Single Molecule Molecular Inversion Probes for High Throughput Germline Screenings in Dystonia.

Background: This study's aim was to investigate a large cohort of dystonia patients for pathogenic and rare variants in the ATM gene, making use of a new, cost-efficient enrichment technology for NGS-based screening. Methods: Single molecule Molecular Inversion Probes (smMIPs) were used for targeted enrichment and sequencing of all protein coding exons and exon-intron boundaries of the ATM gene in 373 dystonia patients and six positive controls with known ATM variants. Additionally, a rare-variant association study was performed. Results: One patient (0.3%) was compound heterozygous and 21 others were carriers of variants of unknown significance (VUS) in the ATM gene. Although mutations in sporadic dystonia patients are not common, exclusion of pathogenic variants is crucial to recognize a potential tumor predisposition syndrome. SmMIPs produced similar results as routinely used NGS-based approaches. Conclusion: Our results underline the importance of implementing ATM in the routine genetic testing of dystonia patients and confirm the reliability of smMIPs and their usability for germline screenings in rare neurodegenerative conditions.

Analysis of blood-based gene expression in idiopathic Parkinson disease.

OBJECTIVE: To examine whether gene expression analysis of a large-scale Parkinson disease (PD) patient cohort produces a robust blood-based PD gene signature compared to previous studies that have used relatively small cohorts (≤220 samples). METHODS: Whole-blood gene expression profiles were collected from a total of 523 individuals. After preprocessing, the data contained 486 gene profiles (n = 205 PD, n = 233 controls, n = 48 other neurodegenerative diseases) that were partitioned into training, validation, and independent test cohorts to identify and validate a gene signature. Batch-effect reduction and cross-validation were performed to ensure signature reliability. Finally, functional and pathway enrichment analyses were applied to the signature to identify PD-associated gene networks. RESULTS: A gene signature of 100 probes that mapped to 87 genes, corresponding to 64 upregulated and 23 downregulated genes differentiating between patients with idiopathic PD and controls, was identified with the training cohort and successfully replicated in both an independent validation cohort (area under the curve [AUC] = 0.79, p = 7.13E-6) and a subsequent independent test cohort (AUC = 0.74, p = 4.2E-4). Network analysis of the signature revealed gene enrichment in pathways, including metabolism, oxidation, and ubiquitination/proteasomal activity, and misregulation of mitochondria-localized genes, including downregulation of COX4I1, ATP5A1, and VDAC3. CONCLUSIONS: We present a large-scale study of PD gene expression profiling. This work identifies a reliable blood-based PD signature and highlights the importance of large-scale patient cohorts in developing potential PD biomarkers.

Molecular Signatures of Primary Human Spermatogonial Progenitors and Its Neighboring Peritubular Stromal Compartment.

In-depth understanding of human spermatogenesis requires studying specific molecular signatures and interactions of spermatogonia with other testicular cell populations, for which isolation of pure populations of different cell types is crucial. Here, we describe a technique to simultaneously enrich pure, multiple testicular cell populations, including spermatogonia, endothelial (TECs), and perivascular mesenchymal stem/stromal cells (TMSCs), from testicular tissue by flow cytometry using a combination of defined markers. Immunohistochemical studies, multicolor staining, and cell sorting followed by multiplex quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that spermatogonia were highly enriched in the CD49f+CD49a-HLA-ABC-SSEA-4+ fraction of primary testicular cells. In contrast to spermatogonia, TMSCs and TECs were highly enriched in the CD49f+CD49a+HLA-ABC+CD144- and CD49f+CD49a+HLA-ABC+CD144+subsets, respectively. The delineation was confirmed by the expression of specific stromal and endothelial key markers as well as by the differentiation and angiogenic capacity of the sorted populations. In this article, for the first time, we performed transcriptome profiling of highly enriched, freshly isolated human spermatogonia and compared their expression profile with that of TMSCs. Our RNA sequencing data favor the hypothesis that TMSCs are candidate niche components for spermatogonia. The composite genotype and phenotype of defined testicular cell populations combined with a robust isolation procedure from small biopsies contributes to a better understanding of cellular interactions and for the establishment of efficient culture techniques to maintain spermatogonial progenitors.

