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Additional file 1. National health record systems.

Zusammenfassung Hereditäre Ataxien stellen aufgrund der Vielfalt der möglichen genetischen Ursachen eine große diagnostische Herausforderung für die medizinische Genetik dar. Dieses Problem wird dadurch verstärkt, dass zwar die Zahl der neu identifizierten Gene in den letzten 3 Jahren durch neue Sequenziertechnologien rasant zugenommen hat, häufig jedoch nur wenige Familien weltweit Mutationen in diesen Genen aufweisen, d. h. sie extrem selten sind. Der vorliegende Artikel gibt eine Übersicht über dominante und rezessive Ataxien und berücksichtigt dabei auch die neu identifizierten Ataxie-Gene. Um den Anforderungen einer praktisch-orientierten genetischen Diagnostik gerecht zu werden, versuchen wir dabei auch, Häufigkeitseinschätzungen der betroffenen Genorte zu geben und – sofern möglich – phänotypische Eigenschaften und Biomarker zu definieren, die eine genetische Diagnostik erfolgversprechend leiten können, insbesondere bei rezessiven Ataxien. Diese diagnostischen Indikatoren werden in Form von diagnostischen Pfaden zusammengefasst, die eine Orientierung bei der mehrstufigen genetischen Diagnostik dominanter und rezessiver Ataxien geben sollen. Aufgrund der Vielzahl der Genkandidaten und des großen phänotypischen Überlappungsbereichs wird es in den meisten Fällen jedoch am zeiteffizientesten und kostengünstigsten sein, Panel-Untersuchungen mittels Next-Generation-Sequencing-Technologien durchzuführen.

Background and Purpose— Strokes have especially devastating implications if they occur early in life; however, only limited information exists on the characteristics of acute cerebrovascular disease in young adults. Although risk factors and manifestation of atherosclerosis are commonly associated with stroke in the elderly, recent data suggests different causes for stroke in the young. We initiated the prospective, multinational European study Stroke in Young Fabry Patients (sifap) to characterize a cohort of young stroke patients. Methods— Overall, 5023 patients aged 18 to 55 years with the diagnosis of ischemic stroke (3396), hemorrhagic stroke (271), transient ischemic attack (1071) were enrolled in 15 European countries and 47 centers between April 2007 and January 2010 undergoing a detailed, standardized, clinical, laboratory, and radiological protocol. Results— Median age in the overall cohort was 46 years. Definite Fabry disease was diagnosed in 0.5% (95% confidence interval, 0.4%–0.8%; n=27) of all patients; and probable Fabry disease in additional 18 patients. Males dominated the study population (2962/59%) whereas females outnumbered men (65.3%) among the youngest patients (18–24 years). About 80.5% of the patients had a first stroke. Silent infarcts on magnetic resonance imaging were seen in 20% of patients with a first-ever stroke, and in 11.4% of patients with transient ischemic attack and no history of a previous cerebrovascular event. The most common causes of ischemic stroke were large artery atherosclerosis (18.6%) and dissection (9.9%). Conclusions— Definite Fabry disease occurs in 0.5% and probable Fabry disease in further 0.4% of young stroke patients. Silent infarcts, white matter intensities, and classical risk factors were highly prevalent, emphasizing the need for new early preventive strategies. Clinical Trial Registration Information— URL: http://www.clinicaltrials.gov .Unique identifier: NCT00414583

Zusammenfassung Das bundesweit erste Behandlungs- und Forschungszentrum für Patienten mit seltenen Erkrankungen (SE) ist in Tübingen eröffnet worden. Eine Erkrankung gilt als „selten“, wenn durchschnittlich weniger als 1 von 2000 Personen daran erkrankt ist. In Deutschland leben etwa 3 Mio. Patienten mit SE. Bis zu 80% der SE sind nach derzeitigem Kenntnisstand genetisch (mit)bedingt. Daher sind Humangenetiker bei der Betreuung dieser Patienten gefordert. Bei der Mehrheit der SE handelt es sich um komplexe Erkrankungen mit Befall mehrerer Organsysteme. Dies erfordert eine intensive interdisziplinäre Betreuung, die in der Regel nur von Universitätsklinika geleistet werden kann.

We have estimated the haplotype distribution of mutant and normal phenylalanine hydroxylase (PAH) alleles for 17 Turkish phenylketonuria (PKU) families: 20 normal and 27 mutated PAH alleles could be identified. Of the latter, the most prevalent were associated with haplotype 6 (29.6%), 1 (18.5%) and 36 (11.1%), while the normal alleles were preferentially associated with haplotype 1 (20%). Of the 19 different haplotypes observed, 5 have not been described previously. The haplotype distribution differed significantly from that of the Northern European population. Two of the eight polymorphic sites were in association with PKU. No deletions of exon sequences were found in the families analysed.

