Leukoencephalopathy (LE) is a structural alteration of the cerebral white matter in which myelin suffers the most damage (Kohler et al., 2018). Leukodystrophies can be broadly subdivided into hypomyelinating leukodystrophies, which are characterized by primary deficits in myelin development, and demyelinating leukodystrophies, where myelin develops normally but subsequently undergoes progressive disruption (Vanderver et al., 2015). At present, ~20 distinct disorders are defined as adulthood leukodystrophies (Tillema and Renaud, 2012; Kohler et al., 2018), such as Pelizaeus–Merzbacher disease, adult polyglucosan body disease, and X-linked adrenoleukodystrophy.

The human PLD3 gene, which encodes a single-pass type II membrane protein with two phospholipase D (PLD) phosphodiesterase domains, is located on chromosome 19q13.2 and consists of 13 exons spanning 32 kb. As a member of the phospholipase D family of enzymes that catalyze the hydrolysis of membrane phospholipids, PLD3 has been proven to be involved in the processing of amyloid-beta precursor protein (Fazzari et al., 2017). Recently, two proteins from the PLD family, namely, phospholipase D3 (PLD3) and phospholipase D4 (PLD4), were found to exhibit single stranded DNA (ssDNA) acid 5′ exonuclease activity (Gavin et al., 2018; Cappel et al., 2020). Previous studies revealed that a heterozygous mutation of PLD3 might increase the risk of Alzheimer's disease and spinocerebellar ataxia (Wang et al., 2015; Nibbeling et al., 2017). However, the effect of homozygous mutations in PLD3 is still not clear.

In this study, we identified a novel homozygous mutation (NM_012268.3: c.186C>G/ p.Y62X) of PLD3 by whole exome sequencing in a patient from a consanguineous family with white matter lesions, hearing and vision loss, and kidney disease.

In this study, a novel homozygous mutation (NM_012268.3: c.186C>G/ p.Y62X) of PLD3 was identified in a consanguineous family with leukoencephalopathy syndrome by whole exome sequencing. Furthermore, real-time PCR and Western blot assays confirmed that the homozygous mutation might have led to non-sense-mediated mRNA decay and resulted in loss of function in PLD3. PLD3 is highly expressed in neurons and may regulate early neuronal development in the central nervous system (Wang et al., 2015). Meanwhile, studies found that the function of PLD3 was to catalyze the hydrolysis of membrane phospholipids. In leukoencephalopathy, neurons are damaged, and myelin is also disrupted (Kohler et al., 2018). Myelin, a phospholipids, might also be regulated by PLD3. Recently, PLD3 was found to exhibit ssDNA acid 5′ exonuclease activity (Gavin et al., 2018; Cappel et al., 2020), which was also associated with brain diseases such as Alzheimer's disease and spinocerebellar ataxia (SCA) (Gavin et al., 2018). Hence, in our case, the pathological changes in the patient who presented with high intensity white matter may have resulted from PLD3 deficiency.

Mutations in PLD3 may also lead to endoplasmic reticulum (ER) stress and reduced phospholipase activity (Nibbeling et al., 2017). Further studies revealed that PLD3 mutation can impair O-glycosylation at pT271 in PLD3, which is essential for normalizing antioxidative phospholipid levels and protecting the brain (Demirev et al., 2019). In addition, variants in PLD3 can reduce PLD3 activity and affect amyloid-β levels in a cellular model of Alzheimer's disease, possibly via the autophagy-dependent mTOR signaling pathway (Tan et al., 2019). Recently, PLD3 has also been found to play a crucial role in regulating inflammatory cytokine responses (Gavin et al., 2018). Macrophages from PLD3-deficient mice had exaggerated TLR9 responses (Gavin et al., 2018). Here, in our study, the patient who carried a homozygous mutation of PLD3 presented with white matter lesions, hearing and vision loss, and focal segmental glomerulosclerosis. We speculated that the homozygous mutation (NM_012268.3: c.186C>G/ p.Y62X) of PLD3 may lead to PLD3 deficiency, which may induce ER stress and reduce phospholipase and exonuclease activities in neurons, as well as loss of O-glycosylation at pT271 of PLD3, ultimately damaging neuros in the central nervous system and optic and vestibulocochlear nerves. Simultaneously, PLD3 deficiency may also induce inflammatory cytokine responses in the kidney. Hence, the proband presented with phenotypes not only in the nervous system but also in the kidney. Our studies indicated that homozygous mutation of PLD3 may result in a novel leukoencephalopathy syndrome including white matter lesions, hearing and vision loss, and kidney disease.

Previous genetic studies revealed that variants in PLD3 may increase the risk for late-onset Alzheimer's disease (van der Lee et al., 2015; Tan et al., 2018). However, studies in Belgium found that rare variants in PLD3 do not raise the risk for early-onset Alzheimer's disease (Cacace et al., 2015). Subsequently, Nibbeling et al. identified novel genes (FAT2, PLD3, KIF26B, EP300, and FAT1) in autosomal dominant SCA patients by whole exome sequencing. Functional studies revealed that PLD3 is located in the ER and that the missense mutation p. Leu308Pro of PLD3 may lead to loss of function, which can induce ER stress and reduce phospholipase activity in COS-7 cells (Nibbeling et al., 2017). However, Gonzalez et al. discovered that PLD3 was located in lysosomes but not in the ER and acted as a 5′ exonuclease in HeLa cells. In addition, they also found that loss of PLD3 did not disrupt lipid catabolism and that PLD3 knockout mice did not present cerebellar ataxia phenotypes, which challenged the interpretation of PLD3 mutations as the causative SCA46 gene (Gonzalez et al., 2018). Hence, the identification of additional patients carrying PLD3 mutations will further strengthen the role of PLD3 in brain disease.

In our study, four heterozygous mutation carriers (III-1, III-2, IV-3, and V-1) showed normal physical features, which indicated that heterozygous non-sense mutation of PLD3 might not be the responsible genetic lesion of SCA and Alzheimer's disease. However, homozygous non-sense mutation of PLD3 can lead to white matter lesions, which may develop into Alzheimer's disease in the future. Certainly, we cannot exclude the genetic heterogeneity and incomplete appearance of PLD3. In fact, we enrolled almost 181 patients with white matter lesions in the past 5 years, but we only detected one homozygous mutation of PLD3 in this family. This may be the first case report on a homozygous mutation of PLD3 in patients with leukoencephalopathy syndrome. Our study may reveal a relationship between leukoencephalopathy syndrome and PLD3 homozygous mutation in patients.

In summary, we identified a novel homozygous mutation (NM_012268.3: c.186C>G/ p.Y62X) of PLD3 in a consanguineous family with white matter lesions, hearing and vision loss, and kidney disease. This may be the first case report on a homozygous mutation of PLD3 in patients worldwide. Our study also provided new insights into function of PLD3 in human diseases.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://ncbi.nlm.nih.gov/, PRJNA723675.

The studies involving human participants were reviewed and approved by Affiliated Hospital of Yangzhou University in China. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Y-HL and H-FZ enrolled the samples and performed the Sanger sequencing. J-YJ performed the real-time PCR and Western blot. Y-QW and C-YW enrolled the clinical data. L-LF and LL revised the manuscript and support the project. Y-HL, H-FZ, and J-YJ wrote the draft. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.