Research Article Mutations in pregnancy-associated plasma protein A2 cause short stature due to low IGF-I availability Andrew Dauber1, María T Muñoz-Calvo2,3, Vicente Barrios2,3, Horacio M Domené4, Soren Kloverpris5, Clara Serra-Juhé6, Vardhini Desikan7, Jesús Pozo2,3, Radhika Muzumdar8, Gabriel Á Martos-Moreno2,3, Federico Hawkins9, Héctor G Jasper4, Cheryl A Conover10, Jan Frystyk11,12, Shoshana Yakar13, Vivian Hwa1, Julie A Chowen2,3, Claus Oxvig5, Ron G Rosenfeld14,15, Luis A Pérez-Jurado6 & Jesús Argente2,3,* Abstract DOI 10.15252/emmm.201506106 | Received 27 November 2015 | Revised 18 January 2016 | Accepted 25 January 2016 | Published online 22 February 2016 Mutations in multiple genes of the growth hormone/IGF-I axis EMBO Mol Med (2016) 8: 363–374 have been identified in syndromes marked by growth failure. However, no pathogenic human mutations have been reported in the six high-affinity IGF-binding proteins (IGFBPs) or their regula- Introduction tors, such as the metalloproteinase pregnancy-associated plasma protein A2 (PAPP-A2) that is hypothesized to increase IGF-I bioac- The growth hormone (GH)/insulin-like growth factor (IGF)-I system tivity by specific proteolytic cleavage of IGFBP-3 and -5. Multiple is essential for optimal human growth. GH promotes growth via members of two unrelated families presented with progressive IGF-I production and by direct actions on the growth plate. IGF-I growth failure, moderate microcephaly, thin long bones, mildly circulates bound to six IGF-binding proteins (IGFBPs). After binding decreased bone density and elevated circulating total IGF-I, IGFBP-3, IGF, IGFBP-3, and -5 also bind with the IGF acid labile subunit and -5, acid labile subunit, and IGF-II concentrations. Two different (ALS) to form a ternary complex, which further increases IGF-I’s homozygous mutations in PAPPA2, p.D643fs25* and p.Ala1033Val, half-life (Baxter, 2000). Free IGF-I (fIGF-I) binds its receptor, activat- were associated with this novel syndrome of growth failure. In ing signaling cascades that up-regulate multiple genes fundamental vitro analysis of IGFBP cleavage demonstrated that both mutations to growth (Yakar et al, 2002). Human genetic defects in this axis cause a complete absence of PAPP-A2 proteolytic activity. Size- lead to syndromes marked by impaired growth and have helped to exclusion chromatography showed a significant increase in IGF-I further our understanding of growth physiology (David et al, 2011). bound in its ternary complex. Free IGF-I concentrations were GH receptor (GHR) mutations were shown to cause Laron syndrome decreased. These patients provide important insights into the with extreme growth failure (Amselem et al, 1989). Mutations in regulation of longitudinal growth in humans, documenting the the STAT5B (Kofoed et al, 2003), IGF-I (Woods et al, 1996) and critical role of PAPP-A2 in releasing IGF-I from its BPs. IGF-I receptor (IGF-IR) (Abuzzahab et al, 2003) genes cause varying degrees of pre- and postnatal growth retardation and mutations in Keywords bone; delayed growth; growth hormone; IGF-binding proteins; ALS cause mild short stature (Domene et al, 2004). The first IGF bioavailability mutation in IGF2 affecting pre- and postnatal growth was recently Subject Categories Genetics, Gene Therapy & Genetic Disease identified (Begemann et al, 2015). To date, no human mutations in 1 Cincinnati Center for Growth Disorders, Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA 2 Department of Pediatrics & Pediatric Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación La Princesa, Universidad Autónoma de Madrid, Madrid, Spain 3 Program of Pediatric Obesity, CIBEROBN, Instituto de Salud Carlos III, Madrid, Spain 4 Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE), CONICET, FEI, División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina 5 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark 6 Genetics Unit, Universitat Pompeu Fabra, Hospital del Mar Research Institute (IMIM) & CIBERER. Instituto de Salud Carlos III, Barcelona, Spain 7 Department of Pediatrics, Division of Pediatric Endocrinology, New York Medical College, Valhalla, NY, USA 8 Division of Endocrinology, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA 9 Department of Endocrinology, Hospital Universitario 12 de Octubre, Universidad Complutense de Madrid, Madrid, Spain 10 Division of Endocrinology, Mayo Clinic, Rochester, MN, USA 11 Medical Research Laboratory, Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus, Denmark 12 Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark 13 Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, NY, USA 14 Oregon Health and Science University, Portland, OR, USA 15 STAT5 LLC, Los Altos, CA, USA *Corresponding author. Tel: +34 91 5035912; Fax: +34 91 5035939; E-mail: jesus.argente@uam.es ª 2016 The Authors. Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine Vol 8 | No 4 | 2016 363 EMBO Molecular Medicine PAPPA2 mutations and short stature Andrew Dauber et al the six high-affinity IGFBPs or their regulatory proteins have been 37 ng/ml, respectively. Fasting glucose concentrations were normal reported in association with a monogenic syndrome. We present in both brothers, with mild hyperinsulinemia in Subject II.4 two families with homozygous loss-of-function mutations in (17.1 lU/ml) and a normal insulin level of 7 lU/ml in Subject II.5. PAPPA2, a gene encoding pregnancy-associated plasma protein-A2 Subjects II.3 and II.4 were born mildly small for gestational age, but (PAPP-A2), a protease highly specific for IGFBP-3 and -5 (Overgaard Subject II.5 was within the normal range (Table 1). All three et al, 2001), resulting in a novel syndrome of growth retardation affected siblings had long thin fingers, a small chin, moderate micro- with markedly elevated circulating IGF-I and IGF-II, but decreased cephaly (Table 1) and delayed dental eruption. fIGF-I concentrations and IGF bioactivity. Skeletal surveys in the two affected siblings from family one and subjects II.4 and II.5 in family two showed no signs of overt skeletal dysplasia; however, thin long bones most prominent in the fibulae, Results tibiae, and femurs were found (Fig 1F). Bone age was consistent with chronologic age in all subjects. Patient descriptions DXA scans (QDR 4500, Hologic, Waltham, MA) were performed on the two affected siblings from family one. Bone mineral density Family one consists of four children of Spanish ancestry born to was decreased at the lumbar spine (height adjusted Z-score 2.49 parents with no known consanguinity. Subject II.1 was evaluated at SDS in II.1, 2.0 SDS in II.3). nine years of age for short stature with a height 1.7 SDS below her Micro-CT analysis of a tooth extracted from Subject II.1 in family mid-parental target height (50th–75th percentile) and elevated serum one showed significantly decreased enamel and dentin density IGF-I, ALS, and IGFBP-3 concentrations (Table 1). Spontaneous GH (Fig EV1). No significant cognitive dysfunction or hearing abnor- secretion over eight hours (daytime) was elevated (4.8 ng/ml/8 h; malities were found in the patients from either family. normal 2.8–3.3). Her younger brother’s height (Subject ll.3) was 1.3 SDS below mid-parental target height, with similar elevations in Genetic analysis IGF-I, IGFBP-3, and ALS (Table 1) and spontaneous GH secretion (5.3 ng/ml/8 h). Their birth lengths and weights were normal, Given the two affected siblings of different sex in family one with although smaller than their unaffected sisters (II.2 and II.4; unaffected parents, we hypothesized that the disorder had an auto- Table 1). Subject II.3 was born premature, but was adequate for somal recessive inheritance pattern. After excluding mutations in gestational age, with a normal catch-up growth (Fig 1B). Their the IGF-IR gene, whole-exome sequencing was performed in the growth patterns revealed consistent short stature relative to target affected sister (II.1). We searched for genes with either a single height, with more prominent growth deceleration as they age (Fig 1, homozygous or two heterozygous rare (minor allele frequency Appendix Fig S1). Subtle dysmorphic features consist of small chins, < 1%) nonsynonymous variants present. Five genes met these mild microcephaly (1.37 & 1.65 SDS at birth; 2.03 & 2.19 SDS criteria (Appendix Table S2), four of which fell within a block of at first physical examination), and long fingers and toes (Fig 1A homozygosity on chromosome one, suggesting that the parents have and B). They are prepubertal. Glycemia was normal on oral glucose a distant common ancestor. The novel homozygous frameshift tolerance tests, but with mild fasting hyperinsulinemia (15.1 and mutation in PAPPA2 (c.1927_1928insAT, p.D643fs25*) was high- 27 lU/ml, normal 4–13). The unaffected sisters were within 1 SDS lighted as the likely causal variant, given the loss-of-function nature of their mid-parental target height with normal IGF-I serum concen- of the mutation and the role of the encoded protein, PAPP-A2, in trations (Table 1, Appendix Fig S2). cleaving IGFBPs. This variant was confirmed via Sanger sequencing Family two consists of five children of Palestinian ancestry born to be homozygous in both affected individuals, heterozygous in to parents of normal stature (mid-parental target height 30th percen- both parents and one unaffected sibling (II.4), and absent in the tile) who are first cousins. Siblings II.3, II.4, and II.5 had significant second unaffected sibling (II.2) (Fig 2). postnatal growth retardation with high serum IGF-I and IGFBP-3 Due to the known consanguinity in family two, we assumed that concentrations. When Subject II.3 was evaluated for short stature at the affected children were homozygous carriers of a rare autosomal age 6.8 years of age, her height was 3.5 SDS below her target recessive variant causal for their growth retardation. Genome-wide height and IGF-I was 517 ng/ml (normal 47–217). An elevated genotyping performed in the three affected children identified IGFBP-3 level of 6,400 ng/ml (normal 2,100–4,200) was found later. four large (> 6 MB) shared regions of homozygosity (Appendix She showed peak GH concentrations > 50 ng/ml on both clonidine Table S3). Whole-exome sequencing was performed on Patient II.3. and arginine tests. Her growth chart demonstrates progressive Analysis focused on rare homozygous nonsynonymous variants growth failure, no discernible pubertal growth spurt and a final within the shared regions of homozygosity. Two novel missense adult height of 138.4 cm (3.81 SDS; Fig 1C). She had normal variants met these criteria, only one of which segregated in the pubertal timing, undergoing menarche at age 13.8 years of age, and family (Fig 2): a novel missense variant in PAPPA2 (c.3098C > T, had elevated IGF-I throughout childhood (Appendix Table S1; most p.Ala1033Val). recent value 1,060 ng/ml at age 18 years). Two younger brothers Neither of the two PAPPA2 variants was found in public data- also had progressive growth failure (Fig 1D and E). Subject II.4 was bases representing more than 60,000 exomes and including at least evaluated for growth failure at age 12.5 years of age with an IGF-I 700 individuals from Spain. Notably, there are no individuals in level of 657 ng/ml (normal 93–567) despite being prepubertal. these large datasets with homozygous loss-of-function variants of Subject II.5 was evaluated at 8.3 years of age with an IGF-I level the PAPPA2 gene. Thus, the genetic analyses of these two families of 636 ng/ml (normal 49–351). Their peak GH concentrations identified two novel nonsynonymous mutations in PAPPA2 as the after stimulation testing with clonidine and L-Dopa were 14.7 and likely causal variants. 