The fetus in the age of the genome.

Due to a number of recent achievements, the field of prenatal medicine is now on the verge of a profound transformation into prenatal genomic medicine. This transformation is expected to not only substantially expand the spectrum of prenatal diagnostic and screening possibilities, but finally also to advance fetal care and the prenatal management of certain fetal diseases and malformations. It will come along with new and profound challenges for the normative framework and clinical care pathways in prenatal (and reproductive) medicine. To adequately address the potential ethically challenging aspects without discarding the obvious benefits, several agents are required to engage in different debates. The permissibility of the sequencing of the whole fetal exome or genome will have to be examined from a philosophical and legal point of view, in particular with regard to conflicts with potential rights of future children. A second requirement is a societal debate on the question of priority setting and justice in relation to prenatal genomic testing. Third, a professional-ethical debate and positioning on the goal of prenatal genomic testing and a consequential re-structuring of clinical care pathways seems to be important. In all these efforts, it might be helpful to envisage the unborn rather not as a fetus, not as a separate moral subject and a second "patient", but in its unique physical connection with the pregnant woman, and to accept the moral quandaries implicitly given in this situation.

General Cognitive Abilities and Psychosocial Development in Children and Adolescents Having a Co-Twin with Down Syndrome.

OBJECTIVE: To examine the general cognitive and psychosocial development in children and adolescents having a co-twin with Down syndrome. STUDY DESIGN: A case control study with an individually matched control group was conducted. Participants included families with twins discordant for Down syndrome as well as with typically developing twins. The group of unaffected co-twins aged 4-16 years was compared with a control group of typically developing twins in terms of general cognitive abilities, behavioral problems, and prosocial behavior. The age and sex and the sex composition of the twins were individually matched. The Sijnders-Oomen nonverbal intelligence test was applied to assess children's IQ, and parents completed the Strength and Difficulties Questionnaire. RESULTS: The unaffected co-twins did not differ from typically developing twins with respect to their IQ. Concerning the psychosocial development, significantly heightened values in unaffected co-twins twins were only obtained for the conduct problems scale (P = .01; r = 0.45), neither for the total difficulties score nor for the other behavioral problem scales significant differences were found. CONCLUSIONS: The general cognitive development of the unaffected co-twin of children with Down syndrome is not affected by the presence of their Down syndrome twin. Unaffected co-twins showed increased conduct problems, which is most pronounced in the younger children.

Behavioral and psychological features in girls and women with triple-X syndrome.

Triple-X syndrome is a common sex chromosome aneuploidy, which appears in 1 out of 1,000 females. The aim of our study was to describe the behavioral features of a large group of girls and women with triple-X in comparison to a control group. A total of 72 subjects with triple-X and 69 subjects of an age-matched control group were included. Psychological and behavioral questionnaires were allocated to three age groups, representing a range of ages from young childhood to adulthood. Regarding the females between 4 and 7 years of age, we found significant differences for social problems, attention problems, and school performance. For the age group 8-17 years, we found larger significant differences for the majority of the scales listed in the child behavior checklist. The most significant differences (p < .001) were from total behavior problems, internalizing problems, and four other scales. Young females with triple-X have significantly lower general self-esteem, especially concerning school and family. In the adults, there were significant differences concerning psychological symptoms and distress, with higher scores in the triple-X subjects. Regardless, their mean scores were still in the normal range. We did not find clinical evidence for more than 50% of the triple-X females in any age group, indicating that approximately half of them do not have behavioral problems, and that more than 60% do not differ in their competence from the control group. However, our findings suggest that triple-X influences mental health and the overall well-being of the individuals across their whole life spans.

Predictive genetic testing of at-risk relatives requires analysis of all CCM genes after identification of an unclassified CCM1 variant in an individual affected with cerebral cavernous malformations.

The mutation detection rate for familial cerebral cavernous malformations (CCM) is extremely high, being about 90 % if direct sequencing of the three genes, CCM1, CCM2, and CCM3, is used in conjunction with quantitative analyses to detect larger CCM1-3 deletions/duplications. We here report on an individual who had presented with more than 30 cerebral and spinal cavernous malformations, two intracranial meningiomas, and disease manifestation only in the mid-forties. A CCM1 missense variant of unclear relevance was found during the first sequencing step. Thereafter, direct sequencing of all three CCM genes revealed the typical pathogenic loss-of-function mutation c.598C > T/p.Q200* in the CCM3 gene. Our results demonstrate that mutation analyses of all three CCM genes in the index patient regardless of previous identification of an unclassified CCM1 variant is crucial for reliable predictive testing of at-risk relatives.

