Discussion
To date, five patients have been described to carry homozygousTDP2 variants. A homozygous splice-site variant in TDP2(c.425+1G>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
(Gómez-Herreros et al., 2014; Zagnoli-Vieira et al., 2018). Recently, a
homozygous nonsense variant in the exon 3 of TDP2(c.400C>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 TDP2 exon 5. By using
immunofluorescent detection of γH2AX as an indirect measure of DSBs, we
demonstrated that the novel TDP2 c.636+3_636+6del splice-site
variant (as the c.425+1G>A) profoundly impacts on the
nuclear DSB repair after treatment with etoposide. Although the
functional effect of the c.400C>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>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 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>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 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 in silico panel for cataract (Table S2) was
negative, a finding against a potential multilocus inheritance.
Interestingly, the exon 5 of 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, 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 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 et al. (2013) found a significant enrichment of a rare
240-kb chromosome 22q11.22 microdeletion spanning TOP3B (encoding
the DNA topoisomerase IIIβ) in Finnish patients affected by
schizophrenia and cognitive impairment, whereas mutation of 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 KIAA0319with the 3′ end of the neighbouring gene 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 KIAA0319 -TDP2 with similar size (33.43 kb) was detected
in a female patient (#281899) with delayed speech and infantile spasms.
Although several studies implicated 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 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 ofTDP2 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 (Gómez-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 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 TDP2mutation 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 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 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.