Skewed X-chromosome inactivation in unsolved neurodevelopmental disease cases can guide re-evaluation For X-linked genes

Abstract

Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders.

Fig. 1. Pedigree and variant analysis in the three families with XCI-skewed female cases.

A, F, J Family trees of families 113, NWM24, and 237. We used X-Chromosome polymorphic microsatellites to reconstruct the haplotypes and to phase the pathogenetic variant on the inactive/active X chromosome (percentage indicated below the symbol of tested females; Xi and Xa indicate the less and the most active X chromosomes). Haplotypes are colored to illustrate the segregation and the presence of recombinants. For each marker, and the gene involved, the physical position in Mb is reported (GRCh37/hg19 reference genome). The hyphen above each symbol indicates whenever DNA was available for genetic testing. B, G, K Sanger sequencing used to confirm the variants in TAF1 (NM_004606.5), PHF6 (NM_01015877.2) and KDM5C (NM_004187.5). Representative electropherograms are shown: wild type (wt); mutant hemizygous (mut); mutant heterozygous (mut/wt). C, H, M Multiple sequence alignment of the protein amino acid sequences in different species obtained using Marrvel software for the relevant changed aminoacids (highlighted in yellow; http://marrvel.org/)(hs: Homo sapiens; mm: Mus musculus; rn: Rattus norvegicus; xt: Xenopus tropicalis; dr: Danio rerio; dm: Drosophila melanogaster). D, I, L Tolerance Landscape obtained using MetaDome Web Server visualizes regional tolerance to normal genetic variation (https://stuart.radboudumc.nl/metadome/). The position of the missense change is indicated for each gene. The Tolerance Landscape Y-axis is reported as a color scale from blue (position tolerant to variation, T), to yellow (position neutral to variation, N), to red (position intolerant to variation, I). Below the X- axis, a schematic representation of the known protein domains (pink). E Localization of the pathogenic (red) and likely pathogenic (orange) variants reported in the literature for TAF1 gene in male (upper panel) and female cases (lower panel). Our patient’s change is shown in black (p.(Gly249Arg)).

Fig. 2. Pedigrees and variants analysis in the three families with XCI skewed mothers of affected males.

A, E, H, I, K Family trees of families 236, NWM25, TF110, 234 and NWM127. See legend in Fig. 1. B NGS Coverage of ATRX exons (schematized above) in ES data (upper panel) and with Xdrop enrichment (lower panel) in the II.1 proband from family 236. Xdrop enrichment primers (blue bars below) were designed 5ʹ of the maximum estimated deletion. After enriching DNA for the region, and subsequent Illumina Sequencing, we were able to precisely identify a 5971 bp deletion spanning exons 3 and 4. C. Sanger sequencing validation of the ATRX deletion in II.1 and his mother (I.2) using primers flanking the deleted segment (arrows). The deletion breakpoint is shown in (D). F, H. Sanger sequencing validation of the identified variants. J In family 234, we sequenced the genomic region (gDNA) and the corresponding transcript (cDNA) in one of the probands (II.1) and their mother (I.2). The wild-type allele only was detected in both cases in the cDNA, showing that the pathogenic variant was not expressed and thus located on the inactive X-chromosome. G Multiple alignment of the protein amino acid sequences in different species as described in the legend for Fig. 1.