The subjects were participants in a Dutch national prospective study, running from April
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1 Supplemental Data Subjects The subjects were participants in a Dutch national prospective study, running from April 1, 1994 to April 1, Infants with neonatal screening results indicative of CH-C and subsequent plasma free T 4 (FT 4 ) concentrations <0.93 ng/dl (<12 pmol/liter) and plasma TSH concentrations <15 µu/ml (<15 mu/liter) were enrolled in the study. Of 385,042 infants screened during the study period, 26 met the inclusion criteria. Anterior pituitary function was assessed primarily by stimulation tests. TRH and CRH tests took place on consecutive days, immediately after referral. Arginine and GnRH tests were performed at the age of 3 months, when euthyroid status had been accomplished by T 4 supplementation and, in case of ACTH deficiency, cortisol was supplemented in a physiologic dosage (12 mg/m 2 per day). In most infants with abnormal TRH results, MRI of the brain (with 3D imaging of the hypothalamus-pituitary region) was also performed at the age of 3 months. Perinatal characteristics and early endocrine test results of these subjects are summarized in Table 1. See reference (9) for detailed study design and overview of function test results. In 2001 and 2006/7, all cases were re-evaluated for revised diagnoses, additional morbidity, growth and treatment data. The study protocol was approved by the Dutch Pediatric Endocrine Society and by the Medical Ethics Committees of the participating centers. Parental informed consent was obtained in all cases. Of the 26 infants enrolled in the study, 5 (subjects 22-26) were excluded from the statistical calculations. One was excluded because of transient CH-C, secondary to gestational maternal hyperthyroidism, as a consequence of untreated Graves' disease (16). Three, referred after the age of 3 months, were excluded to avoid possible disturbance of the test results by age differences. One infant with neonatal screening results and plasma TSH and FT 4 concentrations indicative of CH-C, died of cardiorespiratory insufficiency before further
2 endocrine tests could be performed. The 5 excluded cases are described in a separate section below. The remaining 21 subjects underwent a TRH test at a median age of 39 d (range d). Six of the children referred to us during the study period (subjects 1-6), turned out to have false-positive screening results, non-thyroidal illness (NTI) or possibly transient CH-C. All six had normal (type 0) TRH test results (Fig. 1A) and all had normal baseline FT 4 plasma concentrations, i.e ng/dl ( 12 pmol/liter) at the day of testing. All were screened at the appropriate moment and underwent a TRH test at a median age of 44 d (range d). After 5 yr of follow-up none of the 4 living patients had developed signs or symptoms of pituitary hormone deficiencies. The assumed reasons for the discrepancy between their abnormal screening results and the normal function test results were diverse: Subject 1, a girl with TBG deficiency and erroneous initial FT 4 determination, had normal CRH test results and normal baseline IGF-I plasma concentration. Subject 2, a boy with perinatal streptococcal meningo-encephalitis with sinus thrombosis, extensive infarction in the a. cerebri posterior area and slightly dilated ventricles, may have had NTI or transient CH-C due to elevated intracranial pressure or arterial insufficiency at the time of screening. Subject 3, a boy with diminished FT 4 plasma concentration [0.83 ng/dl (10.7 pmol/liter)], without elevated plasma TSH and with normal CRH test results and normal baseline IGF-I plasma concentration had normal TSH and FT 4 concentrations at long-term follow-up. Subject 4, a boy with persistent low to low normal FT 4 values [ ng/dl ( pmol/liter)], normal CRH test results and normal baseline IGF-I and testosterone plasma concentrations had no clinical signs of hypothyroidism. He showed normal growth and psychomotor development at follow-up.
3 Subject 5, a boy with diminished FT 4 plasma concentration [0.89 ng/dl (11.5 pmol/liter)], without elevated plasma TSH, normal CRH test results and normal baseline IGF-I and testosterone plasma concentrations, showed normal growth and psychomotor development and normal TSH and FT 4 concentrations at long-term follow-up. Subject 6, a girl with subependymal hemorrhage and parenchymal hemorrhage in the glandula pinealis region and intractable epilepsy (Ohtahara type), may have had NTI or transient CH-C due to elevated intracranial pressure or arterial insufficiency at the time of screening. The remaining 15 infants (subjects 7-21) had abnormal TRH test results and were found to have permanent CH-C during 10-yr follow-up. Of those infants, 9 (60%) had evidence of glucocorticoid deficiency, 10 (67%) had evidence of GH deficiency and 6 (40%) had evidence of gonadotropin deficiency. None of the subjects studied had symptoms indicating disturbance of posterior pituitary function to date (9). Subjects excluded from the statistical calculations (subjects 22-26) Subject 22, a boy of Chinese descent, born in The Netherlands at 40.9 wk of gestation to a mother with untreated Graves' disease, had abnormal TRH test results at the age of 18 d (type 2 response: TSH peak value of 4.4 µu/ml (4.4 mu/liter). At the day the TRH test took place, FT 4 had spontaneously increased to 0.91 ng/dl (11.7 pmol/liter), just below the cut-off level of 0.93 ng/dl (12 pmol/liter). Therefore, further restoration of thyroid function was awaited. There were no apparent disturbances of other hypothalamus-pituitary endocrine axes. From 5 wk until 13 months of age, TSH and FT 4 plasma concentrations were within the normal range. TRH testing at the age of 13 months demonstrated a normal (type 0) TSH response (9). The course of both TRH tests is depicted in Fig 1C. Investigations at the age of 8.5 yr, including thyroid ultrasound imaging, revealed normal thyroid function and morphology.