364 EMBO Molecular Medicine Vol 8 | No 4 | 2016 ª 2016 The Authors Andrew Dauber et al PAPPA2 mutations and short stature EMBO Molecular Medicine Table 1. Anthropometric and biochemical data. Gestational Birth weight Birth length Age at examination Subject ID Sex age (weeks) (kg) (cm) (years) Height (cm) BMI (kg/m2) HC (SDS) Family 1 II.1 F 37 2.19 (1.49) 48.0 (0.07) 10.3 132.2 (1.10) 13.9 (1.52) 2.03 II.2 F 39 3.35 (0.44) 51.0 (0.94) 7.8 129 (0.87) 14.8 (0.98) +0.02 II.3 M 35 1.99 (1.27) 45.0 (0.55) 5.7 110.4 (0.96) 13.4 (2.03) 2.19 II.4 F 40 3.64 (0.96) 50.0 (0.11) 3.6 103.3 (1.62) 15.6 (0.44) 0.98 Family 2 II.1 F 40 3.26 (0.61) 48.3 (1.30) 22.2 149.3 (2.16) 24.9 (0.76) 0.45 II.2 F 40 3.57 (0.01) 48.3 (1.30) 19.8 153.9 (1.45) 21.7 (0.01) 1.06 II.3 F 41 2.58 (2.16) 45.7 (2.75) 18.6 138.4 (381) 20.9 (019) 2.03 II.4a M 41 2.84 (1.69) 47.0 (2.18) 13.8 139.1 (2.82) 26.7 (1.61) 1.60 II.5 M 39.4 2.82 (1.28) 48.5 (0.94) 9.5 117.7 (3.14) 15.4 (0.65) 1.08 Total IGF-I Total IGF-II Bioactive Free IGF-I BioIGF/Total IGFBP-3 IGFBP-5 ALS PAPP-A2 (lg/l) (lg/l) IGF (lg/l) (lg/l) IGF-I (%) (lg/l) (lg/l) (U/L) (lg/l) Family 1 II.1 957 1,198 0.87 1.39 0.09 5,912 997 3,745 NDb II.2 340 850 2.30 2.08 0.68 3,696 457 2,588 0.13 II.3 882 1,264 1.54 0.27 0.17 4,850 853 3,625 NDb II.4 264 759 3.21 2.16 1.22 3,384 322 2,206 0.25 Family 2 II.1 237 763 2.31 4.34 0.97 3,235 179 1,375 0.34 II.2 490 629 3.90 8.36 0.80 3,007 187 1,412 0.44 II.3a 1,060 921 3.35 1.98 0.32 4,557 760 2,445 0.23 II.4c 935 914 2.70 3.31 0.29 4,792 981 2,959 0.42 II.5 831 946 0.98 0.35 0.12 4,403 645 2,456 0.30 Normal Values Tanner I 91–225 495–911 d Prepubertal 1.58–3.15 2,206–4,200 211–707 753–2,634 0.16–2.69 1.94  0.15 e median 1.23% Tanner V 270–617 560–794 d Pubertal 4.89–9.37 (range 0.46–2.59%) 2,796–5,280 293–1,023 1,260–4,030 0.23–0.80 2.93  0.15 Affected individuals are shaded in light gray. Unaffected individuals are not shaded. Values in parentheses indicate the standard deviation score (SDS) for anthropometric measures. a This subject is an adult. b Zero values in the affected patients are expected because of the frameshift mutation resulting in the absence of the target epitopes of the assay. HC: head circumference. BioIGF: Biologically active IGF. c This subject was Tanner stage 2 at the time of examination and biochemical evaluation with 5-6 cc testes. ND: not detectable. d Normal ranges for bioactive IGF are based on Sorensen et al (2015). e The normal ratio of bioactive IGF to total IGF-I is based on 95 healthy subjects aged 7–25 years. The ratio was not associated with age. In vitro functional effects of mutations in PAPPA2 none of the variants cleaved the PAPP-A substrate, IGFBP-4. Western blotting showed a complete lack of expression for the PAPP-A2 is a metalloproteinase that specifically cleaves IGFBP-3 D643fs mutant (as would be expected with a severely truncated and -5 (Overgaard et al, 2001). To assess the consequences of protein), while expression of A1033V was markedly reduced our subjects’ mutations on PAPP-A2 proteolytic function, recom- compared to WT PAPP-A2. Upon reduction of disulfide bonds, binant variants were compared with wild-type (WT) PAPP-A2 two bands appeared for A1033V, probably caused by partial following transfection of HEK 293 cells. Media from cells trans- intramolecular cleavage (Fig 3B). To further analyze the A1033V fected with cDNA encoding the D643fs or A1033V variants mutant, samples were adjusted for the reduced expression of this showed no proteolytic activity toward IGFBP-3 or -5 under variant and, at equimolar concentrations (Fig 3C), it still showed conditions where media from cells transfected with WT PAPP-A2 no activity, suggesting that neither full-length A1033V nor proteo- cDNA completely cleaved these substrates (Fig 3A). As expected, lytically cleaved A1033V is capable of cleaving the IGFBPs. ª 2016 The Authors EMBO Molecular Medicine Vol 8 | No 4 | 2016 365 EMBO Molecular Medicine PAPPA2 mutations and short stature Andrew Dauber et al A B C D E F Figure 1. Growth charts and photographs of affected subjects. A Family 1, Subject II.1. B Family 1, Subject II.3. C Family 2, Subject II.3. D Family 2, Subject II.4. E Family 2, Subject II.5. F Top Row—Representative X-rays showing narrow fibulae and elongated digits in Subject II.1 of Family 1. Bottom Row—Lower extremities and foot from Subject II.5 and hand from Subject II.4, both from Family 2. In vivo evaluation of IGF-I and IGFBPs range in the four younger patients and in the upper normal range in the Tanner V subject of family two. ALS concentrations were To further delineate the in vivo consequences of our subjects’ muta- elevated in three of five subjects and in the upper limits of normal tions on IGF-I physiology, we measured serum PAPP-A2 concentra- in two. Serum IGFBP-5 concentrations were elevated in four of five tions. As expected, subjects in family one homozygous for the patients. IGF-II concentrations were elevated in all five patients D463fs truncating mutation had no detectable PAPP-A2 (Table 