PMC8276716_01
The clinical profile of the family. (A) Pedigree of the phospholipase D3 (PLD3)-deficient family. The pedigree chart shows five generations of the family. Roman numerals refer to generations. Circles refer to female subjects. Squares refer to male subjects. Solid symbols refer to affected subjects. Crossed-out symbols refer to deceased subjects. The arrow indicates the proband. (B) The MR images of the proband. T2 image shows high intensity of white matter and thalamus (B1, white arrows), high intensity of brain stem and thalamus (B2, black arrows), high intensity of bilateral white matter (B3, white arrows), high intensity of bilateral thalamus (B4, black arrows), and high intensity of brain stem and bilateral cerebellum (B5, black arrows). B6-8 are the diffusion weighted image (DWI) corresponding to B3-5 and shows slightly higher intensity. (C) The CT image of the proband indicating tapering of bilateral optic nerves (red arrows). (D) Pure-tone audiometry (PTA) results of the proband. (E) HE staining of the renal biopsy specimen of the proband (IV-1); the arrows indicate glomerulosclerosis.
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F1
We enrolled a consanguineous family from the Han-Chinese population (Figure 1A). T2 image showed high intensity of white matter and thalamus, high intensity of brain stem and thalamus, high intensity of bilateral white matter, high intensity of bilateral thalamus, and high intensity of brain stem and bilateral cerebellum (Figure 1B). The computed tomography (CT) image of the proband indicated tapering of bilateral optic nerves (Figure 1C). Pure-tone audiometry (PTA) showed severe hearing loss (Figure 1D). In addition, the patient also suffered from chronic kidney disease (CKD) and was diagnosed with focal segmental glomerulosclerosis by renal biopsy in another hospital 10 years ago (Figure 1E). Brain MRI of the proband's parents and her brother also showed no obvious lesions (Supplementary Figure 1).
Genetic analysis of the family. (A) Sanger sequencing analysis of the PLD3 gene in the patients, their family members, and the controls. (B) Real-time PCR determined the messenger RNA (mRNA) levels of phospholipase D3 (PLD3) in white blood cells from healthy controls (five samples), heterozygotes (III-1, III-2, and IV-3), and a homozygote (IV-1, repeated three times). ***p < 0.01. (C) Western blot analysis of the expression of PLD3 in white blood cells from healthy controls (two samples), the heterozygote group (III-1 and IV-3), and the homozygote group (IV-1, repeated two times).
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F2
Table 1). The novel non-sense mutation, resulting in a premature stop codon in exon 5 of the PLD3 gene, was validated in the proband in a homozygous form and existed in the proband's parents and her brother in a heterozygous form (Figure 2A). After synthesizing complementary DNA (cDNA), real-time PCR found that the mRNA level of PLD3 in heterozygous carriers (III-1, III-2, and IV-3) was decreased by ~43% compared with that in healthy controls (five healthy people without PLD3 mutations), and the expression of PLD3 in homozygous patients (IV-1, repeated three times) was reduced by ~90% compared to that in healthy controls (Figure 2B), which indicated that the non-sense mutation may have led to non-sense-mediated mRNA decay. Western blot analysis found that the expression of PLD3 in heterozygote group was decreased dramatically compared to healthy controls, and PLD3 was almost absent in the proband (IV-1) (Figure 2C).