Novel FHL1 mutation in a family with reducing body myopathy.

INTRODUCTION: Reducing body myopathy is a rare X-linked myopathy. It is characterized by intracytoplasmic inclusions that stain with menadione-nitroblue tetrazolium. It is caused by mutations in the FHL1 gene, which encodes the four-and-a-half LIM domain 1 protein (FHL1). METHODS: We performed a clinical, muscle MRI, and histopathological characterization and immunoblot and genetic analysis of the FHL1 protein in a family with 4 individuals affected by reducing body myopathy. RESULTS: We identified a novel missense mutation in FHL1 (c.449G>C; p.C150S). The patients presented with asymmetric proximal weakness and scoliosis. Both of the boys had a more severe course with earlier onset, contractures, and death due to heart failure at 14 and 18 years of age, respectively. MRI revealed fatty infiltration of posteromedial thigh and paraspinal muscles. Histopathological findings showed FHL1-immunoreactive inclusions. Immunoblot analysis revealed a 50% reduction of FHL1 protein. CONCLUSION: In this study we highlighted diagnostic clues in this myopathy and compared our data with the literature.

Genome wide expression profiling identifies specific deregulated pathways in meningioma.

Genome-wide expression signatures improve the understanding of tumor biology. We performed expression profiling of 24 meningioma including 8 of each WHO grade and 2 dura controls analyzing 55.000 transcripts including 18.300 known genes. We compared expression in meningioma vs. dura, expression of low grade (WHO I) vs. higher-grade (WHO II and WHO III) tumors and expression of meningothelial and syncytial meningioma vs. fibroblastic meningioma. Overall expression was significantly decreased in meningioma compared to dura and in meningothelial and syncytial compared to fibroblastic meningioma. Gene expression was exemplarily confirmed by immunohistochemistry using independent samples. Applying our statistical gene set analysis toolkit "GeneTrail", we identified significantly deregulated biochemical pathways using Kyoto encyclopedia of genes and genomes and Transpath databases. Kyoto encyclopedia of genes and genomes pathways with decreased expression in meningioma included cell adhesion molecules (p<0.0001) and cytokine-cytokine receptor interactions (p<0.0001). Pathways with increased expression included several metabolic pathways. Extended expression profiling by a novel statistical gene set enrichment identified pathways that have previously not been associated with meningioma.

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-Hodgkin's 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.

Hypothesis: Possible role of retinoic acid therapy in patients with biallelic mismatch repair gene defects.

A boy showing symptoms of a Turcot-like childhood cancer syndrome together with stigmata of neurofibromatosis type I is reported. His brother suffers from an infantile myofibromatosis, and a sister died of glioblastoma at age 7. Another 7-year-old brother is so far clinically unaffected. The parents are consanguineous. Molecular diagnosis in the index patient revealed a constitutional homozygous mutation of the mismatch repair gene PMS2. The patient was in remission of his glioblastoma (WHO grade IV) after multimodal treatment followed by retinoic acid chemoprevention for 7 years. After discontinuation of retinoic acid medication, he developed a relapse of his brain tumour together with the simultaneous occurrence of three other different HNPCC-related carcinomas. We think that retinoic acid might have provided an effective chemoprevention in this patient with homozygous mismatch repair gene defect. We propose to take a retinoic acid chemoprevention into account in children with proven biallelic PMS2 mismatch repair mutations being at highest risk concerning the development of a malignancy.

Mutations in the UBIAD1 gene on chromosome short arm 1, region 36, cause Schnyder crystalline corneal dystrophy.

PURPOSE: Schnyder crystalline corneal dystrophy (SCCD; MIM 121800) is a rare autosomal dominant disease characterized by an abnormal increase in cholesterol and phospholipid deposition in the cornea, leading to progressive corneal opacification. Although SCCD has been mapped to a genetic interval between markers D1S1160 and D1S1635, reclassification of a previously unaffected individual expanded the interval to D1S2667 and included nine additional genes. Three candidate genes that may be involved in lipid metabolism and/or are expressed in the cornea were analyzed. METHODS: DNA samples were obtained from six families with clinically confirmed SCCD. Analysis of FRAP1, ANGPTL7, and UBIAD1 was performed by PCR-based DNA sequencing, to examine protein-coding regions, RNA splice junctions, and 5' untranslated region (UTR) exons. RESULTS: No disease-causing mutations were found in the FRAP1 or ANGPTL7 gene. A mutation in UBIAD1 was identified in all six families: Five families had the same N102S mutation, and one family had a G177R mutation. Predictions of the protein structure indicated that a prenyl-transferase domain and several transmembrane helices are affected by these mutations. Each mutation cosegregated with the disease in four families with DNA samples from both affected and unaffected individuals. Mutations were not observed in 100 control DNA samples (200 chromosomes). CONCLUSIONS: Nonsynonymous mutations in the UBIAD1 gene were detected in six SCCD families, and a potential mutation hot spot was observed at amino acid N102. The mutations are expected to interfere with the function of the UBIAD1 protein, since they are located in highly conserved and structurally important domains.

