Genetic analysis of 20 patients with hypomyelinating leukodystrophy by trio-based whole-exome sequencing

Abstract

Hypomyelinating leukodystrophies (HLDs) are a rare group of disorders characterized by myelin deficit of the brain-based on MRI. Here, we studied 20 patients with unexplained HLD to uncover their genetic etiology through whole-exome sequencing (WES). Trio-based WES was performed for 20 unresolved HLDs families after genetic tests for the PLP1 duplication and a panel of 115 known leukodystrophy-related genes. Variants in both known genes that related to HLDs and promising candidate genes were analyzed. Minigene splicing assay was conducted to confirm the effect of splice region variant. All 20 patients were diagnosed with HLDs clinically based on myelin deficit on MRI and impaired motor ability. Through WES, in 11 of 20 trios, 15 causative variants were detected in seven genes TUBB4A, POLR1C, POLR3A, SOX10, TMEM106B, DEGS1, and TMEM63A. The last three genes have just been discovered. Of 15 variants, six were novel. Using minigene splicing assay, splice variant POLR3A c.1770 + 5 G > C was proved to disrupt the normal splicing of intron 13 and led to a premature stop codon at position 618 (p.(P591Vfs*28)). Our analysis determined the molecular diagnosis of 11 HLDs patients. It emphasizes the heterogenicity of HLDs, the diagnostic power of trio-based WES for HLDs. Comprehensive analysis including a focus on candidate genes helps to discover novel disease-causing genes, determine the diagnosis for the first time, and improve the yield of WES. Moreover, novel mutations identified in TUBB4A, POLR3A, and POLR1C expand the mutation spectrum of these genes.

Fig. 1

In-house analysis workflow of trio-based exome sequencing data. gnomAD genome aggregation database, ExAC exome aggregation consortium, 1000G 1000 genomes project, ESP6500, exome sequencing project

Fig. 2

Minigene results of POLR3A c.1770 + 5 G > C variant. a Agarose gel image of PCR products. b Sanger sequence results of the PCR products. c Schematic of the minigene and aberrant splicing. WT wild type, MT mutant, m marker

Fig. 3

MRI images of seven patients. Row 1 and row 2 are axial T2-weighted images, showing diffuse hypomyelination in cerebral white matter in all seven patients and atrophy of the basal ganglia in Pt1, Pt2, and Pt3. Pt1 also demonstrated an abnormal signal in the putamen and caudate nucleus. Raw 3 are midsagittal T1-weighted or T2-weighted images, showing atrophy of cerebellum in Pt1 and Pt6. Six patients were diagnosed with HLD6 (Pt1, Pt2, Pt3), HLD7 (Pt4), HLD11 (Pt5, Pt6), and SOX10 related disorder (Pt7), respectively. Y years, M months