Chromosomal microaberrations in patients with epilepsy, intellectual disability, and congenital anomalies.

Epilepsy is a common finding in patients with chromosomal macro- and micro-rearrangements but only few aberrations show a constant pattern of seizures. DNA array-based studies have reported causative copy number variations (CNVs) in 5-30% of patients with epilepsy with or without co-morbidities. The interpretation of many of the detected CNVs remains challenging. In order to identify CNVs carrying epilepsy-related genes we investigated 43 children with various patterns of epileptic seizures, intellectual disability (ID), and minor dysmorphism, using the Illumina® Infinium Human1M-DuoV1 array. In three patients we found likely causative de novo CNVs, i.e. deletions in 1q41q42.12 (3.4 Mb) and 19p13.2 (834 kb), and a mosaic two-segment duplication in 17p13.2 (218 kb) and 17p13.1 (422 kb). In six additional patients there were aberrations (a deletion in one and duplications in five patients) with uncertain clinical consequences. In total, the finding of causative chromosomal micro-rearrangements in 3 out of 43 patients (7%) and potentially causative CNVs in 6 additional patients (14%) with epilepsy and ID but without major malformations confirms the power of DNA arrays for the detection of new disease-related genetic regions.

Modification of risk for cancer as a coincidental finding in DNA array investigation.

The high resolution of modern DNA arrays has the implification of unintended coincidental detection of gene deletions predisposing to late-onset neurological and oncological disorders. Here, we report the case of an 18-year-old girl with mild intellectual disability, facial dysmorphisms, and a microdeletion of approximately 6.3 Mb on 22q12.1q12.3 including NF2, the gene for neurofibromatosis type 2, and CHEK2, a modifier gene for breast cancer. Subsequent magnetic resonance imaging of the brain showed she had already developed bilateral vestibular schwannomas. The challenge of DNA arrays and the consequences for genetic counselling and informed consent will be discussed in the light of this unique case with a microdeletion including both a high risk and a moderate risk cancer predisposition gene.

[Acoustic study of sustained vowels made by patients with recurrent nerve paralysis after thyroidectomy]. / Etude acoustique de voyelles soutenues produites par des patients présentant une paralysie récurrentielle après thyroïdectomie.

The objective of this work is to evaluate the consequences of thyroid surgery on the voice of patients suffering from recurrent paralysis. The consequences of the surgery are evaluated using a corpus of sustained vowels in order to identify the various disruptions that this procedure may produce. This research also looks for possible compensatory and/or readjustment strategies that can be used by a patient alone and with the help of speech therapy. Acoustic measurements considered are fundamental frequency (F0), Harmonics-to-Noise Ratio (HNR), and vowel space area. This is a longitudinal study, as all patients are recorded once a month during three months after surgery. Results reveal a modification of all parameters in the early recording stages. However, time and speech therapy contribute to obtaining expected values of the measured parameters, and thus to improvement of vocal quality.

Parental origin of de novo cytogenetically balanced reciprocal non-Robertsonian translocations.

De novo cytogenetically balanced reciprocal non-Robertsonian translocations are rare findings in clinical cytogenetics and might be associated with an abnormal phenotype. Knowledge of the parental origin and mechanisms of formation is still limited. By microdissection of the derivative chromosomes and their normal homologs from metaphases followed by microsatellite-mediated marker analysis we identified 7 cases of paternal and 3 cases of maternal origin in a cohort of 10 patients with de novo cytogenetically balanced reciprocal non-Robertsonian translocations. Neither in the maternal nor in the paternal group of our study parental age seems to be increased. Together with the data from the literature our results confirm that the majority of de novo cytogenetically balanced reciprocal translocations are of paternal origin, but the preponderance does not appear to be as distinct as previously thought and the paternal age does not seem to be necessarily a major contributing factor.

Parental origin of apparently balanced de novo complex chromosomal rearrangements investigated by microdissection, whole genome amplification, and microsatellite-mediated haplotype analysis.

Complex chromosomal rearrangements (CCRs) are rare findings in clinical cytogenetics. As a result of the high risk of unbalanced segregation, familial cases are even rarer and maternal transmission occurs more frequently than paternal transmission. Analogous to Drosophila and mice, as well as to CCRs involving the Y chromosome or a clinically relevant associated deletion, a preferential origin in spermatogenesis has been assumed but not proven directly and systematically thus far. Here, we investigated three healthy adults, one healthy child, and one child with multiple congenital anomalies and various balanced de novo CCRs. The analyses were performed in each case on 10 copies of a derivative chromosome and their normal homologs by glass-needle microdissection, whole genome amplification (WGA), and microsatellite-mediated haplotype analysis. With respect to the number of chromosomes involved in each case and in all cases together, the number of chromosomal segments in each case and in all cases together, and the number of breakpoints in each case and in all cases together, the conformity for paternal origin of all derivative chromosomes and maternal origin of their normal homologs makes formation in paternal germline more likely than a postzygotic formation with an accidental uniformity. In conclusion, our results confirm the preferential formation of de novo balanced CCRs in the paternal germline.