The phenylketonuria (PKU) dispensary of the Children's Hospital of the Charité, Berlin, GDR, cares for about 140 affected families, representing about one-third of all PKU families in the GDR. Of these families, 15 expressed their desire for an additional child given the availability of a reasonably reliable prenatal diagnosis procedure. They were investigated by linked RFLP analysis applying a phenylalanine-hydroxylase-cDNA probe. Full genetic predictability for prospective fetuses could be obtained for all of them. In eight cases this was possible by the use of one restriction enzyme, and in the remaining seven by a combination of the information from two restriction-enzyme patterns. No recombination between linked RFLP and the PKU phenotype could be observed in 40 meioses from the investigation of eight families with two affected children each.

A human phenylalanine hydroxylase cDNA clone was isolated from a human liver cDNA library. The size of the cDNA insert is approximately 2.4 kb and appears to be a near full length copy of a phenylalanine hydroxylase mRN A. This cDN A was used to probe for Hindlll restriction enzyme polymorphisms in heterozygote typing of families with phenylketonuria. The use of three alleles of this polymorphism, as opposed to the two alleles as previously described, increases the informativity for typing - and therefore also for prenatal diagnosis -in certain families from 75% to 100%.

Mutations in two genes, α-synuclein and parkin, have been identified as some rare causes for familial Parkinson’s disease (PD). α-Synuclein and parkin protein have subsequently been identified in Lewy bodies (LB). To gain further insight into the pathogenesis of PD we investigated the role of neurofilament light (NF-L), another component of LB aggregation. A detailed mutation search of the NF-L gene in 328 sporadic and familial PD patients of German ancestry revealed three silent DNA changes (G163A, C224T, C487T) in three unrelated patients. Analysis of the promoter region of the NF-L gene identified a total of three base pair substitutions defining five haplotypes. Association studies based on these haplotypes revealed no significant differences between PD patients and 344 control individuals. Therefore, NF-L is unlikely to play a major role in the pathogenesis of PD.

In the last few years, the genetic contribution to Parkinson's disease has gained major attention and resulted in the identification of four gene loci in autosomal dominant and autosomal recessive Parkinson's disease. Several mutations in two genes have been shown to be responsible for neuronal cell death in Parkinson's disease. One of the gene products involved, alpha-synuclein, is a major component of Lewy bodies, the neuropathological feature of Parkinson's disease. In contrast, mutations in the parkin gene are associated with parkinsonism without Lewy body pathology. The elucidation of polygenic changes in the dopamine pathway, mitochondrial dysfunction, and of xenobiotic metabolism is technically now possible by means of association and genotype studies. The increasing knowledge of the pathogenesis of Parkinson's disease at a molecular level will have important implications for the development of individual therapeutic strategies to prevent disease progression.

Recently a mutation in the parkin gene has been identified as the cause for an autosomal-recessively inherited form of early onset Parkinson's disease (EOPD). The disease causing minimal deletion has been defined as a homozygous exon 4 loss in the parkin gene among Japanese patients. We investigated 140 sporadic and familial EOPD patients of German ancestry for the exon 4 deletion in the parkin gene. None of our patients exhibited a homo-zygous deletion of exon 4, suggesting a minor role of this mutation for EOPD in Caucasians. Nevertheless a detailed mutation analysis is warranted to explore the overall significance of mutations in the parkin gene in EOPD.

Introduction: Pediatric cardiomyopathy is a heterogeneous disease with a strong genetic component. Molecular genetic analysis has recently been recommended for any symptomatic patient fulfilling the clinical diagnostic criteria. Here, we describe the evaluation of next generation sequencing technology for the comprehensive testing of 46 genes linked to hereditary cardiomyopathy. Methods: We analyzed 48 DNA samples originating from cardiomyopathy patients, the majority of which suffered from familial HCM. The Illumina TrueSight Cardiomyopathy Panel was used for hybridization-based enrichment of target genes, followed by 150 bp paired-end sequencing on a MiSeq-platform. A proprietary workflow was developed to streamline the sequence-data analysis and to list and interpret variants. Results: As expected, we detected causative mutations in commonly affected disease genes, namely, MYBPC3 and MYH7. In patients devoid of variants in these genes, mutations could be detected in rarely affected genes (e.g., TMEM43, MYOZ2). Additional variants suspected to cause or modify the disease were found in 40% of the patients. Conclusions: We have successfully established a fast and cost-efficient genetic testing method for patients with cardiomyopathy. We detected mutations in patients with both classical and ambiguous phenotypes. Therefore, our approach may assist in the collection of information on both well-defined and unacquainted pediatric cardiomyopathic disorders and in revealing their true phenotypic spectrum.

