Molecular Mechanisms of Skewed X-Chromosome Inactivation in Female Hemophilia Patients—Lessons from Wide Genome Analyses

Abstract

Introduction: Hemophilia A (HA) is an X-linked bleeding disorder caused by factor VIII (FVIII) deficiency or dysfunction due to F8 gene mutations. HA carriers are usually asymptomatic because their FVIII levels correspond to approximately half of the concentration found in healthy individuals. However, in rare cases, a carrier may exhibit symptoms of moderate to severe HA primarily due to skewed inactivation of her non-hemophilic X chromosome. Aim: The aim of the study was to investigate X-chromosome inactivation (XCI) patterns in HA carriers, with special emphasis on three karyotypically normal HA carriers presenting with moderate to severe HA phenotype due to skewed XCI, in an attempt to elucidate the molecular mechanism underlying skewed XCI in these symptomatic HA carriers. The study was based on the hypothesis that the presence of a pathogenic mutation on the non-hemophilic X chromosome is the cause of extreme inactivation of that X chromosome. Methods: XCI patterns were studied by PCR analysis of the CAG repeat region in the HUMARA gene. HA carriers that demonstrated skewed XCI were further studied by whole-exome sequencing (WES) followed by X chromosome-targeted bioinformatic analysis. Results: All three HA carriers presenting with the moderate to severe HA phenotype due to skewed XCI were found to carry pathogenic mutations on their non-hemophilic X chromosomes. Patient 1 was diagnosed with a frameshift mutation in the PGK1 gene that was associated with familial XCI skewing in three generations. Patient 2 was diagnosed with a missense mutation in the SYTL4 gene that was associated with familial XCI skewing in two generations. Patient 3 was diagnosed with a nonsense mutation in the NKAP gene that was associated with familial XCI skewing in two generations. Conclusion: Our results indicate that the main reason for skewed XCI in our female HA patients was negative selection against cells with a disadvantage caused by an additional deleterious mutation on the silenced X chromosome, thus complicating the phenotype of a monogenic X-linked disease. Based on our study, we are currently offering the X inactivation test to symptomatic hemophilia carriers and plan to expand this approach to symptomatic carriers of other X-linked diseases, which can be further used in pregnancy planning.

A novel F8 variant in a Chinese hemophilia A family and involvement of X-chromosome inactivation: A case report

“…[13–15] Recently, some reports revealed that XCI might be a modifier of FVIII plasma levels, leading to the low expression of clotting factor levels and bleeding symptoms in HA carriers. [16–18] Correlation between XCI patterns and FVIII levels in HA female carriers with the same F8 variant contributed to further confirm this theory. In this study, we reported a novel variant in the F8 (NM_000132.4): c.6193T > G (p.W2065G) gene in a Chinese HA family.…”

“…[8][9][10] 46, XX female is a mosaic of 2 cell types that expresses both maternal and paternal X chromosomes. Normally, cells have equal inactivation (50:50) of the maternal or paternal X chromosome, but some females have preferential inactivation of 1 X chromosome, which is known as skewed X-chromosome Zhang et al • Medicine (2023) 102: 18 Medicine inactivation (XCI). [11,12] In females, skewed XCI can result in phenotypic heterogeneity of many X-linked disorders, disease severities were positive correlation with the activated ratio of mutated X chromosome.…”

Real-Time Observation of “Soft” Magic-Size Clusters during Hydrolysis of the Model Metallodrug Bismuth Disalicylate

“…The mechanisms underlying this skewed choice towards inactivating the chromosome harboring the normal allele have not been elucidated yet. It has recently been described that additional deleterious mutations on the other X-chromosome may favor the negative selection of cells in which it is expressed, generating skewed XCI and contributing to the expression of a given phenotype [19]. However, such a mechanism was not identified in the proband.…”

“…Skewed X-chromosome inactivation can be seen in the general population [17], with levels of skewing increasing with age [18], as it may have been the case for the proposita's mother. However, it is also a common feature in symptomatic carriers of X-linked mutations, favoring the expression of the mutated allele [19,20], which is the case of the proband.…”

Skewed X-Chromosome Inactivation and Parental Gonadal Mosaicism Are Implicated in X-Linked Recessive Female Hemophilia Patients

“…Therefore, in addition to conventional assays to analyze coagulation function, a combination of genetic analyses (e.g., chromosomal complement, molecular genetic examinations (e.g., mutational screening of the causative genes), XCI assay, linkage analysis) is required to explicit the underlying mechanism in female hemophilia, especially when only one mutation is detected. Notably, a recent study for heterozygous female HA patients indicated that the main reason for skewed XCI in patients was negative selection against cells with a disadvantage caused by an additional deleterious mutation in hemophilia-unrelated genes on the silenced X chromosome (i.e., the X chromosome without the HA mutation) [ 36 ]. If the hypothesis is true, the two X chromosomes of affected females will be expected to be either homozygous or compound heterozygous for hemophilia mutations, or have a hemophilia mutation in one X chromosome combined with a deleterious mutation in hemophilia-unrelated genes on the other X chromosome that will render preimplantation genetic testing infeasible.…”