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\begin{document}

\title{Association of TGFBI variants with Congenital and Juvenile onset open
angle glaucoma.}



\author[1]{Viney Gupta}%
\author[1]{Arnav Panigrahi}%
\author[1]{Bindu Somarajan I}%
\author[1]{Shikha Gupta}%
\author[1]{Koushik Tripathy}%
\author[1]{Abhishek Singh}%
\author[2]{Anshul Sharma}%
\author[1]{Radhika Tandon}%
\author[1]{Amit Das}%
\author[3]{Dibyabhaba Pradhan}%
\author[2]{Arundhati Sharma}%
\author[4]{Tushar Kushwaha}%
\author[4]{Krishna Inampudi K}%
\affil[1]{All India Institute of Medical Sciences}%
\affil[2]{All India Institute of Medical Sciences Department of Anatomy}%
\affil[3]{Indian Council of Medical Research}%
\affil[4]{All India Institute of Medical Sciences Department of Biophysics}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
\textbf{Purpose:} To describe a novel association of \emph{TGFBI}
variants with congenital glaucoma in a family with GAPO (growth
retardation, alopecia, pseudoanodontia, and progressive optic atrophy)
syndrome as well as among unrelated cases of Juvenile onset open angle
glaucoma (JOAG) along with the mechanistic impact of the variants on the
protein. \textbf{Methods:} This study of one family of GAPO with
congenital glaucoma and three unrelated patients of JOAG analysed a
common link to glaucoma pathogenesis. We report ocular features of 3
girls with GAPO syndrome born of consanguineous marriage in a multi-
generation consanguineous family. The proband (a 4year old girl) and her
younger sibling (1year old girl) were operated for bilateral congenital
glaucoma in both eyes. The elder sibling (10year old female) had
features of GAPO syndrome without glaucoma. \textbf{Results}: A genetic
evaluation using whole exome sequencing revealed a homozygous
\emph{ANTXR1} mutation in all three affected siblings with GAPO. No
other mutations were detected in the genes associated with glaucoma. A
rare missense variant in the \emph{TGFBI} gene was shared in the two
siblings with congenital glaucoma and GAPO syndrome. We further found
three other unrelated patients with JOAG with no known glaucoma causing
gene mutations but having three different missense variants in the
\emph{TGFBI} gene. One of these JOAG patients had familial granular
corneal dystrophy. Molecular dynamic simulations of the \emph{TGFBI} and
3-D structural models of three of its variants showed significant
alterations, which could influence \emph{TGFBI} function.
\textbf{Conclusions:} Variations in the \emph{TGFBI} gene could have a
possible role in the pathogenesis of congenital and Juvenile onset open
angle glaucomas that needs further evaluation.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Primary congenital glaucoma (PCG) and Juvenile onset open angle
glaucomas (JOAG) are generally monogenic, with genes having a strong
biological effect playing a role in their pathogenesis. Apart
from\emph{MYOC,} \emph{CYP1B1} and \emph{LTBP2} (Abu-Amero et al. 2011;
Alsaif et al. 2019; Bayat et al. 2008; Chakrabarti et al. 2006; Chen et
al. 2008; Gupta et al. 2018; Saeedi et al. 2018) newer genes associated
with either form of glaucoma include the \emph{CPAMD8} ,
\emph{GPATCH,}and \emph{EFEMP1} .(Ferre-Fern\selectlanguage{ngerman}ández et al. 2017; Mackay et
al. 2015; Siggs et al. 2020) However, a large number of PCG and JOAG
patients do not have a known gene mutation among these genes. (Knight et
al. 2021) There are chromosomal loci where the putative genes associated
with PCG and JOAG are still to be discovered. (Cascella et al. 2015)
With the advent of next-generation sequencing, new gene discovery,
especially for diseases where genetic variations have significant
biological effect on disease causation, is being undertaken.

In this study, we describe a family of 3 siblings with GAPO syndrome, in
whom we found two of them having congenital glaucoma. A WES analysis
pointed towards the Transforming Growth Factor Beta Induced
(\emph{TGFBI} ) gene variant common to both siblings.
Incidentally,\emph{TGFBI} variants were also seen in three unrelated
patients with JOAG, one of whom had familial granular corneal dystrophy.

