Parallel in-depth analysis of repeat expansions in ataxia patients by long-read sequencing.

Instability of simple DNA repeats has been known as a common cause of hereditary ataxias for over 20 years. Routine genetic diagnostics of these phenotypically similar diseases still rely on an iterative workflow for quantification of repeat units by PCR-based methods of limited precision. We established and validated clinical nanopore Cas9-targeted sequencing, an amplification-free method for simultaneous analysis of 10 repeat loci associated with clinically overlapping hereditary ataxias. The method combines target enrichment by CRISPR-Cas9, Oxford Nanopore long-read sequencing and a bioinformatics pipeline using the tools STRique and Megalodon for parallel detection of length, sequence, methylation and composition of the repeat loci. Clinical nanopore Cas9-targeted sequencing allowed for the precise and parallel analysis of 10 repeat loci associated with adult-onset ataxia and revealed additional parameter such as FMR1 promotor methylation and repeat sequence required for diagnosis at the same time. Using clinical nanopore Cas9-targeted sequencing we analysed 100 clinical samples of undiagnosed ataxia patients and identified causative repeat expansions in 28 patients. Parallel repeat analysis enabled a molecular diagnosis of ataxias independent of preconceptions on the basis of clinical presentation. Biallelic expansions within RFC1 were identified as the most frequent cause of ataxia. We characterized the RFC1 repeat composition of all patients and identified a novel repeat motif, AGGGG. Our results highlight the power of clinical nanopore Cas9-targeted sequencing as a readily expandable workflow for the in-depth analysis and diagnosis of phenotypically overlapping repeat expansion disorders.

Intracranial venous hemodynamics in patients with midline dislocation due to postischemic brain edema.

BACKGROUND AND PURPOSE: Cerebral venous pressure is governed by intracranial pressure, cerebral perfusion pressure, and venous outflow resistance. Therefore, changes in venous flow velocities are to be expected because of changes in intracranial pressure and brain tissue dislocation in patients with ischemic stroke and space-occupying brain edema. METHODS: In 21 prospectively recruited patients with middle cerebral artery stroke and postischemic edema, flow velocities in the basal veins, the vein of Galen, the straight sinus, and the P2 segment of the posterior cerebral artery were recorded every 0.9+/-0.5 days during the first 5 days after symptom onset with the use of transcranial color-coded duplex sonography. The midline shift of the third ventricle was determined by B-mode imaging. RESULTS: We observed an initial increase of flow velocity in the basal vein ipsilateral to the lesion, followed by a significant decrease within 5 days after symptom onset and with increasing midline shift in patients with brain herniation. In the straight sinus, flow velocity showed a biphasic U-shaped response to increasing dislocation of the third ventricle, with an initial decrease followed by an increase in the course of mass movement (midline shift 1 to 1.5 cm). A steep increase of flow velocity in the vein of Galen took place with a midline shift >1.5 cm. In the survivors these changes could not be observed. Flow velocity in the P2 segment of the posterior cerebral artery followed a typical course in neither the fatal cases nor the survivors. CONCLUSIONS: Monitoring of flow velocities in the basal cerebral veins and in the straight sinus can provide additional pathophysiological information in patients with space-occupying brain edema after acute stroke.