The clinical phenotypes associated with germline, bi-allelic and pathogenic variants of the WWOX gene are highly heterogeneous. Symptoms range from a mild phenotypic SCAR12 to a severe early infantile WWOX-related epileptic encephalopathy (WOREE syndrome), depending on the type of mutation and its effect on WWOX expression. Therefore, accurate diagnosis and treatment are hampered by the heterogeneous clinical presentation of individuals. WOREE syndrome is characterized by early onset, epilepsy, growth retardation, delayed psychomotor development, and progressive microcephaly. Early-onset epilepsy is a core feature of the neurological phenotype of WOREE syndrome, which is associated with a high early death rate14. In this study, we describe a child from an unrelated Chinese family who presented with growth retardation, early seizure disorder at birth, and followed by global developmental delay. Our patient had the earliest onset of epilepsy, presenting on the 15th day of life. Unfortunately, his multidaily tonic seizures never responded to any pharmacological treatment, and he died at the age of two and a half years due to persistent seizures. Our patient's phenotype was very similar to the patient described by Abdel-Salam et al., who had a homozygous loss-of-function germline variation in the WWOX gene15.
In this study, we identified a proband with three deletions in WWOX gene involving intron 5, exon 6, and exons 6-8 using WGS, and confirmed these findings by gap-PCR and Sanger sequencing. The intron 5 and exon 6 deletions were found in a single allele, and were discontinuous. This complex germline structural variation, which has not been previously reported, involves the same discontinuous deletion of WWOX. The intron 5 deletion has been associated with cancer in the Han Chinese population as a germline polymorphism (Hussaine et al., 2019)2. The exon 6 deletion (c.517_605ddel, H173Afs*67) is a common deletion predicted to cause a frameshift mutation resulting in a truncated protein with loss of function. In our patient, the exon 6-8 deletion was found in the other allele of the WWOX gene. The exon 6-8 deletion (c.517_1056del, His173_Met352del) theoretically produces a protein with normal WW domains, but missing the SDR domain which may have a poor functional outcome.10. Mignot et al. reported a case of a WOREE syndrome patient who had compound heterozygous deletions in WWOX gene that encompassed exon 1-5 and exon 6-8t10. Similarly, in our case, compound heterozygous deletions were found to be the cause of WOREE syndrome, which is consistent with the proposed mechanism of the syndrome. In 2009, Suzuki et al. found that the pathogenic variant of WWOX was associated with epileptogenesis in mice16. However, the pathophysiological mechanism underlying the development of WOREE syndrome due to total or partial loss of function of WWOX has yet to be fully understood. In-depth analysis of WWOX function in the brain is ongoing. Studies in the WWOX knockout mouse model have demonstrated spontaneous bursts of activity in the neocortex, suggesting that WWOX plays a fundamental role in balancing neocortical excitability17
An exon 6 deletion and an intron 5 deletion were both found on a single allele, suggesting that the chromosome underwent two separate breakage and rearrangement events. This also reflects the WWOX gene encompassing FRA16D from introns 5 to 8, indicating that the WWOX gene is prone to breakages and rearrangements. Multiple models have tried to explain the underlying mechanisms behind the FRA16D instability, including longer AT repeats forming a cruciform and stall replication. Replication origins located within CFS sequences are less efficient and probably responsible for replication perturbation along fragile sites 2,18-20. Although CNVs can be identified by multiplex ligation-dependent probe amplification (MLPA), qRT-PCR, array-CGH and WES, the detection results do not affect the correlation analysis between genotype and phenotype. In order to explain how WWOX deletions involving FRA16D were generated, we used 30× WGS combined with PCR to identify the breakpoints. We accurately identified the breakpoint junctions of three deletions. No R-loop forming sequences were found using R-loopDB to screen the sequences flanking the breakpoint junctions of the three deletions. The deletion involving exons 6-8 and another deletion involving intron 5 were presumably generated by the non-homologous end joining (NHEJ) repair pathway, because this model requires one to four base pairs or no homology between proximal and distal breakpoints. The 3´ flanking regions were (AT)n(AC)n repetitive sequences involving exon 6. The deletion might be caused by the abundance of AT-rich DNA forming complex secondary structures that slow down or block the progress of the replication machinery and ultimately leading to chromosome breakage19.
WWOX has been extensively studied for its role in cancer because it localizes to a common fragile site, FRA16D, a genomic region susceptible to chromosomal rearrangements. Different in vitro and in vivo functional studies have indicated WWOX´s role as a tumor suppressor. The reduction or absence of WWOX expression has been associated with several cancers, including breast cancer, thyroid cancer, oral cancer and lung cancer21. WWOX expression is altered by deletion and/or aberrant synthesis in a significant number of non-small cell lung cancer (NSCLC) tumors (51.8%)22. Similar results have been described in breast cancer23. Moreover, a high incidence of exons 6-8 deletions has been reported in Chinese patients with NSCLC (63.6%), suggesting that exons 6-8 deletions might play a role in the tumorigenesis of NSCLC24. In this study, we hypothesized that the heterozygous WWOX exons 6-8 deletion observed in maternal grandfather (I-3, lung cancer patient) caused an abnormal WWOX expression and the consequent clinical phenotype.
In conclusion, we reported compound heterozygous WWOX the discontinuous deletions of intron 5 and exon 6 in one allele and the deletion of exons 6-8 in another allele,which causing a more severe early infantile WOREE syndrome. Furthermore, we confirmed the exact breakpoints of these deletions using WES combined with gap PCR. Our findings extend the mutation spectrum of the WOREE syndrome and support an important role for the WWOX gene in neural development.