Loss-Of-Function Mutations in IGSF1 Cause an X-Linked Syndrome of Central Hypothyroidism and Testicular Enlargement

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Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism and testicular enlargement.

ARTICLE in NATURE GENETICS · NOVEMBER 2012 Impact Factor: 29.65 · DOI: 10.1038/ng.2453 · Source: PubMed

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Paul Le Tissier Juan Pedro Martínez-Barberá The University of Edinburgh University College London

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Thomas Vulsma Michael Gordon Wade

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Available from: Marco Bonomi Retrieved on: 10 August 2015 © 2012 Nature America, Inc. All rights reserved. addressed to J.M.W. ( UCL Institute of Child Health, London, UK. Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada. The Netherlands. Paediatrics, West Middlesex University Hospital, Isleworth, UK. 14 Ca’Granda, Milan, Italy. and Metabolic Disorders, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano, Milan, Italy. Center, Nijmegen, The Netherlands. Molecular Neuroendocrinology, National Institute for Medical Research, Mill Hill, London, UK. Trust Genome Campus, Hinxton, Cambridge, UK. of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands. UK. University, Montréal, Québec, Canada. 1 associated in a increased expression, pituitary thyroid-stimulating deficient its anterior is enlargement 11 8 Using or Congenital Krishna Chatterjee M Alberto Pereira Nienke R Biermasz A Claudia L Ruivenkamp Timothy M E Davis Luca Persani Juan P Martinez-Barbera Peter Voshol Yu Sun enlargement syndrome of central hypothyroidism and testicular mutationsLoss-of-function in Nature Ge Nature Received 15 May; accepted 3 October; published online 11 November 2012; Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Subdivision of Endocrinology, Department of Pediatrics, Erasmus Medical Center–Sophia Children’s Hospital, Rotterdam, The Netherlands. new distinct

IGSF1 in trafficking unrelated

4 membrane Department of Pediatric Endocrinology, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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, , Irene Campi

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cell School of Medicine and Pharmacology, Fremantle Hospital Unit, The University of Western Australia, Perth, Western Australia, Australia. show and 16

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, , Johan T den Dunnen , other hypothyroidism, in 21

, , Natasha M Appelman-Dijkstra

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, , Mehul T Dattani , pituitary low

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10 , , Nadia Schoenmakers

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Department of Clinical Sciences & Community Health, Università degli Studi di Milano, Milan, Italy. , , Wilhelmina H Stokvis-Brantsma 3 , , Jeroen F J Laros

prolactin Institute of Metabolic Science, Metabolic Research Laboratories, Addenbrooke’s Hospital, University of Cambridge, Cambridge, hormone

identified is

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TRH heterologous

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concentrations, in [email protected]

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Neural Development Unit, University College London (UCL) Institute of Child Health, London, UK. , , Jacqueline K White

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expressed

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, , Marjolein Kriek

, , Jan M Wit Department of Endocrinology and Metabolic Disorders, Leiden University Medical Center, Leiden,

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, , Paolo Beck-Peccoz

doi:10.1038/ng.245 16 1

19 [email protected] 6 , Eleonora , P Eleonora Corssmit Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, , , Paul le Tissier 5 , 21 phenotype ( the grandfather (A-I.4) and another male relative (A-II.4) with the same sequence, ence (p.Ala713_Lys721del)c.2137_2163del in two cousinsthe ( ( hypothyroidism tral cen showed (A-I.4) grandfather maternal the of testing Subsequent ing to a late growth spurt and delayed pubic hair development in each. low for testicular size until 15.2 and 14.2 years of age, respectively, lead In contrast, serum testosterone concentration remained inappropriately continued to grow beyond the reference testes but boys, both range in normally ofadvanced growth testistesticular scence, volume ( (GH) hormone growth ( concentration prolactin low and deficiency partial hypothyroidism, central had age, 7.3 at failure growth for referred when (A-III.7), cousin His hypothyroidism by neonatal screening for congenital hyp The index case in family A (A-III.11; & Daniel J Bernard IGSF1 5 , , Thomas Vulsma 1 , , Sarina G Kant 21 18 9 These authors jointly directed this work. Correspondence should be Department of Human Genetics, Radboud University Nijmegen Medical , , Martijn H Breuning Fig. 1 3 2 Department of Pharmacology and Therapeutics, McGill NM_001170961. 7 , Supplementary TableSupplementary a 8 ) or M.T.D. ( cause an X-linked and 10 , , S Neda Mousavy Gharavy , 12 Table Table 4 , , Hongdong Zhu 16 1 , 18 20 4 2 , , Cathy A J Bosch , , Guido C J Hovens Hazard Identification Division, Environmental , , Marlies J Kempers , 21 , , Wilma Oostdijk [email protected] 1 14 1 ). X-chromosome exome sequencing in in sequencing exome X-chromosome ). ). Independent whole-exome , , Jayanti J Rangasami 1 12 ). The same deletion was present in in present was deletion same The ). Endocrine Unit, Fondazione IRCCS 1 Fig. 1 , Raoul , C Raoul Hennekam

1 a ) identified a 27-nt deletion, ) identified IGSF1 ) was diagnosed with central 11 13 Table ). Division of Endocrine 15 , at Xq25 ( 1

Department of , 5 16

, 1 4

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o 15 IGSF1 thyroidism.

sequencing 5 , Department Table

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1 ). - - -

© 2012 Nature America, Inc. All rights reserved. prolactin content below the lower limit of normal. a K-II.3 orchidometer = 0.58 ml by ultrasound testosterone secretion. Testosterone treatment was started at 76 years. reported unilateral macroorchidism, until the enlarged testis was removed after testicular torsion leading to complete infarction at 74 years. The remaining testis was small and soft, with deficient J-III.2 J-III.1 I-III.2 H-III.3 H-III.2 G-I.1 G-III.3 G-III.1 F-II.8 F-IV.2 F-IV.1 E-IV.3 E-IV.1 D-I.3 D-III.4 D-III.3 C-III.1 B-I.4 B-II.11 B-III.8 B-III.7 A-I.4 A-II.4 A-III.7 A-III.11 Case T 65.5 (B-I.4) and 43.3 (B-II.11) years of age ( were mutation only diagnosed with same central hypothyroidism after the genetic screening at harboring relatives affected two but dice, B-III.7 and B-III.8 presented in infancy with prolonged neonatal jaun in (p.Trp977*),also c.2931G>A mutation, nonsense ( B family in hypothyroidism central with B-III.8) and (B-III.7 brothers two of indicated. not F are E and families in deletions the of positions The family). by (labeled mutations identified the by affected residues of positions relative the showing structure, domain protein IGSF1 ( arrow. an by indicated is A (A-III.11) family of (P) Proband confirmed. was mutation the that indicate 1 Figure s r e t t e l +, serum FT4 concentration of 50–99% of the lower limit of normal; ++, serum FT4 concentration of <50% of the lower limit of normal. In all cases, serum TSH levels were normal. able able 1 a b IlI II l IlI II l

1 Clinical Clinical features of individuals (all males) with IGSF1 c.2309G>A c.3596dupT c.3518G>A c.2839T>C c.2588C>T 328-kb deletion 126-kb deletion c.2248delG c.2248delG c.2931G>A c.2137_2163del alteration Nucleotide Fig. 1 Fig. 1 2 3 2 2 mutations identified in individuals with central hypothyroidism. ( hypothyroidism. central with individuals in identified mutations 4 3 b 3 , , 1 Table 4

