Web-accessible application for identifying pathogenic transcripts with RNA-seq: Increased sensitivity in diagnosis of neurodevelopmental disorders.

For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.

Multi-Omics Profiling in Marfan Syndrome: Further Insights into the Molecular Mechanisms Involved in Aortic Disease.

Thoracic aortic aneurysm is a potentially life-threatening disease with a strong genetic contribution. Despite identification of multiple genes involved in aneurysm formation, little is known about the specific underlying mechanisms that drive the pathological changes in the aortic wall. The aim of our study was to unravel the molecular mechanisms underlying aneurysm formation in Marfan syndrome (MFS). We collected aortic wall samples from FBN1 variant-positive MFS patients (n = 6) and healthy donor hearts (n = 5). Messenger RNA (mRNA) expression levels were measured by RNA sequencing and compared between MFS patients and controls, and between haploinsufficient (HI) and dominant negative (DN) FBN1 variants. Immunohistochemical staining, proteomics and cellular respiration experiments were used to confirm our findings. FBN1 mRNA expression levels were highly variable in MFS patients and did not significantly differ from controls. Moreover, we did not identify a distinctive TGF-ß gene expression signature in MFS patients. On the contrary, differential gene and protein expression analysis, as well as vascular smooth muscle cell respiration measurements, pointed toward inflammation and mitochondrial dysfunction. Our findings confirm that inflammatory and mitochondrial pathways play important roles in the pathophysiological processes underlying MFS-related aortic disease, providing new therapeutic options.

Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics.

Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.

Prenatal diagnosis of xeroderma pigmentosum and trichothiodystrophy in 76 pregnancies at risk.

OBJECTIVE: Evaluation of results in a consecutive series of 76 prenatal diagnoses for xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) made since 1977. METHODS: UV-induced DNA repair synthesis was assessed by the autoradiographic measurement of the incorporation of (3)H-thymidine. RESULTS: XP was diagnosed in 19 of the 76 investigated pregnancies at risk; cultured chorionic villus (CV) cells were used in 33 pregnancies with ten affected fetuses and cultured amniocytes in 43 pregnancies with nine affected fetuses. In four cases, CVS results were corroborated by subsequent investigation of amniocytes because maternal cell contamination in the CV cell culture was either present or could not be excluded. Uncertain results in two other cases with intermediate DNA repair capacity and severe maternal cell contamination required further investigation. Median time needed for cell culture and analysis was 25 days. To reduce intra-assay variations, a modification of the DNA repair synthesis assay has recently been developed. In this assay, patients and controls are investigated simultaneously in mixed cultures of cells labelled with polystyrene beads. CONCLUSION: Reliable prenatal diagnosis for XP and TTD can be made by the demonstration of clearly reduced UV-induced DNA repair synthesis due to defective global genome nucleotide excision repair.

The cystathionine beta-synthase variant c.844_845ins68 protects against CNS demyelination in X-linked adrenoleukodystrophy.

The clinical course of X-linked adrenoleukodystrophy (X-ALD) is of unexplained heterogeneity. Major X-ALD phenotypes are the progressive childhood cerebral form (CCALD) with early confluent cerebral demyelination and the adult-onset adrenomyeloneuropathy (AMN). Adult AMN may present with demyelinated foci of the CNS (adrenoleukomyeloneuropathy, ALMN) or without ("pure" AMN). Activated methionine is essential for CNS myelination, and methionine metabolism is important for glutathione synthesis, which may influence neurodegeneration. Cystathionine beta-synthase (CBS) is a key enzyme of methionine metabolism. The CBS variant c.844_845ins68 (p.-) may influence the availability of activated methionine as well as of glutathione. In this study, we analyzed this variant in genomic DNA samples of 86 X-ALD patients. We observed the allele carrying the insertion in 12 of 49 patients without CNS demyelination ("pure" AMN), but in none of the 37 patients with CNS demyelination (CCALD or ALMN; chi(2)=10.531; p=0.001). We conclude that the insertion allele of CBS c.844_845ins68 protected X-ALD patients against CNS demyelination in our study sample. These data suggest that the individual conditions in methionine metabolism may be a disease modifier of X-ALD. Since methionine metabolism can easily be influenced by vitamin and amino acid substitution, this observation could be a basis of novel treatment strategies in this yet untreatable disease. (c) 2006 Wiley-Liss, Inc.

Prenatal diagnosis of the Cockayne syndrome: survey of 15 years experience.

OBJECTIVE: Evaluation of results in a consecutive series of 29 prenatal diagnoses for the Cockayne syndrome. METHODS: Recovery of DNA-synthesis in UV-irradiated cultured fetal cells was measured by scintillation counting of incorporated (3)H-thymidine. Semiquantitative autoradiographic assessment of the recovery of RNA-synthesis (RecRS) was used as an adjunctive method. RESULTS: In 26 of the 29 pregnancies at risk, a definite diagnosis was directly made, based on normal (n = 23) or clearly reduced (n = 3) recovery of DNA-synthesis in UV-irradiated cultured chorionic villus (CV) cells (n = 23) or amniocytes (n = 3). Adjunctive studies were performed in several pregnancies to corroborate the initial results. On three occasions initial results were unreliable, which required investigation of the recovery of RNA-synthesis (n = 2) or even additional amniocentesis (n = 1) to achieve a firm diagnosis. Thus, four affected fetuses were diagnosed in 29 pregnancies at risk (13.8%). CONCLUSION: Reliable prenatal diagnosis of the Cockayne syndrome can be made by the demonstration of a strongly reduced recovery of DNA-synthesis in UV-irradiated cultured chorionic villus cells or amniocytes. Assessment of the recovery of RNA-synthesis was needed as an adjunctive method in rare cases of poor cell growth and DNA-synthesis.

Prenatal diagnosis of citrullinemia and argininosuccinic aciduria: evidence for a transmission ratio distortion in citrullinemia.

BACKGROUND: In the course of 25 years, we have experienced a high rate of affected fetuses in the prenatal diagnosis of citrullinemia. METHODS AND RESULTS: Ninety-one pregnancies at 1 in 4 risk were tested; 36 were diagnosed as affected (39.5%; P = 0.0015). The high rate of positive diagnoses was found both after chorionic villus sampling (24/68 = 35.3%) and amniocentesis (12/23 = 52.2%) despite the completely different and independent techniques used. Using exactly the same (indirect) enzyme assay for argininosuccinic aciduria on chorionic villi and a similar method on amniotic fluid, the expected rate of affected fetuses was found: 13/53 = 24.5%. Technical and genetic causes for the unexpected results were excluded by confirmatory studies performed on independent fetal material, which was available for 27 of the 36 fetuses affected with citrullinemia. Biochemical confirmation was obtained in the 27 cases, whereas in 18 fetuses homozygosity or compound heterozygosity for disease-causing mutations were retrospectively demonstrated in the stored fetal cells. CONCLUSION: The results suggest the occurrence of preferential transmission of the mutant allele. An explanation for this phenomenon may be found in a protective role of argininosuccinic acid synthetase deficiency in mutant sperm cells against the possibly detrimental or apoptotic effect of nitric oxide produced normally from arginine by nitric oxide synthase.