1). (Table 1). As PAPP-A2 cleaves IGFBP-3 and -5 thereby releasing In family two, PAPP-A2 concentrations were detectable at the low IGF-I from the ternary complex, we hypothesized that fIGF-I concen- end of the normal range, but with no difference between the affected trations would be decreased in the affected subjects, despite the individuals and their heterozygous siblings. elevated total IGF-I concentrations. Indeed, fIGF-I concentrations Serum concentrations of total IGF-I were elevated above the were low in four of five patients (Table 1), suggesting that they normal range in all affected subjects and normal in their unaffected have a functional defect in the ability to liberate IGF-I from its bind- siblings (Table 1). IGFBP-3 concentrations were above the normal ing partners. To substantiate this finding by an independent 366 EMBO Molecular Medicine Vol 8 | No 4 | 2016 ª 2016 The Authors Andrew Dauber et al PAPPA2 mutations and short stature EMBO Molecular Medicine A * * mRNA ATG c.1927_1928insAT c.3098C>T TAA * * 1 2 3 4 5 67 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Prot p.D643fs25X p.A1033V * * Signal Laminin_G_3 Notch Fibronectin_t3 Sushi Notch Peptidase_M43 (wt) (wt) B Fam1 642 G D C C C Fam2 1032D A A L I 1 2 G G A G A C TG C TG C I 1 2 II 1 2 3 4 II 1 2 3 4 5 I1: p.D643fs (het) II1: p.D643fs (hom) I1: p.A1033V (het) II3: p.A1033V (hom) 642 G D fsT/C A/C 642 G D fs T A 1032D 1032D A L A/V A L V G G A G A T A C TG C T G G A G A T A C TG C T C TG C T G C Figure 2. PAPPA2 gene and family pedigrees. A Schematic representation of the genomic structure of the PAPPA2 gene on chromosome band 1q24, the encoded mRNA and the protein with its relevant functional domains. Red stars indicate the location in the exons, mRNA and protein where the subjects’ mutations are found. B Pedigree and representative Sanger sequencing trace of the mutation identified in family 1. C Pedigree and representative Sanger sequencing trace of the mutation identified in family 2. method, the potential of the patients’ serum to stimulate the IGF-IR Discussion in vitro was assessed. IGF bioactivity was lower in the three prepu- bertal patients compared to unaffected prepubertal subjects, with We present two families with a novel syndrome of progressive the percentage of bioactive IGF (bioactive IGF/total IGF-I) reduced growth failure with markedly elevated serum concentrations of in all affected patients (Table 1). IGF-I, IGFBP-3 and -5 resulting from loss of PAPP-A2 function. To further confirm that defects in PAPP-A2 function can cause PAPP-A2, a member of the pappalysin family of metzincin metallo- this unique phenotype, we demonstrated that mice carrying a proteinases, is closely related to PAPP-A, a protease that targets targeted deletion of Pappa2 (Conover et al, 2011) also have mark- IGFBP-4 (Oxvig, 2015). While PAPP-A has been extensively studied edly decreased fIGF-I concentrations, despite a > 50% increase in due to its biomarker roles (Bayes-Genis et al, 2001; Malone et al, total IGF-I concentrations (Appendix Table S4). 2005), the function of PAPP-A2 in human physiology is largely Lastly, measurement of the amount of IGF-I bound in ternary unknown. Prior in vitro work has shown that PAPP-A2 specifically complexes in the serum of subjects of family one showed a marked cleaves IGFBP-3 and -5 with no activity toward other IGFBPs increase in IGF-I as part of the ternary complex in all affected (Overgaard et al, 2001). We demonstrate that loss of PAPP-A2 function subjects (Fig 4A). Moreover, in these patients, the overall capacity leads to increased serum concentrations of IGFBP-3 and -5 resulting to form ternary complexes was greatly increased compared to in increased total IGF-I concentrations. Total IGF-II is also elevated, prepubertal controls (Fig EV2A and B). consistent with the increased ALS and IGFBP-3 concentrations. ª 2016 The Authors EMBO Molecular Medicine Vol 8 | No 4 | 2016 367 EMBO Molecular Medicine PAPPA2 mutations and short stature Andrew Dauber et al A Figure 3. In vitro characterization of mutated PAPP-A2. A Assessment of proteolytic activity present in culture media of human embryonic kidney 293T cells transfected with cDNA encoding PAPP-A2, PAPP-A2(D643fs) (family 1), or PAPP-A2(A1033V) (family 2). Radiolabeled substrates tested include IGFBP-3, IGFBP-4, and IGFBP-5. Cleavage was visualized by autoradiography following SDS–PAGE. Positions of intact IGFBPs (i) and cleavage products (c) are indicated. B Western blots following nonreducing or reducing SDS–PAGE of media from transfected cells, as indicated. Polyclonal antibodies raised against the N-terminal portion of PAPP-A2 were used for detection. C Comparison of proteolytic activity similar to the experiment of panel (A), except that equimolar concentrations of PAPP-A2 and PAPP-A2(A1033V) were used. The variant from Family 1, carrying a frameshift mutation N-terminal to the proteolytic domain of PAPP-A2, did not show any detectable expression. All gels and blots are representative of three independent experiments. to an inability of the abnormal or deficient PAPP-A2 to liberate IGF-I from its binding partners resulting in decreased fIGF-I. The primary B site of action for PAPP-A2 is unknown. Unlike PAPP-A, PAPP-A2 is not membrane bound and does not require the presence of IGF-I for its proteolytic function (Overgaard et al, 2001). We do not know whether the growth failure is due to a systemic effect or a defect in PAPP-A2 function at the tissue level, in particular at the growth plate. It is also possible that aspects of the patients’ phenotype could be due to direct effects of the increased IGFBPs. Interestingly, GH is elevated in the patients, resulting in increased concentrations of all GH-dependent factors (IGF-I, IGFBP-3, IGFBP-5, and ALS). We hypothesize that the rise in GH is due to decreased negative feed- back consequent to low fIGF-I concentrations (Fig 4B). Thus, our study indicates that PAPP-A2 is a key regulator of human growth and IGF-I bioavailability by regulating the proportion of IGF-I that is either free or bound to its IGFBPs. Mouse models of PAPP-A2 deficiency have striking similarities to our patients, as Pappa2/ mice recapitulate the human biochemi- cal phenotype with elevated total IGF-I and low fIGF-I concentra- tions. Pappa2/ mice exhibit a trend toward decreased birth weight and significant postnatal growth retardation (Conover et al, C 2011; Christians et al, 2013), which correlates with our findings as the patients were born mildly small for gestational age and exhibited the most prominent growth failure postnatally. Indeed, progressive postnatal growth retardation occurred in all affected individuals, becoming more prominent with age. While the range in the absolute heights of the affected patients is wide, subjects in family one are quite discordant from their target mid-parental height and it is yet to be seen if their growth failure becomes progressively worse with age, as seen in family two. PAPP-A2 is responsible for the proteoly- sis of IGFBP-5 during pregnancy resulting in increased fIGF-I (Yan et al, 2010), but how this affects fetal growth and development in normal individuals and in our subjects is unknown. Lack of PAPP- A2 led to decreased length of the mandible, skull, femur, pelvic girdle, and tailbone in mice (Christians et al, 2013) and severely reduced cranial cartilages in zebrafish (Kjaer-Sorensen et al, 2014). Our subjects had small chins, long thin fingers, thin femurs and moderate microcephaly. In the two subjects undergoing DXA, one showed mild osteoporosis and the other osteopenia. Mice However, this increase in total IGFs was not accompanied by an with blunted IGF-I activity in osteoblasts show defects in bone elevated ability of serum to activate IGF-IR in vitro (IGF bioactivity). mineralization during postnatal growth (Zhang et al, 2002). Trans- We hypothesize that this relative decrease in IGF bioactivity is due genic mice overexpressing IGFBP-3 (Modric et al, 2001) have 368 EMBO Molecular Medicine Vol 8 | No 4 | 2016 ª 2016 The Authors Andrew Dauber et al PAPPA2 mutations and short stature EMBO Molecular Medicine A II.1 II.2 II.3 II.4 Prepubertal control B GH ↑↑ Feedback ↓ Binding to IGF-I ↑↑↑ IGF receptor ↓ IGF-II ↑↑↑ No PAPP-A2 IGF-I activity IGFBP-3 ↑↑↑ ALS ↑↑↑ X fIGF-I ↓↓↓ GH IGFBP-5 ↑↑↑ Ternary complex↑↑ Figure 4. IGF-I neutral chromatography and proposed mechanism of action of PAPP-A2 deficiency. A Concentrations of endogenous IGF-I in the different fractions obtained by neutral chromatography on a Superdex 200 column. Serum samples from the four siblings of family 1 were analyzed and compared to a nonrelated prepubertal control, as all siblings were prepubertal. The amount of IGF-I detected in the fractions corresponding to the ternary complex (TC) was markedly greater in the homozygous affected subjects (II.1 and II.3) compared to the unaffected siblings (II.2 and II.4) and the prepubertal control. No IGF-I was detected in the fractions corresponding to the binary complex (BC) or fIGF-I in any of the subjects. This is most likely due to the fact that under physiological conditions 80-85% of IGF-I circulates in the TC and after the separation procedure, the amount of IGF-I in the BC or free was below the level of detection of our IGF-I assay. These results clearly demonstrate that in the affected patients the amount of IGF-I circulating in the TC is markedly elevated. B Cartoon of hypothesized mechanism underlying growth failure due to lack of PAPP-A2. There is an increase in the formation of ternary complexes, due to decreased proteolysis subsequent to the lack of PAPP-A2 activity. This results in reduced concentrations of fIGF-I in serum and most likely at specific target tissues. This also decreases the negative feedback effect of IGF-I on growth hormone (GH) production, contributing to the increased circulating concentrations of GH. Increased serum GH concentrations cause increased IGF-I, IGFBP-3, and ALS concentrations. This increase in IGFBP-3 and ALS would then contribute to further ternary complex formation and an increase in total IGF-I and IGF-II concentrations. ª 2016 The Authors EMBO Molecular Medicine Vol 8 | No 4 | 2016 369 EMBO Molecular Medicine PAPPA2 mutations and short stature Andrew Dauber et al modest pre- and postnatal growth retardation despite high IGF-I Whole-exome sequencing was performed in the probands of both concentrations, presumably reflecting reduced fIGF-I, as seen in our families at each institution as previously described (Argente et al, patients. The mild fasting hyperinsulinemia in three of five patients 2014; de Bruin et al, 2015). is possibly due to the elevated GH concentrations. The gene encoding PAPP-A2 was located in a quantitative trait Serum assays locus for body size in mice (Christians et al, 2006) and PAPP-A2 was confirmed to be partially responsible for the effect of this locus ELISAs were used to measure serum concentrations of acid labile (Christians et al, 2013). A SNP located in close proximity to human subunit (ALS; Mediagnost, Reutlingen, Germany), IGF-I, free IGF-I, PAPPA2 (rs1325596) was recently identified in a large