GAPO syndrome is an extremely rare genetic disorder, with only about
sixty cases reported to date. The ocular features of GAPO syndrome
include puffy eyelids, sparse eyelash hair, poliosis, hypertelorism,
ptosis, strabismus, nystagmus, megalocornea, keratoconus, keratopathy,
optic atrophy, myelinated retinal nerve fiber layer and congenital
glaucoma (buphthalmos) (Bozkurt et al. 2013). The diagnosis of GAPO is
usually made early due to the characteristic premature `geriatric'
appearance of the patients, the presence of cardinal features like
alopecia and pseudoanodontia, as well as the associated features like
dwarfism, prominent supraorbital ridges, frontal bossing depressed nasal
bridge, long philtrum, and a wide open anterior fontanelle. Late onset
alopecia has also been frequently documented (Goloni-Bertollo et al.
2008; Rim \& Marques-de-Faria 2005). Patients with GAPO have a reduced
life span and usually die in their third or fourth decades of life due
to generalized interstitial fibrosis of the lungs and atherosclerosis
(Gagliardi et al. 1984; Mullaney et al. 1997).

The invariable consanguinity in these families with GAPO points towards
an autosomal recessive mode of inheritance caused by mutations
in\emph{ANTXR1} on chromosomal position 2p14, a protein essential for
intracellular actin assembly (Bayram et al. 2014; Stranecky et al.
2013). Defective production of this protein leads to altered cell
adhesion properties leading to excessive deposition of extracellular
matrix (ECM) secondary to reduced turnover, resulting in the various
phenotypic features of this syndrome (Stranecky et al., 2013; Wajntal et
al. 1990). While there are reports of the occurrence of glaucoma among
patients with GAPO syndrome, (Goyal et al. 2014; Ilker et al. 1999;
Kocabay \& Mert 2009; Manouvrier-Hanu et al. 1987; Mullaney et al.,
1997; Sayli \& Gul 1993) it is not known whether mutations
in\emph{ANTXR1} gene itself or some other (glaucoma associated) genes,
lead to the development of glaucoma. This report analyses the genetic
background of two (out of three) children with GAPO syndrome and
congenital glaucoma.

\textbf{SUBJECTS AND METHODS}

A multi generation consanguineous family presented to us, where four
girls, three of whom were affected with GAPO, out of whom two also had
congenital glaucoma (\textbf{Figure 1} ). The family belonged to North
India with a strong tradition of consanguinity. The three cases of the
family described here are of the three girls with features of GAPO
syndrome.

\textbf{Case 1 (Proband)}

A 4year old girl, born of a consanguineous marriage, presented to our
glaucoma clinic with chief complaints of watering and an asymmetric
corneal enlargement of the right eye (RE). Physical examination revealed
progressive frontal alopecia with a receding hairline, frontal bossing,
high forehead, prominent supraorbital ridges, depressed nasal bridge,
anteverted nostrils, long philtrum, umbilical hernia, sparse eyebrows,
and an appearance of premature aging (\textbf{Figure 2A1-4} ). Intraoral
examination revealed the absence of a regular dentition pattern
(\textbf{Figure 2A3} ); a radiograph of the teeth revealed impacted
dentition, confirming pseudoanodontia. Her weight (12kg) and height
(92cm) were below the 3rd percentile. The patient's mental status was
found to be normal, and there was no developmental delay.

Examination under anesthesia (EUA) revealed RE enlarged corneal
diameters with an IOP of 30mmHg, with corneal edema precluding a
detailed anterior and posterior segment evaluation. Her left eye (LE)
had a clear cornea with an IOP of 16mmHg. The patient was taken up for
RE trabeculectomy with mitomycin. On subsequent EUA (post-operatively),
when the cornea had cleared up, the anterior segment evaluation revealed
the presence of Haab's striae and peripheral keratopathy with
irido-corneal adhesions that were seen on an ASOCT examination in the RE
(\textbf{Figure 2A3, 2A4} ). Her optic disc examination showed a
vertical cup disc ratio (CDR) of 0.4:1 with temporal disc pallor in RE;
while LE showed a healthy disc with a CDR of 0.2:1. Eventually, she
developed increased IOP in her LE, for which she was taken up for LE
trabeculectomy with mitomycin, three years after the RE surgery. Her
IOPs have been controlled in both eyes (BE) ever since.

