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\begin{document}

\title{Two Novel Biallelic Variants in TECPR2 and FA2H Genes Causing
Complicated Hereditary Spastic Paraplegia in Iranian families from lur
Ethnicity: Case Series.}



\author[1]{Masoud  Edizadeh}%
\author[2]{Negar  Chegeninezhad }%
\author[1]{Soheila Akbari}%
\author[3]{Maryam  Salehirad}%
\author[2]{Rezvan  Pakmanesh}%
\author[1]{Shokoufeh Ahmadipour}%
\author[4]{Kourosh  Hayatigolkhatmi}%
\author[1]{Hamidreza khodadadi}%
\affil[1]{Lorestan University of Medical Sciences}%
\affil[2]{Madar Medical Genetics Center}%
\affil[3]{Madar Medical Genetics center}%
\affil[4]{European Institute of Oncology}%


\vspace{-1em}



  
  \date{May 20, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
We herein report first Iranian families with spastic paraplegia 35 and
49 and claim that TECPR2 gene causes complicated spastic paraplegia 49
with or without sensory autonomic neuropathy. In addition, we show how
coexistence of SPG49 and griscelli syndrome can lead to misdiagnosis.%
\end{abstract}%



\sloppy


\textbf{1 Introduction}

Hereditary spastic paraplegia (HSPs) refers to a heterogeneous group of
rare inherited neurodegenerative disorders. They are generally
characterized by progressive and length-dependent degeneration of distal
retrograde axons of the corticospinal tracts (CST) and posterior columns
of the spinal cord.\textsuperscript{1-3}Clinically, these conditions
share the primary symptoms of progressive spasticity, hyperreflexia and
mild weakness of the lower limbs in the ``Pure'' form. In the
``complicated/complex'' form, additional symptoms such as peripheral
nerve involvement, extrapyramidal disturbances, cerebellar ataxia,
polyneuropathy, cognitive impairment, optic atrophy and seizures might
be added.\textsuperscript{2, 4, 5}

So far, at least 76 clinical types of HSPs and around 80 corresponding
genes with different patterns of inheritance have been
reported.\textsuperscript{1, 2}Almost, 21 HSP-associated genes involved
in the autosomal recessive (AR) form of these disorders. One of the
complicated HSPs with AR inheritance pattern is spastic paraplegia 35
(SPG35, MIM 612319), also known as fatty acid hydroxylase-associated
neurodegeneration (FAHN). The condition is caused by pathogenic
alterations in \emph{FA2H} , located on chromosome 16q23.1. This gene
encodes endoplasmic reticulum (ER) enzyme fatty acid 2-hydroxylase. The
enzyme is a membrane-bound protein with NADPH-dependent mono oxygenase
activity, converting free fatty acids to 2-hydroxy fatty acids (hFAs),
which, subsequently are incorporated into membrane sphingolipids as
essential components of myelin. These compounds show particular temporal
expression pattern and are unessential in early development, but are
required as the individual matures.\textsuperscript{6-9}Considering the
substantial role of the enzyme in the maintenance of the myelin sheath
around neuronal axons, deficiency of its coding gene can manifest
diverse demyelinating phenotypes such as dysmyelinating, leukodystrophy
associated cognitive decline, dysarthria, spastic paraparesis with or
without dystonia and neurodegeneration with brain iron accumulation
(NBIA).\textsuperscript{10-14}Although the frequency of \emph{FA2H}
mutations in patients with HSPs has been obscure at least in Asia, it
was considered as the second most common subtype of
AR-HSP.\textsuperscript{5}SPG35 has early onset and shows heterogeneous
neuroimaging patterns such as a variable degree of white matter lesions
(WMLs), thin corpus callosum, cortical and cerebellar atrophy and iron
accumulation in the globus pallidus.\textsuperscript{4, 7, 10, 15}In
addition, genetic alterations in \emph{TECPR2} , tectonin beta-propeller
repeat containing 2 (TECPR), have been reported with another complicated
form of autosomal recessive HSP called Spastic Paraplegia 49
(SPG49).\textsuperscript{16} This gene encodes a protein which contains
two main domains, tryptophan-aspartic acid repeat (WD repeat)~and TECPR,
and plays a significant role in autophagy
process.\textsuperscript{17-19} The disorder characterized by
developmental delay, generalized hypotonia, microcephaly, short stature
and dysmorphic faces. Affected individuals evolved progressive
spasticity in the lower body muscles. However, in 2016 three more
affected individuals with additional autonomic-sensory neuropathy
features have been introduced.\textsuperscript{20} Therefore, Heimer et
al reclassified disorder as a new subtype of hereditary
sensory-autonomic neuropathy, adding controversy to the exact class of
this disorder. So far, only three pathogenic variations have been
reported in \emph{TECPR2} , implying that SPG49 is a rare genetic
disorder with unknown frequency and heterogenous
phenotype.\textsuperscript{16, 20}

We herein report two Iranian Lur families with typical features of HSPs.
Subsequent genomics and bioinformatics analyses have suggested novel
pathogenic variants in TECPR2 and FA2H genes.