1 4 6 5 2 2 h g 1 5 5 1 p.Ser770Asn p.Glu1200Argfs*3 p.Trp1173* p.Cys947Arg p.Ser863Phe p.Glu750Lysfs*28 p.Glu750Lysfs*28 p.Trp977* p.Ala713_Lys721del Amino-acid alteration 3 and 7 2 8 6 6 ). g Arr Xq26.1q26.2 (130386267–130512002)x0 (hg19). 7 4 8 Supplementary Table Supplementary 4 3 7 9 8 5 9 c 8 10 Age at sonographic determination of testicular volume. 6 11 6 5 10 3 8 7 12 Fig. 1 4 11 1 9 5 13 8 7 12 0 Italy Italy Italy Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands Netherlands UK UK UK UK Netherlands Netherlands Netherlands Netherlands b 6 P 14 ). 1 9 f 11 Origin Estimated on the basis of Prader orchidometer (30 ml by Prader orchidometer = 18.3 ml by ultrasound, 2 ml by Prader 13 IGSF1 1 9 15 12 0

1 14 13 ) identified a identified ) 16 IGSF1 0 15 14 ( 1 Fig. 1 Age atdiagnosis hypothyroidism variants

1 10.6 years 2 weeks 3 weeks 14.1 years 3 weeks 63 years 2.5 weeks 5 weeks 57.5 years 3 weeks 3 weeks 6.5 years 2.5 weeks 2 weeks 61 years 1 week 3 weeks 3 weeks 65.9 years 43.3 years 7 weeks 4 weeks 74.1 years 51.5 years 7.3 years 3 weeks of central c ). ). signal sequenc N-terminal NH - c Family CandD:c.2248delG,p.Glu750Lysfs*28 Family A:c.2137_2163del,p.Ala713_Lys721del Family K:c.2309G>A,p.Ser770Asn Family J:c.3596dupT,p.Glu1200Argfs*3 Family H:c.2839T>C,p.Cys947Arg Family G:c.2588C>T,p.Ser863Phe Family E:126-kbdeletion,arrXq26.1q26.2(130386267–130512002)×0(hg19) Family B:c.2931G>A,p.Trp977* Family I:c.3518G>A,p.Trp1173* Family F:328-kbdeletion,arrXq26.1q26.2(130310905–130639353)×0(hg19)

2 h Arr Xq26.1q26.2 (130310905–130639353)x0 (hg19). zd y aeseii cnrl yohriim ( hypothyroidism central male-specific by ized character K) and J (families families Italian two and C–I) (families tests at diagnosis are shown in shown are diagnosis at tests the basis of thyroxine (T4) and TSH concentrations). Thyroid function detected through neonatal screening for congenital hypothyroidism (on Supplementary1 Fig.

We found additional additional found We a d ) Pedigree of family A. Small horizontal lines above individuals individuals above lines horizontal Small A. family of ) Pedigree Sonographic testicular volume in comparison to references at the same age

hypothyroidism e Central ++ ++ + + + + + + + + + + + + + + + + + + + + + + + + Hydrophobic linker a

DVANCE ONLINE PUBLICATION ONLINE DVANCE a Internal signal deficiency ). Most Dutch and Italian (family J) cases were cases J)Italian (familyandDutch Most ). Prolactin sequence b ) Pedigree of family B. ( B. family of ) Pedigree + + + + + + + + + + − + − + − + + + − + − + − − + − IGSF1 b

A Supplementary Table2 Supplementary variants in seven Dutch families families Dutch seven in variants C + Extracellular domain Age (years) K D 26.54 16.69 15.93 18.36 87.49 23.08 27.52 58.24 12.70 22.37 20.57 62.75 10.46 16.60 66.37 43.30 10.52 67.70 52.41 21.36 17.64 0.16 3.26 9.44 3.79 7.95 G H B c IGSF1 CT c volume (ml)(reference) 0.58/0.58 (0.30–0.65) 0.75/0.80 (0.32–0.70) ) Schematic of the the of ) Schematic 21.5/21.4 (8.5–18.3) 21.7/21.7 (6.7–15.3) 22.7/22.7 (8.5–18.3) 25.5/25.4 (8.5–18.3) 44.6/48.2 (8.5–18.3) 19.6/21.6 (8.5–18.3) i. 1 Fig. >18/>18 (8.5–18.3) >18/>18 (8.5–18.3)

0.8/0.8 (0.32–0.70) 1.8/1.5 (0.45–0.92) 1.1/1.0 (0.55–1.87) 11.8/38 (8.5–18.3) 34/25.5 (8.5–18.3) 4/large (8.5–18.3) Right/left testicular 18/18 (7.8–16.2) I 21/16 (8.5–18.3) 68/37 (8.5–18.3) 32/29 (8.5–18.3) 30/26 (8.5–18.3) 1/0.9 (0.5–1.35) 12.2/8.4 (4–13) 1/1 (0.55–1.87) Nature Ge Nature J 19/17 (8–16.5) 21/20 (7.3–16) D Transmembrane c , Cytoplasmic domain . Mean (s.d.) Mean . Table domai 8 b . +, serum e Self- n n COOH 1 etics and f e f f

d f -

© 2012 Nature America, Inc. All rights reserved. (67 years,agereference 18–54years)andnormalinF-II.8(46years, agereference30–53years). before thestartoftestosterone substitutiontherapy(at76years). ranges foryoungeragegroupsareindicated. for testosteroneinboys(10thto90thpercentile) accordingtovonSchnakerburg references obtained in 1980 (ref. a T4 R/,L-thyroxinetreatment;IU,International Units. K-II.3 J-III.2 H-III.2 F-IV.2 F-IV.1 B-III.7 J-III.1 I-III.2 H-III.3 G-I.1 G-III.3 G-III.1 F-II.8 E-IV.3 E-IV.1 D-I.3 D-III.4 D-III.3 C-III.1 B-I.4 B-II.11 B-III.8 A-I.4 A-II.4 A-III.7 A-III.11 Case T ( cases shown). not (data carriers heterozygous female 20 3 Table TRH was normal (36.3% versus 41.8% in controls) to response FT3 but the (2.7%)), 23.9% = (s.e.m.) (mean for controls FT4 serum (mean (s.d.) = 14.2% (7.9%)) was lower than that reported of increment the subjects, eight In 5)). (ref. mU/l 4.1–13.9 or 4) (ref. mU/l (3.7–12.5 males for ranges reference reported the of half lower the in is which mU/l, 8.5 and 4.3 between were concentrations TSH mU/l) (14–37 controls matched hypothyroidism A similar range was observed in previous reports on congenital central Table 65.5 years TRH standard old), tests were ( conducted considerable differences in the extent of hypothyroidism. was in the lower half of the reference range. Withinrange families, for we ageobserved (range of 83–121%) IGSF1 (s.d.) serum triiodothyronine (T3) concentration thanin in ninecontrols 1-(3.94 toversus 3-week-old5.73; Mann-Whitney test 0.8–6.0 mU/l, mean 2.6 mU/l). The FT4/TSH ratio was therefore lower TSH concentrations were within the reference range in alllower values,cases all ranged(range between 67–94%of of the reference range. Serum limit of the reference range for age and assays. serumfree thyroxineExcept (FT4) concentration for was 79% (13%)two of the lower infants with Nature Ge Nature Height isexpressedass.d.score(SDS)fornational referencedataforTheNetherlands able able 2 Serum prolactin concentrations were decreased in 18 of 26 26 of 18 in decreased were concentrations prolactin Serum (0.4– subjects older eight and old) weeks (2.5–5 infants eight In

-deficient infants was 98% (12%) of the lower limit of the reference 2 Table ). In the infants, the peak TSH concentration was 4.5–16.0 mU/l.

