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\begin{document}

\title{Characterization of a novel loss-of-function variant in \emph{TDP2} in
two adult patients with spinocerebellar ataxia autosomal recessive 23
(SCAR23)}



\author[1]{Edoardo Errichiello}%
\author[2]{Guido Zagnoli-Vieira}%
\author[3]{Romana Rizzi}%
\author[3]{Livia Garavelli}%
\author[2]{Keith Caldecott}%
\author[1]{Orzetta Zuffardi}%
\affil[1]{University of Pavia}%
\affil[2]{University of Sussex}%
\affil[3]{Azienda USL di Reggio Emilia}%


\vspace{-1em}



  
  \date{May 05, 2024}


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\selectlanguage{english}
\begin{abstract}
\emph{TDP2} encodes a 5'-tyrosyl DNA phosphodiesterase that is required
for the efficient repair of double-strand breaks (DSBs) induced by the
abortive activity of TOP2. To date, only two homozygous variants in
\emph{TDP2} have been reported in five patients from three unrelated
pedigrees with spinocerebellar ataxia 23 (SCAR23). By whole-exome
sequencing, we identified a novel \emph{TDP2} splice-site variant
(c.636+3\_636+6del) in two Italian siblings (aged 39 and 43) showing
progressive ataxia, intellectual disability, speech delay, refractory
seizures, and various physical anomalies. The variant caused exon 5
skipping with consequent nonsense-mediated mRNA decay and defective
repair of TOP2-induced DSBs, as demonstrated by the functional assays
performed on the patients' fibroblasts. Our findings further support the
pathogenic role of biallelic loss-of-function variants of \emph{TDP2} in
SCAR23 pathogenesis. Considering the adult age of our patients and their
extensive follow-up, our study delineates in more detail the clinical
phenotype related to the loss of TDP2 activity.%
\end{abstract}%



\sloppy


\textbf{KEYWORDS}

\emph{TDP2} , topoisomerase II (TOP2), double-strand breaks (DSBs),
spinocerebellar ataxia, intellectual disability, epilepsy, speech delay,
whole-exome sequencing (WES)

The impairment of DNA repair systems and accumulation of DNA double
strand breaks (DSBs) are involved in the pathogenesis of different
neurodegenerative diseases, such as Alzheimer's disease and amyotrophic
lateral sclerosis (Madabhushi et al., 2014). \emph{TDP2} (6p22.3)
encodes the enzyme tyrosyl-DNA phosphodiesterase 2 that is required for
efficient repair of topoisomerase II (TOP2)-induced DSBs by
non-homologous end-joining (NHEJ) (Cortes Ledesma et al., 2009;
G\selectlanguage{ngerman}ómez-Herreros et al., 2013). In addition, \emph{TDP2} has been shown to
be involved in neural development and maintenance by regulating
brain-specific gene transcripts (Gómez-Herreros et al., 2014), and it is
expressed throughout the brain of adult mice, especially in the dentate
gyrus of the hippocampus and in the dopaminergic \emph{substantia
nigra}neurons, where it exerts a proapoptotic function by interacting
with Parkinson's disease-associated DJ-1/PARK7 mutant proteins
(Zucchelli et al., 2009).

Biallelic inactivating variants of \emph{TDP2} have been identified in
five individuals with intellectual disability, seizures and ataxia,
defining a new nosological entity known as spinocerebellar ataxia
autosomal recessive 23 (SCAR23; OMIM \#616949) (Gómez-Herreros et al.,
2014; Zagnoli-Vieira et al., 2018; Ciaccio et al., 2019). TOP2-induced
DSBs have been implicated in genome instability and oncogenic chromosome
translocation (Gómez-Herreros et al., 2017). On the other hand, the use
of ``TOP2 poisons''~\selectlanguage{english} \selectlanguage{english}such as etoposide and doxorubicin~ \selectlanguage{english}has been
suggested in clinical practice as adjuvant therapy to sensitize cancer
cells toward TOP2 poisons (Kont et al., 2016).

