Skewed X chromosome inactivation in the patient
Because the patient was a girl, we expected that she would express both wildtype and mutant transcripts in her tissues. However, she only expressed the defective frameshift BCAP31 transcript and her clinical features resembled those of X-linked recessive male patients carryingBCAP31 mutations. Haplotype phasing with linked-read sequencing data around the BCAP31 gene showed that the de novo allele arose from the paternal X chromosome while the wild type allele came from the maternal X chromosome (Figure 4). To explain the patient’s BCAP31 expression pattern and clinical features, we hypothesized that uneven X chromosome inactivation (XCI) occurred in the patient. We performed the HUMARA (Human Androgen Receptor Amplification) assay to assess the methylation ratio of maternal and paternal X chromosome in WBCs, with the knowledge that XCI patterns between blood and other analyzed tissues were highly correlated (Echevarria et al., 2016). The androgen receptor allele profile of triplicate experiments showed skewed X chromosome inactivation, with the maternal X chromosome (carrying the wildtype BCAP31 allele) inactivated in 88% ± 2% of the WBCs (Figure 5). The HUMARA study confirms that the paternal X chromosome (carrying the de novo mutation) was active in the majority of the cells and explained the patient’s “X-linked recessive” phenotype.