Discussion
In the present study, we reported an X-linked myopia case and identified
the variant associated with HM in a Southern Chinese family. A
heterozygous/hemizygous novel nonsense ARR4 mutation,
c.569C>G (p.S190*), was found to be associated with HM and
co-segregated within affected family members.
Furthermore, it was found that the
unstable mutant ARR4 protein induced an ER stress response and was
rapidly degraded via the proteasome degradation pathway. Combining with
previous studies, we found that ARR4 may mediate a disease with
pattern of inheritance which is contrary to the conventional genetics.
ES has been shown to be remarkable at detecting variants associated with
monogenic disorders. Notably, compared to 21-25% diagnostic rate in
proband-only ES, a diagnostic rate of 30-37% was observed among
affected individuals when ES was applied to testing trios(Farwell et
al., 2015; Valencia et al., 2015). Consequently, trio-based ES analysis
was used in this study involving a HM family. Following bioinformatic
analysis, the ARR4 c.569C>G (p.S190*) mutation was
identified as the explanation of the proband’s phenotype as evidenced by
not being present in public databases and a high amino acid
conservation. The ARR4 gene, located on chromosome Xq13.1, has
been linked to abundant expression in retinal tissue. To the best of our
knowledge, because only three HM families have been identified harboring
mutations in ARR4 worldwide(Xiao et al., 2016), it is easy to
omit pathogenic ARR4 variants due to the lack of clinical
associations to this gene. EXOMISER, an exome analysis toolkit, provided
an effective way for prioritizing patients’ candidate genes based on
modified phenotypic databases associated with zebrafish models(Smedley
et al., 2015).
As shown in Figure 2B, of the three previously described pathogenicARR4 mutations, two were missense mutations, while the remaining
variant was a nonsense mutation. When including c.569C>G
(p.S190*), domain predictions indicated that all four mutations may
affect the arrestin C-/N-terminal domains (Figure 2B). Previous studies
have demonstrated that both the C- and N-terminal domains play a
critical role in binding to the receptor molecule(Gurevich & Benovic,
1993; Hirsch, Schubert, Gurevich, & Sigler, 1999). Alignment of
multiple ARR4 amino acid sequences have shown that the serine at codon
190 is highly conserved (Figure 2C).
Arrestin is an important family of proteins that can desensitize
G-protein coupled receptors (GPCRs). Rhodopsin belongs to a class of
GPCRs that can sense external light signals and transmit these to cells
that produce vision. In mammals, there are two types of visual
arrestins: arrestin-1 (ARR1 , also called
S-antigen[SAG ] or 48K protein)(Kuhn, Hall, & Wilden, 1984;
Pfister et al., 1985; Vishnivetskiy et al., 2018; Wacker, Donoso,
Kalsow, Yankeelov, & Organisciak, 1977), and arrestin-4 (ARR4 ,
also called cone arrestin [CAR ], or X-arrestin
[ARRX ])(Craft, Whitmore, & Wiechmann, 1994; Murakami,
Yajima, Sakuma, McLaren, & Inana, 1993). After being activated by
light, opsins are phosphorylated by rhodopsin, and arrestin-1 and
arrestin-4 can bind to phosphorylated opsins to inhibit the transduction
of downstream signals(Deming et al., 2015). In murine models, these two
vision-inhibiting proteins are required for normal inactivation of
rhodopsin in cone cells(Nikonov et al., 2008). Misfolded proteins can be
recognized on the endoplasmic reticulum and transported to the
proteasome for degradation(Griciuc, Aron, & Ueffing, 2011). With
elevated expression of ER-stress-related markers that can be observed,
the mutant ARR4 protein was found to mediate an ER stress. Consistent
with this finding, it was shown that the mutant proteins were degraded
via the proteasome pathway due to the proteasome inhibition effects
observed on mutant APRE19 cells.
The pathogenicity of the ARR4 gene was predicted and its
association with HM in female patients were described for the first time
in 2016. Consequently, an X-linked, female-limited pattern of
inheritance was considered for ARR4 -associated HM cases; a
statement strengthened by the observation that none of the hemizygous
male family members were diagnosed with HM(Xiao et al., 2016). However,
in our study it was demonstrated that the proband’s father was affected
with HM and possessed pathogenic ARR4 mutation. This suggested
that the hereditary pattern of the ARR4 gene may not be X-linked,
female-limited. HM is a complex, heterogeneous disease which is
influenced by environmental and genetic factors. Occurring in females,
an X chromosome is compacted during X-chromosome inactivation, which
results in the random silencing of one of the X chromosomes(Galupa &
Heard, 2018). X-chromosome inactivation could however not explain the
phenotypes observed in this study since a nonsense mutation was
identified in an affected male hemizygote, while unaffected male
hemizygotes carried of a missense mutation in other families. So we
speculate that the mechanism of loss of function caused by null variants
may be more detrimental for the ARR4 gene. Both Arr1 andArr4 genes are expressed in mouse cone cells. If only one of the
two genes is knocked out, the recovery time will slow down after being
stimulated by saturated strong light, but knockout of both genes will
greatly increase the delay of recovery time, which indicates that there
may be functional compensation between Arr1 and Arr4 in mice(Nikonov et
al., 2008). So it becomes reasonable to question that if there is also a
complementary mechanism in human retina, which leads to the occurrence
and absence of diseases in different hemizygous individuals? Further
proof is needed in the future. In Craniofrontonasal syndrome, the female
heterozygote of EFNB1 gene located on X chromosome is usually
patient, but the non-mosaic hemizygous male is usually not affected or
has only mild symptoms(Niethamer et al., 2020). Some studies believe
that this is a kind of ”cellular interaction” caused by the mosaic state
of EPHRIN-B1 protein, and the mechanism is not clear(Twigg et al., 2004;
Wieacker & Wieland, 2005; Wieland et al., 2004). Polydactyly was found
in female heterozygous mice (Efnb1 +/-), but not in hemizygous
males (Efnb1 -) and homozygous females (Efnb1 -/-).
Meanwhile, six sporadic male patients with the mosaic state were found
to be severely affected. Interestingly, different proportion of mosaic
mutations of EFNB1 can be detected in various tissues of them
including peripheral blood(Twigg et al., 2013). However, we reviewed the
exome sequencing results of high-myopia male patients in this study, and
found that the sequencing depth of c.569C>G (p.S190*) site
was 183X, and all of them were pathogenic variant G bases, which seemed
to be inconsistent with the conclusion in the above study. However, the
phenotype of high myopia caused by ARR4 mutation is limited at
the present. So, it’s unknown that if ARR4 mutation is also
affected by ”cellular interference” like EFNB1 , resulting in both
normal and affected hemizygous males? This is the goal of our research
in the future.