Heterozygous Deletion of the SHOX Gene Enhancer in two Females With Clinical Heterogeneity Associating With Skewed XCI and Escaping XCI

Abstract

Skewed X-chromosome inactivation (XCI) plays an important role in the phenotypic heterogeneity of X-linked disorders. However, the role of skewed XCI in XCI-escaping gene SHOX regulation is unclear. Here, we focused on a heterozygous deletion of SHOX gene enhancer with clinical heterogeneity. Using SNP array, we detected that the female proband with Leri-Weill dyschondrosteosis (LWD) carried an 857 kb deletion on Xp22.3 (encompassing SHOX enhancer) and a 5,707 kb large-fragment deletion on Xq25q26. XCI analysis revealed that the X-chromosome with the Xq25q26 large-fragment deletion was completely inactivated, which forced the complete activation of the other X-chromosome carrying SHOX enhancer deletion. While the Xp22.3 deletion locates on the escaping XCI region, under the combined action of skewed XCI and escaping XCI, transcription of SHOX gene was mainly from the activated X-chromosome with SHOX enhancer defect, involving in the formation of LWD phenotype. Interestingly, this SHOX enhancer deletion was inherited from her healthy mother, who also demonstrated completely skewed XCI. However, the X-chromosome with SHOX enhancer deletion was inactivated, and the normal X-chromosome was activated. Combing with escaping XCI, her phenotype was almost normal. In summary, this study was a rare report of SHOX gene enhancer deletion in a family with clinical heterogeneity due to skewed inactivation of different X-chromosomes, which can help in the genetic counseling and prenatal diagnosis of disorders in females with SHOX defect.

Keywords: HUMARA assay; Leri-Weill dyschondrosteosis; SHOX gene enhancer; clinical heterogeneity; escaping X-chromosome inactivation (XCI); skewed X-chromosome inactivation (XCI).

Figure 1

Photograph of the forearm of the proband II2 and mother I2. (A). Photograph of the forearm of the proband II2 showing a Madelung deformity and bowing of the radius (blue and red arrows). This photograph shows the curve and shortening of the forearm, hand and wrist, the structure of which appears like that of a dinner fork. (B). Photograph of the forearm of the mother I2 showing a normal phenotype.

Figure 2

Characteristics of the 5,707 kb and 857 kb deletions of Xq25q26.3 and Xp22.33, respectively. (A). Results of cfDNA screening of the pregnant proband. cfDNA screening study of the maternal plasma, illustrating an uncertain 6 Mb deletion in the long arm of the X-chromosome (128M–133M), Z score = −19.43. (B and C). SNP array analysis of the fetus (III1) and the couples (II1, II2). (B). The red bar indicates a heterozygous 5,707 kb deletion in Xq25q26.3 (chrX: 127,915,006–133,621,667) in the fetus (III1). (C). The red bar indicates a heterozygous 857 kb deletion in Xp22.33 (chrX: 784,064–1,640,746) in the fetus (III1) and the pregnant proband (II2). (D). Pseudoautosomal region (PAR1) of the X and Y chromosomes. The 857 kb deletion in Xp22.33 (red bar) was located 164 kb downstream of the SHOX gene (chrX: 585,079–620,146), including the evolutionarily conserved CNE9 (blue bar), which was the SHOX gene enhancer. (E). For the proband II2, FISH experiment showed that the Xp22.3 deletion (detected by the probe RP11-1119O18, Spectrum Green) and the Xq25q26 deletion (detected by the probe RP11-313D19, Spectrum Red) located on the different X chromosomes, respectively.

Figure 3

Relative ratio of the Xp22.33 (CNE9 and CRLF2) and Xq25q26.3 (AIFM1 and FRMD7) regions in the family by qPCR. (A and B). Xp22.33 region (CNE9 and CRLF2) was located in PAR1 of the X and Y chromosomes, including the SHOX enhancer. Dosages in normal female were equal, Xp22.33 region dosages in I1 and II1 were normal, and those in I2, II2, III1 with Xp22.33 deletion were half. (C and D) The AIFM1 and FRMD7 were present only in the X-chromosome. The AIFM1 and FRMD7 dosage in I2 were normal, while that in I1, II1, II2, III1 with Xq25q26.3 deletion was half. From the above data, it was elucidated that the Xp22.33 deletion in the proband II2 was inherited from her healthy mother I2, and the Xq25q26.3 deletion occurred de novo.

Figure 4

X-chromosome inactivation (XCI) pattern and linkage analyses were based on the polymorphic CAG repeat in exon 1 of the gene for androgen receptor (HUMARA). (A). A peak of 274 bp for HUMARA was observed by assaying the undigested PCR product of I1 and no peaks were observed for the HpaII digested product. (B). The undigested PCR product of I2 gave two peaks of 280 and 283 bp, each, while only one peak of 280 bp was observed with the HpaII digested product. I2 exhibited 100% skewing of XCI, and the inactivated X-chromosome was linked with the 280 bp peak of the HUMARA PCR products. (C). The undigested PCR product of the proband II2 gave two peaks of 274 and 280 bp. One X-chromosome linked with the 280 bp peak of HUMARA was inhibited from the mother I2 and the other from the father I1. The product of HpaII digestion gave only one peak of 274 bp. II2 also demonstrated 100% skewing of XCI, but the inactivated X-chromosome was linked with the 274 bp peak of AR, which is different from that of the mother I2. (D). Schematic diagram of Xp22.33 and Xq25q26.3 deletions, and HUMARA PCR products in the pedigree. It can be seen that Xp22.33 and Xq25q26.3 deletions in II2 are located on different X-chromosomes. Xp22.33 deletion (SHOX enhancer deletion) of I2 occurred in the X-chromosome, whose allele was linked with the 280 bp peak of HUMARA PCR. The X-chromosome was inactivated and delivered to the proband I2, but her X-chromosome was activated.