The PRUNE1 gene, located on chromosome 1q21.3, encodes a 453 amino acid protein, that is, highly expressed in the human fetal brain and involved in the regulation of neuronal migration and proliferation (4). It harbors 2 domains DHH (20-172 amino acids) and DHHA2 (215-359 amino acids). The DHH super-family can be categorized into two main groups based on their C-terminal motif which is highly conserved within each group. Members of this superfamily have four other motifs that are predicted to be responsible for binding metal cofactors and enzymatic function (20). Most of the described mutations in the PRUNE1 gene so far are clustered in the DHH domain. PRUNE through interaction with glycogen synthase kinase-3 (GSK-3B), a suppressor of neurite outgrowth, synapse formation, and neurogenesis might play a molecular role in brain development (21). Furthermore, PRUNE in a complex interaction network by regulating cellular mobility and stimulating expression of genes involved in metastatic pathways, is associated with cancer metastasis (22, 23). Homozygous or compound heterozygous mutations in the PRUNE1 gene cause the neurodevelopmental disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA) (OMIM#617481) that is characterized by global developmental delay, microcephaly, central hypotonia, spastic quadriplegia, and cerebral and cerebellar atrophy (1).
By WES, we identified the first start loss sequence variant, NM_021222.3:c.3G>A; p.(Met1?), in the PRUNE1 in an Iranian female patient presenting spastic quadriplegic cerebral palsy (CP), central hypotonia, developmental regression, and seizure. Since a start codon mutation is likely to alter the translation initiation codon, the detected variant, p.Met1?, is predicted to abolish translation of the PRUNE1 polypeptide. In this case, the translation will either initiate at a downstream site, or not at all. According to a 3D model of PRUNE, the next closest in frame ATG is at codon 183, thus even if this ATG served as an initiation codon, the start of translation at this site would cause the deletion of the DHH domain from the N-terminus of the mature PRUNE1 and likely to lead to loss of function of PRUNE (Fig. 2c). To date, most of the pathogenic variants in NMIHBA patients are reported in DHH domain (p.Asp106Asn, p.Arg128Gln, p.Gly174* and c.521-2A>G in DHH domain and p.His292Glnfs*3 and p.Arg297Trp in DHHA2 domain) (1, 3, 24-28) (Fig. 2a). The truncating mutations result in lack of the domains and affect either or both of the catalytic DHH or DHHA2 domains and might lead to decreased PRUNE activity.
Homozygous start loss variant, NM_021222.3:c.3G>A; p.(Met1?) in PRUNE1 gene identified in this study, probably leads to the deletion of DHH (Asp-His-His) domain, the active site of the protein, and loss of PRUNE1 function. A change in any of these three amino acids has been indicated to greatly reduce the enzyme’s activity (5). A homozygous variant p.Asp106Asn, which was previously reported in Turkish and Italian patients with microcephaly, spastic quadriparesis, hypotonia, cerebellar atrophy, and neurodevelopmental impairment, altered one of these three conserved amino acids DHH (Asp-His-His) domain (3). Additionally, compound heterozygous (c.G383A; p.R128Q and c.G520T; p.G174X) variants in two affected siblings from the United States with severe developmental delay, regression, seizures, and microcephaly have been reported (3). The main characteristics of these patients are similar to our patients’ phenotypes, but most of the reported patients had progressive microcephaly which did not present in our patient. Similar to over case, in another study, a patient with a homozygous (c.540T>A; p.Cys180*) PRUNE1 variant has been reported who did not exhibit progressive microcephaly (29). Furthermore, Costain et al revealed a homozygous likely pathogenic variant in PRUNE1 (c.521-2A>G: IVS4-2A>G; NM_021222.1), affecting the catalytic DHH domain, in a patient with global developmental delay, infantile spasms, and central hypoventilation without microcephaly (27). Thus, it can be suggested that p.(Met1?) may lead to the lack of DHH domain and in turn reduction or absence of the protein activity.
Additionally, we accomplished a clinical and literature review of all 41 NMIHBA patients reported to date in order to further convey the phenotypic spectrum of NMIHBA (Table 3). Cardinal features the majority of patients include developmental delay DD (87.80%), Intellectual disability (82.93%) with speech disorders (75.61%) and cerebral and cerebellar atrophy (48.78%). Hypotonia (41.46%), spastic quadriplegia (21.95%), microcephaly (60.98%), and seizures (58.54%) are features observed in a sizable number of patients. Radiological results display that most of the patients have delayed myelination, thin corpus callosum, and white matter abnormality, which is not reported in our cases. So, in view of previous reports the clinical severity of the phenotype was variable even across patients harboring the same variant allele. No relationship between presence/absence of particular clinical features with the various mutations was obviously observed. Further reporting of patients with novel mutation enables a better understanding of the phenotypic spectrum and helps in advising newly diagnosed patients.
To conclude, by using targeted-exome sequencing, we identified a novel start loss variant, NM_021222.3:c.3G>A; p.(Met1?) in the PRUNE1 gene in two affected members as a possible cause of NMIHBA in an Iranian family. The putative variant meets the criteria of being pathogenic, but we strongly recommend doing functional analysis in order to further confirm the molecular mechanism of the pathogenesis of this variant. We believe that the identification of novel variants in PRUNE1 may also be helpful in the diagnosis of PRUNE1-related syndrome.