\textbf{2 CASE PRESENTATION}

2.1 Patient A

Two affected siblings (shown in Figure 1) from first cousin Lur parents
were referred to Madar Medical Genetics center. The proband was
3.7-year-old girl who was hospitalized immediately after delivery due to
respiratory distress. Then, until age 1.5 she was repeatedly
hospitalized because of recurrent respiratory infections and decreased
consciousness, diagnosed as pneumonia. She had generalized hypotonia and
moderate developmental delay since she has not acquired ambulation until
the time of study, however, she was able to creep and crawl. She can
stand up only by physical aid. The proband achieved limited level of
communication, using few words to call her parents. Physical examination
illustrated short stature, microcephaly, brachycephaly and synostotic
trigonocephaly. The other clinical features include failure to thrive,
short neck, dysmorphic face, triangular face, severe strabismus, myopia
and amblyopia. The proband experienced three episodes of seizures
following temperatures up to 40, phenobarbital administered in order to
control seizures. She showed aggressive behavior and easy mood changes,
injuring other infants by physically attacking them. Detailed clinical
examination revealed specific facial dysmorphic features such as
protruding ears, absent antihelical fold in left ear, sparce eyebrows,
bulbus nose, wide nasal bridge, flat nasal tip as well as chubby cheeks.
The auditory brain-stem responses (ABR) examination was normal at age 2.
Furthermore, her parents complained about her increased appetite in
addition to gastroesophageal refluxes. Neurological examination showed
the extension of hallux during Babinski test, indicating upper motor
neuropathy. However, unlike previously reported patients, no sign of
sensory-autonomic neuropathy including sleep disturbances, apnea,
decreased pain sensitivity, blood pressure, arrythmia was identified at
the time of study. Laboratory findings indicated elevated levels of
platelets and SGOT. The proband's 2-year-old sister represented with
similar clinical features of the proband (Figure 1). Furthermore, the
affected sisters had silver-gray hairs and partial white patches on
their skin. The younger sister's hair was completely silvery or gray in
majority of parts, while the proband's hair was considerably darker.
Similarly, white eyelashes and light iris were observed in both sisters.
The proband had family history of grisclli appearance since her father
and two of aunts and uncles represented with similar appearance. Since
both siblings had griscelli phenotype in addition to developmental delay
and neurological regression, the disorder was mistakenly diagnosed as
phenylketonuria (PKU).

2.2 Patient B

An 11-year-old male who exhibited a history of gait difficulties,
frequent falls and clumsiness was requited to this study. The proband
(Shown in Figure 2) was the first child born to healthy consanguineous
parents from Lorestan province, Iran. The proband was born naturally and
delivery was uneventful. However, the mother had a miscarriage history,
and she was pregnant for a male fetus at the time of the study. The
process of the childhood development was remarkable, since the proband
was able to hold his neck, sit, communicate and walk at the estimated
time. By the age of 4 years, the first sign of the disorder appeared as
he represented with lower limb spasticity and gait difficulties. The
proband's toes became spastic which led to fixed plantar flexion of the
foot, indicating pes caus. Subsequently, he acquired motor difficulties
including hyperreflexia, tremor and ataxia. Strabismus and poor vision
were also observed in eye examination. The rapid progression of the
disorder resulted in loss of previously acquired developmental
milestones, leading to mild-sever cognitive decline, intellectual
disability and progressive loss of ambulation. The family also
complained about the proband's urinary urgency. Electromyography (EMG)
and nerve conduction velocity~(NCV) evaluations of skeletal muscles at
the age of five, presented no evidence of myopathy or peripheral
neuropathy. The magnetic resonance imaging (MRI) of brain and spinal
cord disclosed very mild abnormal signal intensity in centrum semiovale,
suggesting leukoencephalopathy or periventricular leukomalacia (PVL)
(shown in Figure 2). With this regard, clinical findings proposed mild
spastic paraplegia.

