Novel Mutation With Literature Review WW Domain-Containing Oxidoreductase (WWOX) Gene

Genetic alterations in the WW domain-containing oxidoreductase (WWOX) gene cause autosomal recessive developmental and epileptic encephalopathy, characterized by the onset of refractory seizures in infants, along with severe axial hypotonia and profoundly impaired psychomotor development. It has also been expanded to include metabolism and endocrine systems. Despite its function as a tumor suppressor gene, genetic alterations in WWOX have been found in several metabolic disorders and neural diseases related to brain development. Whole-exome sequencing (WES) was performed on the patient sample. Genomic DNA was fragmented, and the exons of known genes in the human genome, as well as the corresponding exon-intron boundaries,were enriched using Roche KAPA capture technology (KAPA hyperExome Library, WES identifying the homozygous variant c.406A>G in WWOX (OMIM:605131). This variant of WWOX was also observed in the prenatal WES data, indicating that both parents were heterozygous carriers and the detected variant was homozygous. This study highlighted the importance of the human WWOX gene in brain development and the association between WWOX gene mutations and developmental delay. We recommend performing WES as a primary screening before the final diagnosis, particularly in populations with high rates of consanguinity and in clinically challenging cases.


Introduction
The WW domain-containing oxidoreductase (WWOX) gene is a transcriptional regulator that is expressed in the common fragile site FRA16D (16q23.1-q23.2). It encodes for 414 amino acid protein, with two interacting WW domains at the N-terminal with conserved proline and tryptophan residues and a short-chain dehydrogenase/reductase domain at the C-terminus [1][2][3]. The phosphorylation of Tyr33 (by tyrosine kinase, Src) activates WWOX, which then functions as a tumor suppressor gene [1,2]. WWOX performs a range of functions at the cellular, organ, and systemic levels, including metabolism, endocrine system control, and CNS differentiation and functioning. Genetic alterations in WWOX have been found in several metabolic and neural diseases. The lack of functional WWOX protein due to germline mutations impacts brain development. The severity of the disease can be determined based on complete or partial loss of protein functionality [1]. Nevertheless, most tumors show a high rate of loss of heterozygosity upon expression of somatic WWOX alternations [1]. Reduced maturation of oligodendrocytes and myelinated axons and impaired axonal conductivity have been observed in WWOX mutant mice [4]. WWOX-related syndromes have been diagnosed using whole-exome sequencing (WES) to identify the homozygous variant in WWOX (OMIM:605131), which leads to amino acid substitution. Out of 21 bioinformatics in silico experiments, 15 suggested a pathogenic effect of this variant. Additionally, based on in silico predictions, the position of the identified variant led to significant alterations in mRNA splicing owing to an altered splice site. Parallel analysis of prenatal WES data revealed that both parents were heterozygous carriers of the WWOX variant. This confirmed the homozygosity of the detected variant in the index. To the best of our knowledge, this variant has not been reported to date. Classifications of variants were conducted based on ACMG Guidelines (Richards et al.) considering database entries (inc. HGMD), Bioinformatics predictions tools, and literature status. The variant has been detected in 0.0042% of the general population (five heterozygous, 0 homozygous; gnomAD v2,1,1 controis) and this is the first time we detected it in our internal database in a homozygous state.

Case Presentation
The patient is a 21-month-old boy. He was a full-term baby delivered by cesarean section (due to a previous CS) with no history of NICU admission. The mother visited the clinic complaining that her 1-yearold son could not walk. The initial concern began when the mother brought the baby to ER after noticing an objective fever, and decreased activity and oral intake after 33 days of birth. The patient was admitted for six days and was diagnosed with an influenza A infection. He had chronic constipation, intermittently for 1 month on and off by the age of seven months. Even when the baby was one years old, he could not roll from the supine to the prone position or walk. In addition, he could not initiate a sitting or standing position. At 14 months, a mild improvement was observed according to the mother, as the baby could walk one step while holding furniture and he could roll but the concern related to his walking ability persisted. By the age of 15 months, the baby could crawl, pull to stand, sit without support, and provide good head support, but could not walk alone. Currently, at the age of 21 months, he can say 10 words but not complete sentences, climb stairs with assistance, walk, and run with recurrent falling. Upon investigating the family history, his parents were consanguineous; of his first-degree relative with four siblings, three were healthy and one was known to have seizures. His cousin also had a history of cerebral palsy.
Upon physical examination, he was vitally stable with no dysmorphic features or distress, but he had an abnormal gaze around three times, and there was a hyper-pigmented spot on his right thigh. He had good eye contact, and responded to commands and sounds but looked at non-specific objects from different angles. The power for both upper and lower limbs was 4/5 with good reflexes and tone. Bowing of the legs and flat feet were noticed.
Metabolic investigations revealed normal levels of urine organic acids and all plasma amino acids except for slightly elevated alpha aminobutyric acid and a marked decrease in cystine level ( Table 1). Liver profiles, including ALT and GTT, were normal, whereas AST, albumin, total bilirubin, and direct bilirubin levels were high, and globulin level was low. The lipid profile revealed low cholesterol triglyceride levels. Anti-smooth muscle antibody test showed the following results, smooth muscle (<1.20, CMV-IgG <5, CMV IgM negative, HBsAg negative, ANA-IFA negative, and antiHBs 699.89). The TSH and free T4 levels were within the normal range. Creatine kinase was high. 25-OH vitamin D and vitamin B12 levels were high. Abdominal ultrasonography revealed mild hepatosplenomegaly. Liver approximately 6.6 cm, with no focal lesion, and no intra-or extrahepatic biliary duct dilatation was observed. His hepatic and portal veins were patent. The gallbladder was partially contracted and appeared on gross examination. There was no abdominopelvic lymphadenopathy. Brain MRI results were normal.

