Discussion
Collectively, these data support the hypothesis that EFFMP1coding variants can cause JOAG by a mechanism that involves
intracellular protein aggregation and retention. Similarly, JOAG-causing
MYOC mutations also promote formation of intracellular protein
aggregates and the extent of protein aggregation appears to correlate
with disease severity (Patterson-Orazem et al., 2019). Misfolded
myocilin is hypothesized to cause ER stress leading to cellular
dysfunction and potentially apoptosis (Yam et al., 2007), however the
specific cells that are impacted and the underlying molecular events are
not known. Studies of human and mouse MYOC knockouts (Kim et al.,
2001; Pang et al., 2002; Wiggs et al., 2001) and mouse knockins (Kim et
al., 2001); Zode et al., 2011) indicate that MYOC mutations are
gain-of-function. The observation that the retina and optic nerves ofEFEMP1 knockout mice are anatomically normal (McLaughlin et al.,
2007; Stanton et al., 2017; Daniel et al., 2020) suggests thatEFEMP1 loss of function also does not underlie glaucoma
development. MYOC and EFEMP1 are among a group of proteins
expressed in ocular extracellular matrix that also includes
Thrombospondin1 (THBS1 ), and Angiopoietin-like 7 (ANGPTL7 )
among other proteins with potential glaucoma involvement (Wirtz et al.,
2021; Tanigawa et al., 2020). Although the specific mechanisms
underlying the contribution of ECM proteins to glaucoma is not known,
preventing mutant MYOC expression (Jain et al., 2017) or
encouraging secretion of misfolded myocilin can reduce intraocular
pressure in mice (Zode et al., 2011; Zode et al., 2021), and similar
approaches may also be therapeutically useful for patients withEFEMP1 mutations.
In this study we showed that the JOAG associated variants demonstrate
increased intracellular protein retention compared with the MLVT/DHRD
variant p.Arg345Trp. EFEMP1 p.Arg345Trp has recently been shown
to effect cholesterol efflux (Tsai et al., 2021) which may potentially
impact the contribution of this mutation to the characteristic
extracellular protein aggregation observed in this disease (Fu et al.,
2007). While some POAG genomic loci include genes that may influence
cholesterol efflux (ABCA1, ARHGEF12, CAV1/2 ) (Gharahkhani et al.,
2021; Springelkamp et al., 2015; Jacobo-Albavera et al., 2021; Wang et
al., 2014; Okuhira et al., 2010), the role of cholesterol metabolism in
glaucoma is not clear and given the involvement of EFEMP1 in JOAG could
be interesting to explore further. It is also of interest that only the
single missense allele p.Arg345Trp is known to cause MLVT/DHRB, yet at
least 3 protein variants can cause glaucoma. It is possible thatEFEMP1 missense alleles are more likely to effect cells of the
ocular outflow pathway than the retinal pigment epithelial cells
involved in MVLT/DHRB. Interestingly, patients with MVLT/DHRB are not
known to be at increased risk of glaucoma, and the JOAG patients withEFEMP1 mutations do not have any evidence of the MVLT/DHRB
retinal dystrophy (Figure 2).
Additionally, we show that the JOAG EFEMP1 variants demonstrate
increased intracellular retention compared to a possible POAG associated
variant, p.Arg140Trp found in a family with 5 members affected by
adult-onset POAG (Mackay et al., 2015), a finding that may be related to
the less severe and genetically complex adult-onset POAG. Our results
could support a contribution of p.Arg140Trp to POAG in this family,
which is interestingly of African American ancestry. Exome-based studies
of African American POAG cases or families have not yet been completed
and further investigation in this population could reveal additionalEFEMP1 disease-related variants. A stop lost variant
(Ter464Gluext*29) similar to the mutation affecting JOAG family B
(Ter464Glnext*29) has recently been described in 3 members of a Chinese
adult POAG family (Liu et al., 2020), While individuals in this family
are described as affected by POAG, the age of disease onset is the
mid-20s which would meet our diagnostic criteria for JOAG and is
consistent with our results.
EFEMP1 is located on chromosome 2p16 within a genomic region that
was initially identified as a POAG genomic locus (GLC1H) in a linkage
study of a Jamaican family and European Caucasian families from the UK
(Suriyapperuma et al., 2007). Subsequently linkage to this region has
also been observed in four Chinese families, including two with disease
onset before age 40 (Liu et al., 2012; Liu et al., 2008). POAG candidate
association studies have also implicated SNPs in the 2p16 region in
cases and controls from Barbados (Jiao et al., 2009), an
African-American cohort (Liu et al., 2010), Chinese (Chen et al., 2012)
and South Indian cohorts (Balasubbu et al., 2021). Together with our
results, these studies support EFEMP1 as the GLC1H gene and also
could suggest that EFEMP1 is more commonly associated with
glaucoma in non-white populations.
Our findings also suggest a phenotype-genotype spectrum that correlates
the extent of intracellular protein retention with disease phenotype and
extend the EFEMP1 phenotypic spectrum to include severe
childhood-onset glaucoma. The mutations causing JOAG cause more severe
intracellular protein retention compared to the MLVT/DHRD EFEMP1variant and also to a variant found in an adult-onset glaucoma family.
Further study will be required to determine the mechanism underlying
intracellular protein retention as well as the molecular events leading
to elevated IOP and glaucoma.
This study investigates the etiology of JOAG in a collection of affected
families from the Philippines, a population with diverse ancestry
including Asian, African and European Caucasian origins (Larena et al.,
2021). Among the pedigrees that we have collected we find thatEFEMP1 variation is a relatively common cause of childhood
glaucoma (20%), equal to MYOC in this population. Interestingly,EFEMP1 variants have not been observed in any prior studies of
childhood glaucoma which have focused primarily of families with
European Caucasian or Asian ancestry (Allen et al., 2015; Huang et al.,
2018). As well, MYOC and other currently known childhood gene
mutations have infrequently been identified in patients with African
ancestry (Liu et al., 2012). These results suggest that investigation of
diverse populations such as this Filipino cohort will be necessary to
develop a more comprehensive set of childhood glaucoma genes.
The discovery of genes causing childhood glaucoma makes it possible to
use genetic testing to inform genetic counseling for affected families.
Treatment initiated at early stages of disease can delay irreversible
optic nerve degeneration and provide the best chance that an affected
child will maintain useful sight throughout their lifetime. Informed
genetic counseling makes it possible to create surveillance and
treatment plans for mutation carriers and alleviates the burden of
screening family members without disease-causing mutations. Currently
however a molecular diagnosis can only be achieved for approximately
20-25% of cases based on known genes (Allen et al., 2015). Discovery of
novel disease-causing genes such as EFEMP1 is needed to improve
the overall diagnostic yield and effectiveness of childhood glaucoma
genetic testing.
In conclusion we have identified 3 different EFEMP1 coding
variants that segregate with a severe form of glaucoma affecting
children in 3 independent families from the Philippines. Our results
suggest that disease-associated variants cause significant intracellular
EFEMP1 aggregation and retention and that the extent of intracellular
retention appears to be correlated with EFEMP1-related disease
phenotypes. This study further supports a role for EFEMP1 in ocular
extracellular matrix and in regulation of intraocular fluid dynamics and
IOP and provides new opportunities for genetic testing and therapeutic
intervention.