Incidence, manifestations, and diagnosis of pituitary hormone deficiencies in children and adolescents

Childhood is a phase of rapid growth and development that culminates in achieving adult height, sexual maturity, and fertility potential during puberty. The pace of growth and development is carefully regulated and individually modulated by genetic variation and a multitude of environmental factors, including nutrition, nurture, and physical exercise. Both common and rare diseases can compromise this delicate development. A severe but treatable underlying condition is hypopituitarism, a defective central regulation of the endocrine system. Hypopituitarism can manifest as reduced growth, absent puberty, or deficient regulation of metabolism and stress responses, and can even be lethal. However, the phenotypic spectrum of hypopituitarism is highly variable, and includes also partial phenotypes that may go unnoticed and be mislabeled, such as constitutional delay of growth and puberty (CDGP; a late normal genetic variant of puberty) or functional hypothalamic amenorrhea (FHA; the absence of menses due to excessive exercise, decreased caloric intake, or stress). A prompt diagnosis and treatment of hypopituitarism are crucial due to the complications related to the severe phenotypes, but also to timely inform the patient and his/her parents of the cause, prognosis, and treatment options. Hypopituitarism can be either congenital or acquired. In the latter case, another condition, such as a brain tumor, causes the dysfunction of the pituitary, whereas congenital hypopituitarism encompasses syndromic, genetic, and idiopathic diseases. Despite continuous advances in pituitary genetic research, many patients with congenital hypopituitarism remain without a molecular genetic diagnosis. However, in recent years, multiple new candidate genes for hypopituitarism have been published but not investigated in larger patient series. This thesis aimed to elucidate the incidence, etiologies, and diagnostic features of childhood-onset hypopituitarism. In Study I, we investigated the spectrum of childhood-onset combined pituitary hormone deficiency (the deficiency of two or more pituitary hormones, CPHD) in Helsinki University Hospital (HUH) pediatric patients (n=124) between 1985 and 2018. The minimal incidence estimate for congenital CPHD was 1/16,000 (95% confidence interval, 1/11,000-1/24,000). Nearly 40% of the congenital CPHD patients presented for pituitary endocrine investigations due to reduced growth. Their presentation was median 2.2 (interquartile range, 1.2 to 3.6) years later than the first detection of abnormal height growth by the national growth screening. This delay was partially due to the follow-up of growth or investigations and treatment of other causes of growth retardation, including hypothyroidism and infections. However, the early diagnostic opportunity of hypopituitarism had been missed in some patients, suggesting that future studies should further evaluate neonatal screening measures and the functioning of the Finnish growth screening in CPHD diagnostics. A genetic diagnosis had been established in 7/48 (15%) of the patients with congenital CPHD in previous clinical or research investigations (PROP1, SOX2, SOX3, OTX2, TBC1D32 variants; the last mentioned had been identified in Study II of this dissertation). We recruited patients with idiopathic CPHD to genetic investigations. Using whole exome sequencing (WES), we detected a likely pathogenic variant in previously reported CPHD or growth hormone deficiency (GHD) candidate genes in 1/21 patients (heterozygous Sonic hedgehog (SHH) variant (c.676G>A, p.(Ala226Thr)). Additionally, extremely rare (minor allele frequency, MAF ≤0.01% or not reported) VUS were encountered in ISL1, SHH, ALMS1, L1CAM, FGFR1, and CHD7. Altogether five patients harbored rare variants in five to six different genes previously implicated in CPHD/GHD. Study II described Finnish siblings and a patient from a consanguineous Pakistani family with syndromic CPHD or GHD, harboring biallelic loss-of-function variants in TBC1D32, and a fetus from the Pakistani patient’s mother’s terminated pregnancy carrying the same TBC1D32 variant as the affected sibling. In situ hybridization showed TBC1D32 expression at Carnegie stage (CS) 23 in the hypothalamus (regulator of pituitary function) and Rathke’s pouch (origin of the anterior and medial lobes of the pituitary). TBC1D32 was also expressed in the adult human pituitary and hypothalamus cDNA libraries. In previous animal models, TBC1D32 had been shown to be necessary for the correct formation of ciliary structure and localization of GLI2 in the cilia, suggesting that the hypopituitary phenotype of humans with TBC1D32 variants is likely due to the mislocalization of GLI2, an SHH pathway protein that has been shown to cause CPHD. This study strengthened the evidence for TBC1D32 variants as a cause of hypopituitarism, augmented the role of ciliopathies underlying hypopituitarism, and extended the phenotypes related to TBC1D32 mutations. In Study III, we investigated nine female patients and monozygotic twin brothers with partial puberty phenotypes or unexplained secondary amenorrhea for defects in FGFR1, GNRHR, TAC3, and TACR3, genes previously related to pituitary gonadotrophin deficiency (hypogonadotropic hypogonadism, HH) in normosmic patients (nHH). We identified biallelic GNRHR mutations in two (22%) of the females, and compound heterozygous GNRHR mutations in the twin brothers. Female nHH is a challenging diagnosis associated with a significant diagnostic delay. This study strengthened the evidence for the role of GNRHR mutations in partial nHH phenotypes and stressed the importance of carefully excluding HH in patients with suspected functional hypothalamic amenorrhea. In conclusion, the etiologies and manifestations of hypopituitarism form a wide spectrum, where, at the severe end, the potential of diagnostic measures, such as the combination of neonatal cues and reduced growth, and the possibilities of neonatal screening should be further investigated to minimize unnecessary delays to diagnosis. In milder forms, it would be important for clinicians to recognize the possibility of this rare but treatable condition. New genes underlying hypopituitarism have been proposed at a fast pace, and Study II elucidated the role of pathogenic TBC1D32 variants and ciliary function in genetic hypopituitarism. In our cohort of idiopathic CPHD patients investigated by WES, although examining the more recently reported genes, we were able to identify the probable cause of CPHD in only one patient.