Loss-of-Function Variants in TBC1D32 Underlie Syndromic Hypopituitarism

Abstract

Context: Congenital pituitary hormone deficiencies with syndromic phenotypes and/or familial occurrence suggest genetic hypopituitarism; however, in many such patients the underlying molecular basis of the disease remains unknown.

Objective: To describe patients with syndromic hypopituitarism due to biallelic loss-of-function variants in TBC1D32, a gene implicated in Sonic Hedgehog (Shh) signaling.

Setting: Referral center.

Patients: A Finnish family of 2 siblings with panhypopituitarism, absent anterior pituitary, and mild craniofacial dysmorphism, and a Pakistani family with a proband with growth hormone deficiency, anterior pituitary hypoplasia, and developmental delay.

Interventions: The patients were investigated by whole genome sequencing. Expression profiling of TBC1D32 in human fetal brain was performed through in situ hybridization. Stable and dynamic protein-protein interaction partners of TBC1D32 were investigated in HEK cells followed by mass spectrometry analyses.

Main outcome measures: Genetic and phenotypic features of patients with biallelic loss-of-function mutations in TBC1D32.

Results: The Finnish patients harboured compound heterozygous loss-of-function variants (c.1165_1166dup p.(Gln390Phefs*32) and c.2151del p.(Lys717Asnfs*29)) in TBC1D32; the Pakistani proband carried a known pathogenic homozygous TBC1D32 splice-site variant c.1372 + 1G > A p.(Arg411_Gly458del), as did a fetus with a cleft lip and partial intestinal malrotation from a terminated pregnancy within the same pedigree. TBC1D32 was expressed in the developing hypothalamus, Rathke's pouch, and areas of the hindbrain. TBC1D32 interacted with proteins implicated in cilium assembly, Shh signaling, and brain development.

Conclusions: Biallelic TBC1D32 variants underlie syndromic hypopituitarism, and the underlying mechanism may be via disrupted Shh signaling.

Keywords: Sonic Hedgehog signaling; TBC1D32; ciliopathy; hypopituitarism; retinal dystrophy.

Figure 1.

A: The pedigrees of our patients with hypopituitarism and biallelic TBC1D32 variants. I: the Finnish pedigree; II: the Pakistani pedigree. Patients I.3 and I.5 carried compound heterozygous and patients II.8 and II.9 carried homozygous variants in TBC1D32. The parents were heterozygous carriers of the respective variants. B: (i) and (ii) the MRIs of the 2 Finnish patients. Upper row, Patient I.3: Sagittal (a) and coronal (b) T1 images without contrast enhancement. The sella turcica and the pituitary gland are not identifiable. Neurohypophyseal bright tissue is seen near the tuber cinereum (arrow). Lower row, Patient I.5: Sagittal (a) and axial (b) T1 images without contrast enhancement. The sella turcica and the pituitary gland are absent. Potentially neurohypophyseal bright tissue is seen near the tuber cinereum (arrow). (iii) MRI of patient II.8. Upper row, Patient II.8: Sagittal (a) T1 image showing partial agenesis of corpus callosum (CC), small interhemispheric lipoma (L), small anterior pituitary (AP), and small ectopic posterior pituitary (PP). Lower row: coronal image showing dysplasia of the cerebellar vermis (arrow) with an abnormal left cerebellum. The “molar tooth” sign of Joubert syndrome is also shown (filled arrow). C: Clinical photos of the patients. (i) Patient I.3 at 2.4 years of age. Note the prominent forehead and the low-set, posteriorly rotated ears; (ii) Patient I.5 presented with prominent forehead, large anterior fontanelle, and low-set ears in infancy; (iii) Patient I.5 at 10.5 years of age; (iv) Patient II.8 in infancy showing a prominent forehead with hypertelorism, low-set ears, flat nasal bridge and anteverted nares; (v) Patient II.8 at 5.5 years of age.

Figure 2.

Human expression of TBC1D32 mRNA transcripts in transverse brain sections at different developmental stages during embryogenesis. A: There is no clear expression in the hypothalamus, Rathke’s pouch, or elsewhere in the brain when comparing results using the antisense probe and the sense probe at Carnegie stage (CS) 19. B: At CS20 there may be some partial expression in the hypothalamus using the antisense probe; however, staining is very similar to the Rathke’s pouch and the hypothalamus when using the control sense probe, where some background staining is noted. C: At CS23, there is partial expression in the trigeminal ganglia, in Rathke’s pouch, and along the hypothalamus when comparing the antisense and control sense probes. D: There is strong expression in the hindbrain, in particular the thalamus, with some expression also seen in the choroid plexus. Abbreviations: CP, choroid plexus; Hyp, hypothalamus; RP, Rathke’s pouch; T, thalamus; Tri, trigeminal ganglia.

Figure 3.

Reverse transcriptase PCR analysis of TBC1D32 expression. A 310-bp fragment of transcript encoding TBC1D32 was amplified from human pituitary gland cDNA and from hypothalamic cDNA (QUICK-Clone pituitary cDNA, Takarabio, 1.5µl / reaction; Hypothalamus Marathon®-Ready cDNA, Takarabio, 1.5ul / reaction). Human GAPDH was used as a reference gene. The PCR products were visualized on a 1.0% agarose gel. Abbreviations: c, negative control without DNA template; ht, hypothalamus; p, pituitary gland.

Figure 4.

The functional grouping of the high confidence interacting proteins of TBC1D32. Interactome analysis reveals known and novel interactions for TBC1D32. AP-MS and BioID analysis of TBC1D32 identified 81 high-confidence protein–protein interactions (yellow lines represent interaction detected by AP-MS approach; green lines represent interactions detected by BioID approach; overlap of the 2 purification methods is shown with grey lines; known interaction is shown with a dashed line). The interacting proteins are grouped based on their molecular functions/complexes.