DISCUSSION
This family of three children affected with GAPO, two of whom presented
with bilateral congenital glaucoma, allowed us to study the genetic
association of glaucoma with GAPO syndrome.
Glaucoma as an ophthalmological manifestation of GAPO syndrome has been
reported in ten cases to date (Goloni-Bertollo et al., 2008; Goyal et
al., 2014; Ilker et al., 1999; Kocabay & Mert 2009; Manouvrier-Hanu et
al., 1987; Mullaney et al., 1997; Rim & Marques-de-Faria 2005; Sayli &
Gul 1993; Wajntal et al., 1990). Glaucoma in five of these patients, was
early onset open angle glaucoma (Goloni-Bertollo et al., 2008;
Manouvrier-Hanu et al., 1987; Rim & Marques-de-Faria 2005; Sayli & Gul
1993). Primary congenital glaucoma (PCG) is described in five patients
(Goyal et al., 2014; Ilker et al., 1999; Kocabay & Mert 2009;
Manouvrier-Hanu et al., 1987; Mullaney et al., 1997; Sayli & Gul 1993)
presenting with Haab’s striae and buphthalmos. Asymmetric glaucoma, as
seen in Case 1 of GAPO syndrome, at presentation has also been
documented (Goyal et al., 2014; Mullaney et al., 1997), while others
have described a late presentation with end stage glaucoma (Goyal et
al., 2014; Ilker et al., 1999; Sayli & Gul 1993). The pathogenesis of
congenital glaucoma in children with GAPO syndrome is not clearly
understood. Mutations in the ANTRX1 gene have been postulated to
be the cause of glaucoma and the variable expression of ANTRX1mutation could be the reason why the elder sister (case 3) withANTRX1 mutation and GAPO syndrome did not have glaucoma. Even if
we consider a variable expressivity in ANTRX1 as the reason for
one sibling with GAPO syndrome not developing congenital glaucoma in
this family, we believe the presence of the TGFBI variant may
have influenced this variable expression.
The abnormal expression of TGFBI is related to the occurrence and
development of some types of cancers as well as different types of
corneal dystrophies; lattice corneal dystrophies (LCD) and granular
corneal dystrophies (GCD) (Lakshminarayanan et al. 2014), while the role
of TGFBI in glaucoma is not known. The human protein TGFBIp
encoded by TGFBI has four FASI domains, and the mutated amino
acid observed in our two patients corresponds to the FAS1-2 domain of
TGFBIp. However, the TGFBI mutations known for various corneal
dystrophies are located in the FASI-1 and FASI-4 domains (Klintworth et
al. 2004). The TGFBI gene is also within the linkage area mapped
as a glaucoma locus 5q22.1-q32. (Pang et al. 2006). A gene expression
profile of human trabecular meshwork(TM ) tissue by SAGE (serial
analysis of gene expression) has shown a higher expression forTGFBI along with other glaucoma causing genes and genes involved
in typical TM maintenance functions (Liu et al. 2011). A study by Kim et
al., using Alb-hßigh3 transgenic mice showed that over expressed human
βigh3/ (hßigh3)/ TGFBIp in the blood might be involved in anterior
segment morphogenesis and eye development in mice. The phenotype
observed in transgenic mice is similar to human eye disorders such as
anterior segment dysgenesis and Peters’ anomaly (Kim et al. 2007). These
observations support the role of TGFBI in congenital glaucoma as
observed in the two sisters with GAPO. A string analysis also showed
that TGFBI and LTBP2 (associated with congenital glaucoma)
co-express, possibly interacting with MYOC and CYP1B1 .
There is also a report of glaucoma observed in a patient withTGFBI, R124H mutation, though the phenotypic details of this
patient are not known (Jiang & Zhang 2021).These observations suggest
an important role of TGFBI in glaucoma pathogenesis, in conjunction with
other genes.
Further, we also found 3 unrelated JOAG patients with TGFBIvariants in a separate cohort who had no other known glaucoma gene
mutations. One of the patients had GCD. There is no report in the
literature of an association between JOAG and GCD.
These 5 patients, with different diagnosis but all with developmental
glaucoma, having variants in the same TGFBI gene support the role
of TGFBI in glaucoma pathogenesis. A high degree of phenotypic
variability and incomplete penetrance is known for TGFBImutations however the fundamental reason, why different gene variations
cause morphologically distinct phenotypes remains elusive. Our study is
the first to report an association of TGFBI with congenital and
juvenile glaucoma.
The modeling studies of the TGFBI wild type, along with the three
variants A323E, A323V, and 649-frameshift, provided insights into the
effect of these variations on the full-length structure and its
dynamics. While specific aggregation mechanisms cannot be deduced at
this stage, the alteration of the protein dynamics was clearly observed
in the simulation studies. The A323 is located at the interface of
CROPT, and FAS1-1 domains, and perturbation at this position can alter
inter-domain interactions and even global protein structure. For A323V,
the presence of hydrophobic residue valine on the surface can
destabilize the protein, and the same was observed in molecular dynamics
studies. In the case of A323E, the glutamic acid was observed to form
strong intra-domain interactions with K72 and N207 from the CROPT domain
and FAS1-1 domain, respectively. In the case of the frameshift variant
of TGFBI , the sequence at the C-terminal is greatly altered
compared to the WT. The comparative modeling studies revealed that the
C-terminal region forms two alpha-helices packed against each other with
hydrophobic residues, further interacting with the FAS1-4 and FAS1-3
domains. Due to the frameshift, packing of the helices was disturbed,
leading to increased solvent exposure of the C-terminal tail.
Limitations of our study include a small number of patients and our
inability to genotype the parents and siblings of JOAG patients. Also,
gene expression and functional studies using cell-based assays and
transgenic animal models of the mutant are further needed to examine the
effect of these variants on the protein and the downstream targets to
establish a cause-and-effect relationship. Moreover, an interaction
between TGFBI and other genes in the causation of glaucoma also
needs to be explored. Nevertheless, based on the findings of this
preliminary report, TGFBI could be well considered a candidate
gene for glaucoma; however, more studies in a larger patient population
as well as functional studies may be needed to further elucidate its
role in glaucoma pathogenesis.