Deep intronic variant causes aberrant splicing of ATP7A in a family with a variable occipital horn syndrome phenotype.

Genetic variants in ATP7A are associated with a spectrum of X-linked disorders. In descending order of severity, these are Menkes disease, occipital horn syndrome, and X-linked distal spinal muscular atrophy. After 30 years of diagnostic investigation, we identified a deep intronic ATP7A variant in four males from a family affected to variable degrees by a predominantly skeletal phenotype, featuring bowing of long bones, elbow joints with restricted mobility which dislocate frequently, coarse curly hair, chronic diarrhoea, and motor coordination difficulties. Analysis of whole genome sequencing data from the Genomics England 100,000 Genomes Project following clinical re-evaluation identified a deep intronic ATP7A variant, which was predicted by SpliceAI to have a modest splicing effect. Using a mini-gene splicing assay, we determined that the intronic variant results in aberrant splicing. Sanger sequencing of patient cDNA revealed ATP7A transcripts with exon 5 skipping, or inclusion of a novel intron 4 pseudoexon. In both instances, frameshift leading to premature termination are predicted. Quantification of ATP7A mRNA transcripts using a qPCR assay indicated that the majority of transcripts (86.1 %) have non-canonical splicing, with 68.0 % featuring exon 5 skipping, and 18.1 % featuring the novel pseudoexon. We suggest that the variability of the phenotypes within the affected males results from the stochastic effects of splicing. This deep intronic variant, resulting in aberrant ATP7A splicing, expands the understanding of intronic variation on the ATP7A-related disease spectrum.

Narrowing the chromosome 22q11.2 locus duplicated in bladder exstrophy-epispadias complex.

INTRODUCTION: Bladder exstrophy-epispadias complex (BEEC) comprises a spectrum of anterior midline congenital malformations, involving the lower urinary tract. BEEC is usually sporadic, but families with more than one affected member have been reported, and a twin concordance study supported a genetic contribution to pathogenesis. Moreover, diverse chromosomal aberrations have been reported in a small subset of individuals with BEEC. The commonest are 22q11.2 microduplications, identified in approximately 3% of BEEC index cases. OBJECTIVES: We aimed to refine the chromosome 22q11.2 locus, and to determine whether the encompassed genes are expressed in normal developing and mature human urinary bladders. RESULTS: Using DNA from an individual with CBE, the 22q11.2 duplicated locus was refined by identification of a maternally inherited 314 kb duplication (chr22:21,147,293-21,461,017), as depicted in this image. Moreover, the eight protein coding genes within the locus were found to be expressed during normal developing and mature bladders. To determine whether duplications in any of these individual genes were associated with CBE, we undertook copy number analyses in 115 individuals with CBE without duplications of the whole locus. No duplications of individual genes were found. DISCUSSION: The current study has refined the 22q11.2 locus associated with BEEC and has shown that the eight protein coding genes are expressed in human bladders both during antenatal development and postnatally. Nevertheless, the precise biological explanation as to why duplication of the phenocritical region of 22q11 confers increased susceptibility to BEEC remains to be determined. The fact that individuals with CBE without duplications of the whole locus also lacked duplication of any of the individual genes suggests that in individuals with BEEC and duplication of the 22q11.2 locus altered dosage of more than one gene may be important in BEEC etiology. CONCLUSIONS: The study has refined the 22q11.2 locus associated with BEEC and has shown that the eight protein coding genes within this locus are expressed in human bladders.