. e ol dmntae eta hyp central demonstrate could We ). Clinical Clinical and laboratory data Age (years) n 26.54 16.69 18.09 20.52 87.49 23.08 27.52 58.24 12.70 22.37 20.57 62.75 10.46 17.39 66.37 43.29 10.52 86.70 52.41 21.36 17.64 0.16 3.26 9.44 3.79 7.95 etics 1 ). Cases A-III.7, H-III.3, I-III.2 and K-II.3 showed showed K-II.3 and I-III.2 H-III.3, A-III.7, Cases ). 2 , 3 , and concentrations are lower than observed in age-

ADVANCE ONLINE PUBLICATION ONLINE ADVANCE T4 R/ + + + + + + + + + − + + + + + + + − + + + + − + + – 26). Median BMI = 2.1 SDS. Height −0.6 −0.4 −0.6 −0.2 −0.6 −0.7 −2.5 −1.9 −0.5 −0.2 –0.5 –1.2 SDS 1 0.1 0.5 1.3 1.1 1.4 0.1 0.6 1.4 1.0 0.6 0.7 0.2 0.3 1.0 . In seven older cases, T3 concentration e 3 GnRH testandtestosteroneperformedat15.19 years,beforestartoftestosteronesubstitutiontherapy. . At later ages (7.3–63 years), peak peak years), (7.3–63 ages later At . a

SDS 1.1 1.4 1.0 2.0 1.7 2.6 2.5 1.5 3.5 8.6 2.9 2.2 2.8 1.0 7.7 1.1 0.8 2.0 2.1 3.3 2.2 1.9 4.3 2.5 1.8 2.6 BMI b

c Reference ranges for males of >17 years: testosterone of 11–35 nM, LH < 0.1–15 IU/l and FSH < 0.1–10 IU/l. Reference ranges o LH (IU/l) g Because oflowplasma SHBGconcentration,freeandrogenindex(FA =(100×testosterone)/SHBG) washighinD-I.3 thyroidism in 5 of of 5 in thyroidism 6 1.0 , 4.4 1.3 2.5 3.2 2.1 3.7 1.6 6.5 2.7 3.2 3.8 3.5 3.6 15 <1 <1 <1 <1 <1 <1 <1 7 3 − − − ( P

Supplementary Supplementary f Supplementary Supplementary e = 0.002). Mean c FSH (IU/l) et al. 10.7 11.0 10.1 17.9 10.6 3.8 3.4 3.9 4.6 6.9 3.9 8.6 2.5 6.0 4.8 6.3 1.1 2.9 3.8 3.1 1.2 54 <1 2 − − − 7 2 . 3 f e , UK d Reference rangeformalesof>18years:inhibin Bof150–400ng/landAMH5–30 c 2

4 andItaly LH GnRH 13.1 36.5 14.3 31.8 38.7 22.2 30.8 29.6 18.9 (BMI) and pituitary-gonadal hormones for cases are shown in data reference Dutch to (compared delayed years) 13.0 at ng/dl <23 = nM (<0.8 concentration testosterone serum in increase evaluable cases, testosterone production was delayed, as defined a late were in observed testicular morphology by ultrasound. In 10 out of 11 by ml ultrasound) >18.3 orchidometer, advance), in adolescence and adult years macroorchidism (>2 (>30 ml testosterone by Prader serum for testes large relatively low, still was from approximately 11 years of age onward, testosterone serum while volume in testicular increase volume in childhood, testicular normal normal. were MRIs cases, other eight In respectively. lesion, stalk small and callosum corpus the of hypoplasia hygroma, frontoparietal a showing abnormal, was (K-II.3), and GH deficiency (C-III.1) hypothyroidism central III.11), (A- macrocephaly of because performed (MRI), imaging resonance the in presented details are biochemical and Clinical treatment. discontinued had who young subjects three In the in GH. normal were tests biosynthetic stimulation GH with adulthood, treated were and insufficiency, growth tration was normal in most cases. A-I.4, who underwent surgery for surgery underwent who A-I.4, cases. most in normal was tration concen testosterone Plasma +2 children. 13 of out 5 than in (SDS) score s.d. higher and adults 13 of out 11 in 25 than higher was BMI but references, population for average the to close was height Mean 9.4 1.4 25 21 max − − − − − − − − − − − – – The most recent (July 2012) clinical data on height, body mass index involving pattern characteristic a showed development Testicular to e 2 5 . Themean(s.d.)heightis0.0(1.0)SDS. FSH

retardation in childhood, associated with biochemical GH GH biochemical with associated childhood, in retardation GnRH 10.7 17.2 15.3 13.1 14.1 15.4 36.7 22.6 18.7 5.1 9.8 7.8 8.3 − − − − − − − − − − − max – – to e 9 and/or a pubertal growth spurt that was >2 years years >2 was that spurt growth pubertal a and/or Testosterone (nM) <0.3 (0.07–0.28) <0.3 (0.07–0.31) 1.7 (0.14–0.66) 1.1 (1.0–16.3) 9.5 (1.7–27.8) <0.3 (0.2–1.2) <0.3 (0.2–1.2) 4.2 (0.4–9.5) Supplementary Note Supplementary 10.1 11.5 16.9 11.8 13.4 17.2 18.7 16.8 12.7 17.6 5.1 4.8 24 24 19 − − − g f 7 e c f LH, FSHandtestosteroneconcentrations b Inhibin B(ng/l) BMI isexpressedasSDSforDutch 533 (80–300) 257 (80–300) 192 (20–100) 299 (80–300) 111 (20–120) 328 (80–300) 92 (20–120) 97 (20–300) 91 (20–300) 8 ( Table 284 265 338 249 249 141 317 454 152 192 279 199 237 <10 . In three cases, magnetic magnetic cases, three In . − − − 1 0 ). 1 ). No abnormalities No ). abnormalities d s r e t t e l 207 (100–1,000) 134 (100–400) 50.2 (10–100) 35.4 (30–200) 14.0 (10–100) 16.6 (10–100) 97 (100–400) 3.3 (10–100) 97 (30–200) AMH ( µ g/l. Reference 10.8 26.9 0.24 12.4 6.4 7.2 5.9 4.5 5.2 1.4 6.2 7.4 5.6 − − − – Table µ g/l)

2 - .