2.3 Molecular analysis

Whole-exome sequencing (WES) was implemented in Madar Medical Genetics
Center, Khorramabad, Lorestan, Iran to identify the subtype of HSPs and
the corresponding gene, due to heterogenous nature of the disorder. To
this end, the genomic DNA of the probands and their relatives were
extracted from peripheral blood based on an established salting out
protocol.\textsuperscript{21} Only the probands' samples were subjected
to WES. For Patient B, the SureSelect Human All Exon V6 Kit (Agilent
Technologies Inc., Santa Clara, CA, USA) was used to capture exonic
region and paired-end sequencing was carried out on illumine NextSeq
(Illumina Inc., San Diego, CA, USA). For patient A, Human Core Exome Kit
(Twist Bioscience) was used for this purpose. The Burrows-Wheeler
Aligner\textsuperscript{22}was implemented to align the sequence reads
to GRC38 human reference genome. GATK
HaplotypeCaller\textsuperscript{23, 24} tool was utilized to call all
variants within the target regain and annotation was performed using
ANNOVAR. At the next step, all variants with more than 0.01 alle
frequency in 1000genome, genomAD exome and GenomAD genome were removed
and the remaining variants were prioritized according to bioinformatics
predictions, inheritance pattern and clinical information. Once WES data
was analyzed, the co-segregation analysis of the disease-associated
variant was done for understanding the inheritance pattern of the
disorder. For this purpose, specific primers Table 1 were designed using
online tools such as primer3 (https://primer3.ut.ee), oligoanalyzer
(https://eu.idtdna.com/calc/analyzer) and ensembl databases
(https://ensembl.org). After PCR amplification, Sanger sequencing was
performed using Applied Biosystems 3500 Genetic Analyzer and the
sequences were aligned to the reference genome by Codon Code Aligner
(https://www.codoncode.com/aligner).

2.4 Genetic findings and co-segregation study

\textbf{2.4.1 Patient A}

In patient A, following WES, a frameshift variant defined as c.1568delC
(NM\_014844, 14:102434385 GCR38, p.S523Ffs*12) in exon 9 of\emph{TECPR2}
was identified. This variant was not found neither in GenomeAD,
1000genome, Iranome databases nor in our local database for 300 Lur
individuals, being extremely rare. The c.1568delC variant was a single
nucleotide deletion of cytosine, leading to a premature stop codon only
11 amino acids after deletion site (shown in Figure 1).\emph{TECPR2}
encodes 1411 amino acid length protein in canonical isoform
(NM\_014844), which contains three WD and ten TECPR domains. As a
consequence of this frameshift mutation, approximately 63\% of critical
region of original protein is eliminated after translation, remaining
only 37\%. In addition, another homozygous deletion defined as
c.1135\_1136del (NM\_024101, 2:237540378 GRC38, p.D379Cfs*19) in exon 10
of \emph{MLPH} was detected in the proband. This mutation describes the
grisclli appearance of affected siblings, since mutations in this gene
have been reported with Griscelli syndrome, type 3. Genotyping family
members including mother and siblings reveled that affected sister
harbored c.1568delC variant in homozygote manner, while mother and the
healthy elder daughter of the family were carriers (shown in Figure 1).
However, father of the family was unavailable to participate in the
study. As expected, two affected siblings were homozygote for
c.1135\_1136 deletion in \emph{MLPH} , confirming the Griscelli
syndrome, type 3 in the family.

\textbf{2.4.2 Patient B}

As a consequence of WES, a homozygous three-nucleotide deletion in exon
5 of \emph{FA2H} was identified. The c.685\_687delATC mutation was a
non-frameshift deletion of nucleotides 685 to 687 (NM\_024306) which
leads to deletion of a non-polar, uncharged amino acid Isoleucine at
229th residue. For understanding the pattern of the inheritance, the
proband's relatives underwent co-segregation analysis. The results
revealed heterozygotes status of parents and male fetus, suggesting an
AR pattern of inheritance (shown in Figure 2). Since the pathogenicity
of the mutation has not been reported in gene variant public databases,
we applied several variant effect predictor websites to identify its
potential pathogenicity. The predictors predicted the mutation according
to ACMG parameters as a probably pathogenic variant
(http://www.varsome.com/). Our analysis also showed that the nucleotides
and their corresponding amino acid are highly conserved among different
species (up to Chrysochloris Asiatica and considering 4 species) (shown
in Figure 2).

\textbf{3 Discussion}

In the current study, we identified two novel pathogenic homozygous
deletions in \emph{TECPR2} and \emph{FA2H} in two Iranian families, who
were diagnosed with complicated spastic paraplegia. Patient A and her
sibling were homozygous for two frameshift deletions in \emph{TECPR2}and
\emph{MLPH} genes, while patient B was homozygous for non-frameshift
mutation in \emph{FA2H} . Regarding \emph{TECPR2} , in 2012, it was the
first time when a frameshift single nucleotide deletion (c.3416delT) in
exon 16 of \emph{TECPR2} was linked to a complicated form of HSP, named
SPG49.\textsuperscript{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 \emph{TECPR2} were
reported.\textsuperscript{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 \emph{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.\emph{TECPR2} plays a critical role in autophagy by
associating with distinct cellular components such as \emph{COPII} ,
\emph{SEC24D} ,\emph{HOPS} and \emph{BLOC-1} .\textsuperscript{19} The
most possible role of \emph{TECPR2} is that it probably acts as an
anchor point for multiple cellular components. In one example, it
stabilizes\emph{SEC24D} 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
of\emph{TECPR2} in neuronal cell development and maintenance.