A USG abdomen revealed the presence of normal kidneys and ovaries. An
MRI of the brain did not detect any intracranial abnormality. Other
investigations were normal, including complete blood counts, serum and
urine biochemistry, hormonal assays and echocardiography,.

\textbf{Case 2}

The younger sibling of the proband presented at 1 year of age with
complaints of watering BE. She had lush black hair until six months of
age, followed by progressive hair loss. There was a prominent
supraorbital ridge, depressed nasal bridge, high forehead, wide anterior
fontanelle, and a prematurely aged look (\textbf{Figure 2B1-4} ). She
also had pseudoanodontia. Just like her elder sister, she had growth
retardation, with her weight and length below the 3rd percentile for her
age.

An EUA revealed BE horizontal corneal diameter of 13.5mm, along with the
presence of a diffuse corneal haze in LE. She also had a central
keratopathy in RE (\textbf{Fig 2B2} ). Her IOP was 28mmHg in RE and
32mmHg in LE. An ultrasound biomicroscopy (UBM) revealed central corneal
thickening and a thinned out iris and ciliary body. (\textbf{Fig 2B4} ).
She had a cup disc ratio of 0.6:1 in BE. For her BE trabeculectomy with
ab externo trabeculotomy with mitomycin was performed. The surgery was
uneventful, and she maintains normal IOP.

\textbf{Case 3}

The 10year-old elder sister of the proband was also evaluated for GAPO
features. Just like the proband, she was observed to have all the
phenotypic features of this syndrome and reduced eyelashes
(\textbf{Figure 2C1-4} ). The intraoral examination also revealed
pseudoanodontia, and her height (120cm) and weight (20kg) were below the
third centile for her age. She had no developmental delay, and her
mental status was normal. Her visual acuity was 6/6 in both the eyes,
and IOP was 14mmHg and 16mmHg for the RE and LE, respectively. Her
corneae were clear, and gonioscopy revealed wide open angles without any
abnormality. Her optic nerves were found to be normal.

\textbf{Table 1} shows the ophthalmic features of the three siblings
with GAPO syndrome. None of the other family members had any features of
GAPO or glaucoma.

\textbf{Whole Exome Sequencing (WES)}

Blood samples were withdrawn from the four children, the parents, and
the paternal grandmother after informed consent was taken from the
parents of the children. The study was carried out according to the
tenets of the declaration of Helsinki, and Ethics committee approval was
obtained from our Institutes ethics committee. A signed informed consent
according to the guidelines of the Institute Ethics Committee was
obtained.

Genomic DNA was extracted from peripheral blood lymphocytes using
PAXgene Blood DNA Kit (Qiagen, Germany) for genetic evaluation. DNA was
run on 0.8\% agarose gel to check for quality and quantified by
Nanodrop. WES capture was performed using the Sure Select Clinical
Research Exome V2 kit (Agilent Technologies, Santa Clara, CA). Variant
analysis was performed using the GenomeAnalysisTK-3.6 toolkit. The
variant call files (VCF), containing the variant call results, generated
were analyzed using Golden Helix VarSeq Software v.1.2.1 (Bozeman, MT).
VarSeq variants with read depth \textless{}15 and genotype quality score
\textless{}20 were excluded. To identify rare mutations, variant
frequency databases were used to remove variants that are present at
high frequencies among large population groups. The remaining variants
were filtered according to minor allele frequency (MAF) \textless{}0.001
in multiple databases, including Exome Aggregation Consortium (ExAC)
(http://exac.broadinstitute.org/), 1000 Genomes Project
(http://browser.1000genomes.org) and gnomAD
(http://gnomad.broadinstitute.org/).

Variants specifically in the two sisters with glaucoma were filtered
based on only those exonic variants (Non-synonymous missense variants,
frameshift and indels and splice region variants) that were present
exclusively in the two sisters and absent in the other family members
and 20 healthy controls. Inherited variants that were present in both
the affected sisters with congenital glaucoma and one of the parents
were also looked at, for any association with the known glaucoma genes.
Copy number variant (CNV) analysis was performed to look for CNV in the
known genes for glaucoma or others with a strong association with
glaucoma pathogenesis. The CNV's were annotated with RefSeq gene
annotations and Database of Genomic Variants v107. The CNV's were then
classified to assess the pathogenicity using classify CNV. The CNV's
common in both affected individuals were visually inspected using IGV.