© 2012 Nature America, Inc. All rights reserved. IGSF1 CTD was analyzed by cell surface biotinylation. IB, immunoblot; the indicated wild-type and mutant IGSF1 proteins. Membrane expression of ( conditions. Nuclei were stained with DAPI (blue). Scale bars, 10 antibody against IGSF1 CTD under non-permeabilizing and permeabilizing Expression of the IGSF1 CTD was analyzed by immunofluorescence using the weight. ( IGSF1 CTD. Non-specific bands are indicated with an asterisk. MW, resolvedmolecular by SDS-PAGE and immunoblotted using an antibody specific to Proteinthe lysates were deglycosylated with either PNGaseF (P) or EndoH (E),vectors expressing the indicated wild-type (WT) and mutant IGSF1 proteins. ( Figure 3 syndrome a metabolic (data not shown). insulin and concentrations C-peptide did not indicate the presence of glucose, triglyceride, cholesterol, (LDL) lipoprotein low-density and relative to the reference range. Serum high-density lipoprotein (HDL) and AMH concentration was relatively low (decreased in five subjects) Inhibin B concentration in to (elevated tended four high be subjects), subjects. two except all in concentration FSH for than LH for higher hormone (100 releasing (GnRH) above the reference range in six The cases. response to gonadotropin- FSH concentration was always higher than LH concentration and was tions were within reference ranges in the majority of cases, but plasma concentra AMH and B inhibin (FSH), hormone stimulating follicle (LH), hormone luteinizing Plasma normal. was index androgen free low plasma sex hormone–binding globulin of (SHBG) concentration, because the but, concentrations, testosterone plasma low had F-II.8) and (D-I.3 low. obesity trations are severe very with subjects two The of age the at 86.7 years, currently, his inhibin B and and, anti-Müllerian hormone (AMH) concen substitution, testosterone of start the before gonadotropins elevated and concentration testosterone low torsion testicular and in whom the remaining testis was atrophic, had s r e t t e l IP, immunoprecipitation; HRP, horseradish peroxidase. c a

b a ) HEK293 cells were transfected with pcDNA3 or with vectors expressing ) HEK293 cells were transfected with pcDNA3 (empty vector) or with Merge IGSF1 CTD Hormone

b Alterations in IGSF1 impair its plasma membrane trafficking. ) HEK293 cells were transfected with the same constructs as in TSH Mouse E12.5 GH µ g administered intravenously) g administered was Human CS18embryo Prolactin µ m. LH

a . - -

pituitary primordium; primordium; pituitary in Rathke’s developing (the expressed pouch mRNAs are abundantly unrelated individuals)) and were not previously reported. previously not were and individuals)) unrelated 500 of data pilot (GoNL, Netherlands the of Genome and (HGMD) Database Mutation Gene Human the (LOVD), Database Variance (dbSNP, Open databases the Leiden available Project, Genomes 1000 Table ( sequencing Sanger by confirmed were variants All in J.family (p.Glu1200Argfs*3)) (c.3596dupT duplication nucleotide G–I single- a was families there and of mutations, nonsense or members missense had K and Affected gene. annotated other no and affected that sizes different of deletions gene submicroscopic deletion observed F, we and E single-nucleotide families In same (p.Glu750Lysfs*28)). the (c.2248delG harbored D and C Families 10 bars, Scale signal. hormone and CTD IGSF1 of colocalization indicate Arrows pituitary. mouse E18.5 wild-type in performed was gonadotropes) LH, lactotropes; prolactin, somatotropes; GH, thyrotropes; (TSH, hormones pituitary anterior indicated the and ( by detected as progenitors IGSF1 of 2 Figure ( gonadotropes in not but lactotropes and somatotropes thyrotropes, tis membrane plasma the (CTD) to domain traffics C-terminal the only that such proteolysis, lational immunoglobulin loops 5 and 6 targets the protein for obligate cotrans ( tail C intracellular short immun C2-type glycoprotein family b Fig. 2 Fig. kDa MW c 100 150 250 a ) Immunofluorescence using antibodies against the IGSF1 CTD CTD IGSF1 the against antibodies using ) Immunofluorescence 25 37 50 75 Igsf1 13– The identified identified The IGSF1 kDa MW 100 150 250 100 150 250 100 150 250 1 2 1

mRNA in human embryo Carnegie stage 18 Rathke’s pouch pouch Rathke’s 18 stage Carnegie embryo human in mRNA 7 4

pcDNA3 b ( mRNA in mouse embryonic day (E) 12.5 embryo and and embryo 12.5 (E) day embryonic mouse in mRNA ), map to conserved regions of the gene, are not present in in present not are gene, the of regions conserved to map ),

) or in testis (data not shown). not (data testis in or ) Supplementary Fig. 2). Fig. Supplementary IGSF1 is expressed in anterior pituitary gland. ( gland. pituitary anterior in expressed is IGSF1 noe a lsa ebae muolbln super immunoglobulin membrane plasma a encodes P P 4 3 WT pcDNA3 E E WT 6 5 A713_K721 A713_K721 a

S863F 8 7 o DVANCE ONLINE PUBLICATION ONLINE DVANCE lbln op, tasebae oan n a and domain transmembrane a loops, globulin C947R IGSF1 P P

1 9 S863F S770N 11 IB: Total lysates IB: HA Total lysates IB: HA IP: HA Streptavidin-HRP IP: HA E E 1 0 , Fig. 2 Fig. 1 β 1 1 -actin 2 mutations are summarized in in summarized are mutations in situ in 12

W977* 12 has protein IGSF1 canonical The . Fig. 1 Fig. 3 * * IGSF1 CTD a β -acti ) and in adult pituitary gland and tes and gland pituitary adult in and ) Immatur Mature hybridization. Scale bars, 10 10 bars, Scale hybridization. c IGSF1 protein is detected in mouse mouse in detected is protein IGSF1 n ). A hydrophobic linker separating separating linker hydrophobic A ). 1 3 . Human Human . e b W977* S863F A713_K721 WT pcDNA3 Non-permeabilize µ IGSF1 m.

Nature Ge Nature and mouse mouse and d Supplementary Supplementary a ) Expression ) Expression Permeabilize iue 1 Figure µ m. m. n IGSF1 etics Igsf1 d

c - - - .

© 2012 Nature America, Inc. All rights reserved. staining pattern in permeabilized cells with all constructs ( constructs all with cells intracellular strong permeabilized in a pattern staining observed we contrast, In shown). not (data 3b Fig. ( permeabilized cells transfected with vectors expressing IGSF1 ( p.Trp977* protein ciency and with distinct glycosylation patterns compared to wild-type effi poor with membrane plasma the reached proteins mutant that 3b Fig. Supplementary IGSF1 Ala713_Lys721del and the three missense mutants ( ( IGSF1 wild-type for construct the with transfected cells non-permeabilized in observed was staining Membrane CTD. IGSF1 the of terminus N the against expressed proteins by immunofluorescence with an antibody directed with constructs encoding wild-type and mutant IGSF1 and detected the ER. the in retained be to appeared mutants IGSF1 identified the glycosylation, of patterns their of basis the On IGSF1. wild-type to relative truncated were and sugars immature possessed J and ( (p.Glu750Lysfs*28) (p.Glu1200Argfs*3) D and C families in mutants frameshift p.Trp1173*, 11–13; lanes B and I mutants in (p.Trp977*; families The nonsense 6–11). K); (family Ser770Asn and H) (family G; 3 Fig. (family Ser863Phe IGSF1 for observed were patterns migratory ( form immature an as predominantly in given are sequences primer ( and immature endoplasmic reticulum (ER)-resident forms of the CTD ( analysis blot cells. Wild-type IGSF1 migrated as a HEK293 doublet ofheterologous in 140–150 mutants kDa IGSF1 in of protein regulation translational ( region coding identified mutations in families A–D and G–K map to the IGSF1 CTD ( F) and complete with individuals of those to lar was determined by two-tailed Student’s wild-type and and ( ( levels in adult wild-type and in male wild-type and (WT) and hypothyroidism. ( Figure 4 Nature Ge Nature and n d a Fig. 3 Fig. Fig. 3

= 14–16 per genotype). ( ) Serum total T3 concentration in adult wild-type and Tshb mRNA levels To assess plasma membrane trafficking, we transfected HEK293 cells The phenotypes of cases with intragenic mutations are highly simi

