Different types of disease-causing non-coding variants revealed by genomic and gene expression analyses in families with X-linked intellectual disability

The pioneering discovery research of X-linked intellectual disability (XLID) genes has benefitted thousands of individuals worldwide however, approximately 30% of XLID families still remain unresolved. We postulated that non-coding variants that affect gene regulation or splicing may account for the lack of a genetic diagnosis in some cases. Detecting pathogenic, gene-regulatory variants with the same sensitivity and specificity as structural and coding variants is a major challenge for Mendelian disorders. Here, we describe three pedigrees with suggestive XLID where distinctive phenotypes associated with known genes guided the identification of three different non-coding variants. We used comprehensive structural, single nucleotide and repeat expansion analyses of genome sequencing. RNA-Seq from patient-derived cell lines, RT-PCRs, western blots and reporter gene assays were used to confirm the functional effect of three fundamentally different classes of pathogenic non-coding variants: a retrotransposon insertion, a novel intronic splice donor and a canonical splice variant of an untranslated exon. In one family, we excluded a rare coding variant in ARX, a known XLID gene, in favour of a regulatory non-coding variant in OFD1 that correlated with the clinical phenotype. Our results underscore the value of genomic research on unresolved XLID families to aid novel, pathogenic non-coding variant discovery.Michael J. Field, Raman Kumar, Anna Hackett, Sayaka Kayumi, Cheryl A. Shoubridge, Lisa J. Ewans, Atma M. Ivancevic, Tracy Dudding, Byth, Renée Carroll, Thessa Kroes, Alison E. Gardner, Patricia Sullivan, Thuong T. Ha, Charles E. Schwartz, Mark J. Cowley, Marcel E. Dinger, Elizabeth E. Palmer, Louise Christie, Marie Shaw, Tony Roscioli, Jozef Gecz, Mark A. Corbet

Standardization and calibration for breast MRI : the use of a unique air bubble free agar phantom

PURPOSE The routine use of an in vivo phantom could provide the basis for standardization and calibration of DCE-MRI of the breast. Here, it is used to correct for imperfect flip angle profiles to obtain a more accurate T1 quantitation. METHOD AND MATERIALS A unique soft phantom was developed for clinical use. The air bubble-free ultra-pure agar content of the ring-shaped phantom covers a range of contrast agent concentrations (Omniscan, 0 – 1.0 mM). As a reference, an accurate variable flip angle measurement (3D FSPGRE, FA= 3/5/10/15/20/25/30/35/40/50°, TR/TE=25/1.92 ms, NA = 4) was performed on the phantom. A healthy volunteer (no contrast, IRB approval) was scanned in a standard bilateral coil at 1.5 T, with the phantom placed around the breast. Again a variable flip angle measurement was performed (3D FSPGRE, FA = 5/10/20/25/40°, TR/TE=25/1.9 ms, NA = 1). Based on the signal intensities and the previously measured T1 in the phantom, the effective flip angles were simultaneously estimated and used to calculate T1 for the breast. In addition, to ensure that the presence of the phantom does not interfere with the appearance of the breast, clinical T2-w (2D turbo spin echo) and T1-w (ultrafast gradient echo) images were acquired with and without the phantom around the breast. RESULTS Under the assumption of a uniform B1 field of the body coil in the central slices, the effective flip angles were estimated at: 4.8/9.8/21.9/27.3/57.1°, resulting in corrected T1 estimates for fat and parenchyma of 261.4 ± 6.6 ms (without flip angle correction: 344.5 ± 54.3 ms), and 763.0 ± 31.3 ms (without correction: 930.1 ± 154.1 ms), respectively. The quality of the clinical images containing the phantom was found to be adequate by an experienced breast radiologist. Its presence did, however, affect automatic windowing. No discomfort was reported. CONCLUSION This in vivo phantom can be used to improve T1 quantitation. Alternatively, it may be used to quantify T1 without the need for a variable flip angle measurement. A reliable phantom could also improve quantitative measurements of proton density. An improved phantom design is under development and will be used in routine clinical practice. CLINICAL RELEVANCE/APPLICATION The routine use of a reliable clinical calibration phantom could lead to improved standardization of DCE-MRI across different institutions

Aicardi Syndrome Is a Genetically Heterogeneous Disorder

Aicardi Syndrome (AIC) is a rare neurodevelopmental disorder recognized by the classical triad of agenesis of the corpus callosum, chorioretinal lacunae and infantile epileptic spasms syndrome. The diagnostic criteria of AIC were revised in 2005 to include additional phenotypes that are frequently observed in this patient group. AIC has been traditionally considered as X-linked and male lethal because it almost exclusively affects females. Despite numerous genetic and genomic investigations on AIC, a unifying X-linked cause has not been identified. Here, we performed exome and genome sequencing of 10 females with AIC or suspected AIC based on current criteria. We identified a unique de novo variant, each in different genes: KMT2B, SLF1, SMARCB1, SZT2 and WNT8B, in five of these females. Notably, genomic analyses of coding and non-coding single nucleotide variants, short tandem repeats and structural variation highlighted a distinct lack of X-linked candidate genes. We assessed the likely pathogenicity of our candidate autosomal variants using the TOPflash assay for WNT8B and morpholino knockdown in zebrafish (Danio rerio) embryos for other candidates. We show expression of Wnt8b and Slf1 are restricted to clinically relevant cortical tissues during mouse development. Our findings suggest that AIC is genetically heterogeneous with implicated genes converging on molecular pathways central to cortical development.Thuong T. Ha ... Mark A. Corbett ... Jozef Gecz ... Michael Lardelli ... Clare L. van Eyk ... Raman Sharma ... et al

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor beta Signaling

BACKGROUND: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative.METHODS: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology.RESULTS: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocannpal-dependent learning and memory.CONCLUSIONS: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor beta signaling and hippocampal function.Genetics of disease, diagnosis and treatmen