Mayer-Rokitansky-Küster-Hauser syndrome discordance in monozygotic twins: matrix metalloproteinase 14, low-density lipoprotein receptor-related protein 10, extracellular matrix, and neoangiogenesis genes identified as candidate genes in a tissue-specific mosaicism.

OBJECTIVE: To find a potential underlying cause for Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) discordance in monozygotic twins. DESIGN: Prospective comparative study. SETTING: University hospital. PATIENT(S): Our study genetically analyzed 5 MRKHS-discordant monozygotic twin pairs with the unique opportunity to include saliva and rudimentary uterine tissue. INTERVENTION(S): Blood, saliva, or rudimentary uterine tissue from five MRKHS-discordant twins was analyzed and compared between twin pairs as well as within the same individual where applicable. We used copy number variations (CNVs) to identify differences. MAIN OUTCOME MEASURE(S): CNVs in blood, rudimentary uterine tissue, and saliva, network analysis, and review of the literature. RESULT(S): One duplication found in the affected twin included two genes, matrix metalloproteinase 14 (MMP14) and low-density lipoprotein receptor-related protein 10 (LRP10), which have known functions in the embryonic development of the uterus and endometrium. The duplicated region was detected in rudimentary uterine tissue from the same individual but not in saliva, making a tissue-specific mosaicism a possible explanation for twin discordance. Additional network analysis revealed important connections to differentially expressed genes from previous studies. These genes encode several molecules involved in extracellular matrix (ECM) remodeling and neoangiogenesis. CONCLUSION(S): MMP-14, LRP-10, ECM, and neoangiogenesis genes are identified as candidate genes in a tissue-specific mosaicism. The detected clusters provide evidence of deficient vascularization during uterine development and/or disturbed reorganization of ECM components, potentially during müllerian duct elongation signaled by the embryologically relevant phosphatidylinositol 3-kinase/protein kinase B pathway. Therefore, we consider these genes to be new candidates in the manifestation of MRKHS.

Genome-wide UPD screening in patients with intellectual disability.

Uniparental disomy (UPD) describes the inheritance of a pair of chromosomes from only one parent. It may occur as isodisomy, heterodisomy or a combination of both and may involve only chromosome segments. UPD can affect each chromosome. The incidence is estimated to be around 1:3500 in live births. Some parts of chromosomes are subject to 'parent-of-origin imprinting' and the phenotypic effect in UPD syndromes is mainly due to functional imbalance of imprinted genes. Isodisomy can result in mutation homozygosity in autosomal-recessive inherited diseases. UPD causes several well-defined imprinting syndromes associated with intellectual disability (ID). Although knowledge on frequency and size of UPDs in patients with unexplained ID remains largely unknown as no efficient genome-wide screening technique was available for detection of both isodisomic and heterodisomic UPDs. SNP microarrays have been proven to be capable to detect UPDs through Mendelian errors. The correct subclassification of UPD requires child-parent trio experiments. To further elucidate the role of UPD in patients with unexplained ID, we analyzed a total of 322 child-parent trios. We were not able to detect UPDs (isodisomies and heterodisomies) within our cohort spanning whole chromosomes or chromosomal segments. We conclude that UPD is rare in patients with unexplained ID.

UPDtool: a tool for detection of iso- and heterodisomy in parent-child trios using SNP microarrays.