We characterized a novel \emph{TDP2} variant in two adult siblings (aged
45 and 39 years), supporting the causative role of
\emph{TDP2}loss-of-function variants in SCAR23 and further delineating
the related clinical phenotype. We also addressed the open question of
whether\emph{TDP2} may be involved in susceptibility to other
neurological conditions as well as cancer.

\textbf{Clinical phenotypes}

The probands are a 45-year-old male (III-38) and his 39-year-old sister
(III-40), born to declared non-consanguineous parents from the same
small village of Southern Italy (Calabria). The second-born child, who
died at 5 in a car accident, developed normal language skills (Figure
1a). The siblings were affected by global developmental delay (walking
\textgreater{}18 months), ataxia, intellectual disability, speech delay
(first words at 5-6 years), focal drug-resistant epileptic seizures (by
the age of 15) and progressive gait deterioration. Furthermore, they
showed several craniofacial dysmorphisms, hypertrichosis, abdominal
distention, and flatfoot (Table 1). Brain MRI in the brother showed
bilateral mesial temporal sclerosis (documented when the patient was 35
years old) and clinodactyly of the 5\textsuperscript{th} finger (Figure
1b). Both siblings achieved complete pubertal development. Metabolic
tests (urinary amino and organic acids, lactate, pyruvate, ammonium),
echocardiogram, audiometry, and first-level genetic screening
(karyotyping, subtelomeric FISH) were normal. The proposita underwent
bilateral cataract surgery at the age of 31.

The family history was remarkable for other neuromuscular diseases in
the maternal side of the pedigree, whereas no extensive information was
available for paternal relatives. Two cousins (daughters of a mother's
brother; III-1 and III-2) died of congenital muscular dystrophy at the
age of 6 and 2 years, respectively; a 38-year-old cousin (son of a
mother's brother; III-18) was affected by cognitive impairment since the
age of 10, and developed hallucinations, social isolation, and
aggressive mood at the age of 18, being diagnosed as paranoid
schizophrenia (ICD-10); and a 28-year-old cousin (daughter of a mother's
brother; III-28) was affected by epilepsy since the age of 3. The mother
of the probands developed a colorectal adenocarcinoma (TNM: pT3 N1b)
when she was 51 years old.

Patient's data have been submitted to the ClinVar database with
accession number SCV001147007. The study was conducted in accordance
with the Declaration of Helsinki and national guidelines. Written
informed consent was obtained from the patients' guardian for the
publication of this study and any potentially identifying images.

\textbf{Genetic analysis and functional validation in the patients}

Whole-exome sequencing (WES; SureSelect Human All Exome V7) identified
in both siblings a homozygous variant (NM\_016614.3:c.636+3\_636+6del)
within the exon-intron boundary of \emph{TDP2} exon 5, removing 4
nucleotides near the splicing donor site (Figure S1). The variant,
unreported in gnomAD and found in only one carrier individual in the
Bravo/TOPMed dataset (1/125,568, 0.000796\%), was at the heterozygous
state in the healthy parents. Homozygosity mapping detected a large
region of homozygosity (ROH) of ~30 Mb (chr6:10,398,474-40,431,707;
GRCh38/hg38), shared by the affected siblings and spanning
\emph{TDP2}(chr6:24,649,977-24,667,033).