We used Homozygosity mapping to identify long stretches of homozygous
haplotypes in genes associated with glaucoma. Regions of Homozygosity
(ROH) were identified by~using the AutoMap software (Quinodoz et~al.,
https://automap.iob.ch/) on WES data (Quinodoz et al. 2021).

Further functional impact of the protein was predicted by bioinformatic
tools; variant effect predictor (VEP), Mutation Taster, Polyphen, and
FATHMM. The identified variants were confirmed by Sanger sequencing.

\emph{In silico} \textbf{3D-modeling and Molecular dynamic analysis
of\emph{TGFBI} and its variants:}

The nucleotide sequence of \emph{TGFBI} protein was retrieved from NCBI
(ID), and the structure was modeled through comparative modeling using
the Robetta server (Song et al. 2013).The structure of
full-length\emph{TGFBI} was modeled using the coordinates of
PDBID:5NV6(Garcia-Castellanos et al. 2017). For further refinement, the
model was subjected to molecular dynamics (MD) simulation for 200ns. The
refined structure was validated using MolProbablity Server(Williams et
al. 2018). The last frame of the 200ns simulation was used as a template
for modeling the mutant forms of TGFBI, including A323E, A323V, and 649
frameshift. The modeled mutants were then simulated for 200ns each in
order to understand the impact of variations on the structure and
dynamics of the TGFBI protein.

MD simulations of the modeled proteins were done employing GROMACS
software version 2021.5(Van Der Spoel et al. 2005). CharmM36 forcefield
was used for generating the topology, and the system was solvated using
the TIP3P water model(Vanommeslaeghe et al. 2010). Neutralization of the
system was done by the addition of the appropriate number of sodium
ions. Equilibration of the system was then performed under successive
NVT and NPT ensembles for 500ps each. The production simulation was run
using a leap-frog dynamics integrator and a step size of 2fs with the
consideration of periodic boundary conditions in all three dimensions.
The trajectory was analyzed. Principal component (PC) analysis was
performed to understand the global motions of the TGFBI and the three
mutant forms. The covariance matrix was generated from the C\selectlanguage{greek}α \selectlanguage{english}atom
coordinates and diagonalized to obtain the eigenvectors. Since the first
two eigenvectors described the major proportion of the trace of the
covariance matrix, the trajectory was projected along the first two
principal components. Free energy landscape was further generated using
the 2-dimensional projection of the trajectory along PC1 and PC2.

\textbf{RESULTS}

We found a missense homozygous \emph{ANTXR1} gene mutation in all three
children affected with GAPO (NM\_032208.2:c.572T\textgreater{}C,
p.Ile191Thr). This change is novel and predicted to be a disease causing
by mutation taster (0.99), PolyPhen-2 (1.0), and PROVEAN (-4.0). Another
change at this position, c.572T\textgreater{}G, p.Ile572Ser is reported
in the COSMIC database (COSV100362660).

Homozygosity mapping applied to WES data of the consanguineous family,
detected one large (11Mb) homozygous region involving the \emph{TEK}gene
(i.e., chromosome 9) in only one of the two sisters with congenital
glaucoma, among the genes known for glaucoma. However, neither CNV nor
intronic splice region variant was detected in the \emph{TEK} gene in
either of the sisters.

