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. 2016 Dec 8;6(3):295-305.
doi: 10.1016/j.molmet.2016.12.002. eCollection 2017 Mar.

Functional and Clinical Relevance of Novel and Known PCSK1 Variants for Childhood Obesity and Glucose Metabolism

Free PMC article

Functional and Clinical Relevance of Novel and Known PCSK1 Variants for Childhood Obesity and Glucose Metabolism

Dennis Löffler et al. Mol Metab. .
Free PMC article


Objective: Variants in Proprotein Convertase Subtilisin/Kexin Type 1 (PCSK1) may be causative for obesity as suggested by monogenic cases and association studies. Here we assessed the functional relevance in experimental studies and the clinical relevance through detailed metabolic phenotyping of newly identified and known PCSK1 variants in children.

Results: In 52 obese children selected for elevated proinsulin levels and/or impaired glucose tolerance, we found eight known variants and two novel heterozygous variants (c.1095 + 1G > A and p.S24C) by sequencing the PCSK1 gene. Patients with the new variants presented with extreme obesity, impaired glucose tolerance, and PCOS. Functionally, c.1095 + 1G > A caused skipping of exon8 translation and a complete loss of enzymatic activity. The protein was retained within the endoplasmic reticulum (ER) causing ER stress. The p.S24C variant had no functional effect on protein size, cell trafficking, or enzymatic activity. The known variants rs6230, rs35753085, and rs725522 in the 5' end did not affect PCSK1 promoter activity. In clinical association studies in 1673 lean and obese children, we confirmed associations of rs6232 and rs6234 with BMI-SDS and of rs725522 with glucose stimulated insulin secretion and Matsuda index. We did not find the new variants in any other subjects.

Conclusions: We identified and functionally characterized two rare novel PCSK1 variants of which c.1095 + 1G > A caused complete loss of protein function. In addition to confirming rs6232 and rs6234 in PCSK1 as polygenic risk variants for childhood obesity, we describe an association of rs725522 with insulin metabolism. Our results support the contribution of PCSK1 variants to obesity predisposition in children.

Keywords: Children; Obesity; PC1/3; PCSK1; Prohormone convertase 1/3.


Figure 1
Figure 1
Association with obesity and metabolic factors. Carriers for rs6232 (A) and rs6234 (B) had higher BMI-SDS compared to wt. Carriers for rs725522 had higher (C) AUCIns/AUCBG and lower (D) Matsuda Insulin sensitivity indices (ISI) as compared to wt carriers. There were no significant differences in (E) proinsulin and (F) peak insulin levels. (G) Course of blood glucose and (H) insulin levels during oGTT in carriers of rs725522 wt (N = 473) and minor allele (N = 26). Data are given as mean ± SEM. AUC, area under the curve; wt, wild-type.
Figure 2
Figure 2
Functional characterization of the c.1095 + 1G > A mutation. The mutation c.1095 + 1G > A was named as ΔEx8. (A) Schematic representation of exon 8 skipping. Hek293 cells were transiently transfected with expression vectors encoding the Flag-tagged PC1/3 variants (wtcDNA; gwt; c.1095 + 1G > A (ΔEx8) cDNA; gc.1095 + 1G > A (ΔEx8)). (B) PCR products exons 1 to exon 14 showing different sized amplicons for gwt and c.1095 + 1G > A (ΔEx8) variants. EV, empty vector. (C) Immunoblot with antibodies to the Flag epitope showing protein in the cell lysates but not in the supernatants of c.1095 + 1G > A (ΔEx8) variants. (D) Confocal microscopy of CHO cells transfected with Flag-tagged PC1/3 variants wtcDNA (wild-type) and c.1095 + 1G > A cDNA (ΔEx8) as indicated and with dsRedER plasmids shows assembly of PC1/3 in the ER in wild-type and perinuclear localization (which was inhibited by Brefeldin/Monensin (B/M)) treatment. The c.1095 + 1G > A (ΔEx8) variant was detectable in the ER without assembly in perinuclear vesicles. (E) Enzymatic activity was completely abolished in Hek293 cells expressing the ΔEx8 variants compared to wildtype protein.
Figure 3
Figure 3
Functional characterization of the p.S24C mutation. Immunoblot (A + B) with antibodies to the Flag epitope showing protein in the cell lysates and in the supernatants of wild-type (WT) and mutated variant. After Brefeldin/Monensin (B/M) treatment, proteins were retained in the cell. EV, empty vector. (C) Confocal microscopy of CHO cells transfected with Flag-tagged PC1/3 variants wtcDNA (wild-type) and p.S24CcDNA (p.S24C) as indicated and with dsRedER plasmids shows equally subcellular assembly of PC1/3 wt and variants in the ER and perinuclear localization (which was inhibited by Brefeldin/Monensin (B/M) treatment). (D) Enzymatic activity of PC1/3 was retained in p.S24C variant similar to the wild-type.
Figure 4
Figure 4
Endoplasmic reticulum stress. (A) Influence of PCSK1 variants on expression/maturation and release of PC1/3 wild-type (WT) and different variants as indicated by immunoblot analysis (EV = empty vector). (B) UPR activation was assessed via PCR for the IRE1 (XBP1) and ATF (BiP and CHOP) pathway (BFA = BrefeldinA [1 μg/ml]). (C) For analysis of PERK pathway activation, phosphorylated (PeIF2α) and unphosphorylated eIF2α was analyzed by western blotting. (D) Apoptosis was assessed by FACS identification of Annexin-V/Propidium Iodide stained cells. Results were normalized to the apoptosis rate of the wild-type transfected cells (C/E = camptothecin [2 μM]/etoposide [85 μM]).

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