4 Subject 23, a boy not referred and tested before the age of 2 yr despite abnormal neonatal screening results, had abnormal results of tests of thyrotropic, adrenocorticotropic and somatotropic function. Gonadotropic function could not be tested at the age of referral. MRI of the brain showed PPE. Subject 24, a boy of Indian descent, born in India, was screened at the age of 4 months, after the family had moved to The Netherlands. His TRH test, at the age of 138 d, did not show any increase of either TSH (type 2 response) or PRL. In addition, he had complete GH deficiency. Adrenocorticotropic and gonadotropic axes were considered normal. MRI showed marked hypoplasia of the anterior pituitary. Molecular studies revealed 2 (novel) mutations in the POU1F1 gene.* Subject 25, a boy in whom the TRH test could not be carried out in the neonatal period as his pediatrician had already started T 4 -supplementation, was diagnosed with isolated TSHdeficiency by a TRH test at the age of 1 yr, after temporary discontinuation of T 4 - supplementation. Subject 26, a boy who died of cardiorespiratory insufficiency before endocrine tests could be performed, had multiple defects associated with a chromosomal deletion (del 6 p22 pter). Screening results and venous TSH, T 4, FT 4, T 3 and TBG concentrations were strongly suggestive of central hypothyroidism. Other pituitary hormone deficiencies were not demonstrated. Hormone assays All hormone assays regarding the H-P-T axis were performed in the laboratory of endocrinology of the Academic Medical Center, Amsterdam. TSH was measured by ICMA (immunochemiluminescence immunometric assay; Behring, Marburg, Germany); the lower limit of detection was 0.01 mu/liter TSH. The intra-assay coefficient of variation (CV) was 2% at TSH 0.5 mu/liter and 1% at TSH 7.5 mu/liter; the inter-assay variation was 3% at TSH
5 concentration 0.5 mu/liter and 4% at TSH 7.5 mu/liter. PRL was measured by ICMA (Behring, Marburg, Germany); the lower limit of detection was 1 µg/liter. The intra-assay coefficient of variation (CV) was 6.1% at PRL 6.1 µg/liter and 3.2% at PRL 28.7 µg/liter; the inter-assay variation was 8.1% at PRL concentration 6.1 µg/liter and 4.2 % at PRL 28.7 µg/liter T 4 was measured by an in-house RIA (radio immunoassay; AMC, Amsterdam; see ref. 29). The peripheral conversion of thyroxine (T4) into triiodothyronine (T3) and reverse triiodothyronine (rt3); Thesis; Amsterdam:University of Amsterdam); the lower limit of detection was 5 nmol/liter T 4. The intra-assay coefficient of variation (CV) was 2% at T 4 75 nmol/liter and 4% at T nmol/liter; the inter-assay variation was 5% at T 4 concentration 75 nmol/liter and 8% at T nmol/liter. T 3 was measured by an in-house RIA (AMC, Amsterdam; see ref. 29); the lower limit of detection was 0.3 nmol/liter T 3. The intra-assay coefficient of variation (CV) was 3% at T nmol/liter and 4% at T nmol/liter; the inter-assay variation was 7% at T 3 concentration 1.25 nmol/liter and 9% at T nmol/liter. FT 4 was measured by a two step RIA (SPAC ft 4 Fraktion, Byk-Sangtek Diagnostica, Dietzenbach, Germany); the lower limit of detection was 2 pmol/liter FT 4. The intra-assay coefficient of variation (CV) was 4% at FT 4 12 pmol/liter and 6% at FT 4 22 pmol/liter; the interassay variation was 5% at FT 4 concentration 12 pmol/liter and 8% at FT 4 22 pmol/liter. TBG was measured by RIA (Eiken Chemical, Tokyo, Japan); the lower limit of detection was 30 nmol/liter TBG. The intra-assay coefficient of variation (CV) was 2% at TBG 250 nmol/liter and 4% at TBG 540 nmol/liter; the inter-assay variation was 6% at TBG concentration 250 nmol/liter and 8% at TBG 540 nmol/liter. Magnetic resonance imaging of the brain To evaluate pituitary morphology, MRI studies were performed under general anesthesia, using a 1.5 Tesla Siemens Magnetom. Transversal (5 mm), sagittal (3 mm) and
6 coronal (3 mm) T1-weighted spin-echo images were obtained. In the transversal plane also proton density as well as T2-weighted turbo-spin echo sequences were used. In addition, a T1-weighted 3D-series (MPRage) was taken. Fourteen patients underwent MRI of the brain around the age of 3 months. Eight (57%) had posterior pituitary ectopia (PPE), a picture characterized by a hypoplastic anterior pituitary lobe, located in the sella turcica, invisible or very thin pituitary stalk, and an ectopic posterior pituitary lobe, located at the median eminence in the floor of the third ventricle. Six patients (43%) had additional cerebral abnormalities, such as agenesis of the corpus callosum, bilateral periventricular nodular heterotopia, and/or hydrocephalus (9). All patients with PPE had MPHD (see Table 2). * Hendriks-Stegeman BI, Augustijn KD, Bakker B, Holthuizen P, van der Vliet PC, Jansen M 2001 Combined pituitary hormone deficiency caused by compound heterozygosity for two novel mutations in the POU domain of the Pit1/POU1F1 gene. J Clin Endocrinol Metab 86:
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