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.