SHORT syndrome (OMIM 269880) is a rare autosomal-dominant disorder characterized by short stature, hyperextensibility of joints, hernias, ocular depression, ophthalmic anomalies (Rieger anomaly, posterior embryotoxon, glaucoma), teething delay, partial lipodystrophy, insulin resistance and facial dysmorphic signs. Heterozygous mutations in PIK3R1 were recently identified in 14 families with SHORT syndrome. Eight of these families had a recurrent missense mutation (c.1945CT; p.Arg649Trp). We report on two unrelated patients with typical clinical features of SHORT syndrome and additional problems such as pulmonary stenosis and ectopic kidney. Analysis of PIK3R1 revealed the mutation c.1945CT; p.Arg649Trp de novo in both patients. These two patients not only provide additional evidence that PIK3R1 mutations cause SHORT syndrome, but also broaden the clinical spectrum of this syndrome and further confirm that the amino acid exchange c.1945CT; p.Arg649Trp is a hotspot mutation in this gene.

Behavioral characterization is an important part of establishing novel animal models, but classical behavioral tests struggle to reveal conclusive results due to problems with both reproducibility and validity. On the contrary, automated homecage observations are believed to produce robust outcomes that relate more to natural animal behavior. However, information on the behavior of background strains from such observations, which could provide important reference material, is rare. For this reason, we compared the behavior of the commonly used Lister Hooded, Lewis, Fischer 344 and Wistar rats during 70 h of exposure to an automated homecage system at 2, 4 and 6 months of age. We found considerable strain differences in metabolic parameters, novelty-induced and baseline activity-related behavior as well as differences in the development of these parameters with age. The results are discussed in terms of advantages and disadvantages of the system compared to classical behavioral tests, as well as the system's ability to recreate common findings in literature.

Die Analyse molekularer Mechanismen beim Ubergang vom duktalen in situ Karzinom (DCIS) der Brust zum invasiven duktalen Karzinom (IDC) tragt zum Verstehen der Vorgange bei der Tumorprogression bei. Die Ziele dieses Projektes sind (1) die Identifikation und Validierung potentieller prognostischer Marker und (2) die Identifikation von Markern, die DCIS mit aggressivem Potential charakterisieren. 40 formalinfixierte, in Paraffin eingebettete (FFPE) „reine”DCIS Tumorproben ohne IDC Komponente (keine Metastasen oder Rezidiv 5 Jahre nach OP), sowie 32 DCIS/IDC Tumore wurden ausgewahlt. Gewebeschnitte wurden angefertigt, Hamatoxilin-Eosin gefarbt und die Epithelzellen mittels Lasermikrodissektion (LCM) isoliert. 200ng RNA wurde extrahiert, auf den „Whole Genome DASL Array“ (Illumina) hybridisiert und bioinformatisch ausgewertet. Die Expression ausgewahlter RNAs und wurde mittels qRT-PCR in unabhangigen Gewebeproben validiert. Es wurden 993 Transkripte, die zwischen DCIS und IDC desselben Tumors differentiell exprimiert sind und 1138 Transkripte, die zwischen „reinen“ DCIS und DCIS aus DCIS/IDC-Tumoren differentiell exprimiert sind, identifizieren. Die Validierung der differentiellen Expression von 9 Kandidatengenen wurde anhand zwei Probensets durchgefuhrt (1) die technische Validierung (15 DCIS/IDC Tumore, 15 „reine“ DCIS) (2) unabhangige Validierung (26 DCIS/IDC Tumore, 17 „reine“ DCIS). MMP11 und Col10a1 sind im IDC stark exprimiert wahrend die Expression in DCIS aus DCIS/IDC Tumoren geringer war. In „reinen“ DCIS konnten MMP11 und Col10a1 wenig oder gar nicht detektiert werden. Wir konnten progressions-spezifische Kandidatengene mittels LCM und Microarray Analyse aus FFPE Mammakarzinomgewebe identifizieren. MMP11 und Col10a1 sind Progressionsmarker, die zwischen hoch und niedrig Risiko DCIS unterscheiden konnen.

Die jungsten Entwicklungen in der genetischen Forschung, die zur Identifizierung der genetischen Grundlagen einer Vielzahl von Erkrankungen gefuhrt haben, fuhren dazu, dass humangenetische Kenntnisse fur eine Vielzahl von klinischen Disziplinen immer relevanter werden.