genome-wide IGFBP-3, IGFBP-5, and PAPP-A2 (Ansh Labs, Webster, TX, USA). association study as being correlated with height in the general All assays were performed according to the manufacturers’ instruc- population (Lango Allen et al, 2010; Wood et al, 2014). Thus, tions. Briefly, in all determinations, after incubation of the serum common genetic variants in PAPPA2 may have mild effects on samples in a microtiter plate previously coated with the correspond- growth, while rare loss-of-function variants, such as those seen in ing antibody, the wells were washed and the enzyme conjugate was our subjects, have a more pronounced effect. added. At the end of the incubation period and after washing, a In conclusion, we have identified a new syndrome in two unre- substrate solution was added until the color developed adequately. lated families with loss-of-function variants in PAPPA2 resulting in Measurements were taken at 450 nm with an automatic microplate progressive growth failure, moderate microcephaly, thin long bones, analyzer. a marked elevation of total IGF-I concentrations due to impaired The PAPP-A2 ELISA employed is a two-site assay based on speci- proteolysis of IGFBP-3 and -5, and reduced fIGF-I. These patients fic monoclonal antibodies. Epitope mapping of the two antibodies represent the first human cases of diminished IGF-I bioavailability has been published earlier (Kloverpris et al, 2013). The catching due to defects in IGFBP regulation, indicating that PAPP-A2 is a key antibody maps to the SCR3 module and the detecting antibody maps regulator of IGF-I bioavailability and that deregulated IGFBP proteol- to the SCR1 module of PAPP-A2. All SCR modules are located ysis can have relevant consequences in human physiology. C-terminal to the site of mutations. It remains to be seen whether postnatal growth in these children Serum insulin concentrations were measured by an immunora- can be improved. One potential treatment includes human recombi- diometric assay from DIAsource ImmunoAssays (Louvain-la-Neuve, nant IGF-I. It is possible that increasing total IGF-I in the circulation Belgium). Samples were dispensed, and a 125I-anti-insulin could shift the equilibrium of the GH-IGF system such that there is monoclonal antibody was added into the tubes coated with another also an increase in free/bioactive IGF-I, which could result in anti-insulin monoclonal antibody. After incubation and washing, increased systemic growth. This hypothesis is now being tested as the bound fraction was counted in a gamma-counter. the prepubertal patients are under hrIGF-I therapy. In all assays, the intra- and inter-assay coefficients of variation The results reported here also emphasize the potential impor- were lower than 10%. Normal ranges were obtained by measuring tance of proteins that interact with PAPP-A2 (Kloverpris et al, 2013) concentrations in groups of Tanner Stage I or V healthy controls that have been previously shown to affect human height (Lango recruited from the clinic at the Hospital Infantil Universitario Niño Allen et al, 2010). Thus, patients currently labeled as idiopathic Jesús (n = 50 in each group). short stature with elevated IGF-I and IGFBP-3 concentrations could Bioactive IGF concentrations were measured by kinase receptor conceivably obtain a specific diagnosis after undergoing testing for activation assay (KIRA) as previously described (Chen et al, 2003; mutations in PAPPA2, or possibly in proteins that regulate the activ- Reinhard et al, 2013). Briefly, this assay measures the ability of IGF ity of this protease. Indeed, the data presented here indicate that in serum to phosphorylate the IGF-I receptor (IGF-IR) in an in vitro- understanding PAPP-A2 regulation and function will have important based model employing human embryonic cells transfected with implications in both the clinical diagnosis of growth retardation and cDNA of the human IGFR gene. The ability of serum to phosphory- other areas of IGF-I biology. late the IGF-IR in vitro is compared to a serial dilution of IGF-I. This assay also detects IGF-II and pro-IGF-II activation of the IGF-IR (cross-reactivity 12% and 2%, respectively), but the cross-reactivity Materials and Methods of proinsulin and insulin is negligible (< 1%). Thus, the output of this assay is referred to as IGF bioactivity as both IGF-I and IGF-II Studies were approved by the Institutional Review Boards at the can activate the IGF-IR. The detection limit of this assay is Hospital Infantil Universitario Niño Jesús and Cincinnati Children’s < 0.08 lg/l. Hospital Medical Center. Written informed consent was obtained from all subjects or their legal guardians and the experiments Chromatography for endogenous IGF-I complex profiles conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Levels of complex-bound endogenous IGF-I were assayed by neutral Belmont Report. Full consent was also obtained for the publication size-exclusion chromatography and the capacity to form such of the patients’ pictures. complexes as previously described (Domene et al, 2004). Briefly, to determine the IGF-I complex profile, we performed neutral chro- Genetic analysis matography of 300 ll of serum on Superdex 200. Two-ml fractions were collected and those corresponding to the ternary (six frac- In family two, homozygosity mapping analysis was implemented tions), binary (seven fractions) and free IGF-I (one fraction) were on genome-wide SNP data using PLINK (Purcell et al, 2007). collected and dried in a speed vacuum, reconstituted in 100 ll of 370 EMBO Molecular Medicine Vol 8 | No 4 | 2016 