Partial 7q11.23 deletions further implicate GTF2I and GTF2IRD1 as the main genes responsible for the Williams-Beuren syndrome neurocognitive profile.

BACKGROUND: Williams-Beuren syndrome (WBS) is a developmental disorder with multisystemic manifestations mainly characterised by vascular stenoses, distinctive craniofacial features, mental retardation with a characteristic neurocognitive profile, and some endocrine and connective tissue abnormalities, caused by a recurrent deletion of 1.55 Mb including 26-28 genes at chromosomal region 7q11.23. The analysis of clinical-molecular correlations in a few reported atypical patients has been useful to propose several deleted genes as main contributors to specific aspects of the WBS phenotype. PATIENTS AND METHODS: Two additional families with partial phenotypes and atypical 7q11.23 deletions were studied. Deletions were precisely defined at the nucleotide level, and the expression levels of some affected and flanking genes were assessed in lymphoblastoid cell lines. RESULTS: Affected individuals presented variable cardiovascular and connective tissue manifestations, subtle craniofacial features, normal visuospatial construction abilities with low average IQ and no endocrine abnormalities. The deletion in family NW1 encompassed 817 kb with 11 genes (CLDN3-GTF2IRD1), and 610 kb with 14 genes (VPS37D-RFC2) in family NW2. All deleted genes in typical and atypical deletions revealed low expression levels in lymphoblastoid cell lines, except for GTF2IRD1. CLIP2 was also underexpressed in all patients despite being outside the deletion in NW2, while no other flanking non-deleted gene showed significantly different expression compared to controls. CONCLUSIONS: Along with previously reported cases, clinical-molecular correlations in these two families further confirm that the functional hemizygosity for the GTF2I and GTF2IRD1 genes is the main cause of the neurocognitive profile and some aspects of the gestalt phenotype of WBS.

Two sporadic spinal neurofibromatosis patients with malignant peripheral nerve sheath tumour.

We analyzed two unrelated male patients in whom neurofibromatosis type 1 (NF1) was not suspected until they presented with malignant peripheral nerve sheath tumours (MPNSTs) in their thirties and forties, respectively. Patient A presented with progressive peroneus paresis due to a rapidly growing MPNST in the thigh. MRI examination revealed multiple symmetrical spinal neurofibromas in this patient as well as in patient B who presented at the age of 42 with paraparesis and an MPNST at spinal level L4. Dermal features in both patients were strikingly mild, therefore both patients were considered belonging to the NF1-subform of spinal neurofibromatosis (SNF). The novel NF1 mutations identified, i.e. splice mutation, c.7675+1G > A, in patient A and two alterations, p.Cys1016Arg and p.2711delVal, located in trans in patient B support the notion that the phenotype of SNF may be related to mutations with possible residual functionality. The MPNSTs of both patients showed LOH affecting chromosome 17 including the NF1 locus. Furthermore, a truncating TP53 mutation was identified in the tumour of patient A. Both alterations are frequent findings in NF1-associated MPNSTs. To our knowledge these are the first MPNST patients with the clinical phenotype of SNF. The clinical course observed in these two patients suggests that nodular plexiform neurofibromas and spinal-nerve-root neurofibromas which may be asymptomatic for a long time and, hence, unrecognized in SNF patients bear the risk for malignant transformation.

First non-mosaic case of isopseudodicentric chromosome 18 (psu idic(18)(pter --> q22.1::q22.1 --> pter) is associated with multiple congenital anomalies reminiscent of trisomy 18 and 18q- syndrome.

Isopseudodicentric chromosome 18 is very rare and results in a combination of partial trisomy and partial monosomy of chromosome 18. We report here a hypotrophic newborn with a lateral cleft lip and palate and multiple craniofacial dysmorphisms, a combined heart defect, unilateral hypoplasia of the kidney, bilateral aplasia of thumbs, and generalized contractures. Cytogenetic analysis revealed an isopseudodicentric chromosome 18 with breakpoint in 18q (46,XX,psu idic(18)(pter --> q22.1::q22.1 --> pter)). The isopseudodicentric chromosome 18 was observed in 100% of blood lymphocytes and umbilical cord fibroblasts, thus indicating a non-mosaic finding of the isopseudodicentric chromosome in the child. An elongated derivative chromosome 18 had also been found prenatally in amniotic cells. In contrast, a terminal deletion (18q-) was detected in placental cell cultures. The breakpoint was mapped to a 0.9 Mb region on 18q22.1 (located 64.8-65.7 Mb from the telomere of the p-arm) by a novel quantitative PCR approach with SYBR green detection. The results indicate an identical breakpoint of the isopseudodicentric chromosome 18 in the child and the 18q- chromosome in the placenta. To our knowledge this is the first report that a fetus carrying an isopseudodicentric chromosome 18 with breakpoint in 18q (46,XX,psu idic(18)(pter --> q22.1::q22.1 --> pter)) in non-mosaic form can be viable, but is associated with severe congenital malformations of the child.

Adhesive backing foil interactions affecting the elasticity, adhesion strength of laminates, and how to interpret these properties of branded transdermal patches.

Standard tensile strength and peel adhesion tests were carried out to investigate interactions of pressure-sensitive adhesives (PSAs) with several backing foils used for transdermal patches. Seven branded transdermal patches (Alora, Cutanum, Estraderm MX 50, Estraderm TTS 50, Fem7 -50 micrograms, Menorest, Oesclim) were included in the investigation. Their skin adhesion measured in several clinical trials was compared with the results of the laboratory measurements according to PSTC-1 (Peel Adhesion for Single Coated Tapes 180 degrees Angle, Pressure Sensitive Tape Council, Illinois, 1996), such as Young's modulus at 3% elongation and peel adhesion to stainless steel. Data obtained for the PSA-coated backings (laminates) show increasing elasticity with increasing PSA thickness. Interactions of PSAs with backing foil became evident in significant changes in Young's modulus by low PSA thickness, as seen for the silicone adhesive. The Young's moduli of the laminates were found to be influenced not only by the elasticity of the backing foil but also by the chemical structure of the PSA. There was no correlation between the elasticity and peel adhesion of both the laminates and the branded patches. Likewise, for the branded patches the peel adhesion to stainless steel does not correlate with skin adhesion values obtained from clinical trials. The Young's modulus of the branded patches was between 4 N/mm2 (Oesclim) and 501 N/mm2 (Fem7). For the branded transdermal patches no correlation was found between Young's modulus and both the peel force on stainless steel and the skin adhesion reported in studies.

Live-cell microscopy of single nuclear chromosomes and genome compartments: evaluation of labeling procedure and imaging conditions.

BACKGROUND: Single chromosomes and genome compartments in nuclei of living mammalian cells can be analyzed microscopically after specific labeling with fluorescent dyes. This is achieved by incorporating fluorescent nucleotides into the chromosomal DNA during replication (Zink et al.: Hum Genet 102:241-251, 1998; Manders et al.: J Cell Biol 144:813-821, 1999; Sadoni et al.: J Cell Biol 146:1211-1226, 1999). We characterized the potential artificial impact of this approach on chromosome structure and dynamics. We also evaluated potential sources of artifacts in corresponding live-cell imaging. MATERIALS AND METHODS: The subchromosomal distribution of labeled DNA was analyzed, and the fate of labeled nucleotides within cell nuclei was studied. Cell-cycle parameters were used to analyze cell function after incorporation of fluorescent nucleotides. The influences of phototoxic effects on cell division and morphology were studied. RESULTS: Fluorescent nucleotides were only incorporated for a restricted time period during S-phase, and a uniform labeling of chromosomal DNA could not be achieved. Fluorescent nucleotides incorporated into the DNA showed no or only mild effects on cell growth. Cell-cycle parameters and cellular morphology were valuable indicators for proper cell function during live-cell imaging. CONCLUSIONS: There is no indication for a substantial impairment of cellular functions if fluorochromes are covalently linked to chromosomal DNA. The controls we present for proper cell function during the imaging period are of general importance, as appropriate controls for live cell microscopy have not yet been well-defined.

Additional dark G-band in the p-arm of chromosome 19 due to a paracentric inversion with a breakpoint in the pericentromeric heterochromatin.

Paracentric inversions in chromosome 19 have rarely been described. Here we present an inv(19)(p11p13.1) with a breakpoint in the pericentromeric heterochromatin which leads to an additional dark G-band in the p-arm of chromosome 19. The rearranged chromosome segregated in two generations of a family without any phenotypic effects. A detailed characterization of the inv(19) by molecular cytogenetic techniques is presented.

AcroM fluorescent in situ hybridization analyses of marker chromosomes.

