[Concepts for the return of secondary genetic findings in medical diagnostics and research]. / Konzepte zur Mitteilung genetischer Zusatzbefunde in der medizinischen Diagnostik und Forschung.

High-throughput sequencing of whole genomes is technically already at a high level and is being discussed as a cost-effective alternative to other targeted, analytical procedures for clinical diagnosis of heritable disorders. On the other hand, with whole genome and whole exome sequencing, there is a high likelihood of uncovering secondary findings not associated with the primary aim of the investigation. This article tries to outline the current scientific and technical status of whole genome and whole exome sequencing and of the national and international recommendations concerning the handling of secondary genetic findings which are already available, above all in the research-related context and less so in the clinical context.

Autosomal dominant Parkinson's disease in a large German pedigree.

OBJECTIVE: While several genes have been identified to cause Parkinson's disease (PD), monogenic forms explain only a small proportion of cases. We report clinical and genetic results in a large family with late-onset autosomal dominant PD. METHODS: Thirty-eight family members of a five-generation Northern German PD family underwent a detailed neurologic examination, and transcranial sonography was performed in fifteen of them. Comprehensive mutation analysis of known PD-causing genes and a genome-wide linkage analysis were performed. RESULTS: Late-onset definite PD was found in five subjects with a mean age at onset of 63 years. Another six individuals presented either with probable/possible PD or with subtle parkinsonian signs. Six members with a mean age of 79 years had an essential tremor phenotype. Mode of PD inheritance was compatible with autosomal dominant transmission. One of three examined patients with definite PD demonstrated an increased area of substantia nigra hyperechogenicity upon transcranial sonography. Comprehensive linkage and mutational analysis excluded mutations in known PD-causing genes. Genome-wide linkage analysis suggested a putative disease gene in an 11.3-Mb region on chromosome 7p15-21.1 with a multipoint LOD score of 2.0. CONCLUSIONS: The findings in this family further demonstrate genetic heterogeneity in familial autosomal dominant late-onset PD.

First prenatally detected small supernumerary neocentromeric derivative chromosome 13 resulting in a non-mosaic partial tetrasomy 13q.

Neocentromeres are functional centromeres located in non-centromeric euchromatic regions of chromosomes. The formation of neocentromeres results in conferring mitotic stability to chromosome fragments that do not contain centromeric alpha satellite DNA. We present a report of a prenatal diagnosis referred to cytogenetic studies due to ultrasound malformations such as large cisterna magna, no renal differentiation, hypotelorism and ventriculomegaly. Cytogenetic analysis of GTG-banded chromosomes from amniotic fluid cells and fetal blood cells revealed a de novo small supernumerary marker chromosome. Molecular cytogenetic studies using fluorescence in situ hybridization and comparative genomic hybridization showed this marker to be an inverted duplication of the distal portion of chromosome 13q which did not contain detectable alpha satellite DNA. The neocentromeric constriction was located at band 13q31. The presence of a functional neocentromere on this marker chromosome was confirmed by immunofluorescence with antibodies to centromere protein-C. The anatomopathologic study revealed a female fetus with facial dysmorphisms, low set ears and renal dysplasia. Ten small supernumerary neocentromeric chromosomes originating from the distal region of chromosome 13q have been reported to date. There are only three additional cases described with the location of the neocentromere in band 13q31. This is the first reported case detected prenatally.

Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

Molecular cytogenetic identification and characterization of a de novo supernumerary neocentromeric derivative chromosome 13.

We report a young girl with microphthalmia, conductive deafness, aortic isthmus stenosis, laryngomalacia, and laryngeal stenosis carrying a de novo supernumerary neocentromeric derivative chromosome 13. For the precise identification and characterization of the eu- and heterochromatic content of the marker chromosome, straightforward molecular cytogenetic analyses were performed, such as chromosome microdissection, FISH with different probes (e.g. wcp, alphoid centromeric probes, BAC), centromere-specific multicolor FISH (cenM-FISH), and multicolor banding (MCB). The analyses demonstrated that the marker consisted of an inverted duplication (partial tetrasomy) of the distal portion of chromosome 13 that was separated from the endogenous chromosome 13 centromere. Using an all-centromere probe and multicolor cenM-FISH, no alpha-satellite DNA hybridization signal was detectable on any portion of the derivative chromosome. The presence of a functional and active neocentromere on the derivative chromosome 13 was confirmed by positive immunofluorescence signals with CENP-C antibodies. BAC-FISH confirmed the cytogenetic localization of the neocentromere in band 13q31.3. Thus the patient had a mosaic conventional karyotype mos 47,XX,+inv dup(13)(qter-->q21.3::q21.3-->q31.3-->neo-->q31.3-->qter)[6]/46,XX [49].

First small supernumerary ring chromosome carrying 10q euchromatin in a patient with mild phenotype characterized by molecular cytogenetic techniques and review of the literature.

We report the identification and characterization of the first supernumerary ring chromosome 10 containing a considerable proportion of 10q euchromatin by microdissection and reverse painting in a female patient presenting with short stature. Fluorescence in situ hybridization studies showed that the marker chromosome originates from chromosome 10 and includes the euchromatic bands p11.2 and q11.2. The supernumerary marker chromosome 10 was found in 14% of the peripheral blood lymphocytes analyzed. This constitutional mosaic could be confirmed in oral mucosa cells as a second cell system (16%) by interphase FISH using an alphoid centromeric probe for chromosome 10. Parental karyotypes were normal, uniparental disomy for the normal chromosomes 10 could be excluded by microsatellite analysis. The karyotype of the patient detected in peripheral blood cells can be described as mos 47,XX,+mar.rev ish r(10)(p11.2q11.2)(wcp10+,cep10+)/46,XX.

