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Case Reports
. 2013 Dec;57(6):759-67.
doi: 10.1097/MPG.0b013e3182a8ae6c.

Exome Sequencing Finds a Novel PCSK1 Mutation in a Child With Generalized Malabsorptive Diarrhea and Diabetes Insipidus

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Free PMC article
Case Reports

Exome Sequencing Finds a Novel PCSK1 Mutation in a Child With Generalized Malabsorptive Diarrhea and Diabetes Insipidus

Michael Yourshaw et al. J Pediatr Gastroenterol Nutr. .
Free PMC article

Abstract

Objectives: Congenital diarrhea disorders are a group of genetically diverse and typically autosomal recessive disorders that have yet to be well characterized phenotypically or molecularly. Diagnostic assessments are generally limited to nutritional challenges and histologic evaluation, and many subjects eventually require a prolonged course of intravenous nutrition. Here we describe next-generation sequencing techniques to investigate a child with perplexing congenital malabsorptive diarrhea and other presumably unrelated clinical problems; this method provides an alternative approach to molecular diagnosis.

Methods: We screened the diploid genome of an affected individual, using exome sequencing, for uncommon variants that have observed protein-coding consequences. We assessed the functional activity of the mutant protein, as well as its lack of expression using immunohistochemistry.

Results: Among several rare variants detected was a homozygous nonsense mutation in the catalytic domain of the proprotein convertase subtilisin/kexin type 1 gene. The mutation abolishes prohormone convertase 1/3 endoprotease activity as well as expression in the intestine. These primary genetic findings prompted a careful endocrine reevaluation of the child at 4.5 years of age, and multiple significant problems were subsequently identified consistent with the known phenotypic consequences of proprotein convertase subtilisin/kexin type 1 (PCSK1) gene mutations. Based on the molecular diagnosis, alternate medical and dietary management was implemented for diabetes insipidus, polyphagia, and micropenis.

Conclusions: Whole-exome sequencing provides a powerful diagnostic tool to clinicians managing rare genetic disorders with multiple perplexing clinical manifestations.

Conflict of interest statement

The authors report no conflicts of interest.

Figures

Figure 1
Figure 1
Exome sequencing results. A, Aligned pileup of sequenced fragments at chr5:95746544 with 30 forward strand reads and 41 reverse strand reads of the C variant and 2 low-quality reads of the reference G allele on fragment 3′ ends. Visualized by the Integrative Genomics Viewer (39). B, Sanger sequencing validation of variant. C, Structure of PC1/3 showing locations of previously identified mutations and the novel Y343X. Adapted from (38) and (40).
Figure 2
Figure 2
Functional characterization and visualization of wild-type (WT) prohormone convertase 1/3 (PC1/3) complementary DNA and PC1/3 containing the Y343X nonsense mutation. HEK293 cells were transiently transfected with empty pcDNA3 (not shown), WT PC1/3, and Y343X PC1/3. A, Enzymatic activity of secreted recombinant PC1/3 proteins in conditioned medium was compared by measuring maximum cleavage rates using the fluorogenic substrate pyr-RTKR-amc during a 1-hour kinetic assay. Three replicates per condition were assayed in triplicate and are shown as the mean ± standard deviation, P<0.0017 (2-tailed). B, Western blot of cell lysates and media from transfected HEK293 cells, using amino terminal–directed PC1/3 primary antiserum for detection of recombinant PC1/3 proteins. The data shown represent 1 of 3 independent experiments. β-actin shows equivalent loading of cell extracts.
Figure 3
Figure 3
Absence of prohormone convertase 1/3 (PC1/3)-positive enteroendocrine cells in small bowel mucosa. A, Hematoxylin and eosin (H&E)–stained proprotein convertase subtilisin/kexin type 1 mutant showing normal morphology; original magnification ×200. B, Chromogranin A immunohistochemistry of mutant tissue showing a normal pattern of enteroendocrine cells; original magnification ×400. C, PC1/3 immunohistochemistry showing complete absence of PC1/3-positive enteroendocrine cells in the mutant tissue; original magnification ×400. D, PC1/3 immunohistochemistry showing normal PC1/3 enteroendocrine cells in a healthy control; original magnification ×400.

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