3 Discussion
In the current study, we identified two novel pathogenic homozygous deletions in TECPR2 and FA2H in two Iranian families, who were diagnosed with complicated spastic paraplegia. Patient A and her sibling were homozygous for two frameshift deletions in TECPR2and MLPH genes, while patient B was homozygous for non-frameshift mutation in FA2H . Regarding TECPR2 , in 2012, it was the first time when a frameshift single nucleotide deletion (c.3416delT) in exon 16 of TECPR2 was linked to a complicated form of HSP, named SPG49.16 All five affected individuals in the study were from the same ethnic group and shared similar clinical features of generalized hypotonia, developmental delay, progressive spasticity in lower body, dysmorphic features, and recurrent respiratory infections. Since the fact that all individuals were from unrelated Jewish Bukharin families and harbored similar genetic variation, the c.3416delT mutation was considered as a founder mutation in that specific ethnic group. There had not been any TECPR2 related phenotype until 2016, when three affected individuals with c.C566T (p.Thr189Ile) and c.1319delT (p.Leu440Argfs*19) mutations in TECPR2 were reported.20 Despite having similar clinical features of previously reported individuals, all the patients showed extra clinical manifestations of autonomic neuropathy. The authors, as a conclusion, believed that this disorder should be classified as a form of HSAN not HSP, owing to the fact that none of complicated forms of HSP shows autonomic neuropathy. Interestingly, two affected siblings in current study represented with identical clinical manifestations to those who were previously reported in the 2012 study, showing no sign of autonomic neuropathy except having grescilli appearance due to c.1135_1136del genetic alteration in MLPH . Moreover, detailed clinical and paraclinical examinations revealed additional symptoms including elevated levels of platelets and SGOT in blood and triangular face. All in all, because of diverse multisystem signs and symptoms of this disorder, affecting autonomic neurons in limited individuals, we believe that it should still be classified as complicated SGP49 with or without sensory autonomic neuropathy. Another interesting point is that co-incidence of two monogenic disorders in an individual can sometimes misguide clinicians in diagnosis, as in our study, we initially misdiagnosed the disorder as PKU with combination of immunodeficiency.TECPR2 plays a critical role in autophagy by associating with distinct cellular components such as COPII , SEC24D ,HOPS and BLOC-1 .19 The most possible role of TECPR2 is that it probably acts as an anchor point for multiple cellular components. In one example, it stabilizesSEC24D to ensure the efficient function of endoplasmic reticulum in exporting several secretory elements. In general, more functional studies are required to throw light on the exact mechanism ofTECPR2 in neuronal cell development and maintenance.
Turning to FA2H , the detected variant was predicted as likely pathogenic, due to the protein changing effect of the deletion of conservative amino acid Isoleucine (p.I229del). FA2H contains seven exons and encodes an integral membrane enzyme of smooth endoplasmic reticulum (ER), which catalyzes galactosylceramide and sulfatide hydroxylation in the myelin sheath. The enzyme contains two conserved domains. First, is the N- terminus cytochrome b5-like heme-binding domain (residues 15–85), responsible for the redox activity of the enzyme. Second, is the sphingolipid fatty acid hydroxylase domain, spanning residues 124-366 in the C-terminal. The identified deletion is located in the latter domain. The domain is known as sterol desaturase domain, composed of a catalytic di-iron cluster and four transmembrane domains, mediating anchoring the enzyme to the ER membrane.1, 4, 25With this regard, the variant may interfere with the catalytic activity of the protein. In previous reports, several pathogenic alterations, which are scattered along the sterol desaturase domain, including p.V149L, p.L130F, p.R235C and p.H260Q, have been examined. The result indicated their pathogenic effects on the hydroxylase activity of the FA2H. The enzyme activity of p.V149L and p.R235C variants each separately was 60%-80%, p.L130F approximately 52% and p.H260Q nearly 0%.5, 26, 27However, additional consequences of FA2H variants in post transcriptional level, such as p. R154C variant, which, reduces the mRNA or protein stability should not be excluded. In such a case, western blot for measuring protein abundance and enzyme-linked immunosorbent assay (ELISA) or gas chromatography–mass spectrometry for quantifying the enzyme activity are applied.10Since we were unable to determine the enzyme activity and protein abundance in our patient, we predicted the variant’s pathogenicity and its effect on protein structure in silico. Our findings showed on the surface that the mutation is pathogenic and leads to change of the helix structure of the enzyme to a moderate extent, proposing a possible role of the conserved amino acid Isoleucine in the catalytic activity. Further functional studies in terms of patient’s cell culture or animal models are necessary to confirm our findings.
The SPG35 affected patients show a complicated genotype-phenotype correlation. Similar to the majority of cases with early onset spastic paraplegia (mean age of onset 5.76 years ±3.20; 36 patients out of 3828), our proband revealed early onset spastic paraplegia at age 2. Dystonia, with a high prevalence among the SPG35 patients (89.7%; 26 patients out of 29), was also observed in the proband. SPG35 is considered as a subtype of neurodegeneration with brain iron accumulation (NBIA).10, 29According to some research, iron accumulation is not always present in all of the patients, whilst the affected individuals share the same mutation.10, 11In our proband’s MRI, no sign of brain iron deposition was found. This is suggestive of the phenotype being associated with iron accumulation as a consequence of FA2H variation seeming variable in the patients, or T2 MRI not being a fully conclusive technique for detection of iron deposition. However, progressing cognitive decline and epileptic seizures as typical symptoms of SPG35 which are seen in almost 90% and 30% of patients, respectively.1, 30
In summary, WES analysis allowed us to identify two homozygous deletions, p.I229del and c.1568delC in F2AH and TECPR2respectively, in two patients with symptoms of complicated spastic paraplegia. Nonetheless, functional studies in terms of enzyme testing, for assessing the enzyme activity in patients and also animal models are required to validate such a deduction. Further investigations are needed to precisely describe the range of phenotypes in the SPG35 and SPG49 cases in Iran.