A novel IGSF1 mutation in a large Irish kindred highlights the need for familial screening in the IGSF1 deficiency syndrome

Summary Objective Loss‐of‐function mutations in IGSF1 result in X‐linked central congenital hypothyroidism (CeCH), occurring in isolation or associated with additional pituitary hormone deficits. Intrafamilial penetrance is highly variable and a minority of heterozygous females are also affected. We identified and characterized a novel IGSF1 mutation and investigated its associated phenotypes in a large Irish kindred. Design, Patients and Measurements A novel hemizygous IGSF1 mutation was identified by direct sequencing in two brothers with CeCH, and its functional consequences were characterized in vitro. Genotype‐phenotype correlations were investigated in the wider kindred. Results The mutant IGSF1 protein (c.2318T > C, p.L773P) exhibited decreased plasma membrane expression in vitro due to impaired trafficking from the endoplasmic reticulum. Ten hemizygous males and 11 heterozygous females exhibited characteristic endocrine deficits. Ireland operates a TSH‐based CH screening programme, which does not detect CeCH; therefore, genetic ascertainment preceded biochemical diagnosis of moderate CH in five of seven boys as well as their 75‐year‐old grandfather. Clinical features potentially attributable to hypothyroidism were variable; normal free T3 (FT3) and low/low normal reverse T3 (rT3) concentrations suggested that preferential deiodination of FT4 to FT3 may help maintain tissue euthyroidism in some individuals. However, neonatal jaundice, delayed speech or growth, and obesity were observed in seven subjects in whom diagnosis was delayed. Conclusions As observed with other IGSF1 mutations, p.L773P results in variably penetrant IGSF1 deficiency syndrome. Our observations emphasize the need for multi‐generation genetic ascertainment in affected families, especially where TSH‐based CH screening programmes may fail to detect CeCH at birth.


| INTRODUC TI ON
Central congenital hypothyroidism (CeCH) is a rare entity affecting up to one in 16 000 individuals, 1  in TBL1X or IGSF1 may present as isolated TSH deficiency. 2,[4][5][6] Since the initial description of IGSF1 mutations in eleven European kindreds, larger studies have substantiated the complex nature of the IGSF1 deficiency syndrome as well as confirming the relatively frequent occurrence of IGSF1 mutations in CeCH cases. 4,7 IGSF1 encodes a transmembrane immunoglobulin superfamily glycoprotein that undergoes co-translational proteolysis such that only its seven carboxyterminal immunoglobulin loops are expressed extracellularly at the plasma membrane. 8 The majority of previously reported IGSF1 mutations adversely affect trafficking and membrane localization of this carboxy-terminal domain 9 ( Figure 1A). IGSF1 is abundantly detected at mRNA level in Rathke's pouch and adult pituitary gland 4 ; however, a paucity of reliable antibodies has hampered expression studies in humans. In rodents, differential antibody usage has yielded divergent results; IGSF1 protein has been detected in all cells of the Pou1f1 (Pit1) lineage in murine and rat pituitaries using one custom IGSF1-CTD antibody 4,10 ; however, a different, commercially available anti-IGSF1 antibody (Genetex) localized IGSF1 to thyrotropes and gonadotropes in rats, but not somatotropes or lactotropes. 11 Despite clinical and murine data supporting a role for IGSF1 in regulation of TRH action in the pituitary, its molecular function remains undefined. 4,12,13 Hormone deficiencies associated with IGSF1 mutations may involve all cells of the POU1F1 lineage. Hemizygous males almost universally exhibit central hypothyroidism and 60% have basal hypoprolactinaemia; a minority (~15%) exhibit transient childhood growth hormone (GH) deficiency. 9 Endocrine evaluation of heterozygous females usually reveals a milder phenotype with FT4 concentrations in the lower tertile of the normal range, although 18% do exhibit overt central hypothyroidism and 22% have subnormal basal prolactin. 7,9 Affected boys exhibit delayed pubertal testosterone rise and growth spurt, associated with preserved testicular growth and development of macroorchidism from late adolescence onwards. 4,9 Additional features include raised BMI, and mildly elevated or highnormal adult IGF-1 concentrations. 7,9 Biochemical and physiological severity of CeCH are variable in IGSF1 deficiency (including in congenic mouse strains), and some individuals tolerate lifelong thyroid hormone deficiency without apparent adverse consequences, whereas others present symptomatically early on. 4,9,11 Here, we describe a large Irish kindred in which two male siblings with CeCH were found to harbour a novel missense IGSF1 mutation. In vitro evaluation of the mutant IGSF1 protein demonstrated reduced plasma membrane expression. Family screening identified eight additional hemizygous males and 11 heterozygous females who subsequently underwent endocrine evaluation. Tissue manifestations of hypothyroidism reflect the pleiotropic effects of thyroid hormone and in childhood may include growth retardation, delayed bone age or neurodevelopmental milestones, and prolonged neonatal jaundice. Affected children and adults may also exhibit bradycardia, hypothermia, overweight and dyslipidaemia and may describe constipation and fatigue. 14 Biochemical severity of hypothyroidism was usually moderate in our kindred and some cases appeared to tolerate hypothyroidism remarkably well, whereas seven individuals exhibited adverse sequelae potentially attributable to thyroid hormone deficiency. Our observations support the notion that although some individuals are apparently asymptomatic despite significant central hypothyroidism, family screening in this context remains crucial in enabling prompt diagnosis in cases where growth and development may otherwise be impaired. normal free T3 (FT3) and low/low normal reverse T3 (rT3) concentrations suggested that preferential deiodination of FT4 to FT3 may help maintain tissue euthyroidism in some individuals. However, neonatal jaundice, delayed speech or growth, and obesity were observed in seven subjects in whom diagnosis was delayed.

