PMC10031778_01
Findings of brain MRI in patients. (A, E) The ventricular system was enlarged (arrowhead in A), the sulci and cisterns were widened, the brain stem morphology was poor (arrowhead in E), and lacked a clear high signal indicating the neurohypophysis in patient 1. (B, F) The MRI finding presented as an abnormally small neurohypophysis without encephalatrophy in patient 2. (C, G) Atrophic changes of the brain was found and lacked a clear high signal indicating the neurohypophysis in patient 5. (D, H) No signs of brain atrophy and a normal high signal of neurohypophysis (arrowhead in H) were displayed in patient 6.
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During the follow-up, hearing impairment occurred and signs of encephalatrophy were found by MRI ( Figures 1A, E ). Patient 2 was diagnosed at the age of 4 years and 9 months, initially diagnosed with T1DM; MRI results showed that the neurohypophysis was abnormally small ( Figures 1B, F ). Patient 5, aged 7 years and 3 months, was initially diagnosed with T1DM, neurogenic bladder, optic atrophy, dilated cardiomyopathy, severe anaemia, cardiac insufficiency, and brain atrophy ( Figures 1C, G ). Routine examination before operation found that blood glucose was increased and no abnormality was found in brain MRI ( Figures 1D, H ).
Pedigrees of families with WFS1 variants. (A) The pedigree of family 1. Patient 1 and cousin (Patient 4) carry the same pathogenic variation and have similar clinical symptoms. Patient 3 only shows non-insulin-dependent diabetes; patient 2 carries two heterozygous variants, with a light clinical phenotype. (B) The pedigree of family 2. In addition to typical diabetes, optic atrophy, and neurogenic bladder, patient 5 also had a rare cardiomyopathy phenotype. Family members carrying the variant were asymptomatic. (C) The pedigree of family 3. The parents of patient 6 were heterozygous carriers and had no clinical symptoms at present.
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The distribution of variation in families is shown in  Figure 2 .
Localization of variation site (P/LP) in protein region and prediction of protein structure in amino acid residue region. The amino acids encoded by c.1348, c.1329, and c.1350-1356 were located in the fourth transmembrane region of WFS1 protein. The amino acids encoded by c.1381 were located in the non-transmembrane region of the cytoplasm. The amino acid encoded by c. 2081 was located in the COOH terminal domain of the endoplasmic reticulum. The c.1381A>C and c.1329C>G variations did not affect the formation of the alpha-spiral structure.
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The amino acid encoded by c.2081delA (p.Glu694Glyfs*16) was located in the COOH terminal domain of the ER ( Figure 3 ), which may have affected the protein function. The online software SWISS-MODEL predicted that the protein structure of the amino acid residues encoded by c.1381A>C(p.Thr461Pro) and c.1329C>G(p.Ser443Arg) variants can form an alpha-spiral structure, but the local subspace structure was abnormal ( Figure 3 ).
Analysis results of differentially expressed genes and splicing abnormalities by RNA sequencing. (A) Volcano plot showing gene expression differences between patients and their parents. Differentially expressed genes with an adjusted p <0.05 and a fold-change FC >= |2| are depicted in red (up-regulated) and blue (down-regulated). (B) Heatmap of all differentially expressed genes. Gene expression levels were quantified by transcript per million and scaled by gene. Red indicates higher expression level while blue indicates lower. (C) Gene Ontology (GO) enrichment scatter plot of differential expressed genes. The significance of GO term enrichment is represented by adjusted p-value and mapped to the scatter plot by point colour, while point size indicates the number of candidate genes annotated with a GO term. (D) Gene Set Enrichment Analysis (GSEA) showed that ER stress related genes exhibited little differential expression. (E) Sashimi plot of HLA-DRB1 gene of family 1 patient and their parents. Exon 1 of HLA-DRB1 were not transcribed in patients (Patient 1 and Patient 2), compared with their parents as normal transcripts. The last sashimi plot shows the average level of junction reads from 755 GTEx whole blood samples. (F) The expression level of HLA-DRB1 in different samples. The expression level of HLA-DRB1 was lower in other patients with WFS1 variants and T1DM, compared to the parent group without diabetes.
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Differential gene expression analysis revealed that there were 80 candidate differentially expressed genes in the patient group (P), which contained 47 down-regulated genes and 33 up-regulated genes ( Figures 4A, B ). According to the Gene Ontology enrichment analysis, the differentially expressed genes were mainly enriched to biological processes related to immune function like antigen binding ( Figure 4C ). Gene set enrichment analysis showed that differentially expressed genes were not significantly enriched in ER stress related genes ( Figure 4D ). Compared with the average reads of 755 whole blood samples from healthy normal humans in GTEx (genetic tissue expression) datasets, the junction reads in the front segment of the transcripts in the patient group were relatively low ( Figure 4E ). T1DM group. The expression level of the patient group was lower than that of the parent group, and higher than that of the T1DM group ( Figure 4F ).