UNLABELLED: UPDtool is a computational tool for detection and classification of uniparental disomy (UPD) in trio SNP-microarray experiments. UPDs are rare events of chromosomal malsegregation and describe the condition of two homologous chromosomes or homologous chromosomal segments that were inherited from one parent. The occurrence of UPD can be of major clinical relevance. Though high-throughput molecular screening techniques are widely used, detection of UPDs and especially the subclassification remains complex. We developed UPDtool to detect and classify UPDs from SNP microarray data of parent-child trios. The algorithm was tested using five positive controls including both iso- and heterodisomic segmental UPDs and 30 trios from the HapMap project as negative controls. With UPDtool, we were able to correctly identify all occurrences of non-mosaic UPD within our positive controls, whereas no occurrence of UPD was found within our negative controls. In addition, the chromosomal breakage points could be determined more precisely than by microsatellite analysis. Our results were compared with both the gold standard, microsatellite analysis and SNPtrio, another program available for UPD detection. UPDtool is platform independent, light weight and flexible. Because of its simple input format, UPDtool may also be used with other high-throughput technologies (e.g., next-generation sequencing). AVAILABILITY AND IMPLEMENTATION: UPDtool executables, documentation and examples can be downloaded from http://www.uni-tuebingen.de/uni/thk/de/f-genomik-software.html.

A high-throughput resequencing microarray for autosomal dominant spastic paraplegia genes.

Hereditary spastic paraplegias (HSP) are a heterogeneous group of neurological disorders. Insidiously progressive spastic weakness of the lower extremities is the common criterion in all forms described. Clinically, HSP is differentiated into pure (uncomplicated) and complex (complicated) forms. While pure HSP is predominantly characterized by signs and symptoms of pyramidal tract dysfunction, additional neurological and non-neurological symptoms occur in complicated forms. Autosomal dominant, autosomal recessive, and X-linked modes of inheritance have been described and at least 48 subtypes, termed SPG1-48, have been genetically defined. Although in autosomal dominant HSP families 50-60% of etiologies can be established by genetic testing, genotype predictions based on the phenotype are limited. In order to realize high-throughput genotyping for dominant HSP, we designed a resequencing microarray for six autosomal dominant genes on the Affymetrix CustomSEQ array platform. For validation purposes, 10 previously Sanger sequenced patients with autosomal dominant HSP and 40 positive controls with known mutations in ATL1, SPAST, NIPA1, KIF5A, and BSCL2 (32 base exchanges, eight small indels) were resequenced on this array. DNA samples of 45 additional patients with AD spastic paraplegia were included in the study. With two different sequencing analysis software modules (GSEQ, SeqC), all missense/nonsense mutations in the positive controls were identified while indels had a detection rate of only 50%. In total, 244 common synonymous single-nucleotide polymorphisms (SNPs) annotated in dbSNP (build 132) corresponding to 22 distinct sequence variations were found in the 53 analyzed patients. Among the 22 different sequence variations (SPAST n = 15, ATL1 n = 3, KIF5A n = 2, HSPD1 n = 1, BSCL2 n = 1, NIPA1 n = 0), 12 were rare variants that have not been previously described and whose clinical significance is unknown. In SPAST-negative cases, a genetic diagnosis could be established in 11% by resequencing. Resequencing microarray technology can therefore efficiently be used to study genotypes and mutations in large patient cohorts.

A combination of transcriptome and methylation analyses reveals embryologically-relevant candidate genes in MRKH patients.

BACKGROUND: The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is present in at least 1 out of 4,500 female live births and is the second most common cause for primary amenorrhea. It is characterized by vaginal and uterine aplasia in an XX individual with normal secondary characteristics. It has long been considered a sporadic anomaly, but familial clustering occurs. Several candidate genes have been studied although no single factor has yet been identified. Cases of discordant monozygotic twins suggest that the involvement of epigenetic factors is more likely. METHODS: Differences in gene expression and methylation patterns of uterine tissue between eight MRKH patients and eight controls were identified using whole-genome microarray analyses. Results obtained by expression and methylation arrays were confirmed by qRT-PCR and pyrosequencing. RESULTS: We delineated 293 differentially expressed and 194 differentially methylated genes of which nine overlap in both groups. These nine genes are mainly embryologically relevant for the development of the female genital tract. CONCLUSION: Our study used, for the first time, a combined whole-genome expression and methylation approach to reveal the etiology of the MRKH syndrome. The findings suggest that either deficient estrogen receptors or the ectopic expression of certain HOXA genes might lead to abnormal development of the female reproductive tract. In utero exposure to endocrine disruptors or abnormally high maternal hormone levels might cause ectopic expression or anterior transformation of HOXA genes. It is, however, also possible that different factors influence the anti-Mullerian hormone promoter activity during embryological development causing regression of the Müllerian ducts. Thus, our data stimulate new research directions to decipher the pathogenic basis of MRKH syndrome.

Utilization of AFFX spike-in control probes to monitor sample identity throughout Affymetrix GeneChip Array processing.

Microarrays evolved from a highly specialized technique into a standard molecular biology method that is widely used for whole-genome gene expression profiling. One of the most important aspects of this method is sample identity, that is, whether the expression profile recorded from an array actually derives from the indicated sample. Several potential steps in the protocol exist where a mix-up of samples may occur. With the increasing size of microarray studies, it is important to ensure that each expression profile is assigned to the correct sample. Errors at this level almost certainly lead to erroneous results and can even cause a complete failure of the microarray study. We developed a system that utilizes probes already present on commercially available Affymetrix arrays to unambiguously correlate the recorded expression profile with the input sample RNA. A set of eight spike-in controls were generated, which can be added to sample RNA in different combinations to generate an "on-chip identifier" that passes through the entire array processing protocol and results in a sample-specific hybridization pattern. This pattern can then be used to monitor whether each array was hybridized with the correct sample. The spike-in controls did not have any negative effect on RNA integrity or any detectable influence on the expression values of the remaining probes on the array; therefore, these controls represent an inexpensive and easily adaptable system to guarantee high-quality results from microarray experiments.

Inhibition of glutathione-S-transferase as a treatment strategy for multidrug resistance in childhood rhabdomyosarcoma.

Multidrug resistance (MDR) is a common problem in the treatment of childhood rhabdomyosarcoma (RMS). A complete reversal of MDR is currently not possible. The aim of this study was to investigate the role of glutathione-S-transferase (GST) as mechanism of MDR in childhood RMS and to analyze possible reversal strategies. Female athymic mice underwent xenotransplantation with embryonal or alveolar RMS cells and were treated with vincristine. Gene expression analysis using Affymetrix HU-Gene 1.0 arrays revealed 2314 differentially expressed genes between the groups in alveolar RMS and 1387 in embryonal RMS. Ingenuity pathway analysis revealed a cluster of 5 overexpressed genes of the GST family in animals treated with vincristine, putative mediating the development of MDR. In order to analyze possible GST activity after chemotherapy with other commonly used drugs (doxorubicin, topotecan), cell culture experiments with alveolar and embryonal RMS cells were carried out. Specific GST activity was quantified using the clorodinitrobenzol conjugation with glutathione. Increased GST activity was found after incubation with cytotoxic agents in all cell lines. Highest induction of GST activity was found in embryonal RMS (up to 12-fold). After incubation with the GST inhibitors, tumor cell viability was decreased depending on the type of tumor cell and inhibitor used. We detected a novel mechanism for MDR in childhood RMS mediated via genes and proteins of the GST family. Reversal of these effects may be achieved by GST inhibitors in part. The GST family represents a promising target for further treatment strategies in childhood RMS.

Array-based resequencing assay for mutations causing hypertrophic cardiomyopathy.