No pathogenic variants or CNV were detected that co-segregated with
glaucoma in the two girls with congenital glaucoma (Case 1 and 2) at any
of the known loci for Mendelian forms of glaucoma (GLC1A, GLC1B,
GLC1D-Q) or PCG (GLC3A-D) including the known causative genes
namely,\emph{MYOC} (GLC1A), \emph{CYP1B1} (GLC3A), \emph{WDR36}
(GLC1G),\emph{ASB10} (GLC1F), \emph{OPTN} (GLC1E), \emph{NTF4}
(GLC1O),\emph{TBK1} (GLC1P), \emph{LTBP2} (GLC3C), \emph{FOXC1, PITX2}
,\emph{TEK,} and \emph{PAX6.} Exonic variants specific to the two
sisters with congenital glaucoma and the variants they inherited from
either of the parents are listed in \textbf{Suppl Table 1.}

Four missense variants exclusive to the two sisters with GAPO and
congenital glaucoma (case 1 and case 2); \emph{PTPN4} , \emph{PCDHB4,
GGT2} and \emph{TGFBI,} were observed. The variants on \emph{PTPN4}
and\emph{PCDHB4} were rejected as they were found to be benign.
The\emph{GGT2} variant was not considered as it was earlier reported
that hGGT2 does not encode a functional enzyme and therefore is unlikely
to play a role in glaucoma pathogenesis. A missense heterozygous variant
in\emph{TGFBI} , NM\_000358.3:c.968C\textgreater{}T
NP\_000349.1:p.Ala323Val \textbf{(Fig 1.a)} was found, which is reported
to be damaging by the variant prediction tools PROVEAN (-2.5),
FATHMM-MKL (0.825), PolyPhen-2 (0.89) and disease causing by Mutation
Taster (0.99). The variant is highly conserved with a Genomic
evolutionary rate profiling (GERP) value of 5.65. Sanger sequencing
confirmed heterozygous missense p.Ala323Val mutation in both case 1 and
case 2 and was absent in case 3 and other family members. The gnomAD
frequency of the variant was found to be 0.001450. The
\emph{Clinvar}star rating score, extracted for the variant, was found to
be 1/4 with two submissions, one for corneal dystrophy.

We then looked for the presence of \emph{TGFBI} gene variants in a
cohort of 40 patients with JOAG (diagnosed before 25 years of age) in
whom WES was previously done. We found two \emph{TGFBI} variants present
in two unrelated JOAG patients. Neither of these 2 patients had any
other mutations in the known glaucoma genes. In one patient,
a\emph{TGFBI} heterozygous missense variant was found in the same codon
observed in the described cases of GAPO, but with a different amino acid
residue.; NM\_000358.3:c.968C\textgreater{}A, NP\_000349.1:p.Ala323Glu.
The second was a novel variant in the other JOAG patient. This was a
null variant (frameshift mutation); NM\_000358.3:c.1944del,
NP\_000349.1:p.Ser649LeufsTer22. These variants were of uncertain
significance with minor pathogenic evidence and likely pathogenic
respectively, as per ACMG classification. The former was damaging by
Mutation taster (0.99), PolyPhen-2 (0.89), and PROVEAN (-2.5). The
gnomAD frequency of NM\_000358.3:c.968C\textgreater{}A was found to be
0.00003269, and the frequency of (NM\_000358.3):c.1944del is not
reported in gnomAD. A third patient with granular corneal dystrophy
referred to the glaucoma clinic was also found to have JOAG. This
patient had familial granular dystrophy of the cornea with 2 other first
degree relatives (a brother and his daughter) also affected with
granular dystrophy but without having glaucoma or raised IOP
(\textbf{Fig 3} ). WES analysis of this patient revealed a
heterozygous\emph{TGFBI} mutation NM\_000358.3:c.1663C\textgreater{}T,
NP\_000349.1:p.Arg555Trp which is reported to be pathogenic
in\emph{Clinvar} with 3 submissions associated with granular corneal
dystrophies (GCD). (\textbf{Table 2} )