Igsf1 (relative to WT) Supplementary Fig. 3b Fig. Supplementary 0.5 1.0 1.5 a a 0 b ; lanes 8–10) and the other two missense mutants (Cys947Arg mutants (Cys947Arg and two ; missense the other 8–10) lanes ). We did not detect secreted proteins in the culture medium medium culture the in proteins secreted detect Wenot ). did , lanes 3 and 4)

) ) or the other truncated forms of the protein ( Table ∆ Igsf1 Igsf1 i. 3 Fig. ex1 n mice ( Igsf1 etics ∆ ∆ ex1 WT ex1 Fig. 3 Fig. 1 a Fig. Fig. 1 c ), suggesting loss of IGSF1 function in all cases. The The cases. all in function IGSF1 of loss suggesting ), mice have several characteristics of central ) Pituitary mice ( . ebae inl wr nvr eetd n non- in detected never were signals Membrane ). ∆ ex1 n Genotype

= 6 per genotype). ( Igsf1 mice ( a ADVANCE ONLINE PUBLICATION ONLINE ADVANCE 1 c , lane 2), reflecting the mature N-glycosylated N-glycosylated mature the reflecting 2), lane , Supplementary Fig. 3a Fig. Supplementary 3 Fig. 3 Fig. ). ). We and post- expression examined therefore n . In contrast, IGSF1 Ala713_Lys721del (family A; = 6 per genotype). ( ); however, cell surface biotinylation showed showed biotinylation surface however,cell ); ∆ e ex1 Igsf1 Tshb ) Igsf1 n Supplementary Fig. 3a Fig. Supplementary Trhr = 5 per genotype). Statistical significance mice ( b ), confirming ), ). A similar pattern was observed with with observed was pattern similar A ). ∆ mRNA levels in 12-week-old wild-type ∆ ex1 mRNA levels in 12-week-old wild-type ex1 Supplementary Table 5 Table Supplementary mice ( n = 6 per genotype). ( t test in each panel. f b ) Supplementary Fig. 3a Fig. Supplementary Trh TSH (µg) n 0.8 0.2 0.4 0.6 Fig. 3 Fig. = 18–22 per genotype). 0 mRNA levels in 12-week-old b IGSF1

) Pituitary TSH content expression of the mutant the of expression , lanes 3–5 and 12–14) 12–14) and 3–5 lanes , a , lanes 5–7). Similar Similar 5–7). lanes , deletion (families E (families deletion WT Igsf1 , lanes 17–19) and and 17–19) lanes ,

c Supplementary Supplementary P Genotype ) Serum TSH ∆ <0.0014 ex1 mice Fig. ) migrated migrated ) Igsf1 Fig. Fig. 3 Fig.

3 , lanes , lanes

b ∆ and ex1

3 a b - - ,

e reduced, and hypothalamic pituitary idea, this Consistent with signaling. ( ( secretion and ( controls in the majority of cases, and thyroid histology appeared normaldeficient males ( Fig. 5b ( TSH and proteins. Thus, the identified mutations proteins. impair Thus, block or the identified substantially trafficking of the IGSF1 CTD, consistent with a loss of protein identified human enlargementtesticular and variable prolactin and GH deficiency. The in mutations loss-of-function unresolved. are cases our in ment Fig. 5a mal testicular size and FSH concentration (ref. cases six ( only in range reference the exceeded but LH of that than in vivo and binding recent more by challenged been has coreceptor inhibin an as IGSF1 of role putative the However, secretion. FSH enhanced therefore, and, activity inhibin of loss to linked be might cases our coreceptor display several characteristics of central hypothyroidism. weeks; 12 at g 0.3 males were heavier than their Finally,control shown). not littermates (data altered not (29.9 was expression ( controls to relative mice deficient including genes, hormone-encoding Tshb pituitary various for levels in func thyroid tion and pituitary examined we hypothyroidism, central trafficking. membrane plasma CTD IGSF1 c Supplementary Fig. 5c Fig. Table Fig. Trhr mRNA levels (relative to WT) Serum TSH (mU/l) In summary, our data delineate a new X-linked disorder in which which in disorder X-linked new a delineate data our summary, In inhibin as a pituitary to function hypothesized initially was IGSF1 and function IGSF1 of loss between link causal a establish To 0.5 1.0 1.5 100 150 200 250 50 0 0 Supplementary Fig. 5a Fig. Supplementary , did not differ between controland between differ not did ,

4b content was significantly reduced in adult in reduced significantly was content 4 a ). Whereas circulating T3 concentrations were decreased in data , 2 e ) in , ). Moreover, male c ). ). Therefore, at present, the mechanisms of testicular enlarge Igsf1 ). Pituitary prolactin content was unaffected ( 15 WT Igsf1 17 WT , 18 , dfcet mice -deficient 1 9 P , Fig. 4b Fig. Genotype Fig. 4 1 P . In our cases, serum FSH concentration was higher higher was concentration FSH serum cases, our In . Genotype <0.0001 -deficient mouse pituitaries suggested impaired TRH 9 <0.0015 IGSF1 , raising the possibility that macroorchidism in in macroorchidism that possibility the raising , P = 0.004). Thus, adult male male adult Thus, 0.004). = d ), T4/FT4 concentrations were similar to those of , Igsf1 c , defects impair either expression or membrane Igsf1 d ) in spite of normal normal of spite in ) and data not shown). Reduced TSH synthesis Igsf1 ∆ ∆ Trh ex1 ex1 ). In contrast, both pituitary and serum serum and pituitary both contrast, In ). 1 -deficient mice are fertile and have nor IGSF1 7 mRNA levels were increased, in ( upeetr Fg 4 Fig. Supplementary d f Fig. 4e Fig. Trh mRNA levels (relative to WT) Serum total T3 (ng/dl) cause central hypothyroidism, hypothyroidism, central cause 100 150 200 0.5 1.0 1.5 2.0 50 0 0 Igsf1 , f ). Pituitary Pituitary ). Tshb -deficient mice ( mice -deficient Trhr 17 and WT WT Igsf1 Igsf1 mRNA expression mRNA mRNA levels weremRNAlevels P P Genotype Genotype ± s r e t t e l <0.0192 <0.0009 -deficient males males -deficient 0.4 versus 28.2 Supplementary Supplementary Supplementary -deficient mice mice -deficient Igsf1 Gnrhr . mRNA ). Igsf1 Igsf1 -deficient -deficient