BACKGROUND: Dissecting the complex genetic basis of hypertrophic cardiomyopathy (HCM) may be key to both better understanding and optimally managing this most prevalent genetic cardiovascular disease. An array-based resequencing (ABR) assay was developed to facilitate genetic testing in HCM. METHODS: An Affymetrix resequencing array and a single long-range PCR protocol were developed to cover the 3 most commonly affected genes in HCM, MYH7 (myosin, heavy chain 7, cardiac muscle, beta), MYBPC3 (myosin binding protein C, cardiac), and TNNT2 [troponin T type 2 (cardiac)]. RESULTS: The assay detected the underlying point mutation in 23 of 24 reference samples and provided pointers toward identifying a G insertion and a 3-bp deletion. The comparability of array-based assay results to conventional capillary sequencing was > or =99.9%. Both techniques detected 1 heterozygous variant that was missed by the other method. CONCLUSIONS: The data provide evidence that ABR can substantially reduce the high workload previously associated with a genetic test for HCM. Therefore, the HCM array could facilitate large-scale studies aimed at broadening the understanding of the genetic and phenotypic diversity of HCM and related cardiomyopathies.

Gene expression changes in a transgenic mouse model overexpressing human wildtype and mutant torsinA.

Primary torsion dystonia is an autosomal-dominantly inherited, neurodevelopmental movement disorder caused by a GAG deletion (ΔGAG) in the DYT1 gene, encoding torsinA. This mutation is responsible for approximately 70% of cases of early-onset primary torsion dystonia. The function of wildtype torsinA is still unknown, and it is unsolved how the deletion in the DYT1 gene contributes to the development of the disease. To better understand the molecular processes involved in torsinA pathology, we used genome-wide oligonucleotide microarrays to characterize gene expression patterns in the striatum of mouse models overexpressing the human wildtype and mutant torsinA. By this approach we were able to detect gene expression changes that seem to be specific for torsinA pathology. We found an impact of torsinA, independent from genotype, on vesicle trafficking, exocytosis, and neurotransmitter release in our mouse model. In addition, we were able to identify several new pathways and processes involved in the development of the nervous system that are affected by wildtype and mutant torsinA. Furthermore, we have striking evidence from our gene expression data that glutamate receptor mediated synaptic plasticity in the striatum is the affected underlying cellular process for impaired motor learning in human ΔGAG torsinA transgenic mice.

Nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3: in vivo evidence.

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat in the MJD1 gene resulting in an expanded polyglutamine repeat in the ataxin-3 protein. To study the course of the disease, we generated transgenic mice for SCA3 using full-length ataxin-3 constructs containing 15, 70, or 148 CAG repeats, respectively. Control mice (15 CAGs) were phenotypically normal and had no neuropathological findings. However, mice transgenic for ataxin-3 with expanded polyglutamine repeats were severely affected by a strong neurological phenotype with tremor, behavioral deficits, strongly reduced motor and exploratory activity, a hunchback, and premature death at 3 to 6 months of age. Neuropathological examination by immunohistochemical staining revealed ubiquitin- and ataxin-3-positive intranuclear inclusion bodies in a multitude of neurons. Directing ataxin-3 with 148 CAGs to the nucleus revealed an even more pronounced phenotype with more inclusions and earlier death, whereas mice transgenic with the same construct but attached to a nuclear export signal developed a milder phenotype with less inclusions. These studies indicate that nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3 in vivo.

Whole genome expression analyses of single- and double-knock-out mice implicate partially overlapping functions of alpha- and gamma-synuclein.

