Genotype-phenotype correlations J Med Genet: first published as 10.1136/jmedgenet-2017-105113 on 30 July 2018. Downloaded from ORIGINAL ARTICLE Mutations in IRS4 are associated with central hypothyroidism Charlotte A Heinen,1,2 Emmely M de Vries,1 Mariëlle Alders,3 Hennie Bikker,3 Nitash Zwaveling-Soonawala,2 Erica L T van den Akker,4 Boudewijn Bakker,5 Gera Hoorweg-Nijman,6 Ferdinand Roelfsema,7 Raoul C Hennekam,8 Anita Boelen,1 A S Paul van Trotsenburg,2 Eric Fliers1 ► Additional material is ABSTRact In this study, we used ‘whole’ exome sequencing published online only. To view Background Four genetic causes of isolated congenital (WES) to identify the genetic cause of isolated CeH please visit the journal online (http:// dx. doi. org/ 10. 1136/ central hypothyroidism (CeH) have been identified, in two unrelated families. We identified mutations in jmedgenet- 2017- 105113). but many cases remain unexplained. We hypothesised insulin receptor substrate 4 (IRS4) in both families. the existence of other genetic causes of CeH with a Sanger sequencing in other cases of CeH identified For numbered affiliations see Mendelian inheritance pattern. IRS4 mutations in three families. The IRS family end of article. Methods We performed exome sequencing in two acts as interface between tyrosine kinase receptors, families with unexplained isolated CeH and subsequently including the insulin, leptin and insulin-like growth Correspondence to Sanger sequenced unrelated idiopathic CeH cases. We factor 1 (IGF-1) receptors, and multiple intracel- Dr. A S Paul van Trotsenburg, 6 Department of Paediatric performed clinical and biochemical characterisation of lular signalling pathways. Although the precise Endocrinology, Academic the probands and carriers identified by family screening. function of IRS4 is unclear, its discrete expression Medical Center, Emma We investigated IRS4 mRNA expression in human in rat hypothalamic nuclei suggests a specialised Children’s Hospital, University neuroendocrine function(s).7 of Amsterdam, Amsterdam, The hypothalamus and pituitary tissue, and measured serum Netherlands; thyroid hormones and Trh and Tshb mRNA expression in a. s. vantrotsenburg@ amc. uva. nl hypothalamus and pituitary tissue of Irs4 knockout mice. METHODS Results We found mutations in the insulin receptor Senior authors AB, ASPvT and EF Patient acquisition contributed equally. substrate 4 (IRS4) gene in two pairs of brothers with CeH (one nonsense, one frameshift). Sequencing of IRS4 in In contrast to TSH-based neonatal congenital hypothyroidism (CH) screening programmes, the Received 10 January 2018 12 unrelated CeH cases negative for variants in known Dutch thyroxine (T4)+TSH+T4 binding glob- Revised 27 May 2018 genes yielded three frameshift mutations (two novel) Accepted 12 June 2018 ulin (TBG)-based screening effectively detects in three patients and one male sibling. All male carriers Published Online First 30 July primary CH and congenital CeH.8 9 Many of these 2018 (n=8) had CeH with plasma free thyroxine concentrations children are treated by the Department of Paedi- http://jmg.bmj.com/ below the reference interval. MRI of the hypothalamus atric Endocrinology of the Academic Medical and pituitary showed no structural abnormalities (n=12). Center, University of Amsterdam.10 We studied 24-hour thyroid-stimulating hormone (TSH) secretion two pairs of brothers: 18-year-old A.III.4 and profiles in two adult male patients showed decreased his 14-year-old brother A.III.5 (figure 1A), and basal, pulsatile and total TSH secretion. IRS4 mRNA 12-year-old B.III.4 and his 22-year-old maternal was expressed in human hypothalamic nuclei, including half-brother B.III.3 (figure 1B). The first three the paraventricular nucleus, and in the pituitary gland. on September 28, 2021 by guest. Protected copyright. were detected by neonatal screening and were diag- Female knockout mice showed decreased pituitary Tshb nosed with congenital CeH at the age of 2 weeks. mRNA levels but had unchanged serum thyroid hormone B.III.3, who was born before optimisation of the concentrations. Dutch neonatal screening programme for CeH, Conclusions Mutations in IRS4 are associated with had normal screening results and was diagnosed isolated CeH in male carriers. As IRS4 is involved in leptin after presenting with short stature and delayed signalling, the phenotype may be related to disrupted tooth eruption at age 12 years (table 1). In all four, leptin signalling. the diagnosis CeH was supported by thyrotro- pin-releasing hormone (TRH) stimulation testing (online supplemental table 1), while additional endocrine testing demonstrated normal func- INTRODUCTION tioning of the other hypothalamic–pituitary axes. © Author(s) (or their Central hypothyroidism (CeH) is