First systematic CGH-based analyses of ancient DNA samples of malformed fetuses preserved in the Meckel Anatomical Collection in Halle/Saale (Germany).

We present the first data on our comparative genomic hybridization (CGH)-based strategy for the analysis of ancient DNA (aDNA) samples extracted from fetuses preserved in the Meckel Anatomical Collection in Halle, Germany. The collection contains numerous differently fixed ancient samples of fetal malformations collected from the middle of the 18th to the early 19th century. The main objective of this study is to establish a "standard" aDNA extraction and amplification protocol as a prerequisite for successful CGH analyses to detect or exclude chromosomal imbalances possibly causative for the malformations described for the fetuses.

Prenatal diagnosis and molecular cytogenetic characterization of an unusual complex structural rearrangement in a pregnancy following intracytoplasmic sperm injection (ICSI).

We report on a balanced complex chromosomal aberration detected in a fetus after amniocentesis. The pregnancy was achieved after intracytoplasmic sperm injection. GTG-banding revealed a complex structurally rearranged karyotype with a translocation between chromosomes 5 and 15 and an additional paracentric inversion in the der(15) between bands 5q11.2 and 5q15. Ag-NOR staining showed an interstitial active nuclear organizer region in the der(15). Molecular cytogenetic analyses using whole-chromosome-painting probes, comparative genomic hybridization, and multicolor banding did not point to further structural aberrations or imbalances. Therefore, a complex rearrangement with three breakpoints has occurred, and the karyotype can be described as 46,XX,der(5)t(5;15) (q11.2;p12),der(15)t(5;15)(q11.2;p12)inv(5)(q11.2q15).

Delineation of a complex karyotypic rearrangement by microdissection and CGH in a family affected with split foot.

We report on a male patient and members of his family with additional material in chromosome 3. This derivative chromosome 3 was transmitted from his mother who had a complex rearrangement between chromosomes 2, 3, and 7. It was possible to delineate her chromosomal rearrangement by microdissection and reverse painting and to exclude these aberrations from being responsible for neonatal deaths and several abortions in this family. Two members of this family suffer from ectrodactyly or split hand/foot malformations (SHFM) of the feet which possibly correlates with the derivative chromosome 7 containing a breakpoint in the SHFM1 critical region involving several homeobox genes.

Characterization of the first supernumerary tricentric ring chromosome 1 mosaicism by conventional and molecular cytogenetic techniques.

We report on the conventional cytogenetic and fluorescence in situ hybridization (FISH) results obtained for a 3.5-year-old girl with developmental and language delay and a supernumerary ring chromosome mosaicism in 8% of T-lymphocytes analyzed. Using different conventional and molecular cytogenetic techniques as YAC hybridization and comparative genomic hybridization, we could show that the extra tricentric ring chromosome consists of three heterochromatic blocks with inserted euchromatic material. Additionally, chromosome microdissection followed by FISH analysis demonstrated that the small tricentric ring chromosome consisted of material from the pericentromeric region of chromosome 1q21. Thus, the patient has a mosaic of normal cells and cells with partial pentasomy of the pericentromeric region of chromosome 1. So far, 19 cases with single supernumerary marker chromosome 1 have been published, but no tricentric ring chromosome 1 is, to our knowledge, reviewed in the literature. In this study, we compare the clinical features of our patient with cytogenetically comparable cases described in the literature. We introduce a hypothesis for the formation of a tricentric ring chromosome: starting with a monocentric ring, sister chromatid exchange leading to the formation of a tetracentric ring, which underwent intrastrand recombination generating the tricentric ring.

Significance of gene expression analysis in uveal melanoma in comparison to standard risk factors for risk assessment of subsequent metastases.

PURPOSE: This study was undertaken to identify and compare the prognostic value of gene expression, chromosomal, and clinico-pathological data for the prediction of subsequent metastases in patients with primary uveal melanoma. PATIENTS AND METHODS: For comparison of different sets of predictor variables diagonal linear discriminant analysis was used. Chromosomal events were assessed by comparative genomic hybridization and gene expression profiling by microarray. Twenty-eight patients with a median follow-up of 68 months were analyzed, of whom 12 had developed subsequent metastases. RESULTS: Diagonal linear discriminant analysis with crossvalidation of gene expression data detected 42 genes as differentially expressed in metastasizing vsnon-metastasizing uveal melanomas in all 28 cases. Comparing quantitative scores of discriminant analysis, grouping precision was significant better with gene expression profiling compared to comparative genomic hybridization (P=0.01) and to clinical data (P=0.001). Two published gene lists associated with monosomy 3 and metastatic tumor growth were used as classifier for discriminant analysis and yielded superior classification in patients with and without subsequent metastases than chromosomal or clinico-pathological data. CONCLUSION: In our patient cohort gene expression profiling of primary uveal melanoma tissue was superior to clinical-pathological and chromosomal analysis to assess for the risk of subsequent metastases.