Mutations in HID1 Cause Syndromic Infantile Encephalopathy and Hypopituitarism

Abstract

Objective: Precursors of peptide hormones undergo posttranslational modifications within the trans-Golgi network (TGN). Dysfunction of proteins involved at different steps of this process cause several complex syndromes affecting the central nervous system (CNS). We aimed to clarify the genetic cause in a group of patients characterized by hypopituitarism in combination with brain atrophy, thin corpus callosum, severe developmental delay, visual impairment, and epilepsy.

Methods: Whole exome sequencing was performed in seven individuals of six unrelated families with these features. Postmortem histopathological and HID1 expression analysis of brain tissue and pituitary gland were conducted in one patient. Functional consequences of the homozygous HID1 variant p.R433W were investigated by Seahorse XF Assay in fibroblasts of two patients.

Results: Bi-allelic variants in the gene HID1 domain-containing protein 1 (HID1) were identified in all patients. Postmortem examination confirmed cerebral atrophy with enlarged lateral ventricles. Markedly reduced expression of pituitary hormones was found in pituitary gland tissue. Colocalization of HID1 protein with the TGN was not altered in fibroblasts of patients compared to controls, while the extracellular acidification rate upon stimulation with potassium chloride was significantly reduced in patient fibroblasts compared to controls.

Interpretation: Our findings indicate that mutations in HID1 cause an early infantile encephalopathy with hypopituitarism as the leading presentation, and expand the list of syndromic CNS diseases caused by interference of TGN function. ANN NEUROL 2021;90:149-164.

FIGURE 1:

Clinical phenotype and MRI images of patients with bi-allelic HID1 variants. (A) Patient 1:III-2 at age 3 months (A1, A2). T2-weighted sagittal (A3) and coronal (A4) brain MR images at age 4 months indicate hypoplasia of the corpus callosum and a small sella turcica (white arrows). (B) Patient 1:III-3 at age 2 months (B1, B2), at age 2 years (B3), and at age 3 years (B4). T2-weighted sagittal and coronal brain MR images age 4 months (B5, B6) and at age 3 years and 2 months (B7, B8) show a relatively small pituitary gland and a thin and dysplastic corpus callosum (B5 white arrows). Note also distinct cortical atrophy and enlargement of the inner ventricles evolving during the course of disease (B7, B8). An unspecific signal alteration of the ventral part of the pons (B7, white arrow) is also marked. (C) Patient 2:IV-3 at age 3 years (C1) and at age 21 years (C2, C3). T1-weighted brain MR image displays a relatively small pituitary gland and a small corpus callosum. No generalized brain atrophy is visible. (D) Patient 3:IV-1 at age 3 years (D1, D2). T2-weighted brain MR image displays a thin corpus callosum (D3, white arrows). (E) Patient 4:II-2 at age 8 months (E1, E2), T2 (E3). T1-weighted (E4) sagittal brain MR images show a thin corpus callosum. T1-weighted coronal image depicts enlarged lateral ventricles and suggests gyral atrophy (F) Patient 5:II-1 at age 2 weeks (F1), at age 20 months with cushingoid face (F2) and at age 3 years (F3). Sagittal T1-weighted MR image at age 18 months indicates a dysplastic corpus callosum and a small pituitary gland (F4), while a coronal FLAIR image depicts mild gyral atrophy (F5). Consent was obtained from parents/legal guardians for use of clinical photographs shown in this Figure (including parental consent for inclusion of the identifiable (non-obscured) facial photographs).

FIGURE 2:

HID1 variants. (A) Pedigrees of families 1 to 5. Family members who underwent genetic testing for the respective HID1 variants are indicated with black and white symbols. Grey-and-white symbols correspond to family members not tested for the familial HID1 variant. (B) The human HID1 gene (NM_030630) consists of 19 exons on the minus strand of chromosome 17 (hg19 coordinates: chr17:72946837–72968854). HID1 variants found in the families reported are highlighted. Missense variants are indicated white with black background. (C) Alignment of the protein sequences in vicinity to amino acids D28, R433 and L533, between human and homologous HID1 proteins. R433, L533 and adjacent amino acids are widely conserved between species, D28 is also highly conserved except for nematoda.

FIGURE 3:

Neuropathological findings of patient 1:III-3. (A) Gross examination of a coronal brain section of patient 1:III-3 at age 3 years shows cerebral atrophy (brain weight 702 g), enlargement of the lateral ventricles and a thin corpus callosum. The cortical grey matter is well distinguished from the white matter. (B) Klüver Barrera (KB) staining displays normal cortical architecture in the frontal cortex and cerebellum, and no signs of de- or hypomyelination (magnification ×40 1st column; ×400 2nd column). Activated glial cells are numerous in frontal cortex as confirmed with antibodies against GFAP (magnification ×400).