A comparative modeling of the \emph{TGFBI} variants (p.Ala323Val,
p.Ala323Glu and p.Ser649LeufsTer22) was performed since the available
crystal structures lack the C-terminal region of the protein. The
modeling results showed \selectlanguage{greek}α-\selectlanguage{english}helical region in the C-terminal, as shown
in\textbf{Figure 4} . The model was subjected to 200ns MD simulations
for refinement. During this simulation, the C-terminal region of the
protein was gradually stabilized through interactions with the FAS1-4
and FAS1-3 domains. The last frame of this simulation was therefore used
as a template for the generation of the variants A323E, A323V, and 649
frameshift, which were then subjected to MD simulations to understand
the effect of mutations on the local and global structure of\emph{TGFBI}
and its variants. Structural deviation, compactness of the proteins, and
residue fluctuations for 200ns simulations were calculated to assess the
root mean square deviation (RMSD), radius of gyration (RG), and root
mean square fluctuation (RMSF), respectively (\textbf{Supplementary
Figure 1} ). MD simulations followed by Principal Component Analysis
(PCA) were performed to study the overall motions of the \emph{TGFBI}
and these 3 variants. Projection of the trajectory along PC1 and PC2
depicted correlated motions of various domains of the protein, as shown
in \textbf{Figure 5} . We found that \emph{TGFBI}fluctuated between an
elongated and a banana-like shape due to bending between FAS1-2 and
FAS1-3 domains. The bending movement was more prominent in the variants
compared to wild-type (WT) \emph{TGFBI,}majorly in the FAS1-2 and FAS1-3
domains. The impact of the three variants was also evident on the Free
Energy Landscape (FEL) since the free energy well was shallow and
distorted compared to the deeper well of WT \emph{TGFBI} (\textbf{Figure
6} ).

\textbf{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 \emph{ANTRX1} gene have been postulated to
be the cause of glaucoma and the variable expression of
\emph{ANTRX1}mutation could be the reason why the elder sister (case 3)
with\emph{ANTRX1} mutation and GAPO syndrome did not have glaucoma. Even
if we consider a variable expressivity in \emph{ANTRX1} as the reason
for one sibling with GAPO syndrome not developing congenital glaucoma in
this family, we believe the presence of the \emph{TGFBI} variant may
have influenced this variable expression.

The abnormal expression of \emph{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 \emph{TGFBI} in glaucoma is not known. The human protein TGFBIp
encoded by \emph{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 \emph{TGFBI} mutations known for various corneal
dystrophies are located in the FASI-1 and FASI-4 domains (Klintworth et
al. 2004). The \emph{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
for\emph{TGFBI} 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-h\selectlanguage{ngerman}ßigh3 transgenic mice showed that over
expressed human \selectlanguage{greek}β\selectlanguage{english}igh3/ (h\selectlanguage{ngerman}ßigh3)/ 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 \emph{TGFBI} in congenital
glaucoma as observed in the two sisters with GAPO. A string analysis
also showed that \emph{TGFBI} and \emph{LTBP2} (associated with
congenital glaucoma) co-express, possibly interacting with \emph{MYOC}
and \emph{CYP1B1} . There is also a report of~glaucoma observed in a
patient with\emph{TGFBI,} 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
\emph{TGFBI}variants 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 \emph{TGFBI} gene support the role
of \emph{TGFBI} in glaucoma pathogenesis. A high degree of phenotypic
variability and incomplete penetrance is known for \emph{TGFBI}mutations
however the fundamental reason, why different gene variations cause
morphologically distinct phenotypes remains elusive. Our study is the
first to report an association of \emph{TGFBI} with congenital and
juvenile glaucoma.

The modeling studies of the \emph{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 \emph{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 \emph{TGFBI} and other genes in the causation of glaucoma also
needs to be explored. Nevertheless, based on the findings of this
preliminary report, \emph{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.

\textbf{Data Availability:} Data supporting the findings of the study
are available from the corresponding author (VG) on request\textbf{.}

\textbf{Author Contributions:}

Concept, Design: Viney Gupta, Bindu Somarajan

Data Collection: Shikha Gupta, Arnav Panigrahi, Koushik Tripathi, Anshul
Sharma, Abhishek Singh, Dibyabhaba Pradhan, Tushar Kushwaha, Radhika
Tandon, Amit Das, Krishna K Inampudi.

Manuscript editing: Arundhati Sharma, Krishna K Inampudi

Overall Responsibility\textbf{:} Viney Gupta

\textbf{Funding:~} This study was supported by funding from the Indian
Council of Medical Research, New Delhi, India (Grant no.
ISRM/12(58)/2019).

\textbf{Ethics approval: The study was approved by the Institute Ethics
Committee, AIIMS, New Delhi, India}

\textbf{Conflict of interest:} None of the authors have any conflict of
interests.

