We reasoned that unbalanced XCI could be a guide for re-evaluating clinical and molecular data in undiagnosed NDD cases. To test XCI, we set up a multiplex fluorescent PCR simultaneously analyzing the methylation status of three independent polymorphic markers on the X-chromosome. This assay allowed us to increase informativeness to > 98%, compared to 80% using the standard HUMARA.(13)
The analysis of 91 females NDD patients and 186 mothers of male NDD patients, previously undiagnosed by CMA and trio-ES, showed a significant enrichment of subjects with extremely unbalanced XCI, defined as a > 90:10 XCI ratio (28/277, 10%) in line with a similar study.(9) This suggested that several of our undiagnosed cases could be attributed to a gene on the X chromosome. A proof of principle came from the re-evaluation of available trio-ES data: focusing on X-linked coding regions, we identified variants likely causative of a disease in 7/28 cases, solving 25% of skewed XCI cases. In our original survey of 575 NDD cases, we had 9 patients with X-linked variants classified as class 4 or 5, and 12 with class 3 variants. Considering the 28 cases with skewed XCI, we may estimate that X-linked genes account from 6.4 to 8.5% (9 + 28/575; 21 + 28/757) in our survey. These figures agree with a recent evaluation of the burden of X-linked coding variation based on 11,044 Developmental Disorder patients, who estimated X-linked causes in 6.0% of males and 6.9% of females.(34)
We previously missed the seven variants on the X chromosome because of three reasons: the gene was unknown at the time of the analysis (OTUD5, PDZD4, and ZMYM3), the variant was a structural rearrangement missed by ES (ATRX) or overlooked because apparently inconsistent with X-linked segregation, since both males and females were affected (TAF1, PHF6, and KDM5C).
In the former category, LINKage-specific-deubiquitylation-deficiency-induced embryonic defects (LINKED) syndrome, has been associated to pathogenic OTUD5 variants only in 2021,(31) whereas PDZD4 and ZMYM3 have been presently only proposed as a disease gene.(33, 35)
The microdeletion identified in ATRX (family 236) highlights the importance of searching for genomic rearrangements, exploiting exome data, or performing genome sequencing. In this case the deletion was missed by CMA due to lack of array probes in the deleted tract. The strong clinical suspicion of ID-hypotonic facies syndrome (MIM# 309580) prompted us to analyze the coverage of all ATRX exons on ES data and to finally identify the deletion of exon 3–4.
We also chose to locate the precise breakpoints using a novel method based on the enrichment for targeted resequencing by the Xdrop technology, which combines high-resolution droplet PCR (dPCR) with droplet sorting and Multiple Displacement Amplification in droplets (dMDA).
This approach proved successful to fine-mapping the deletion breakpoints, narrowing it down from a large putative region of ≈ 20 kb between exon 2 and 5. Given the flexibility of this technology, we expect that it might be useful in the future in other similar cases where the large size of the involved region hampers the efficient use of traditional assays for the characterization of structural variations at the single-base resolution. Alternatively, to achieve the same results, we would have required either a genome sequencing (with higher costs) or a very large sets of PCR-based assays and a laborious work to map the whole 20 kb region, also because the deletion maps within a region rich in repeated sequences. The availability of the deletion boundaries allowed to set up a simple PCR test to segregate the variant in the family.
The X chromosome is often underestimated in the diagnosis of female NDD patients, because of the common perception that females are less susceptible to X-linked conditions.(17) Although many X-linked conditions show a profound sex-linked bias, given the specific mechanism of inheritance, an increasing number of X-linked disease has been described to occur similarly in both female and male patients.(17)
In families 113 and NWM24, we identified a missense variant in a female for TAF1 and PHF6; we overlooked/ignored these variants at first ES reading because inconsistent with an X-linked recessive disease. However, literature data reported females with phenotypes consistent with variants in those genes. In TAF1, the completely skewed XCI is consistent with other recently described cases where the phenotype, different between females and males, is uniform within each sex.(36) XCI unbalance is favoring the wild type allele in both the mother (mild phenotype) and her affected daughter, leaving unclear the pathogenic mechanism. We can speculate that (i) 5% of the pathogenic allele is enough to cause the phenotype or (ii) that the XCI pattern is different in affected tissues such as brain, where the pathogenic allele is for some reason more expressed than in blood.
In family NWM24, the phenotype associated with PHF6 is consistent with literature data reporting two females carrying the de novo p.Cys305Phe, a few amino acids distant from our proband’s variant.(24, 37)
Family 237 is another example of X-linked gene complexity: three females carrying a missense variant in KDM5C, but we detected skewed XCI towards the deleterious allele only in the one with the disease phenotype. Segregation analysis showed skewing towards the deleterious allele. KDM5C is known to escape XCI and thus the role of skewing in the phenotype is not clear.(38)
Among the various causes of female susceptibility to X-linked conditions, XCI certainly plays a key role at the penetrance level. Although XCI mechanism has been known for a very long time now, evaluation of its influence on the phenotype remains challenging.
In some cases, the presence of skewed XCI in more easily explainable by the selection of cells that inactivate the mutated allele, expressing only the wild type allele and gaining a selective advantage at the first stages of development.(10) Examples are given by mothers heterozygous for OTUD5, ATRX, ZMYM3, PDZD4 variants, who are protected against the deleterious effect of an X-linked pathogenetic variant by skewed XCI. In females affected by X-linked conditions, XCI can modulate the expression of phenotype;(39) likely, there are several mechanisms that may underlie disease and skewing that currently escape our understanding and are not always easily identifiable.
In 20 XCI skewed cases, we did not identify any potentially causative variant. We hypothesize that the phenotype could be explained by variants in coding regions not covered by exome or noncoding variation such as deep intronic variant affecting splicing or regulatory regions.
Overall, our data suggest that XCI testing may be a simple and inexpensive analysis to help focusing the reanalysis of exomic data on the X chromosome.