Investigations value Value
Alpha aminobutyric High

Discussion
WWOX gene alterations can cause severe problems. It is expressed in the common fragile site FRA16D (16q23.1 -q23.2), prone to high frequencies of loss of heterozygosity and homozygous deletion [5]. WWOX expression has been uniformly detected in neurons and glial cell types. Moreover, progenitor oligodendrocytes are considered to have higher WWOX expression compared to a mature myelinated oligodendrocyte. WWOX is involved in the development and differentiation of these specialized cells. Stable expression of WWOX in microglial cells has been observed from the analysis of RNA-seq data available in the public database [6][7][8]. Severe neural diseases, metabolic disorders, and early death can result from a homozygous null mutation in the WWOX gene [9]. The present case report study reported the first case of a WWOX-related phenotype. This WWOX variant is found in 0.0042% of the general population (5 heterozygous, 0 homozygous; gnomAD v2,1,1 controis); to the best of our knowledge, first time detected in a homozygous state. It has been reported that 87% of the variants in gnomAD appeared 1-109 times, with an average of 14 individual alleles per variant. None of these variants were observed in a homozygous state [3]. Pathogenic variants in WWOX cause autosomal recessive developmental and epileptic encephalopathy 28 (DEE28; OMIM:6162111), characterized by the onset of refractory seizures in infants. Affected individuals have severe axial hypotonia and severely impaired psychomotor development. More severely affected patients have acquired microcephaly, poor or absent visual contact, and retinal degeneration leading to early death. From the available information, the patient phenotype appears partially supportive of DEE28. In the present study, our patient presented with a global developmental delay and no early seizure disorder despite a family history of seizures and cerebral palsy in his brother and cousin, respectively. Additionally, the MRI was normal, with no progressive microcephaly or bilateral optic atrophy. In addition, our patient had mild hepatosplenomegaly and a high abnormal level of creatinine kinase (CK), cholestasis, and chronic constipation that have not been reported in WWOX patients; nevertheless, the symptoms in our patient are considered less severe than the other variants that have been reported previously. A case in the Emirates, for instance, described an infant with an early seizure disorder associated with global developmental delay, progressive microcephaly, delayed psychomotor development, and spastic quadriplegia [10]. This phenotype is very similar to another case reported in an Egyptian family; however, it is considered a more lethal case as the patient died at the age of 16 months [11]. The patients in the other two reported families (Saudi and Palestinian) did not develop progressive microcephaly, and the severity of the disease was considerably less than that in the Emiratis and Egyptian cases. Furthermore, the case of the Saudi Family lacked spasticity and the deep tendon reflexes were diminished, whereas the case of the Palestinian had spasticity with exaggerated reflexes in addition to the ataxia (  WWOX is critical for homeostasis in vivo and cell death. The point or homozygous nonsense mutations in the WWOX gene affect embryonic neural development, resulting in autosomal recessive cerebellar ataxia and epilepsy, growth retardation, microcephaly with seizure, retinal degeneration, and early death at 16 months of age [9]. As shown in our case, the lipid profile was low, which can be attributed to the WWOX gene, as it has been reported to be involved in the regulation of lipid homeostasis and metabolism [13,14]. Thus, WWOX plays a crucial role in neural development and lipid metabolism. The lack of functional WWOX can lead to severe neural diseases and metabolic disorders. Moreover, the severity of phenotypes depends on the genetic makeup of the patient. Unfortunately, the number of cases with WWOX mutations is low; therefore, it remains uncertain whether some WWOX mutations have a unique phenotype. WWOX genotypes appeared to correlate with the severity and onset of seizures and spasticity in the studied cases. However, studies on more patients are needed to establish accurate genotypephenotype correlations. Given the function of WWOX as a signaling protein involved in different proteinprotein interactions, the disruption of neuronal pathways could explain the susceptibility to seizures [4].