Different bioinformatic tools predicted donor splice-site loss due to
the c.636+3\_636+6del variant. In agreement with the
\emph{in-silico}predictions, fibroblasts' analysis revealed that the
variant caused the skipping of \emph{TDP2} exon 5, leading to the
insertion of a premature stop codon within six amino acid residues
(p.His174GlufsTer6) and consequent nonsense-mediated mRNA decay (Figure
2a and Figure 2b). Accordingly, immunoblotting failed to detect TDP2
protein in the fibroblasts of both siblings, totally overlapping the
expression pattern previously observed in the fibroblasts of the 850BR
patient (Zagnoli-Vieira et al., 2018) harbouring the
c.425+1G\textgreater{}A variant in \emph{TDP2} (Figure 2c). Furthermore,
cells from affected individuals carrying either the c.636+3\_636+6del or
the c.425+1G\textgreater{}A variants revealed defective repair of
TOP2-induced DSBs relative to control cells, as measured by the
persistence of \selectlanguage{greek}γ\selectlanguage{english}H2AX immunostaining, an indirect marker of DSBs, after
treatment with the TOP2 poison etoposide (Figure 2d). According to the
ACMG criteria (Richards et al., 2015), the variant was classified as
pathogenic.

\textbf{Pedigree segregation and somatic analysis}

By Sanger sequencing, we detected the c.636+3\_636+6del variant at the
heterozygous state in one apparently healthy paternal uncle as well as
in the cousin with schizophrenia (III-18). In this latter, the variant
was inherited from the apparently unaffected father and was also present
in the younger healthy brother (Figure 1a). WES analysis in III-18
excluded the presence of additional pathogenic variants in known
schizophrenia-associated genes (Table S1) as well as copy number
variations (CNVs). DNA from other family members was not available for
the analysis. Considering the crucial role of TDP2 in NHEJ and
maintenance of genome stability and the development of a colorectal
cancer in the patients' mother, we were prompted to examine whether a
second somatic hit in \emph{TDP2} may have occurred. However, any
pathogenic coding variant was detected in the DNA obtained from the
tumour sample.

\textbf{Discussion}

To date, five patients have been described to carry
homozygous\emph{TDP2} variants. A homozygous splice-site variant in
\emph{TDP2}(c.425+1G\textgreater{}A) has been reported in three Irish
siblings and in an additional patient from the United States who shared
the same haplotype, suggesting a founder effect (G\selectlanguage{ngerman}ómez-Herreros et al.,
2014; Zagnoli-Vieira et al., 2018). Recently, a homozygous nonsense
variant in the exon 3 of \emph{TDP2}(c.400C\textgreater{}T; p.Arg134Ter)
has been detected in a 11-year-old Italian girl (Ciaccio et al., 2019).

In this study we identified a novel homozygous spice-site variant
disrupting the correct splicing of \emph{TDP2} exon 5. By using
immunofluorescent detection of \selectlanguage{greek}γ\selectlanguage{english}H2AX as an indirect measure of DSBs, we
demonstrated that the novel \emph{TDP2} c.636+3\_636+6del splice-site
variant (as the c.425+1G\textgreater{}A) profoundly impacts on the
nuclear DSB repair after treatment with etoposide. Although the
functional effect of the c.400C\textgreater{}T (p.Arg134Ter) variant was
not established, it is expected to lead to nonsense-mediated mRNA decay,
as in the case of the c.425+1G\textgreater{}A and the c.636+3\_636+6del
splice-site variants.

The five previous and our current patients share several clinical
features, mainly craniofacial and neurological (Table 1). Particularly,
5 out of 7 patients with \emph{TDP2} inactivation showed speech
impairment, which may be considered a recurrent feature in SCAR23 along
with drug-resistant epileptic seizures and intellectual disability --
shared by all described cases, progressive ataxia (6/7), and
craniofacial dysmorphisms (6/7) (Table 1). We previously observed
neutropenia in a 6-year-old child carrying the c.425+1G\textgreater{}A
variant (Zagnoli-Vieira et al., 2018). Interestingly, TDP2 is highly
expressed in the hemolymphoid system and interacts with CD40, a member
of the tumour necrosis factor (TNF) receptor family that plays a
critical role in several immunological processes (Pype et al., 2000),
and mutations of \emph{CD40} gene have been associated to an autosomal
recessive form of immunodeficiency with hyper IgM and neutropenia (OMIM
\#606843; Ferrari et al., 2001). Nevertheless, haematological
manifestations have not been reported in other patients and do not seem
to be a pathognomonic signature for SCAR23, at least based on current
observations in patients.