characterised Levothyroxine (LT4) treatment was started in all. employer(s)) 2018. Re-use by thyroid hormone deficiency due to insuffi- permitted under CC BY-NC. No While A.III.5, B.III.3 and B.III.4 have grown and commercial re-use. See rights cient production of thyroid-stimulating hormone developed normally, A.III.4 had a delayed pubertal 1 and permissions. Published (TSH). Congenital CeH is often part of multiple growth. Testosterone-primed growth hormone by BMJ. pituitary hormone deficiency, but isolated TSH (GH) stimulation tests were normal. With sponta- To cite: Heinen CA, deficiency occurs in 20%–25% of cases. Known neous progression of puberty, his growth improved, de Vries EM, Alders M, et al. genetic causes of congenital isolated CeH include resulting in a normal adult height (online supple- J Med Genet mutations in TSHB, TRHR, IGSF1 and TBL1X.2–5 mental table 2). All four patients tested negative 2018;55:693–700. However, many cases remain unsolved. for mutations in TSHB, TRHR, IGSF1, and TBL1X. Heinen CA, et al. J Med Genet 2018;55:693–700. doi:10.1136/jmedgenet-2017-105113 693 Genotype-phenotype correlations J Med Genet: first published as 10.1136/jmedgenet-2017-105113 on 30 July 2018. Downloaded from Figure 1 Pedigrees of five families with IRS4 mutations. Probands are indicated by an arrow; small horizontal lines indicate that DNA sequence analysis was performed. Black filled symbols represent mutation carrying individuals. (A) Pedigree of family A, (B) family B, (C) family C, (D) family D and (E) family E. Genetic analyses Endocrine measurements Genomic DNA isolation was performed as described previously.4 Plasma free T4 (FT4) and prolactin concentrations were WES and variant calling were conducted using BWA-MEM measured by fluoroimmunoassay using the Delfia 1232 Fluo- (0.7.5), GenomeAnalysisTK-2.8–1-g932cd3a, Cartagenia 3.0. rometer (Wallac, Turku, Finland), while total T4, triiodothy- and the SeqCap EZ Human Exome Library v3.0 kit (NimbleGen; ronine (T3) and reverse T3 (rT3) were measured by an inhouse Roche, Madison, Wisconsin, USA) on a HiSeq2000 (Illumina, radioimmunoassay (RIA).13 Plasma TSH, luteinizing hormone San Diego, California, USA). Variants were filtered and analysed (LH), follicle-stimulating hormone (FSH) and 17β-oestradiol as described previously.5 Candidate variants were confirmed by were measured by electrochemiluminescence assay using the Sanger sequencing. WES was performed in the four probands; Roche cobas e602 (Roche Diagnostics, Mannheim, Germany). Sanger sequencing was performed in all their available first-de- Plasma TBG and serum thyroglobulin were measured by a gree and second-degree relatives and in other patients with idio- RIA (BRAHMS, Thermo-Scientific Hennigsdorf, Germany). pathic isolated CeH. Written informed consent was obtained in Plasma testosterone was measured by an inhouse method using all cases. the Acquity ultra-performance liquid chromatography-tandem mass spectrometry system (Waters, Milford, Massachusetts, http://jmg.bmj.com/ USA). Serum sex hormone-binding globulin (SHBG) was Phenotyping measured with immunoluminometric assay (ILMA) using the Clinical studies Architect iSR2000 (Abbott Laboratories, Diagnostics Division, Mutation carriers underwent assessment of growth and devel- Illinois, USA). Serum IGF-1 and GH was measured with elec- opment, biochemical evaluation of hypothalamus–pituitary axes trochemiluminiscence assay using the Liaison (Diasorin, S.P.A., and glucose homeostasis, thyroid ultrasound and pituitary MRI. Saluggia, Italy). Plasma insulin, insulin growth factor binding Two patients (A.III.4 and B.III.3) underwent 24-hour blood protein 3, adrenocorticotropic hormone and cortisol were on September 28, 2021 by guest. Protected copyright. sampling to assess TSH secretion, as described earlier.11 Testicular measured by ILMA using the Immulite 2000 (Siemens Medical ultrasound was performed in all male mutation carriers aged 12 Solutions, Camberley, UK). years or older. Mutation carriers additionally underwent hearing Plasma total cholesterol was measured with enzymatic colori- assessment by pure tone audiometry (PTA) or otoacoustic emis- metric assay using the Roche cobas c502, while triglycerides and sion testing. PTA was performed in a soundproof booth, using glucose were measured with enzymatic colorimetric assay using a manual audiometer (Madsen Electronics, Taastrup, Denmark) the Roche cobas c702 (both from Roche Diagnostics). Plasma with TDH-39 headphones, calibrated according to ISO-389–1, leptin concentrations were measured with a RIA from Millipore with adequate masking.12 (Billerica, Massachusetts, USA). Figure 2 Schematic of IRS4 and the positions of the mutations. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain;