate to ask whether this diagnostic ‘offer’ can be refused, and whether an autonomous decision-making process is still possible. Reproductive autonomy, however, is of utmost importance in PND. This is because PND differs from other diagnostic procedures in medicine insofar as most of the conditions tested cannot be cured or substantially alleviated. In these cases, the mother’s only option is to decide whether to accept the child’s impairment or to terminate the pregnancy. Consequently, the main argument for offering prenatal genetic testing is to enhance the reproductive autonomy of the pregnant woman. Studies have investigated the quality of informed consent in currently available multi-step procedures for PND. In a representative German survey, women were asked what prompted them to opt for PND: 25% stated that their physician wanted it; 36% thought that PND is an almost mandatory part of routine maternal care; and 16% had not given consent to the performed PND or could not remember giving consent. Studies in many other countries have provided similar results. In France, a survey of 305 pregnant women — most of whom had undergone maternal serum screening — revealed that nearly half of the women were uninformed about the procedure. There is already a need to improve the quality of informed consent for existing multi-step prenatal genetic examinations — a need that will be even more pressing if NIPD becomes a one-step procedure. We argue that a new organizational setting for NIPD, which from the patient’s point of view is clearly distinguishable from the setting for therapy-aimed prenatal care, could allow for a decision-making process to take place prior to NIPD and thus secure the reproductive autonomy of the pregnant woman. Women need to understand that there is a decision to be taken, that NIPD is not part of routine maternity care, and that the main aim of NIPD is to enable the pregnant woman to decide whether to live with an impaired child or to terminate the pregnancy. Therefore, a two-step approach with counselling in the first stage and decision-making and specific testing, where appropriate, in the second stage is essential for ensuring a value-consistent shared decision-making process that also takes into account the welfare of the unborn child.

Frequent mitotic errors in tumor cells of genetically micro-heterogeneous glioblastomas

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity as to both histomorphology and genetic changes, displaying a wide variety of numerical chromosome aberrations the most common of which are monosomy 10 and trisomy 7. Moreover, GBM in vitro are known to have variable karyotypes within a given tumor cell culture leading to rapid karyotype evolution through a high incidence of secondary numerical chromosome aberrations. The aim of our study was to investigate to what extent this mitotic instability of glioblastoma cells is also present in vivo. We assessed the spatial distribution patterns of numerical chromosome aberrations in vivo in a series of 24 GBM using two-color in situ hybridization for chromosomes 7/10, 8/17, and 12/18 on consecutive 6-µm paraffin-embedded tissue slides. The chromosome aberration patterns were compared with the histomorphology of the investigated tumor assessed from a consecutive HE-stained section, and with the in vitro karyotype of cell cultures established from the tumors. All investigated chromosomes showed mitotic instability, i.e., numerical aberrations within significant amounts of tumor cells in a scattered distribution through the tumor tissue. As to chromosomes 10 and 17, only monosomy occurred, as to chromosome 7 only trisomy/polysomy, apparently as a result of selection in favor of the respective aberration. Conversely, chromosomes 8, 12, and 18 displayed scattered patterns of monosomy as well as trisomy within a given tumor reflecting a high mitotic error rate without selective effects. The karyotypes of the tumor cell cultures showed less variability of numerical aberrations apparently due to clonal adaptation to in vitro conditions. We conclude that glioblastoma cells in vivo are characterized by an extensive tendency to mitotic errors. The resulting clonal diversity of chromosomally aberrant cells may be an important biological constituent of the well-known ability of glioblastomas to preserve viable tumor cell clones under adaptive stress in vivo, in clinical terms to rapidly recur after antitumoral therapy including radio- or chemotherapy.

Malignant odontogenic myxoma of the maxilla: case with cytogenetic confirmation.

An odontogenic myxoma of the maxilla with an aggressive clinical course is presented. The tumour arose in a 53-year-old patient, recurred two times after extended maxillectomy and ultimately caused the patients death by uncontrollable local disease with infiltration of the cranial cavity. Microscopically, the tumour showed histological features of a low grade malignant myxosarcoma with cellular areas, enhanced mitotic activity and nuclear pleomorphism. Cytogenetic analysis revealed an unexpectedly aberrant hypertetraploid chromosome complement, that was considered as incompatible with the usual karyotypic patterns of benign tumours.

Monosomy 1p is correlated with enhanced in vivo glucose metabolism in meningiomas

The 2-[18F]fluoro-2-deoxy-D-glucose (FDG) uptake of 25 human meningiomas was preoperatively evaluated in vivo by positron-emission tomography (PET). After surgery, meningioma biopsies were analyzed cytogenetically. Five meningiomas showed partial monosomy for chromosome 1p additional to other typical chromosome aberrations. This aberrant karyotype was correlated with increased FDG uptake. Three of five meningiomas with monosomy 1p were classified as grade II according to WHO, while only one of 20 tumors without monosomy 1p was classified as grade II. Thus, monosomy 1p and elevated FDG uptake in PET are to be regarded as cytogenetic and metabolic parameters for the aggressiveness of meningiomas.