Non-synonymous mutations affecting both alleles of PCSK1 (proprotein convertase 1/3) are associated with obesity and impaired prohormone processing. We report a proband who was compound heterozygous for a maternally inherited frameshift mutation and a paternally inherited 474kb deletion that encompasses PCSK1, representing a novel genetic mechanism underlying this phenotype. Although pro-vasopressin is not a known physiological substrate of PCSK1, the development of central diabetes insipidus in this proband suggests that PCSK1 deficiency can be associated with impaired osmoregulation.ISF and SOR were supported by the Wellcome Trust, the MRC Centre for Obesity and Related Disorders and the UK NIHR Cambridge Biomedical Research Centre.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S1096719213001145#

Bochukova, Elena

Candela, Ninfa

Farooqi, I Sadaf

Fox, Joyce

Frank, Graeme R

Jovanovic, Zorica

Levine, Jeremiah

O'Rahilly, Stephen

Papenhausen, Peter R

PubMed

Graeme R. Frank

Joyce Fox

Ninfa Candela

Zorica Jovanovic

Elena Bochukova

Jeremiah Levine

Peter R. Papenhausen

Stephen O'Rahilly

I. Sadaf Farooqi

Crossref

Molecular Genetics and Metabolism

Severe obesity and diabetes insipidus in a patient with PCSK1 deficiency

Frank, GR

Fox, J

Candela, N

Jovanovic, Z

Bochukova, E

Levine, J

Papenhausen, PR

O'Rahilly, S

Farooqi, IS

Queen Mary Research Online

Molecular Genetics and Metabolism 110 (2013) 191–194Contents lists available at ScienceDirectMolecular Genetics and Metabolismj ourna l homepage: www.e lsev ie r .com/ locate /ymgmeBrief CommunicationSevere obesity and diabetes insipidus in a patient with PCSK1 deficiencyGraeme R. Frank a, Joyce Fox b, Ninfa Candela c, Zorica Jovanovic d, Elena Bochukova d, Jeremiah Levine c,Peter R. Papenhausen e, Stephen O'Rahilly d, I. Sadaf Farooqi d,⁎a Cohen Children's Medical Center, Division of Pediatric Endocrinology, New Hyde Park, NY 11042, USAb Cohen Children's Medical Center, Division of Human Genetics, New Hyde Park, NY 11042, USAc Cohen Children's Medical Center, Division of Pediatric Gastroenterology, New Hyde Park, NY 11042, USAd University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge CB2 2QQ, UKe National Director, Cytogenetics, Laboratory Corporation of America, Research Triangle Park, NC 27709, USA⁎ Corresponding author.E-mail address: isf20@cam.ac.uk (I.S. Farooqi).1096-7192 © 2013 The Authors. Published by Elsevier Ihttp://dx.doi.org/10.1016/j.ymgme.2013.04.005a b s t r a c ta r t i c l e i n f oArticle history:Received 8 February 2013Received in revised form 3 April 2013Accepted 3 April 2013Available online 17 April 2013Keywords:ObesityProhormonesNon-synonymous mutations affecting both alleles of PCSK1 (proprotein convertase 1/3) are associated withobesity and impaired prohormone processing. We report a proband who was compound heterozygous fora maternally inherited frameshift mutation and a paternally inherited 474kb deletion that encompassesPCSK1, representing a novel genetic mechanism underlying this phenotype. Although pro-vasopressin isnot a known physiological substrate of PCSK1, the development of central diabetes insipidus in this probandsuggests that PCSK1 deficiency can be associated with impaired osmoregulation.© 2013 The Authors. Published by Elsevier Inc. Open access under CC BY-NC-SA license.1. IntroductionProprotein convertases (PCs) are a family of serine endoproteasesthat cleave inactive pro-peptides into biologically active peptides [1].Two family members, Proprotein Convertase Subtilisin/Kexin types 1and 2 (PCSK1 and PCSK2) are selectively expressed in neuroendocrinetissues where they cleave a broad but specific set of prohormonesincluding pro-opiomelanocortin (POMC), prothyrotrophin releasinghormone (TRH), proinsulin, proglucagon, and progonadotrophinreleasing hormone (GnRH) [2–9]. Congenital deficiency of PCSK1has previously been reported in three unrelated probands with severehyperproinsulinemia, malabsorptive diarrhea, hypogonadotropichypogonadism, partial central defects in the adrenal and thyroidaxes and severe obesity [10–12]. At least some of these phenotypescan be explained by the known or suggested involvement of PCSK1in the processing of proinsulin, proopiomelanocortin, proglucagon,proGnRH and proTRH [1]. We describe the fourth patient withPCSK1 deficiency whose phenotype, in addition to the above, includedcentral diabetes insipidus.2. Research design and methodsDirect nucleotide sequencing