| MATERIAL S AND ME THODS
The study was approved by Cambridge South REC (MREC 98/5/24) and includes additional measurements undertaken as part of routine clinical follow-up with consent from patients and/or next of kin.

| Clinical measurements
All biochemical measurements were performed in CPA (Clinical Pathology Accreditation) and INAB (Irish National Accreditation Board) accredited laboratories, using local automated assays, and results were compared to local reference ranges (age and genderspecific where appropriate). Auxological measurements were performed by trained auxologists during clinical visits, and SDS scores were computed according to published reference data [15][16][17] using GrowthXP Endo, a children growth monitoring commercial programme.
Relevant medical history was acquired by review of patients' notes or direct questioning. Unless otherwise stated, reference ranges refer to 95% confidence intervals, and variance is reported as standard deviation score. Sleeping heart rate was compared to a locally-generated reference range for healthy males and depicts minimum and maximum. Basal metabolic rate was measured using ventilated hood indirect calorimetry as described in. 18

| Testicular volume assessment
Calculation of ultrasonographic testicular volume was computed as previously described and compared with published reference data. 19 Testicular volumes in children were assessed clinically by a trained paediatric endocrinologist using the Prader Orchidometer.

| In vitro analysis of the mutant IGSF1 protein
The L773P mutation was introduced into the human myc-IGSF1-HA expression vector described in 8 using the QuikChange mutagenesis protocol and the following primer set:

| Pathogenicity of mutation
The missense variant identified was absent from published databases

| Additional genetic ascertainment and endocrine evaluation
Family screening identified an additional eight hemizygous males (five children and three adults) and eleven heterozygous females.
Baseline endocrinology and auxology was assessed, and dynamic endocrine testing was performed in a subset of individuals (Figures 2-5; Tables 1 and 2; Supporting Information Tables S1-S3).

| Thyroid function
All evaluable male cases exhibited central hypothyroidism (FT4 ranging from 6.9 to 10.2 pmol/L, mean Z-score −3.3 ± 0.4) although biochemical penetrance was variable. In all cases, FT3 concentrations were maintained within the reference range (mean Z-score −0.8 ± 0.8) despite a subnormal FT4, and in three evaluable cases, this was achieved at the expense of low/low normal reverse T3 concentrations (Figures 2 and 4). Assessment of physiological thyroid status is challenging due to a paucity of sensitive and specific bio-

| Prolactin
Prolactin concentrations were usually in the lower half of the reference range (hemizygotes; mean Z-score −1.

| Growth hormone
Two male cases had either low normal (3i) or mildly subnormal in the mid-normal range (mean Z score 0.04 ± 0.9), but were more variable in the hemizygous male children (mean Z score −1.46 ± 0.46) (Tables 1 and 2; Supporting Information Tables S1 and S2).  Table S3).
Affected individuals in our kindred exhibit classical endocrine manifestations of IGSF1 deficiency, and characterization of the p.L773P mutation in vitro demonstrates deficits in trafficking of the protein from the endoplasmic reticulum to the cell surface, as has been described for previously-reported pathogenic IGSF1 mutations. 4,7,9 In keeping with previous reports, biochemical penetrance of thy- in the context of basal hypoprolactinaemia ( Figure 5, Cases 3a and 3e). 22,23,26 The role of IGSF1 in prolactin production remains unclear and is likely to involve pathways separate from the TRHR, as TRHR signalling is not required for normal basal prolactin production. 2,31 Female hypoprolactinaemia had no apparent effect on pregnancy, although one individual experienced difficulties with lactation.
Although IGSF1 expression has been reported in murine and rat somatotrophs, its role in GH dynamics may be complex. GH deficiency occurs rarely in IGSF1 deficiency; however, IGF-1 is usually in the upper part of normal range in IGSF1 deficient adults and may be associated with acromegaloid features consistent with mild GH excess. 7,9 In our kindred, one individual had GH deficiency and another had insufficient GH concentrations on stimulation testing with normal IGF-1 and growth velocity. However, the potential for puberty to be delayed in the context of IGSF1 deficiency may complicate interpretation of these results. Two adult heterozygotes had mildly elevated IGF-1 concentrations (Cases 1d and 2a) and one hemizygote (Case 1a) exhibited acromegaloid facies with inappropriately preserved IGF-1 given his hypothyroidism. Cortisol production was not evaluated systematically in our kindred; however, transient neonatal hypocortisolism has also been reported in IGSF1 deficiency. 9 In this context, it is noteworthy that 3a exhibited neonatal hypoglycaemia; although this is a recog- suggesting that formal studies are needed to investigate the benefits of levothyroxine therapy in apparently asymptomatic adults, and to delineate apparent compensatory mechanisms preventing overt tissue hypothyroidism in such individuals.