ª 2016 The Authors Andrew Dauber et al PAPPA2 mutations and short stature EMBO Molecular Medicine PBS buffer 0.05 M, NaCl 0.15 M, pH 7.4, BSA 0.5%, and IGF-I was by reversed-phase high-pressure liquid chromatography (RP-HPLC) extracted by alcohol-acid treatment and cryoprecipitation. The IGF-I on a Discovery BIO Wide Pore C5 column (4 × 250 mm, Sigma), as concentration in each fraction was then determined by QLIA (Immu- described (Gyrup & Oxvig, 2007). Protein purity was assessed by lite, Siemens) according the manufacturer’s instructions. SDS–PAGE, and quantification of purified proteins was done by amino acid analysis. The purified IGFBPs were labeled with 125I Mutagenesis (Amersham Biosciences), and cleavage reactions were carried out as previously described for PAPP-A (Winn et al, 2009). In brief, media Recombinant PAPP-A2 and variants thereof were generated by harvested from cells transfected with empty vector or human transfection of mammalian cells with WT or mutated cDNAs. PAPPA2 cDNA or variants thereof were diluted 1:25 and mixed with 125 Mutated proteins were compared to WT PAPP-A2 in their abilities to I-IGFBP-5 (10 nM) in 50 mM Tris–HCl, 100 mM NaCl, 1 mM proteolytically cleave radiolabeled IGFBP-3, -4, and -5 and by CaCl2, pH 7.5. For wild-type PAPP-A2, this dilution corresponds to a Western blotting following nonreducing or reducing SDS–PAGE. concentration of 90 pM PAPP-A2 dimer. To assess activity toward Plasmid constructs encoding variants of human PAPP-A2 were IGFBP-3 and -4, culture media were diluted 1:10, corresponding to a made by overlap extension PCR (Ho et al, 1989) using human PAPP-A2 concentration of 225 pM. Variants of all experiments in PAPPA2 cDNA contained in plasmid pPA2 (Overgaard et al, 2001) which the IGFBPs were preincubated with IGF-I (100 nM; GroPep as a template. Outer primers were 50 -CTGTATGTGGATGGCACTC Bioreagents) prior to the addition of PAPP-A2 were also carried out. AGG-30 and 50 -GCAACTAGAAGGCACAGTCGAG-30 . Overlapping Following 3 h of incubation at 37°C, the reactions were terminated mutated sets of internal primers were 50 -CTTTGACGACGGAGAT by the addition of hot SDS–PAGE sample buffer supplemented with ACTGCTG-30 and 50 -CAGCAGTATCTCCGTCGTCAAAG-30 for mutant 25 mM EDTA. Substrate and cleavage products were separated by PAPP-A2(D643fs), and 50 -GAGGATAGAGATTGATGTAGCACTCCTG nonreducing SDS–PAGE and visualized by autoradiography using a ACTTCTC-30 and 50 -GAGAAGTCAGGAGTGCTACATCAATCTCTATC storage phosphor screen (Molecular Dynamics) and a Typhoon CTC-30 for mutant PAPP-A2(A1033V). The mutated fragments were imaging system (GE Healthcare). To assess specific activity toward digested with XbaI and XhoI and swapped into pPA2 to generate the IGFBPs, cleavage reactions of 6 h were carried out using plasmids pPA2(D643fs) and pPA2(A1033V), respectively. Plasmid equimolar concentrations of PAPP-A2 wild-type and mutant PAPP- DNA was prepared by GenElute HP Plasmid Miniprep Kit (Sigma). A2(A1033V). All constructs were verified by sequence analysis. Western blot analysis Cell culture and transfection Proteins separated by nonreducing or reducing SDS–PAGE were Human embryonic kidney 293T cells (293tsA1609neo, ATCC CRL- blotted onto a PVDF membrane (Millipore), blocked with 2% 3216) (Pear et al, 1993) were maintained in high-glucose DMEM Tween-20, and equilibrated in 50 mM Tris–HCl, 500 mM NaCl, supplemented with 10% fetal bovine serum, 2 mM glutamine, 0.1% Tween-20, pH 9.0 (TST). The blots were incubated overnight nonessential amino acids, and gentamicin (Invitrogen). The cells are at room temperature with an antiserum raised against the N-term- routinely tested for mycoplasma contamination. For transient trans- inal laminin G-like module of PAPP-A2 diluted 1:10,000 in TST fection, 6.0 × 106 cells were plated onto 10-cm dishes and trans- containing 0.5% fetal bovine serum (Winn et al, 2009). The blots fected 18 h later by calcium phosphate coprecipitation using 10 lg were incubated for 1 h at room temperature with polyclonal swine plasmid DNA (Overgaard et al, 2000). In addition to plasmid anti-rabbit IgG-HRP (DAKO, P0217) diluted 1:2,000 in TST contain- constructs encoding PAPP-A2 or mutated variants, constructs ing 0.5% fetal bovine serum. All washing between the steps was encoding human IGFBP-3 (Laursen & Oxvig, 2005), human IGFBP-4 done with TST. The blots were developed using enhanced chemilu- (Boldt et al, 2001), or human IGFBP-5 (Overgaard et al, 2001) were minescence (ECL Prime, GE Healthcare), and images were captured used. Culture supernatants were harvested 48 h post-transfection and analyzed using an ImageQuant LAS 4000 instrument (GE and cleared by centrifugation, or the cells were further cultured in Healthcare). For quantification of mutant PAPP-A2 (A1033V), a serum-free medium (CD293, Invitrogen) to facilitate purification of standard curve was generated based on serial dilutions of wild-type the secreted proteins. Concentrations of WT PAPP-A2 in the media PAPP-A2 of known concentration. were determined by a two-site ELISA (Laursen et al, 2007). Pappa2 KO mice Assays for proteolytic activity All procedures involving mice complied with the standards stated in Purification of recombinant proteins (IGFBP-3, -4, and -5) was the “Guide for the Care and Use of Laboratory Animals” and were carried out by affinity chromatography on a 1 ml HisTrap HP approved by the Institutional Animal Care and Use Committee of column (GE Healthcare). Serum-free media were diluted 1:1 in Mayo Clinic. 