Our patients manifested eye anomalies, particularly early-onset
bilateral cataracts in the proposita, a feature never described in
SCAR23. The extended \emph{in silico} panel for cataract (Table S2) was
negative, a finding against a potential multilocus inheritance.
Interestingly, the exon 5 of \emph{TDP2} is predicted to contain a 21-bp
transcription factor binding site for PAX6 (chr6:24,654,475-24,654,495,
GRCh38/hg38; V\$PAX6\_01, Transfac Matrix Database v.7.0), whose
deficiency is associated with distinct ocular phenotypes including
cataract (OMIM \#106210). Although more functional studies are needed to
support this hypothesis, \emph{TDP2} could be involved in the gene
regulatory circuitry of PAX6/SOX2 that orchestrates lens development and
neurogenesis.

TDP2 has been recognized as a key player for neural homeostasis because
of its role in preventing DNA damage led by abortive TOP2 activity.
Curiously, biallelic inactivation of \emph{TDP1} causes spinocerebellar
ataxia with axonal neuropathy (SCAN1; OMIM \#607250) (Takashima et al.,
2002), further supporting the central role of topoisomerases and their
partner tyrosyl-DNA phosphodiesterases in neurodevelopment. Moreover,
Stoll \emph{et al.} (2013) found a significant enrichment of a rare
240-kb chromosome 22q11.22 microdeletion spanning \emph{TOP3B} (encoding
the DNA topoisomerase III\selectlanguage{greek}β\selectlanguage{english}) in Finnish patients affected by
schizophrenia and cognitive impairment, whereas mutation of
\emph{TOP1}(encoding the DNA topoisomerase I) has been implicated in
autism spectrum disorders (King et al., 2013; McKinnon, 2016).
Interestingly, a small inherited duplication in 6p22.3 (37.89 kb)
joining the first noncoding exon of \emph{KIAA0319}with the 3' end of
the neighbouring gene \emph{TDP2} (exons 4--7) was identified in an
Irish patient with autism spectrum disorder (ASD) (Holt et al., 2012).
In Decipher, another inherited heterozygous duplication of
\emph{KIAA0319} -\emph{TDP2} with similar size (33.43 kb) was detected
in a female patient (\#281899) with delayed speech and infantile spasms.
Although several studies implicated \emph{KIAA0319} in dyslexia
susceptibility (Francks et al., 2004; Cope et al., 2005; Harold et al.,
2006; Paracchini et al., 2006; Dennis et al., 2009; Eicher et al.,
2014), a condition frequently associated with ASD, we cannot rule out
the involvement of \emph{TDP2} , a gene that ``keeps the brain healthy''
(McKinnon, 2014), as susceptibility factor in the development of
schizophrenia when combined with other genetic and environmental
factors. In this regard, we detected the c.636+3\_636+6del variant at
the heterozygous state in the DNA of the probands' cousin suffering from
schizophrenia. Although the variant was inherited from the father, with
no apparent neurobehavioral abnormalities, a possible involvement
of\emph{TDP2} may not be completely ruled out and merits further
investigations.