of the PCSK1 gene was carried outas previously reported [10]. SNP microarray analysis was performednc. Open access under CC BY-NC-SA licusing the Affymetrix 6.0 platform using 500 ng of total genomic DNA.Data was analyzed using Affymetrix Genotyping Console Browserv.3.01. Multiplex Ligation-independent Probe Amplification (MLPA)probes in the PCSK1 gene region (chr5:95751875-95774445) weredesigned following MRC-Holland (The Netherlands) recommendations(http://www.mlpa.com/) and sequences are available upon request.MLPA Hybridization, ligation and PCR were carried out using 200 ng ofgenomic DNA and the SALSA-MLPA kit (MRC-Holland, TheNetherlands),according to the manufacturer's instructions. The MLPA PCR products(1 μl) were mixed with 0.5 μl GeneScan™-500 ROX™ size standard(Applied Biosystems, UK) and 10 μl of HiDi formamide (AppliedBiosystems, UK) and separated on an ABI 3130 genetic analyzer (AppliedBiosystems, UK) and electrophoresis data extracted using GeneMappersoftware v4.0 (Applied Biosystems, UK).3. ResultsThe male proband weighed 3.8 kg at birth. Diarrhea began atthree days of life and persisted despite oral feeding with multipleformula changes. Endoscopy and flexible sigmoidoscopy revealeda grossly normal esophagus, stomach and duodenum, and scatteredulcers in the sigmoid colon. Pathology reported eosinophils presentin the duodenum, stomach and colon and eosinophilic cryptitisin the colon consistent with “allergic colitis”. Due to continueddiarrhea and failure to thrive, parenteral nutrition together withcontinuous nasogastric feeds of an amino-acid based formulawas instituted at 2 months of age. Watery diarrhea, however,continued. At 3 months the patient was admitted for sepsis associ-ated with his intravenous catheter. Within 2 h of central lineense.Fig. 1. A. Growth chart of proband demonstrating marked failure to thrive over first 2months; stabilization of weight gain from 2 months (with introduction of parenteralnutrition and an amino acid based formula) and excessive weight gain from 1 year.B. Family tree with chromatograms. Wildtype (Wt); deletion (del). C. MLPA confirma-tion of 474kb deletion of 5q15-q15 (95669703-96143955). Patient's MLPA traces arein red overlayed on the control MLPA traces in black. MLPA probes for genes in theregion of interest are detailed below. Two of the MLPA probes are positioned withinthe PCSK1 gene and appear to be deleted in the patient (arrows), and two are on eitherside of the deleted region on chromosome 5q15 (distal and proximal probes), and theyare identical to the normal control.192 G.R. Frank et al. / Molecular Genetics and Metabolism 110 (2013) 191–194removal, his blood glucose fell to 33 mg/dl, with a low serumcortisol of 11.60 μg/dl (normal > 18 μg/dl) and a normal growthhormone (13.8 ng/ml). Thyroid function was consistent withcentral hypothyroidism (TSH 1.60 μIU/ml) with free thyroxinelevels of 0.78 ng/dl (normal 0.9–1.8 ng/dl) and a low testosterone(28 ng/dl) and micropenis noted on clinical examination. In viewof these findings, he was diagnosed with partial hypopituitarismand started on hydrocortisone and thyroxine replacement therapy.Monthly testosterone injections for 3 months resulted in thenormalization of his penile size. Persistent polydipsia and polyuriawere noted in the third year of life. Following restriction offluid overnight, his serum Na was 145 mEq/l, Cl 107 mEq/l andosmolality was 304 mOsm/kg (urine specific gravity was 1.010and osmolality 386 mOsm/kg), confirming the diagnosis of diabe-tes insipidus. He was started on oral desmopressin which led toa marked improvement in symptoms and electrolyte abnormalities.From 12 months, rapid weight gain began despite an apparentaverage caloric intake of ~70 kcal/kg/day. Some solid foods wereintroduced for the first time at about 14 months, and this resulted inimproved stool consistency and less frequent diarrhea. By 24 monthsold, the childwas grossly obese (Fig. 1A). The amino-acid based formulawas discontinued at 3 years of age.In view of the combination of chronic diarrhea, rapid weightgain and selective hypopituitarism, the possibility of PCSK1 defi-ciency was entertained. A nonfasting plasma insulin was low(0.2 μU/ml) but proinsulin was >200 μU/ml. Direct nucleotidesequencing of PCSK1 revealed an apparently homozygous singlebase pair deletion which results in a frameshift after Pro 341, withthe introduction of 91 abnormal amino acids terminating in apremature stop codon (W342GfsX92). The