FIGURE 4:

Pituitary Gland. (A) Analysis of the pituitary gland of patient 1:III-3 shows normal adenoid morphology. Staining with antibodies against pituitary hormones depicts moderate ACTH reduction, while FSH, HGH, LH, Prolactin (Prol) and TSH are strongly reduced compared to the control (inlay) (magnification 200×). (B) Immunofluorescence analysis with antibodies against HID1 shows a cytoplasmic expression in neuronal cells of the frontal cortex and cerebellum and in adenoid cells of the pituitary gland in patient and control (magnification ×630, white bars correspond 50 μm).

FIGURE 5:

HID1 expression. (A) Expression of HID1 transcript in different human tissues. Data from the Genotype-Tissue Expression (GTEx) Project were downloaded from the R2: Genomics Analysis and Visualization Platform. Gene and transcript expression are shown in transcripts per million (TPM) (for calculation see methods section). HID1 is highly expressed in different parts of the brain, with highest expression in cerebellum. High expression levels are found in most of the secretory tissues also, with highest expression in the pituitary gland and the pancreas. Boxplots of log2-transformed TPM values for HID1. The boxplots of each category “BRAIN”, “SECRETORY TISSUES”, “GI”, “REPRODUCTIVE”, “CARDIOVASCULAR” and “DIVERSE” are sorted by their median values in descending order. (B, C) Expression level of HID1 in human brain (merged data of different brain regions). Data at different time points of fetal ages were downloaded as RPKM (reads per kilobase of exon model per million mapped reads) values (BrainSpan) or signal intensities (Kang et al.) respectively. Expression values of HID1 at different weeks post-conceptionem were analyzed. Pearson’s correlation analysis was used for calculation of the correlation coefficients and corresponding P-values of RPKM values or signal intensities respectively and ages. (D) Compared to an age matched control HID1 mRNA expression is lower in liver tissue and two brain regions (CB and Str) in patient 1:III-3. Fold expression was calculated with the integrated qBase module of the Bio-Rad CFX Manager 3.1 (3.1.1517.0823) software. For comparison of data from only one patient with one control, statistical significance was not calculated. qPCR experiments were done in triplicate.

FIGURE 6:

HID1 and Golgi staining of fibroblasts. Double immunofluorescence staining of cultured fibroblast cells from patients and family members in comparison to a control patient. Representative confocal micrographs of a control individual (Ctrl, A1–A3), single-allelic HID1 variant family members (family-1,1:II-1; B1–B3; 1:II-2, C1–C3), and bi-allelic HID1 variant patients 1:III-3 (D1–D3) and 4:II-2 (E1–E3) stained with rabbit polyclonal antibodies against human HID1 (green) and mouse monoclonal antibodies against 58K Golgi marker (red). The overlay (yellow) shows a co-localization of HID1 and the Golgi apparatus in all fibroblasts. Scale bar = 35 μm.

FIGURE 7:

Extracellular acidification rate (ECAR) dependent on HID1 function. (A) Analysis of the mean relative ECAR change demonstrates that addition of 22.5 mM KCl was not sufficient to induce a significant increase of ECAR in any individual, while addition of 45 and 90 mM KCl induced a significantly higher increase in controls than in the affected individual 4:II-2. A parametric t-test was used to analyze the differences between the affected individuals and controls. Mean SEM are shown. *p < 0.05; **p < 0.01. (B–G) Baseline ECAR and relative change in (ECAR) in fibroblasts and PC12 cells after addition of potassium chloride (KCl). (B) Analysis of the relative change in ECAR over time shows lower values at all time points after addition of 45 mM KCl for the affected bi-allelic individual (4:II-2; n = 16 wells) compared to two controls (each n = 8 wells). (C) Analysis of the mean relative change of ECAR directly after 45 mM KCl addition reveals a significantly lower value in patient 4:II-2 compared to controls (time point 6 compared to time point 5). (D) Relative change in ECAR in the affected bi-allelic individual (1:III-3; n = 32 wells), heterozygous parents (father 1:II-1; n = 16 wells and mother 1:II-2; n = 16 wells) and two controls (each n = 14 wells) over time. (E) Mean relative change directly after 45 mM KCl addition is significantly lower in 1:III-3 compared to controls and heterozygous parents (time point 6 compared to time point 5). (F) Relative change in ECAR in PC12 wild-type and HID1 knockout cells over time. (G) Mean relative change directly after 90 mM KCl addition is significantly lower in HID1-knockout compared to wild-type cells (time point 6 compared to time point 5). A parametric t-test was used to analyze the differences between the affected individuals and controls. Mean SEM are shown. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.