Favorably skewed X-inactivation accounts for neurological sparing in female carriers of Menkes disease

Abstract

Classical Menkes disease is an X-linked recessive neurodegenerative disorder caused by mutations in ATP7A, which is located at Xq13.1-q21. ATP7A encodes a copper-transporting P-type ATPase and plays a critical role in development of the central nervous system. With rare exceptions involving sex chromosome aneuploidy or X-autosome translocations, female carriers of ATP7A mutations are asymptomatic except for subtle hair and skin abnormalities, although the mechanism for this neurological sparing has not been reported. We studied a three-generation family in which a severe ATP7A mutation, a 5.5-kb genomic deletion spanning exons 13 and 14, segregated. The deletion junction fragment was amplified from the proband by long-range polymerase chain reaction and sequenced to characterize the breakpoints. We screened at-risk females in the family for this junction fragment and analyzed their X-inactivation patterns using the human androgen-receptor (HUMARA) gene methylation assay. We detected the junction fragment in the proband, two obligate heterozygotes, and four of six at-risk females. Skewed inactivation of the X chromosome harboring the deletion was noted in all female carriers of the deletion (n = 6), whereas random X-inactivation was observed in all non-carriers (n = 2). Our results formally document one mechanism for neurological sparing in female carriers of ATP7A mutations. Based on review of X-inactivation patterns in female carriers of other X-linked recessive diseases, our findings imply that substantial expression of a mutant ATP7A at the expense of the normal allele could be associated with neurologic symptoms in female carriers of Menkes disease and its allelic variants, occipital horn syndrome, and ATP7A-related distal motor neuropathy.

Fig. 1

ATP7A deletion breakpoint. A map of the genomic DNA containing ATP7A exons 12 through 15 is shown. (a) Sequencing of the mutant allele from the proband led to identification of the breakpoints shown. (b) Primers (arrows) were designed to flank the region of the deletion to amplify a 664-bp junction fragment associated with the mutant allele. See Methods section for details.

Fig. 2

Molecular characterization of family with history of Menkes disease. (a) Three-generation family pedigree. Fully shaded squares indicate affected males; half-shaded circles indicate females heterozygous for ATP7A exon 13/14 deletion. Ratios underneath female symbols indicate the respective X-inactivation patterns (for female heterozygotes, ratios denote the percentage of cells with the mutant allele inactivated: percentage of cells with the normal allele inactivated). (b) Junction fragment assay. A 664-bp junction fragment is detected in four of six at-risk females: II-4, II-6, II-7, and II-8 (top panel). The fragment is not amplified in an unrelated normal female control (C). Lowermost panel: all family members, except the male proband (III-1), show amplification of ATP7A exon 13 (291 bp). L, ladder of molecular weight markers; B, blank.

Fig. 3

X-inactivation analyses. (a) The proband’s grandmother (I-1) has HUMARA allele sizes of 269 and 287 bp (top left panel). Individual II-4, and all other of the proband’s maternal aunts who are heterozygous for the exon 13/14 deletion, inherited allele 281 from their father and allele 269 from their mother (top right panel). In contrast, the non-carrier females in this family (II-1 and II-3) inherited the 287-bp HUMARA allele from their mother (data not shown). These results indicated that the 269-bp HUMARA allele originates from the X chromosome harboring the ATP7A exon 13/14 deletion. (b) Digestion of genomic DNA from each subject with the methylation-sensitive restriction endonuclease, HpaII, prior to polymerase chain reaction amplification yields only allele 269, representing the methylated, inactive X chromosome (bottom panels) and indicating completely skewed (100:0) inactivation of the X chromosome bearing the ATP7A deletion.