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.

Keywords: X-chromosome inactivation; carrier; hemophilia; monogenic disease; mutation; non-random; whole-exome sequencing.

Figure 1

Analysis of patient 1 and her family members for the PGK1 mutation c.1061_1062delCT. (A)—patient 1, (B)—mother, (C)—brother.

Figure 2

Segregation of F8 and PGK1 mutations in the pedigree of patient 1, demonstrating three generations of skewed inactivation of the X-chromosome bearing the mutant PGK1 gene (%XCI is indicated in parentheses). Patient 1 (II-1), her mother (I-1) and her daughter (III-1) demonstrate skewed inactivation of the same X chromosome. Patient 1 presents with HA phenotype due to a F8 mutation inherited from her father.

Figure 3

Analysis of family members of patient 2 for the SYTL4 mutation c.1655A > C. (A)—patient 2, (B)—father, (C)—mother, (D)—sister 1, (E)—sister 2, (F)—brother.

Figure 4

Segregation of F8 and SYTL4 mutations in the pedigree of patient 2, demonstrating two generations of skewed inactivation of the X chromosome bearing the mutant SYTL4 gene (%XCI is indicated in parentheses). Both patient 2 (II-1) and her mother (I-1) demonstrate skewed inactivation of the same X chromosome. Patient 2 presents with HA phenotype due to de novo F8 intron 22 inversion, which occurred on her paternal X-chromosome and skewed inactivation of the X chromosome bearing the mutant SYTL4 gene inherited from her mother. The patient’s son inherited the X chromosome bearing the F8 intron 22 inversion.

Figure 5

Analysis of family members of patient 3 for the NKAP mutation c.175 C> T. (A)—patient 3, (B)—mother, (C)—sister, (D)—brother 1, (E)—brother 2.

Figure 6

Segregation of F8 and NKAP mutations in the pedigree of patient 3, demonstrating two generations of skewed inactivation of the X-chromosome bearing the mutant NKAP gene (%XCI is indicated in parentheses). Patient 3 and her sister are both obligatory HA carriers carrying the F8 intron 22 inversion mutation. Patient 3 also carries the NKAP mutation and demonstrates complete inactivation of the X chromosome bearing the NKAP mutation, thus presenting with the HA phenotype. The patient’s sister demonstrates random XCI and is an asymptomatic HA carrier. Their mother, who is a carrier of the NKAP mutation, demonstrates skewed XCI.