DGAT1 mutation is linked to a congenital diarrheal disorder

Abstract

Congenital diarrheal disorders (CDDs) are a collection of rare, heterogeneous enteropathies with early onset and often severe outcomes. Here, we report a family of Ashkenazi Jewish descent, with 2 out of 3 children affected by CDD. Both affected children presented 3 days after birth with severe, intractable diarrhea. One child died from complications at age 17 months. The second child showed marked improvement, with resolution of most symptoms at 10 to 12 months of age. Using exome sequencing, we identified a rare splice site mutation in the DGAT1 gene and found that both affected children were homozygous carriers. Molecular analysis of the mutant allele indicated a total loss of function, with no detectable DGAT1 protein or activity produced. The precise cause of diarrhea is unknown, but we speculate that it relates to abnormal fat absorption and buildup of DGAT substrates in the intestinal mucosa. Our results identify DGAT1 loss-of-function mutations as a rare cause of CDDs. These findings prompt concern for DGAT1 inhibition in humans, which is being assessed for treating metabolic and other diseases.

Figure 1. Mutation in DGAT1 segregates with CDD.

(A) Pedigree of the affected family, indicating diarrheal phenotype and DGAT1 genotype. T, wild type; C, mutant. (B) RFLP assay of genomic DNA confirming mutation presence. PCR product digested with Fnu4HI yields 165 and 32 bp for reference and 122, 43, and 32 bp for mutant allele. Numbers refer to the family members indicated in A. (C) Predicted splicing patterns for wild-type and mutant alleles. (D) RT-PCR analysis of mRNA from blood of the proband, unaffected sibling, and parents. Numbers refer to exons. The asterisk indicates the mutated nucleotide (“T” for wild type and “C” for the mutant allele). Mutant DGAT1 allele yields Δ8 mRNA. Numbers refer to the family members indicated in A. (E) DGAT1 protein diagram showing predicted transmembrane domains (gray), putative catalytic residues (*), and Δ8 region (Δ, diagonal lines).

Figure 2. Δ8 mutant allele yields an unstable protein, resulting in loss of DGAT1 activity.

DGAT1 cDNAs were expressed in DGAT1-KO or DGAT1,2-KO MEFs. (A) DGAT1 activity (accumulation of [¹⁴C]-TG) was absent in lysates from Δ8-expressing cells, despite the presence of mRNA. All lysates synthesized [¹⁴C]-diacylglycerol ([¹⁴C]-DAG) (internal control). (B) Intact Δ8-expressing cells do not accumulate TG, as measured by thin-layer chromatography in DGAT1,2-KO cell lines after incubation with oleic acid (200 mM). Free cholesterol (FC) was used as a loading control. (C) Immunoblot showing that proteasome inhibition (MG132) rescues Δ8 protein expression in DGAT1,2-KO MEFs. (D) MG132-treated DGAT1,2-KO MEFs expressing Δ8 do not accumulate [¹⁴C]-TG after a 2-hour treatment with 200 mM [¹⁴C]-oleic acid. (E) qPCR showing that humans lack DGAT2 expression in the small intestine. Act., activity; Duo., duodenum; Jeju., jejunum; Ile., ileum.