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease. The function of alpha-synuclein has not been deciphered yet; however, it might play a role in vesicle function, transport, or as a chaperone. alpha-Synuclein belongs to a family of three proteins, which includes beta- and gamma-synuclein. gamma-Synuclein shares 60% similarity with alpha-synuclein. Similar to alpha-synuclein, a physiological function for gamma-synuclein has not been defined yet, but it has been implicated in tumorgenesis and neurodegeneration. Interestingly, neither alpha- (SNCA(-/-)), gamma- (SNCG(-/-)), nor alpha/gamma- (SNCA_G(-/-)) deficient mice are present with any obvious phenotype. Using microarray analysis, we thus investigated whether deficiency of alpha- and gamma-synuclein leads to similar compensatory mechanisms at the RNA level and whether similar transcriptional signatures are altered in the brain. Sixty-five genes were differentially expressed in all mice. SNCA(-/-) mice and SNCG(-/-) mice shared 84 differentially expressed genes, SNCA(-/-) and SNCA_G(-/-) expressed 79 genes, and SNCG(-/-) and SNCA_G(-/-) expressed 148 genes. For many of the physiological pathways such as dopamine receptor signaling (down-regulated), cellular development, nervous system function, and cell death (up-regulated), we found groups of genes that were similarly altered in SNCA(-/-) and SNCG(-/-) mice. In one of the pathways altered in both models, we found Mapk1 as the core transcript. Other gene groups, however, such as TGF-beta signaling and apoptosis pathways genes were significantly up-regulated in the SNCA(-/-) mice but down-regulated in SNCG(-/-) mice. beta-synuclein expression was not significantly altered in any of the models.

Genome-wide expression profiling of the retinoschisin-deficient retina in early postnatal mouse development.

PURPOSE: The Rs1h knockout mouse displays retinal features typical for X-linked juvenile retinoschisis (RS). Consequently, this mouse line represents an excellent model to study early molecular events in RS. METHODS: Whole genome expression profiling using DNA-microarrays was performed on total RNA extracts from retinoschisin-deficient and wild-type murine retinas from postnatal days 7, 9, 11, and 14. Quantitative real-time RT-PCR (qRT-PCR) analysis of additional time points facilitated the refinement of the temporal expression profile of differentially regulated transcripts. Differential protein expression was confirmed by Western blot analysis. RESULTS: Based on biostatistic and knowledge-based DNA-microarray analyses we have identified differentially regulated retinal genes in early postnatal stages of the Rs1h-deficient mouse defining key molecular pathways including adhesion, cytoskeleton, vesicular trafficking, and immune response. A significant upregulation of Egr1 at P11 and several microglia/glia-related transcripts starting at P11 with a peak at P14 were identified in the diseased retina. The results provided evidence that macrophage/microglia activation precedes apoptotic photoreceptor cell death. Finally, the role of Egr1 in the pathogenesis of Rs1h-deficiency was investigated, and the results indicated that activation of the MAPK Erk1/2 pathway occurs as early as P7. Analyses of Rs1h(-/Y)/Egr1(-/-) double-knockout mice suggest that Egr1 upregulation is not a prerequisite for macrophage/microglia activation or apoptosis. CONCLUSIONS: The findings imply that microglia/glia activation may be triggering events in the photoreceptor degeneration of retinoschisin-deficient mice. Furthermore, the data point to a role of Erk1/2-Egr1 pathway activation in RS pathogenesis.

A rapid microarray based whole genome analysis for detection of uniparental disomy.

To date, uniparental disomy (UPD) with phenotypic relevance is described for different chromosomes and it is likely that additional as yet unidentified UPD phenotypes exist. Due to technical difficulties and limitations of time and resources, molecular analyses for UPD using microsatellite markers are only performed in cases with specific phenotypic features. In this study, we carried out a whole genome UPD screening based on a microarray genotyping technique. Six patients with the diagnosis of both complete or segmental UPD including Prader-Willi syndrome (PWS; matUPD15), Angelman syndrome (AS; patUPD15), Silver-Russell syndrome (SRS; matUPD7), Beckwith-Wiedemann syndrome (BWS; patUPD11p), pseudohypoparathyroidism (PHP; patUPD20q) and a rare chromosomal rearrangement (patUPD2p, matUPD2q), were genotyped using the GeneChip Human Mapping 10K Array. Our results demonstrate the presence of UPD in the patients with high efficiency and reveal clues about the mechanisms of UPD formation. We thus conclude that array based SNP genotyping is a fast, cost-effective, and reliable approach for whole genome UPD screening.