Turning to \emph{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). \emph{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.\textsuperscript{1, 4, 25}With 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\%.\textsuperscript{5, 26,
27}However, additional consequences of \emph{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.\textsuperscript{10}Since 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 \selectlanguage{ngerman}±3.20; 36 patients out of
38\textsuperscript{28}), 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).\textsuperscript{10, 29}According to some
research, iron accumulation is not always present in all of the
patients, whilst the affected individuals share the same
mutation.\textsuperscript{10, 11}In 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.\textsuperscript{1, 30}

In summary, WES analysis allowed us to identify two homozygous
deletions, p.I229del and c.1568delC in \emph{F2AH} and
\emph{TECPR2}respectively, 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.

\textbf{ACKNOWLEDGEMENTS}

Authors are grateful for participants and their families for their
participation and cooperation in this study.

\textbf{CONFLICT OF INTEREST}

The authors declare no competing interests.

\textbf{AUTHOR CONTRIBUTIONS}

M. Edizadeh performed WES data analysis, variant interpretation,
bio-informatics measurements and wrote the manuscript. N. Chegeninezhad
performed WES, Sanger sequencing and co-segregation analysis. S. Akbari
made clinical diagnosis. R. Pakmanesh designed primers and extracted DNA
from peripheral blood samples. M. Salehirad performed PCR and Sanger
sequencing. S. Ahmadipour made patient work-up and management. K.
Hayatigolkhatmi wrote and revised the manuscript. H. Khodadadi
supervised the study, revised and finalized the manuscript, supported
the study financially.

\textbf{ETHICAL APPROVAL}

This study was approved by the ethics committee of Lorestan University
of Medical Sciences (IR.LUMS.REC.1399.328) and adhered to tenets of the
Declaration of Helsinki. Prior to study, written informed consent form
was obtained from the participants' parents for publication of this
study.

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Figure 1. Pedigree and molecular findings of family A. A: There are two
affected siblings (IV-2 and IV-3) with clinical features of complicated
hereditary spastic paraplegia, indicated with blue color. Proband's
father, aunt and uncles (III-1,2,3) represented with Griscelli
appearance, green color. C: The nucleotide and amino acid sequences in
the mutation site (c.1568delC, p.S523Ffs*12) are shown. In normal
protein, TCT codon codes serin residue at 523th amino acid, while
c.1568delC changes this amino acid to phenylalanine and creates
premature stop codon exactly 11 amino acids after mutated residue,
causing truncated protein. B and D illustrate nucleotide sequences
in\emph{TECPR2} and \emph{MLPH} respectively. Mother is carrier for both
mutations, while the affected sibling is homozygote for identified
alterations. In addition, the mutation in TECPR2 gene was not found in
the healthy elder sibling, however, the mutation in MLPH gene was
identified in heterozygote manner.

Figure 2. Pedigree and molecular findings of family B. A: MRI imaging at
age 5 which shows very mild abnormal deep white matter periventricular
(yellow arrows). B: Pedigree of the family. The proband has been
indicated by the arrow. C: The mutation segregation by Sanger sequencing
has been done for the proband's relatives. As can be seen in sequence
chromatograms, his parents (III-3 and III-4) are heterozygotes for c.
del685-687 and the proband (IV-1, colored in red) is homozygous. D: The
deletion of three nucleotides leads to non-frameshift deletion of highly
conserved amino acid Isoleucine at 229th residue as shown by the
orthologous sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi). E:
Schematic representation of 7 exons of the FA2H and its two highly
conserved domains; cytochrome b5-like heme-binding domain (residues
15--85) and sterol desaturase domain (residues 124-366). The identified
variant in this study was shown.

\textbf{Table 1. Primers}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Gene & Forward Primer & Reverse Primer\tabularnewline
FA2H & GCTATGCACCGGTCCTCA & GCTATGCACCGGTCCTCA\tabularnewline
TECPR2 & TTAGCTGCCCTCAAACCCAT & ACATCTTCCCTCGCTCCATT\tabularnewline
MLPH & GGTCTTAGATAGCCAGGGGT & TTGGTGTGCTCCTGTGGG\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Fig/Fig}

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\end{figure}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fih/Fih}

\end{center}
\end{figure}

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