function. mRNA Fig. 4 Fig. ∆ ∆ ex1 ex1 Igsf1 Igsf1 ± a - - - - -

© 2012 Nature America, Inc. All rights reserved. A.S.P.v.T., W.O., S.G.K., N.R.B., N.M.A.-D., A.M.P., M.H.B., M.T.D.,R.C.H., Y.S., J.T.d.D., M.K., N.S. and K.C. developed the exome sequencing protocol. M.T.D.). (to Charity Children’s Hospital Street Ormond Great the and M.B.); to (GR-2008-1137632 IRCCS Italiano Auxologico Istituto and Health of Ministry Italian the of Younggrant the Investigator (D.G.D.B.); Foundation Research Growth Dutch T.M.E.D.); the (to Fellowship Practitioner Australia of Council Research Medical P.l.T.); to and Health National a U117570590 (MRC; Council Research Medical UK the and K.C.) and N.S. (to Centre Research Biomedical Cambridge Research Health of Institutes J.P.M.-B.), National the to 086545 and J.K.W.;078432 and E.C. to 084361, WT077157/Z/05/Z K.C.; and N.S. to Wellcome Trust (095564 the from grants D.J.B.); Award (to Senior Boursier Chercheur (FRSQ) Québec du Santé en Recherche la de Fonds a and 341801-07 Grant Discovery NSERC B.B.), Award (to Research Doctoral (NSERC) Council Research Engineering and Sciences National a Y.S.); (to Council Scholarship China the from grant a by part in supported was work Our participating. for families their and subjects We the thank families. UK the from data of analyses bioinformatics preliminary and sequencing undertook who URLs), Hub (see Informatics and Sequencing Region Eastern The We data. acknowledge clinical providing in Radetti G. and Graaff de L.C.G. Schroor, E.J. J. Gosen, Zwaveling, N. of help the We samples. acknowledge We S. thank references. age providing for and AMH and B inhibin serum measuring for We F.J. Y. thank advice. and Jong and de Rijke de support technical for Meerten M. E. Escudero, A. Moreno, J.C. Bikker, H. We thank also respectively. RNA, pituitary and testis human for Amsterdam) of University Center, Medical P. and (Academic University) Bisschop (McGill Kimmins S. and CTD IGSF1 to antibody the for P. mice, in TSH Scheiffele and FT4I T3, T4, measuring for DK15700) grant (NIH) Health of Institutes National US by supported Chicago, of University (The laboratory Refetoff the from Liao X.-H. thank authors The Note: and LOVD3 database: Accession codes. the pa the of in version available are references associated any and Methods Me lovd.nl/igsf g SeattleSeqAnnota com/gmap http://samtool ww gov www.10 www.ncbi URLs. and pituitary in function. testicular IGSF1 for role relevant clinically and unexpected completely a uncover observations our Collectively, benefit. clinical of substantial thyroxinebe will treatmentmembers family of affected and screening biochemical cases, future of ascertainment genetic ing risk, which can be reversed by thyroxine treatment cardiometabolic adverse with associated is hypothyroidism clinical sub untreated but severe, less was hypothyroxinemia cases, other In cases, with thesome attendant in risk hypothyroxinemia of neurodevelopmentalprofound with delay, associated if was untreated. function perhaps secondary to impaired TRH receptor signaling. Loss of IGSF1 Igsf1 s r e t t e l AUTHOR CONTRIBUTIONS A enetics.bwh.harvard.

cknowledgments

Losekoot, E. Endert, J.W.A. Smit, R.van Rijn and E.L. and Rijn J.W.A. R.van Endert, Smit, E. Losekoot, w.umd.be/HSF / th ht ; UniProt,

Supplementary deletion deletion decreases pituitary and circulating TSH content in mice, tp://databases.lovd. RefSeq, RefSeq, od 00genomes.org .nlm.nih.gov/project

Tran X. and s / 1 ; SeattleSeq Annotation, Annotation, SeattleSeq ; . http://www.un s.sourceforge.net http://www. X-chromosome sequencing data were deposited at the pe

tion131 information http://database / ; BWA, r .

Zhou for assistance with collection of mouse serum serum mouse of collection with assistance for Zhou edu/pph2 / ; HapMap Project, / ; SIFT, nl/shared/individual http:/

ncbi.nlm.nih.gov/Ref is iprot.org

available s/SNP / /bio-bwa.sourceforge s.lovd.nl/shared/ind http://sift / ; ; LOVD IGSF1 database, GMAP, ;

/ in / ; 1000 Genomes Project, ; Project, 1000 Genomes ; Human Splicing Finder, http:/

the .jcvi.org http:

online h /snp.gs.washington.e ttp://research-pub.g //hapmap.ncbi.nlm.ni s/0000020

van Persijn-van Persijn-van van ersion / Seq ; PolyPhen-2, 20– ividuals/0000020 / 2 .net ; dbSNP, ;

2 of . . Thus, follow

the 9 / http://www . ; SAMtools,

pape

onli Aten, Aten, http:// http:// r http:// http://

ene. du/ ne ne h. 8 - - .

1. reprints/index.htm at online available is information permissions and Reprints Published online at The authors declare no competing interests.financial and M.T.D. conceived the andstudy. supervised M.T.D., D.J.B.R.C.H., and J.M.W. prepared the manuscript. D.J.B., J.M.W., K.C. and the delineation of the subject phenotypes. Y.S., B.B., N.S., A.S.P.v.T., K.C., E.P.C., M.H.B., andA.C.S.H.-K. R.C.H., M.T.D. contributed to clinical evaluations T.M.E.D., D.G.D.B.,A.C.S.H.-K., J.J.R., S.G.K., N.R.B., N.M.A.-D., A.M.P., G.C.J.H., experiments. A.S.P.v.T., W.O., W.H.S.-B., T.V., M.J.K., L.P., I.C., M.B., P.B.-P., and C.A.L.R. C.A.J.B. performed and analyzed the microarray and hybridization H.Z., J.P.M.-B. outcarried the IGSF1 expression studies in mouse and human embryos. performed measurements of pituitary TSH and prolactin content. S.N.M.G. and phenotyping. E.C., J.K.W. and M.G.W. performed mouse T4 measurements. P.l.T. andcollection construction of the figures. P.V. and M.G.W. contributed to mouse the all mouse model, supervised and analyzed pituitary gene expression; and prepared figures. D.J.B. generated analyses; maintained the mouse colony, all mouse collected tissues and plasma, the vectors expressing mutant IGSF1 and performed all associated biochemical bioinformatics analyses, mutational analysis and genotyping. B.B. generated J.M.W. the designed clinical research studies. Y.S., J.F.J.L. and N.S. performed N.S., L.P., I.C., M.B., P.B.-P., H.Z., T.M.E.D., K.C., D.G.D.B.A.C.S.H.-K., and 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING

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26 , © 2012 Nature America, Inc. All rights reserved. sequences are listed in in listed are sequences (primer to mutagenesis site-directed (Thermo; alteration) Phusion p.Ala713_Lys721del the create or QuikChange using construct the into introduced were Mutations (P. CTD IGSF1 Scheiffele). against antibody polyclonal with detection facilitate to protein mouse the match to Stratagene) (QuikChange, modified was P.Scheiffele) (from construct Myc-IGSF1-HA human the by constructs. Expression for four generations. at is known least relationship no familial although lies, deletion the around Mb (rs929590 to23 rs16979902), of suggesting a commonhaploblock ancestral same origin for the these fami identified analysis this array (Affymetrix) according to the manufacturer’s protocol. In familiesMicroarrays. C and D, Biosystems Applied the system. 3730xl with 96-capillary sequenced and primer reverse or forward pmol 10 of with mixed and (Qiagen) kit purification PCR QIAquick the with in listed are sequences (primer protocol manufacturer’s the following (Finnzyme) DNA polymerase analysis. sequence Sanger variants, using public databases to identify plausible candidates. onpreviously focused unknown,shared homozygous hemizygousorX-linked filtered out all variants present in databases and the UK 10K genomesshared projectexonic andand splice-site variants were taken forward to the filtering these step.variants. For family B,We we compared variants from the two brothers; only on applied was procedure filtering same The deletions. and insertions larger of identification enables and alignment gap allows GMAP as SAMtools, and promisingresults.We therefore variantgeneratedthe examined list byGMAP allele frequency of >1% in populations of European ancestry, which yielded no database,dbSNP, Project,HapMapLOVDGenomesand1000 an Projectwith We then filtered out all variants present in the local, in-house exome sequencing basis of their function; only exonic and splice-site variants were taken forward. Filtering strategy. Toolkit Analysis Genome the Hub using Informatics and Sequencing Region Eastern at the undertaken were analyses and preliminary for sequencing experiments, out Whole-exome mutation screening. variants. filtered were all Project Genomes annotate 1000 to the and applied dbSNP131 in Variants was 131 Annotation SeattleSeq calling). by (alignment) detected GMAP v3. 2011-03-28 (ref. 0.1.9 SAMtools with performed was Burrows- by (single-nucleotide variant (SNV) and insertion and/or deletion (indel)) hg19 calling genome (ref. reference (BWA)0.5.8 to Aligner Wheeler aligned were reads data, exome annotation. and calling variant mapping, Read Biosystems). (Applied 4 system SOLiD the with performed was Sequencing Technologies). (Agilent kit 50Mb Exon All Human in SureSelect the using undertaken shown was sequencing is An reads. reads of paired-end 51-bp overview generated sequencing IIx Analyzer Genome that DNA was hybridized with half of the except amount of suggested protocol, probe. Illumina manufacturer’s the to according library capture nucleotide oligo chromosome X Agilent’s SureSelect using out carried was enrichment Exome enrichment and next-generation sequencing. Italy). (Milan, Italiano Auxologico Istituto IRCCS the and Netherlands) The Street Hospital (London, UK), Erasmus University Medical Center (Rotterdam, The Netherlands), Anglia and (Amsterdam, Oxford MREC Center (Cambridge, Medical UK), Great Academic Ormond Netherlands), The (Leiden, Center Medical University Leiden of committees ethics the by approved was study The consent. informed written provided participants All protocol. facturer’s manu the following reagents, associated and (Qiagen) system LS Autopure isolation. DNA O Nature Ge Nature Centre). Innovation Québec Genome and University (McGill sequencing NLIN E E M n We performed analysis on the cytogenetic whole-genome 2.7M 2.7M whole-genome We on cytogenetic the analysis performed E etics TH DNA was isolated from human whole blood using the the using blood whole human from isolated was DNA In family A, variants shared by both boys were classified on the OD Supplementary Table 5 Supplementary S The epitope for the antibody to IGSF1 CTD encoded to encoded CTD IGSF1 for antibody the epitope The upeetr Tbe 4 Table Supplementary PCR was performed using Phire Hot Start II II Start Hot Phire using performed was PCR upeetr Tbe 1 Table Supplementary 28). BAM file manipulation and variant variant and manipulation file BAM 28). 3 1 . 29). The deletion in family A was was A family in deletion The 29). 3 ). Constructs were verified by verified were Constructs ). 0 and SAMtools (variant 0.1.9 ). Products were purified purified were Products ). For the X-chromosome X-chromosome the For X-chromosome exome . Whole-exome Whole-exome . - -

washed three times with PBS. Secondary antibody (1:600 dilution in 5% BSA BSA 5% in dilution (1:600 antibody Secondary PBS. with times three washed CTD (1:500 dilution in 5% BSA in PBSX) was applied to cells for 2 1 h. for Cells werePBSX in BSA 5% in incubated and (PBSX) PBS Triton in 0.3% in X-100 incubated PBS, in times PFA, three 4% using washed aqueous medium with DAPI. Under permeabilizing conditions, cells using mounted were and PBS fixed with times three washed were cells and h, 1 for cells to applied was PBS) in BSA 5% in dilution (1:600 antibody for 1 Secondary h. were Cells with then three times washed PBS and in incubated 5% BSA in PBS (PFA). paraformaldehyde 4% with fixed and DMEM serum-free with times at 37 °C in 5% CO to IGSF1 CTD (1:500 dilution in serum-free DMEM) was applied to cells for 2 h immunofluorescence. Cell were blots described previously as Protein performed instructions. manufacturer’s the to according Biolabs), and (New EndoH England PNGaseF with were Protein lysates deglycosylated 10 mM NaPO fluoride, RIPA using mM 50 NaCl, mM (150 sodium buffer transfection after Protein blotting. vector.expression of ng 100 with transfected and well per cells 50,000 with coverslips glass on plates 24-well in plated were cells HEK293 immunofluorescence, For tions. manufacturer’s instruc the following (Invitrogen) 2000 Lipofectamine using vector expression of ng 200 with transfected and plates 6-well in plated were culture. Cell (IMAGE). Human embryonic material was provided by the Human Human the by Wellcome and Trust 082557. Grant provided Council G0700089 Grant Research Medical was by supported Resource, material Biology Developmental embryonic Human (IMAGE). described situ In (McGill institutional with guidelines. federal and University) accordance in performed was collection blood Animals. Plus-ECL Lightning in Western the washes (PerkinElmer). using kit 10-min with visualized was times signal three and TBST, washed was Membrane (Vector) min. kit 30 Vectastain for the from reagents TBST B in and A of milk each 2.5% drops in two with incubated was protein membrane for blocking, processed After then blotting. were Lysates instructions. manufacturer’s to the according Sigma) (E6779, gel affinity anti-HA EZview using out was carried immunoprecipitation and Lysates s). collected was 10 for supernatant W (9 centrifuged, were sonication by lysed mM 1 and NaCl, mM X-100) 150 Triton 7.5, 1% pH EDTA, Tris, mM (50 buffer in lysis in glycine mM 100 harvested with PBS, times three washed EZ-link-sulfo-NHS-LC-biotin °C, 4 at mg/ml min 30 for 0.5 of (Thermo) ml 1 in incubated and PBS biotinylation. surface Cell mounted. and PBST with times three washed h, 1 for dilution) (1:600 rabbit to antibody secondary goat 488–conjugated Alexa and buffer) blocking in dilution (1:400 Red Streptavidin–Texas in incubated goat dilution (1:150 in for buffer) blocking 1 h, washed three times with PBST, three washed with times PBST, to antibody were in horse secondary incubated biotin-conjugated Sections °C. 4 at overnight buffer) blocking in dilution, 1:200 (sc7805) prolactin and (sc10365) GH and dilution, 1:400 (sc7824), LH and CTD, TSH (sc7815) and dilution, 1:500 (from Santa Biotechnology) Cruz (IGSF1 antibodies primary with incubated were Sections (PBST). Tween-20 described 5 with sliced sections with and sacrificed, embryo were heads were E18.5 fixed at overnight mice in 4% Pregnant PFA E0.5. at 4 °C considered and was embedded in paraffin, detected immunofluorescence. Pituitary conditions. non-permeabilizing under mounted and PBS with times three for applied were was 1 washed h, and in PBSX) cells hybridization.

