title: Characterization of a New DGKE Intronic Mutation in Genetically Unsolved Cases of Familial Atypical Hemolytic Uremic Syndrome.
abstract: OBJECTIVE
  Genetic and acquired abnormalities causing dysregulation of the complement alternative pathway contribute to atypical hemolytic uremic syndrome (aHUS), a rare disorder characterized by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute kidney failure. However, in a substantial proportion of patients the disease-associated alterations are still unknown.
  
  
  METHODS
  Whole-exome and whole-genome sequencing were performed in two unrelated families with infantile recessive aHUS. Sequencing of cDNA from affected individuals was used to test for the presence of aberrant mRNA species. Expression of mutant diacylglycerol kinase epsilon (DGKE) protein was evaluated with western blotting.
  
  
  RESULTS
  Whole-exome sequencing analysis with conventional variant filtering parameters did not reveal any obvious candidate mutation in the first family. The report of aHUS-associated mutations in DGKE, encoding DGKE, led to re-examination of the noncoding DGKE variants obtained from next-generation sequencing, allowing identification of a novel intronic DGKE mutation (c.888+40A>G) that segregated with disease. Sequencing of cDNA from affected individuals revealed aberrant forms of DGKE mRNA predicted to cause profound abnormalities in the protein catalytic site. By whole-genome sequencing, the same mutation was found in compound heterozygosity with a second nonsense DGKE mutation in all affected siblings of another unrelated family. Homozygous and compound heterozygous patients presented similar clinical features, including aHUS presentation in the first year of life, multiple relapsing episodes, and proteinuria, which are prototypical of DGKE-associated aHUS.
  
  
  CONCLUSIONS
  This is the first report of a mutation located beyond the exon-intron boundaries in aHUS. Intronic mutations such as these are underreported because conventional filtering parameters used to process next-generation sequencing data routinely exclude these regions from downstream analyses in both research and clinical settings. The results suggest that analysis of noncoding regions of aHUS-associated genes coupled with mRNA sequencing might provide a tool to explain genetically unsolved aHUS cases.
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Caterina
authors: C
authors: Mele
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Mathieu
authors: M
authors: Lemaire
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Paraskevas
authors: P
authors: Iatropoulos
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Rossella
authors: R
authors: Piras
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Elena
authors: E
authors: Bresin
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Serena
authors: S
authors: Bettoni
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: David
authors: D
authors: Bick
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Daniel
authors: D
authors: Helbling
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Regan
authors: R
authors: Veith
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Elisabetta
authors: E
authors: Valoti
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Roberta
authors: R
authors: Donadelli
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Luisa
authors: L
authors: Murer
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Maria
authors: M
authors: Neunhäuserer
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Matteo
authors: M
authors: Breno
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Véronique
authors: V
authors: Frémeaux-Bacchi
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Richard
authors: R
authors: Lifton
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material. giuseppe.remuzzi@marionegri.it.
authors: Giuseppe
authors: G
authors: Remuzzi
authors: Due to the number of contributing authors,the affiliations are provided in the Supplemental Material.
authors: Marina
authors: M
authors: Noris
mesh_terms: D000293:Adolescent; D065766:Atypical Hemolytic Uremic Syndrome / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002648:Child; D004252:DNA Mutational Analysis; D019852:Diacylglycerol Kinase / Q000235:genetics* / Q000378:metabolism; D005260:Female; D056726:Genetic Association Studies; D020022:Genetic Predisposition to Disease; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007223:Infant; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011237:Predictive Value of Tests; D012307:Risk Factors
chemical_list: C526464:DGKE protein, human; D019852:Diacylglycerol Kinase
keywords: complement; diacylglycerol kinase epsilon; genetics; hemolytic uremic syndrome; intronic mutation