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.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.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.The findings in this family further demonstrate genetic heterogeneity in familial autosomal dominant late-onset PD.

Zielsetzung: Basierend auf eigenen Microarray-Untersuchungen mit invasiv-duktalen (IDC) und duktalen in situ (DCIS)-Arealen derselben Mammakarzinome wurden aus 548 Kandidatengenen mittels statistischer Analyse ein Progressionsgenset aus 9 Transkripten optimiert (Schutz et al., Cancer Research, 2006). Dieses Projekt soll (1) das Progressionsgenset an FFPE-Gewebe evaluieren und (2) Gene finden, die aggressive DCIS mit einem hoch-invasivem Potential charakterisieren. Material und Methoden: Da die Kryokonservierung „reiner“ DCIS zur Wahrung der Diagnosesicherheit nur bedingt erfolgen kann, wurde Archivmaterial untersucht: (a) Gewebeproben mit einem „reinen“ DCIS ohne IDC-Komponente (mindestens funf Jahre tumorfrei); und (b) Gewebeproben mit DCIS/IDC-Mischtumoren. Paraffingewebeschnitte (8µm) wurden angefertigt, mit Hamatoxylin-Eosin gefarbt und die Tumorepithelzellen mittels Lasermikrodissektion (LCM) isoliert. 200ng RNA wurden auf den „Whole-Genome Gene Expression DASL-Array“ (Illumina) hybridisiert und bioinformatisch ausgewertet. Fur die Validierung wurde die RNA mithilfe der Ribo-SPIA® Technik (WT-Ovation™ FFPE Systems, NuGen™) linear amplifiziert und die Expression der Kandidatengene mit dem Roche-LightCycler® evaluiert. Ergebnisse: Die differentielle Expression von Kandidatengenen aus dem Progressionsgenset lies sich sowohl anhand von Paraffingewebe der analogen Kryoproben als auch an unabhangigem Archivmaterial bestatigen. Zusatzlich konnten wir Gene identifizieren und evaluieren, die zwischen „reinen“ DCIS und DCIS aus Mischtumoren differentiell exprimiert sind: die Gene Plekhc1 und Col11a1 sind im „reinen“ DCIS niedriger exprimiert als in DCIS aus Mischtumoren. Zusammenfassung: In diesem Projekt haben wir FFPE-Gewebe in Kombination mit LCM und der DASL-Methode erfolgreich eingesetzt um (a) Progressionsmarker zu bestatigen, die wir aus Kryogewebe identifiziert haben, und (b) mogliche Marker fur aggressive DCIS zu finden.

The phenotype in the rd mouse is similar to the clinical presentation of Leber congenital amaurosis (LCA) in humans. Recently a nonsense mutation in the beta subunit of the cGMP phosphodiesterase (Pdeb) gene has been defined as the cause for the rd phenotype in the mouse and has raised the question as to whether mutations in the human PDEB gene might cause LCA. We have previously cloned and characterized the human homologue of the mouse Pdeb gene and have mapped it to chromosome 4p16.3. In this study, a total of 23 LCA families of various ethnic backgrounds have been investigated. Linkage analysis using highly polymorphic (CA)n microsatellites has excluded the PDEB gene as a cause for LCA in 6 families. In the remaining 17 families, we have searched for mutations in the 22 exons of the PDEB gene using single-strand gel electrophoresis (SSGE). Multiple exonic polymorphisms have been determined. However, no DNA changes in the PDEB gene have been identified in our study population which could be causative for the LCA phenotype.

We have evaluated oligonucleotide hybridization and amplification techniques with regard to quantity and quality of genomic DNA that is under investigation in practical forensic case work. In order to obtain sufficient information from analyzing stain material, we use hypervariable simple repeat sequences for individualization, which occur in all eukaryotic genomes. For the analysis of larger amounts of stains (greater than 500 ng DNA) the multilocus probes (CAC)5/(GTG)5* are superior because of their discrimination potential--provided that the hybridizing DNA is of high molecular weight. The less discriminating probes (CT)8 and (GACA)4 are more sensitive (minimal amount: 100ng DNA) and still informative when the DNA is degraded. To increase the sensitivity of forensic stain analysis in special cases we have used the polymerase chain reaction technique to amplify hypervariable simple (gt)n/(ga)m repeat structures from the intron 2 of HLA-DRB genes. Largely independent of the starting amount of DNA and independent of the degradation status, we were able to generate discriminating DNA fragments, which can be used to type (i) microstains and (ii) totally degraded material including human mummy DNA.