The presence of a de novo supernumerary marker chromosome (SMC) poses problems in genetic counseling. The consequences of the additional chromosomal material may range from harmless to detrimental. As the composition of a SMC cannot be deciphered by traditional banding analysis, sophisticated methods are needed for their rapid and detailed analyses. A new strategy is presented, which allows the elucidation of the composition of SMCs in one or two hybridizations. One hybridization, termed AcroM-FISH, involves a newly generated probe mix, which consists of painting probes for all acrocentric chromosomes, centromere probes for chromosomes 13/21, 14/22, 15, and a probe specific for rDNA, each labeled with a specific combination of fluorochromes. This probe mix is sufficient to characterize approximately 80% of all SMCs. For the other 20% of SMCs, chromosomes can be analyzed in a second hybridization by multicolor karyotyping, for example, multiplex FISH (M-FISH), to check for the presence of euchromatin of other chromosomes. The potential of AcroM-FISH was tested in various applications.

Classifying by colors: FISH-based genome analysis.

In recent years a fascinating evolution of different multicolor fluorescence in situ hybridization (FISH) technologies could be witnessed. The various approaches to cohybridize multiple DNA probes in different colors opened new avenues for FISH-based automated karyotyping or the simultaneous analysis of multiple defined regions within the genome. These developments had a remarkable impact on microscopy design and the usage of highly sensitive area imagers. In addition, they led to the introduction of new fluorochromes with appropriate filter combinations, refinements of hybridization protocols, novel probe sets, and innovative software for automated chromosome analysis. This paper attempts to summarize the various multicolor approaches and discusses the application of the individual technologies.

New concepts to improve resolution and sensitivity of molecular cytogenetic diagnostics by multicolor fluorescence in situ hybridization.

BACKGROUND: Routine application of multicolor fluorescence in situ hybridization (M-FISH) technology for molecular cytogenetic diagnostics has been hampered by several technical limitations. First, when using chromosome-specific painting probes, there is a limit in cytogenetic resolution of approximately 2-3 Mb, which can mask hidden structural abnormalities that have a significant clinical effect. Second, using whole chromosome painting probes, intrachromosomal rearrangements cannot be detected and the exact localization of breakpoints is often not possible. METHODS: We suggest the use of multiplex-labeled region or locus- specific probes in combination with an optimal probe design to improve the sensitivity and resolution of the M-FISH technology. To allow the application of this assay in routine diagnostics, we developed a multipurpose image analysis system. RESULTS: goldFISH was applied to the study of cryptic translocations in mental retardation patients and to the study of high-resolution breakpoint mapping in non-small cell lung cancer patients. For an individual with mental retardation, who had an apparently normal karyotype by G-banding, we detected an unbalanced translocation involving chromosomes 2 and 7. CONCLUSIONS: In combination with optimally designed probe kits, goldFISH overcomes most of the present limitations of the M-FISH technology and results in virtually 100% reliability for detecting interchromosomal and intrachromosomal rearrangements.

A new strategy for the detection of subtelomeric rearrangements.

We present a new strategy for the detection of subtelomeric rearrangements. This approach is based on two hybridizations with different probe sets. The first set consists of microdissected subtelomeric probes (each 5-10 megabases in size) labeled combinatorially employing 7 different fluorochromes. With this set, subtelomeric interchromosomal exchanges can be detected in a 24-color experiment. The second set comprises a second generation of subtelomeric PAC-, P1- and BAC-clones. Probes for p- and q-arms are labeled with two different colors. This second set detects small deletions; in addition it provides regional information, so that translocated material identified by the first probe set can be assigned to the p- or q-arm of a chromosome. The test has been evaluated in a blind study on a series of subtle translocations and deletions.

Analysis of chromosomal alterations in non-small cell lung cancer by multiplex-FISH, comparative genomic hybridization, and multicolor bar coding.

Lung cancer has a considerable impact on morbidity and mortality throughout the world. Despite extensive effort, no lung cancer-specific cytogenetic changes, such as lineage-specific translocations or inversions, have been described to date. In this study we used multiplex fluorescence in situ hybridization (M-FISH), comparative genomic hybridization, and multicolor bar coding to analyze eight cell lines derived from non-small cell lung cancers. M-FISH did not identify any balanced translocations, which are the dominating feature in leukemias and lymphomas. Instead, M-FISH unraveled an enormous number of numerical and structural aberrations, with each tumor having its own "private" pattern of chromosomal changes. In contrast, comparative genomic hybridization demonstrated similarities between tumors, because each cell line shared some chromosomal segments that were commonly gained or lost. One of these involved chromosome 12. Chromosome 12 specific bar code probe sets were constructed and used to demonstrate that breaks on chromosome 12 occur preferentially within specific bands. With the progressive use of higher resolution approaches, more information can be gained about the chromosomal alterations in cancer.