mutation is locatedwithin a region that encodes the catalytic domain and thus wouldbe expected to abolish PCSK1 function. The proband's motherwas found to be heterozygous for the same mutation. However,no PCSK1mutation was found in his father. SNP microarray analysisrevealed a 474kb interstitial deletion of 5q15-q15 (95669703-96143955) which contains three genes, PCSK1, calpastatin andendoplasmic reticulum aminopeptidase 1. This deletion was con-firmed by Multiplex Ligation-independent Probe Amplification(MLPA) and was also found in the patient's father (Figs. 1B and C).Thus, the patient had inherited a large deletion that encompassedthe PCSK1 gene and also a frameshift mutation within the generesulting in the loss of both alleles and the phenotype of PCSK1deficiency. Common heterozygous variants in PCSK1 have beenassociated with late-onset obesity [13]. However, both parentsof this proband were of normal weight and had no evidence ofintestinal or endocrine abnormalities. While these findings do notpreclude a more subtle effect of some PCSK1 variants on bodyweight, the loss of both PCSK1 alleles appears to be necessary forthe complete phenotype.4. DiscussionWe describe the fourth patient with PCSK1 deficiency due to thecombination of a maternally inherited frameshift mutation leadingto a premature stop and a paternal deletion on chromosome 5encompassing PCSK1 and two adjacent genes. This patient differssignificantly from the three previous cases all of which involved ho-mozygosity or compound heterozygosity for missense or splice sitemutations [10–12]. In this case one allele was deleted and the otherdisrupted by a 91 amino acid insertion followed by a prematurestop codon occurring within the catalytic domain. While someresidual PCSK1 enzymatic activity could conceivably have persistedin the three previously reported cases it seems highly likely thatthis patient represents the null phenotype of PCSK1.The development of central diabetes insipidus in our patientsuggests that PCSK1 may be involved in the full functioning or centralsensing of osmolality in humans. Whether this is due to the failure ofprovasopressin processing or to some other malfunction of osmo-reception or vasopressin production or release is unknown. Vasopres-sin is synthesized in hypothalamic neurons expressing both PCSK1and PCSK2 [5]. In mice, both PCSK1 and PCSK2 are involved in theprocessing of pro-vasopressin to vasopressin, suggesting that a degreeof redundancy may exist in vivo [6,14,15]. In summary we report thefourth patient with PCSK1 deficiency. The finding of diabetes insipidusin this patient, and clinical features suggestive of this diagnosis in onepatient previously reported by us [12], suggest that this potentiallyclinically significant and treatable abnormality should be sought infuture patients presenting with this syndrome.Conflict of interestNone of the authors have a conflict of interest.AcknowledgmentsISF and SOR were supported by the Wellcome Trust, the MRCCentre for Obesity and Related Disorders and the UK NIHR CambridgeBiomedical Research Centre.Fig. 1 (continued).193G.R. Frank et al. / Molecular Genetics and Metabolism 110 (2013) 191–194References[1] N.G. Seidah, The proprotein convertases, 20 years later, Methods Mol. Biol. 768 (2011)23–57.[2] P. Schaner, R.B. Todd, N.G. Seidah, E.A. Nillni, Processing of prothyrotropin-releasinghormone by the family of prohormone convertases, J. Biol. Chem. 272 (1997)19958–19968.[3] D.Q. Barge-Schaapveld, S.M. Maas, A. Polstra, L.C. Knegt, R.C. 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English

Non-synonymous mutations affecting both alleles of PCSK1 (proprotein convertase 1/3) are associated with obesity and impaired prohormone processing. We report a proband who was compound heterozygous for a maternally inherited frameshift mutation and a paternally inherited 474kb deletion that encompasses PCSK1, representing a novel genetic mechanism underlying this phenotype. Although pro-vasopressin is not a known physiological substrate of PCSK1, the development of central diabetes insipidus in this proband suggests that PCSK1 deficiency can be associated with impaired osmoregulation

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Severe obesity and diabetes insipidus in a patient with PCSK1 deficiency.

http://dx.doi.org/10.1016/j.ymgme.2013.04.005

Severe obesity and diabetes insipidus in a patient with PCSK1 deficiency.

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