Severe infantile isolated exocrine pancreatic insufficiency caused by the complete functional loss of theSPINK1gene

Abstract

Exocrine pancreatic insufficiency (EPI) is rare in children, with most if not all cases occurring as part of syndromic conditions such as cystic fibrosis and Shwachman–Diamond syndrome. Here we report two cases, both presenting with severe EPI around 5 months of age. Characterized by diffuse pancreatic lipomatosis, they otherwise exhibited no remarkable deficiencies in other organs. Novel non‐identical homozygous variants (a deletion removing the entire SPINK1 gene and an insertion of a full‐length inverted Alu element into the 3′‐untranslated region of the SPINK1 gene) resulting in the complete functional loss of the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor) were identified in each patient. Having correlated our findings with current knowledge of SPINK1's role in exocrine pancreas pathophysiology, we propose that complete and partial functional losses of the SPINK1 gene are associated with quite distinct phenotypes, the former causing a new pediatric disease entity of severe infantile isolated EPI.

Aluinsertion-mediated dsRNA structure formation with pre-existingAluelements as a novel disease-causing mechanism

“…We have previously analyzed the functional impact of the SPINK1 Alu -Ins event using a cell culture-based full-length gene expression assay (FLGEA). We established that it caused a complete loss of SPINK1 mRNA expression in transfected HEK293T cells, an observation which was corroborated by three lines of evidence 7 . First, reverse transcription-PCR (RT-PCR) of mRNA from the SPINK1 Alu -Ins homozygote-derived cultured lymphocytes yielded no SPINK1 transcripts.…”

“…We surmised that SPINK1 Alu -Ins may have formed secondary structures with either or both of these two pre-existing Alu elements in the mutant pre-mRNA, thereby hindering the recognition of the 3’ splice site of intron 3. However, the aberrantly spliced transcripts would have to have been degraded by mRNA decay mechanisms such as nonsense-mediated mRNA decay (NMD) 18 , in order to explain the non-detection of SPINK1 mRNA sequences from either mutant-transfected HEK293T cells or patient-derived lymphocytes observed in our previous study 7 .…”

“…1 ). This partial reduction failed to account for the previously observed complete functional loss of SPINK1 7 , obliging us to look for other potential mechanisms.…”

“…To test the above hypothesis, we firstly re-performed the cell culture-based FLGEA for the previously constructed SPINK1 wild-type (WT) and Alu -Ins mutant (Mut) expression vectors 7 but this time in the presence of cycloheximide, a known NMD inhibitor. RT-PCR of mRNAs from HEK293T cells transfected with the WT construct yielded two bands ( Fig.…”

“…Alu elements continue to be amplified in the human genome through long interspersed element-1 (LINE-1 or L1)-mediated retrotransposition 2,3 , which is also an important cause of human genetic disease 3–5 . In part due to detection bias 6 , disease-causing Alu insertions have invariably been found to be located within the coding or proximal intronic regions of affected genes until very recently, when a full-length Alu insertion (henceforth termed SPINK1 Alu -Ins) was identified in the 3’-untranslated region (3’-UTR) of the SPINK1 gene (OMIM# 167790) in a patient presenting with a new pediatric disease entity, termed severe infantile isolated exocrine pancreatic insufficiency (SIIEPI) 7 . This new finding would not have come to light had the gene’s 3’-UTR not been included in the mutational screen and had the Alu -Ins variant not been present in the homozygous state.…”