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In vitro and in silico evidence against a significant effect of the SPINK1 c.194G>A variant on pre-mRNA splicing
  1. Hao Wu1,2,3,4,
  2. Arnaud Boulling2,3,
  3. David N Cooper5,
  4. Zhao-Shen Li1,4,
  5. Zhuan Liao1,4,
  6. Jian-Min Chen2,3,6,
  7. Claude Férec2,3,6,7
  1. 1Department of Gastroenterology, Changhai Hospital, the Second Military Medical University, Shanghai, China
  2. 2Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France
  3. 3Etablissement Français du Sang (EFS)—Bretagne, Brest, France
  4. 4Shanghai Institute of Pancreatic Diseases, Shanghai, China
  5. 5Institute of Medical Genetics, School of Medicine, Cardiff University, Cardiff, UK
  6. 6Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale (UBO), Brest, France
  7. 7Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Centre Hospitalier Universitaire (CHU) Brest, Hôpital Morvan, Brest, France
  1. Correspondence to Dr Jian-Min Chen, INSERM U1078 and EFS—Bretagne, 46 rue Félix Le Dantec, Brest 29218, France; Jian-Min.Chen{at}univ-brest.fr Dr Zhuan Liao, Department of Gastroenterology, Changhai Hospital, the Second Military Medical University, 168 Changhai Road, Shanghai 200433, China; liaozhuan{at}smmu.edu.cn

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We read with interest the recent publication of Beer and Sahin-Tóth1 reporting that exonic variants affecting pre-mRNA splicing contribute to the genetic burden in chronic pancreatitis. One particular variant, affecting the last nucleotide of exon 3 of the SPINK1 gene, c.194G>A, was found to cause an ∼80% reduction in SPINK1 mRNA expression as compared with the wild type in a minigene assay performed in human embryonic kidney 293T (HEK293T) cells. The SPINK1 sequence inserted into the minigene expression vector however comprised only exon 1, exon 2, exon 3, intron 3 and exon 4 of the four-exon gene.1 It should be noted that the potential effect of c.194G>A as a missense mutation (p.Arg65Gln) on protein function has previously been analysed; engineered expression of the full-length mutant coding sequence in Chinese hamster ovary cells and HEK293T cells showed a consistent 50%–60% reduction in protein secretion as compared with the wild type.2 ,3

We recently analysed the functional consequences of 24 SPINK1 intronic variants in relation to their associated mRNA splicing phenotypes4 ,5 by means of a full-gene splicing assay in which the full-length 7 kb SPINK1 genomic sequence (including all four exons plus all three introns of the gene) was cloned into the pcDNA3.1/V5-His-TOPO vector.6 This full-length gene expression system has already proved itself in practice by accurately representing the in vivo situation in the context …

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