Recessive mutations in diacylglycerol kinase ɛ (DGKE) can cause atypical haemolytic uraemic syndrome (aHUS)—a common cause of acquired acute renal failure in children. New research by Giuseppe Remuzzi and colleagues extends this association, with the identification of a novel aHUS-causative intronic mutation in DGKE located well beyond the exon–intron boundaries.

The researchers performed whole-exome and whole-genome sequencing in two families (one from Italy and one from the USA) with infantile recessive familial aHUS of unknown genetic origin. “Analysis of the data using conventional variant filtering parameters did not reveal any obvious candidate mutations in the Italian family, and only a heterozygous nonsense DGKE mutation in the US family that alone could not explain the disease,” says researcher Marina Noris. “Given that deep intronic mutations can result in abnormal splicing, we examined the intronic variants in the whole-exome sequencing data of the Italian family.”

The DGKE locus was analysed in detail because of similarities with regard to the mode of disease inheritance, age of onset, and clinical phenotype between the affected patients of the two families and those with known DGKE aHUS-causative mutations. A novel DGKE mutation located in intron 5 (c.888+40A>G), was identified and segregated with the disease in a recessive pattern.

Sequencing DGKE mRNA obtained from blood leucocytes of the two affected siblings in the Italian family, demonstrated that the intronic mutation affected splicing of exon 5, resulting in the production of three aberrant DGKE isoforms. The wild-type DGKE mRNA was absent.

Further analyses showed that the most abundant DGKE mRNA isoform retained an additional 39 nucleotides that immediately follow exon 5 and encoded a DGKE protein that was 13 amino acids longer than the wild-type. Structural 3D modelling predicted that this 13 amino acid insertion would affect DGKE kinase activity. The other two isoforms were not detected at the protein level. Strikingly, the same mutation was found in compound heterozygosity with the nonsense DGKE mutation in the three affected siblings of the US family.

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“We now plan to design next-generation sequencing panels that target the introns of DGKE and other complement-associated aHUS genes, to identify novel disease-associated mutations,” says Noris. “Sequencing intronic regions of known disease-associated genes followed by analysis of the mRNA sequence could be a valuable tool to resolve aHUS cases with an unknown genetic basis.” The researchers propose that this study might be an example for other rare diseases where exon screening fails to identify any causative mutation.