3 3 Igsf1 3 4 . Nonspecific . binding Nonspecific was blocked using 5% BSA in PBS with 0.2% using riboprobes from human human from riboprobes using For protein blotting and cell surface biotinylation, HEK293 cells cells HEK293 Forbiotinylation, protein surface and blotting cell 4 , , 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). -deficient mice have been described previously described been have mice -deficient Whole-cell extracts were prepared from transfected cells 24 h 2 . Cells . were to Cells transferred room temperature, three washed

µ situ Underconditions, antiserum non-permeabilizing Transfected cells were washed three times with with times three washed were cells Transfected m thickness. Sections were treated as previously previously as treated were Sections thickness. m hybridization was performed as previously previously as performed was hybridization The morning when a vaginal plug was was plug vaginal a when morning The 3 2 . IGSF1 cDNA clone 30387876 30387876 clone cDNA

h. Antiserum to IGSF1 IGSF1 to Antiserum h. doi:10.1038/ng.2453 1 7 . Tissue and and Tissue . -

© 2012 Nature America, Inc. All rights reserved. scribed as previously described previously as scribed 1.44 and below, and mm 2.5 dorsoventral-interaural mm, to 0.94 3.94 coordinates following anteroposterior-interaural the to according blocks hypothalamic 3-mm and PCR. Quantitative described as (FT4I) index T4 free the as expressed and uptake T4 resin to T4 total of ratio the from estimated were described previously as measured was concentration TSH RIAcoated-tube (Siemens adapted Medical Solutions for Diagnostics) mouse. Peptide Program). In Chicago, total T4 serum and T3 levels were bymeasured described interassay and intra- previously published were used; variations was assay II Gen the concentration, AMH For Coulter). (Beckman methods immunometric enzyme using estimated were B concentrations and AMH inhibin Serum respectively. and 5.1%, 3.7% were using Immulite luminescence intra- 2000 and (Siemens); interassay variations by chemi was analyzed Insulin respectively. and were 10.8%, 9.2% variations interassay and intra- RIA; by in-house analyzed was concentration tosterone tes Plasma (GH). and and 6.2% 3.8% (prolactin) and 5.3% 3.4% (TSH), 5.7% tions, respectively, were 5.1% and 6.8% (FT4), 7.7% and 11.3% (FT3), 4.2% and intra- and interassay varia 1232 Fluorometer using (PerkinElmer); the Delfia by were fluoroimmunoassay and analyzed GH concentrations prolactin TSH, FT3, FT4, respectively. 7.8%, and 6.3% were variations interassay and intra- Plasma T3 <5%. concentration were was measured by variations in-house radioimmunoassay (RIA); interassay and intra- E170; Roche a on analyzed were concentrations FSH and LH Plasma measurements). (other Center Medical Academic the at AMH) Radiochemistry and and B Endocrinology of (inhibin Laboratory the Center and Medical Erasmus at the Laboratory in investigated Endocrine were samples follow-up, At departments. participating the of laboratories the in determined were concentrations prolactin and T3 measurements. concentration Hormone mouse of 18–20 exons ning RNA (13 Each Kimmins. S. from obtained was RNA testicular Human purposes. research for information clinical and material brain human of use and autopsy brain a for permission formal with accordance in obtained was and 02-055) (NBB; RNAblotting. doi:10.1038/ng.2453 Mouse pituitary TSH and PRL content measurement was previously previously was measurement content PRL and TSH pituitary Mouse 3 7 and used reagents supplied by A.L. Parlow (National Hormone and Human pituitary RNA was from the Netherlands Brain Bank Bank Brain Netherlands the from was RNA pituitary Human µ g) was blotted as previously described previously as blotted was g)

mm lateral. RNA (2 RNA (2 lateral. mm Mouse RNA was extracted from whole pituitaries pituitaries whole from extracted was RNA Mouse Igsf1 3 . 2 . Quantitative PCR (qPCR) was (qPCR) PCR . Quantitative 35 3 9 , . 3 6 µ . g) was extracted and reverse tran reverse and extracted was g) At diagnosis, serum FT4, TSH, TSH, FT4, serum diagnosis, At 1 7 , using a probe span a probe , using 3 8 . Free T4 levels levels T4 Free .

performed performed 4 0 - - - - - :

35. 34. 33. 32. 31. 30. 42. 41. 40. 39. 38. 37. 36. 29. 28. as defined was Statistics. were levels 2 the using Transcript protocol. manufacturer’s of to those normalized the and using (Invitrogen) HRM, UDG 6200 SuperMix qPCR ( primer of each Green pmol 0.4 SYBR Platinum using

imnm, .. d Ln vn inare, .. Otn BJ, e og FH & F.H. Jong, de B.J., Otten, R.F., Wijngaarden, van Lind de E.P., Siemensma, Gaston-Massuet, C. differentially is (Ptx1) 1 homeobox Pituitary J. Drouin, Y.& Gauthier, C., Lanctôt, of expression activin-stimulated mediate SMAD3 and SMAD2 Both D.J. Bernard, A. and McKenna, variants complex of detection SNP-tolerant and Fast S. Nacu, & T.D. Wu, Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real- using data expression gene relative T.D.of Schmittgen, Analysis & K.J. Livak, of quantitative years Twenty-five W.M. Freeman, & K.E. Vrana, H.D., VanGuilder, K.B.J. Franklin, & G. Paxinos, R.E. Weiss, Pohlenz, J. L. McGuinness, M.E. Kevenaar, Li,H. Burrows-Wheeler with alignment read short accurate and Fast R. Durbin, & H. Li, oknKeea AC Tsiua fiue n os ih rdrWli syndrome: Prader-Willi with boys in failure Testicular A.C. Hokken-Koelega, humans. and USA mice Sci. in tumors pituitary to rise gives cells stem development. pituitary during expressed hormone Endocrinol. Mol. follicle-stimulating the (2010). data. sequencing DNA next-generation analyzing reads. short in splicing (2009). 2078–2079 time quantitative PCR and the 2 the and PCR quantitative time analysis. expression gene for PCR 2001). Diego, San Press, (Academic edn hormone. thyroid to resistant hormone. thyroid to sensitivity thyrotroph and concentration thyrotropin in differences strain serum: in (2003). 720–731 vesicles secretory syndrome. ovary polycystic with women in (2012). E452–E459 hormones. reproductive of studies longitudinal transform. et al.et In In −

∆ 108 Bioinformatics ∆ Figure Figure TheSequence Alignment/Map format andSAMtools. et al. et al. et C P T , 11482–11487 (2011). 11482–11487 , < 0.05 and was determined by two-tailed Student’s by two-tailed determined was and 0.05 < t al. et method et al. et Improved radioimmunoassay for measurement of mouse thyrotropin

t al. et Mice deficient in the steroid receptor co-activator 1 (SRC-1) are (SRC-1) 1 co-activator receptor steroid the in deficient Mice 18 n vitro in 4 et al. h Gnm Aayi Tokt a aRdc faeok for framework MapReduce a Toolkit: Analysis Genome The , 606–623 (2004). 606–623 , Thyroid and all supplementary figures, statistical significance significance statistical figures, supplementary all and Variants in the in Variants Rpl19 Autosomal dominant growth hormone deficiency disrupts deficiency hormone growth dominant Autosomal Bioinformatics Supplementary Table 6 Supplementary Increased Wingless (Wnt) signaling in pituitary progenitor/ 41

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(encoding ribosomal protein L19) and analyzed and L19) protein analyzed ribosomal (encoding 2 9 , 1754–1760 (2009). 1754–1760 , . EMBO J. EMBO , 1265–1271 (1999). 1265–1271 , h Mue ri i Seetxc Coordinates Stereotaxic in Brain Mouse The n vivo in − ∆ Biotechniques ∆ C β T ACVR1 method. uui i mue oaorp cells. gonadotrope mouse in subunit

26 18 n rngnc mice. transgenic in Endocrinology , 873–881 (2010). 873–881 , , 1900–1904 (1999). 1900–1904 , Hum. Reprod. Hum. gene are associated with AMH levels AMH with associated are gene Methods

. ln Edcio. Metab. Endocrinol. Clin. J. 44 eoe Res. Genome ) on a Corbett Rotor-Gene Rotor-Gene ) on a Corbett , 619–626 (2008). 619–626 ,

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