20 mM NaH2PO4, 150 mM NaCl, pH 7.4 (PBS) and loaded onto the Pappa2 KO mice are on a C57BL/6 background. They were column with a flow rate of 1 ml/min. The column was washed with generated at Lexicon Pharmaceuticals Inc. (The Woodlands, TX). 20 column volumes of 50 mM NaH2PO4, 1 M NaCl, 20 mM imida- Pappa2 KO and wild-type littermates were housed in a standard zole, 0.05% Tween-20, pH 7.4, followed by five column volumes of pathogen-free mouse facility at Mayo Clinic with ad libitum access PBS. The proteins were eluted with 50 mM NaH2PO4, 300 mM to food and water. Serum total and free IGF-I were measured in imidazole, pH 7.4, and dialyzed against 20 mM HEPES, 150 mM sixteen-week-old wild-type males (n = 9), wild-type females NaCl, pH 7.4. Prior to iodination, the IGFBPs were further purified (n = 12), KO males (n = 8), and KO females (n = 7). ª 2016 The Authors EMBO Molecular Medicine Vol 8 | No 4 | 2016 371 EMBO Molecular Medicine PAPPA2 mutations and short stature Andrew Dauber et al to JAC), Centro de Investigación Biomédica en Red Fisiopatología de Obesidad The paper explained y Nutrición (CIBEROBN), Instituto de Salud Carlos III (JA), Fundación Endocrino- Problem logía y Nutrición (JA), the Catalan Government (2014SGR1468 and ICREA Syndromes marked by growth failure have been associated with Acadèmica to LAPJ), the Eunice Kennedy Shriver National Institute of Child mutations in multiple genes of the growth hormone (GH)/IGF-I axis. Health & Human Development of the National Institutes of Health (Award However, a large percentage of cases of familiar short stature Number K23HD07335 to AD), The Danish Council for Independent Research continue to be classified as idiopathic. Identification of the specific (FNU), and the Novo Nordisk Foundation (CO). A CIBER for Rare Diseases cause underlying impaired longitudinal growth would most likely improve treatment protocols for the affected individual. (CIBERER) fellowship supported CSJ. The funding sources had no role in the design or interpretation of the results or in the decision to submit these data Results for publication. The content is solely the responsibility of the authors and Here, we present two families with homozygous loss-of-function does not necessarily represent the official views of the National Institutes of mutations in PAPPA2, a gene encoding pregnancy-associated plasma Health. protein-A2 (PAPP-A2). PAPP-A2 is a protease highly specific for the cleavage of two of the six high-affinity IGF-binding proteins (IGFBP), IGFBP-3 and -5. In the circulation, most IGF-I is bound in ternary Author contributions complexes in conjunction with the acid labile subunit (ALS) and IGFBP3 AD and JA designed and conducted the experiments, analyzed the data, pheno- or IGFBP5 and must be released to act upon its target sites. The PAPP- typed the patients, and wrote the manuscript. VB, HMD, SK, CSJ, FH, HGJ, CAC, A2 produced by both mutated genes lacks the ability to cleave either JF, SY, VH, JAC, CO, and LAPJ conducted the experiments, analyzed the data, IGFBP3 or IGFBP5. This could explain the observed increase in the and reviewed the manuscript. MTMC, GÁM, JP, VD, and RM phenotyped the amount of IGF-I bound in ternary complexes and the decrease in free or bioactive IGF-I in these patients, which in turn most likely underlies patients, analyzed the data, and reviewed the manuscript. RGR analyzed the their significantly decreased growth. Reduced levels of bioactive IGF-I data and reviewed the manuscript. would also reduce the ability of this hormone to act on the pituitary to reduce GH secretion. This would result in the increased GH secretion Conflict of interest found in these patients, which in turn could explain the increased Drs. Dauber, Hwa, and Argente have a patent application pending entitled circulating levels of GH-dependent proteins such as IGFBP-3 and ALS. Moreover, mild modifications in bone mineral density and bone “Enzyme replacement therapy of PAPP-A2 (Application No: 62/126,831)” morphology were observed in these patients. relevant to this work. Otherwise, the authors have no conflict of interest to declare. Impact This is a novel syndrome of growth retardation that presents with For more information progressive growth failure with markedly elevated circulating IGF-I, http://exac.broadinstitute.org/ IGF-II, IGFBP-3, IGFBP-5, and ALS, but decreased fIGF-I levels and IGF http://evs.gs.washington.edu/EVS/ bioactivity. Analysis of PAPP-A2 in patients presenting with a similar phenotype will lead to a new specific diagnosis in a subset of patients http://geevs.crg.eu/ now classified as idiopathic. Moreover, the data presented here indi- http://bioinfo.cipf.es/ apps-beta/exome-server/beta/ cate that understanding PAPP-A2 regulation and function will not only have important implications in the clinical diagnosis of growth retardation, but also in other areas of IGF-I biology. 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J plasma protein-A2 in insulin-like growth factor (IGF) binding Biol Chem 277: 44005 – 44012 protein-5 proteolysis during pregnancy: a potential mechanism for increasing IGF bioavailability. J Clin Endocrinol Metab 95: License: This is an open access article under the 1412 – 1420 terms of the Creative Commons Attribution 4.0 Zhang M, Xuan S, Bouxsein ML, von Stechow D, Akeno N, Faugere MC, License, which permits use, distribution and reproduc- Malluche H, Zhao G, Rosen CJ, Efstratiadis A et al (2002) Osteoblast- tion in any medium, provided the original work is specific knockout of the insulin-like growth factor (IGF) receptor gene properly cited. 374 EMBO Molecular Medicine Vol 8 | No 4 | 2016 ª 2016 The Authors