Apart from their role in normal brain function, topoisomerases, when
altered and mis-expressed, may play a critical role in chromosome
instability (CIN), a hallmark of carcinogenesis (Liu et al., 2019).
Clinically, a large arsenal of topoisomerase inhibitors has been used in
personalized cancer therapy to suppress DNA replication of tumour cells
(Chen et al., 2015). Several studies showed that TDP2-dependent repair
of DSBs protects cells from genome instability and chromosome
translocations (G\selectlanguage{ngerman}ómez-Herreros et al., 2013, 2017; Canela et al., 2019).
Moreover, TDP2 has been suggested as a promising target for anticancer
therapy, further underlying its possible role in carcinogenesis.
According to the COSMIC database, 94 different variants in \emph{TDP2} ,
mostly missense substitutions (76.7\%), have been detected in 103 out of
35,902 tumour samples (0.28\%) tested from different anatomical regions,
with an overall enrichment for the large intestine (28/2331, 1.2\%).
These data raise the question of whether SCAR23 patients and
\emph{TDP2}mutation carriers may be more prone to develop tumours and
need specific oncological follow-up. Although more observations are
needed, we excluded the presence of a second coding hit in the tumour
sample of the patients' mother, weakening a possible involvement of
\emph{TDP2} as a tumour suppressor gene in colorectal carcinogenesis. In
this regard, it should be also considered that other redundant
mechanisms are available to repair TOP2-induced DSBs, such as the
Mre11-dependent homologous recombination (Hoa et al., 2016).

In summary, our results strongly suggest that biallelic loss-of-function
variants in \emph{TDP2} are associated with SCAR23. Whether the
accumulation of other classes of variants, such as missense
substitutions in crucial TDP2 functional domains, especially the
C-terminal exonuclease-endonuclease-phosphatase (EEP) catalytic domain
(aa 117-351), might be associated with distinct phenotypes or degrees of
severity needs further investigations.

\textbf{CONFLICT OF INTERESTS}

The authors have no conflicting interests to declare.

\textbf{DATA AVAILABILITY STATEMENT} The data that supports the findings
of this study are available in the supplementary material of this
article. Any other data will be made available on request from the
corresponding author.

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\textbf{FIGURE LEGENDS}

\textbf{FIGURE 1.} (a) Family pedigree showing a miscellany of
neurological phenotypes in the last generation of the family. +/+
homozygous wild-type; +/- heterozygous carrier; -/- homozygous mutant
for the \emph{TDP2} c.636+3\_636+6del variant. (b) Clinical features of
the probands (III-38 and III-40), showing highly overlapping facial
dysmorphisms, large hands, clinodactyly of the
5\textsuperscript{th}finger, and flatfoot.

\textbf{FIGURE 2} . Functional analysis on the probands' fibroblasts.
(a) RT-PCR shows exon 5 skipping at the RNA level in the probands
compared to control pools; NTC: no template control. (b) Sequencing
analysis of the exon 5 skipped band in the RT-PCR. The junction between
exon 4 and exon 6 creates a frame shift leading to a premature
termination codon within six amino acid residues. (c) TDP2
immunoblotting showing no detectable TDP2 expression in either Italian
(novel mutation) or US patients (previous mutations); 1BR: control human
fibroblast cell line; 850BR: TDP2-mutated patient primary human
fibroblasts; III-38, III-40: probands' fibroblasts; YJR1504: primary
fibroblast control cell line. (d) Repair kinetics of yH2AX foci after
25\selectlanguage{greek}µ\selectlanguage{english}M etoposide treatment showing decreased repair of TOP2-induced DSBs
in affected cells (probands and 850BR) compared to control cell line
(1BR). Data are the mean (\selectlanguage{ngerman}±s.e.m.) of four independent experiments.

\textbf{FIGURE S1.} WES analysis and segregation in the family. WES
results visualized on the Integrative Genomics Viewer (IGV) in the
probands and parents (top left panel). NGS findings were confirmed by
Sanger sequencing (top right panel), which was also used for segregation
analysis in other family members (bottom panel).

\textbf{Hosted file}

\verb`Figure 1.tif`  available at \url{https://authorea.com/users/301643/articles/431493-characterization-of-a-novel-loss-of-function-variant-in-tdp2-in-two-adult-patients-with-spinocerebellar-ataxia-autosomal-recessive-23-scar23}

\textbf{Hosted file}

\verb`Figure 2.tif`  available at \url{https://authorea.com/users/301643/articles/431493-characterization-of-a-novel-loss-of-function-variant-in-tdp2-in-two-adult-patients-with-spinocerebellar-ataxia-autosomal-recessive-23-scar23}

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