Histopathologic indicators of recurrence in meningiomas: correlation with clinical and genetic parameters.

Meningiomas in general are circumscribed slow-growing tumors. However, despite gross total resection, tumor relapse and patients' outcome are still an issue. Risk stratification based on histomorphology alone remains problematic. This study explored the independent prognostic value of potential risk factors among 206 patients who underwent meningioma resection and followed-up until death or a median of 44 months. The statistical analysis considered clinical data, histomorphologic parameters, cytogenetic findings, Ki-67 immunoreactivity, and activity of tissue non-specific alkaline phosphatase (ALPL). Recurrence-free survival estimates were computed and prognostic factors were identified using Cox proportional hazards model. Independent predictors of recurrence included (1) anaplasia; (2) mitotic index > or =20/10 high-power fields; (3) subtotal tumor resection; (4) loss of short arm of chromosome 1 (1p-); and (5) Ki-67 labeling index (LI) >12%. Among totally resected WHO grade I meningiomas, neither histopathologic nor clinical parameters were predictive, whereas 1p- was the only independent prognostic factor. ALPL did not reach significance in the multivariate modeling, however, the fast and low-cost histochemical detection of ALPL expression could be proved as a highly sensitive screening method for 1p-. In particular, biologically aggressive meningiomas of histologically benign or "borderline" phenotype could be therefore identified by ALPL detection followed by 1p in situ hybridization.

Interstitial loss and gain of sequences on chromosome 22 in meningiomas with normal karyotype.

In nearly half of sporadic low grade meningiomas no chromosome aberration can be detected. In the majority of the other half chromosome 22 is lost. In higher grade meningiomas this loss is followed by characteristic secondary chromosome aberrations. Regarding the molecular findings in Schwannomas, homozygous loss or mutation of the NF2 gene located on chromosome 22, was supposed also to be the primary event in meningioma development. However, in nearly all high grade but in only a minority of low grade meningiomas the loss of the NF2 protein is observed. Therefore, both the hypothetical combined heterozygous loss of or inactivation of two or more tumour suppressor genes (at least one of them located on chromosome 22) or the homozygous loss of a regulatory gene on chromosome 22 different from NF2 was discussed. In search for microdeletions or/and structural recombinations of chromosome 22 we investigated primary cell cultures of 43 meningiomas by conventional G-banding (26 without, 17 with loss of chromosome 22). Twenty-seven tumours were analysed with spectral karyotyping (SKY) and 16 with fluorescence in situ hybridisation (FISH) with DNA probes for the chromosomal regions of 22q11.2, 22q11.23q12.1, 22q12.1 and 22q13.3. SKY analysis confirmed G-banding data for chromosome 22 and could specify marker chromosomes and translocations containing material from chromosome(s) 22. Confirming our assumption microdeletions on chromosome 22 were detected by FISH in 6/8 cytogenetically non-aberrant meningiomas. Surprisingly, in 2/8 cases we observed gains of the 22q13.3 and in 2/8 gains of the 22q12.1 region. Here we present first evidence for an uncommon mechanism during early meningioma development at least for a meningioma subgroup: i) duplication and translocation of sequences from chromosome 22 to different chromosomes. ii) deletion of the original sequences on chromosome 22, resulting in disomy again (only visible as translocation in metaphase FISH). iii) loss of chromosome 22.

Detailed chromosomal characterization of the breast cancer cell line MCF7 with special focus on the expression of the serine-threonine kinase 15.

Although the cell line MCF7 is widely used in breast cancer research, its cytogenetic properties have not been thoroughly investigated so far. As conventional G-banding analysis cannot resolve the complex chromosome aberrations, we investigated MCF7 cells using molecular-cytogenetic methods, with particular attention to the DNA amplification site on chromosome 20q. With spectral karyotyping we found numerous unbalanced chromosome translocations, and with comparative genomic hybridization we detected many quantitative genomic imbalances. Furthermore, we analyzed the amplified region at 20q with the candidate tumour susceptibility gene STK15 in detail by fluorescence in situ hybridization, whole chromosome painting, immunohistochemistry, Western blot and expression analysis. In MCF7 interphase cells we found increased copy number of the STK15 gene associated with overexpression of STK15 mRNA. Accordingly, STK15 protein is overexpressed as compared to normal human fibroblasts in Western blot analysis. Overexpression of STK15 mRNA and protein is disproportionally stronger than that expected from the single additional copy of the STK15 gene. These data indicate that the highly increased level of STK15 protein in MCF7 cannot be explained by gene amplification alone. Apparently, secondary mechanisms of gene up-regulation are involved. This observation may be of general interest with regard to the activation of oncogenes in tumour cells.

Left ventricular systolic dysfunction in asymptomatic Marfan syndrome patients is related to the severity of gene mutation: insights from the novel three dimensional speckle tracking echocardiography.

BACKGROUND: In asymptomatic Marfan syndrome (MFS) patients we evaluated the relationship between the types of fibrillin-1 (FBN1) gene mutation and possible altered left ventricular (LV) function as assessed by three-dimensional speckle tracking echocardiography (3D-STE). METHODS AND RESULTS: Forty-five MFS patients (mean age 24 ± 15 years) and 40 age-matched healthy controls were studied. Genetic evaluation for the FBN1 gene was carried on 32 MFS patients. Gene mutation (n = 15, 47%) was classified as mild when the mutation resulted in nearly normally functioning protein, while mutations resulting in abnormally function protein were considered to be severe (n = 17, 53%). All patients and controls underwent 3D-STE for evaluation of LV function by an echocardiographer blinded to the results of the genetic testing. Compared to controls, MFS patients had significantly lower 3D-STE derived LV ejection fraction (EF, 57.43 ± 7.51 vs. 62.69 ± 4.76%, p = 0.0001), global LV longitudinal strain (LS, 14.85 ± 2.89 vs. 17.90 ± 2.01%, p = 0.0001), global LV circumferential strain (CS, 13.93 ± 2.81 vs. 16.82 ± 2.17%, p = 0.0001) and global LV area strain (AS, 25.76 ± 4.43 vs. 30.51 ± 2.61%, p = 0.0001). Apart from the global LV LS all these parameters were significantly lower in patients with severe gene mutation than in those with mild mutation (p < 0.05). In the multivariate linear regression analysis only the type of mutation had a significant influence on the 3D-STE derived LVEF (p = 0.017), global CS (p = 0.005) and global AS (p = 0.03). CONCLUSIONS: In asymptomatic MFS patients latent LV dysfunction can be detected using 3D STE. The LV dysfunction is mainly related to the severity of gene mutation, suggesting possible primary cardiomyopathy in MFS patients.

New insights into the genetics of glioblastoma multiforme by familial exome sequencing.

Glioblastoma multiforme (GBM) is the most aggressive and malignant subtype of human brain tumors. While a family clustering of GBM has long been acknowledged, relevant hereditary factors still remained elusive. Exome sequencing of families offers the option to discover respective genetic factors.We sequenced blood samples of one of the rare affected families: while both parents were healthy, both children were diagnosed with GBM. We report 85 homozygous non-synonymous single nucleotide variations (SNVs) in both siblings that were heterozygous in the parents. Beyond known key players for GBM such as ERBB2, PMS2, or CHI3L1, we identified over 50 genes that have not been associated to GBM so far. We also discovered three accumulative effects potentially adding to the tumorigenesis in the siblings: a clustering of multiple variants in single genes (e.g., PTPRB, CROCC), the aggregation of affected genes on specific molecular pathways (e.g., Focal adhesion or ECM receptor interaction) and genomic proximity (e.g., chr22.q12.2, chr1.p36.33). We found a striking accumulation of SNVs in specific genes for the daughter, who developed not only a GBM at the age of 12 years but was subsequently diagnosed with a pilocytic astrocytoma, a common acute lymphatic leukemia and a diffuse pontine glioma.The reported variants underline the relevance of genetic predisposition and cancer development in this family and demonstrate that GBM has a complex and heterogeneous genetic background. Sequencing of other affected families will help to further narrow down the driving genetic causes for this disease.

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 chromosome 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.

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.