Legends:

\textbf{Figure 1} : (a)Pedigree chart of the family with three children
affected with GAPO syndrome showing at least three generations of
consanguinity. The half shaded circle depicts the child having only GAPO
syndrome, but not glaucoma. The younger two siblings had glaucoma with
GAPO syndrome depicted as full shaded circles. The ages mentioned in the
pedigree are those at their last follow up.

(b) Chromatograms showing heterozygous,\emph{TGFBI;}
NM\_000358.3:c.968C\textgreater{}T; p.Ala323Val mutation found in the
two girls with congenital glaucoma and GAPO syndrome.

\textbf{Figure 2} : Clinical photographs of Case 1 (A1-4), Case 2 (B1-4)
and Case 3 (C1-4). Front facial profile and side facial profile of the
patients illustrate the presence of a characteristic geriatric
appearance, with alopecia, sparse eyebrows, frontal bossing, prominent
supraorbital ridges, depressed nasal bridge, anteverted nostrils and a
long philtrum. Clinical photograph of the oral cavity shows impaction of
teeth with a reduction in its number. Radiograph of the face (lateral
view with open mouth) reveals a normal number of teeth, confirming
pseudoanodontia. Anterior segment photograph of Case1 (A2) reveals the
presence of megalocornea with prominent iridocorneal adhesions and
keratopathy (blue arrow). Corneal opacities are also seen (black arrows;
magnified inset). AS-OCT image of the same patient (A4) confirms the
iridocorneal adhesions (white arrow). Central keratopathy can be seen in
the anterior segment photograph of RE of Case 2 (B2). An ultrasound
biomicroscopy (UBM) of the same Case revealed central corneal thickening
with thinned out iris and ciliary body (B4).

\textbf{Figure 3} :

(A) Anterior segment photo of the patient with granular corneal
dystrophy and JOAG. (B) Goniophotograph of the same patient showing open
angles.(C) Fundus photograph of the patient showing advanced
glaucomatous optic atrophy (D) Pedigree chart of the patient. Black
shaded square depicts the proband having glaucoma and granular
dystrophy. Grey shaded square and circle indicates affected status with
only granular corneal dystrophy. The ages mentioned in the pedigree are
those at their last follow up.

\textbf{Figure 4:}

Modeled structures of full length \emph{TGFBI} and its variants are
depicted in cartoon representation. The residue substitution at 323
position is magnified along with the C-terminal helices in wild type and
649 frameshift \emph{TGFBI} .

\textbf{Figure 5} :

Porcupine plots showing correlated movements of the protein throughout
the 200ns simulations along PC1. The mutant forms of \emph{TGFB} I were
more dynamic compared to the wild type \emph{TGFBI.}

\textbf{Figure 6:}

Free Energy Landscape (FEL) generated from the projection of trajectory
on PC1 and PC2. The FEL for wild type \emph{TGFBI} was deeper compared
to the shallow FELs of the mutant forms due to decreased stability of
the protein.

\textbf{Supplementary Figure 1:} \textbf{(A)} RMSD plots showing
structural deviation of the wild type and mutant \emph{TGFBI}
proteins\textbf{(B)} Radius of gyration plots showing compactness of the
protein. \textbf{(C)} RMSF plot showing residue fluctuation throughout
the 200ns simulations

Table 1: Ocular features of the 3 girls( with Pedigree notation) with
GAPO Syndrome\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& Case 1 (VI iii) & Case 2 (VI iv) & Case 3 (VI ii)\tabularnewline
\midrule
\endhead
Glaucoma & + & + & -\tabularnewline
Optic Atrophy & - & - & -\tabularnewline
Keratopathy & - & + & -\tabularnewline
Frontal bossing & + & + & +\tabularnewline
Sparse eyebrows & + & + & +\tabularnewline
Sparse eyelashes & + & + & +\tabularnewline
White eyelashes & - & - & -\tabularnewline
Prominent globes & + & + & +\tabularnewline
Swollen eyelids & + & + & +\tabularnewline
Prominent supraorbital ridges & + & + & +\tabularnewline
Epiblepharon & + & - & +\tabularnewline
\bottomrule
\end{longtable}

Table 2: \emph{TGFBI} variants identified in Congenital glaucoma and
JOAG patients.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Subjects\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Variant\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
rs number\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Transcript HGVS coding\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Amino acid change\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
gnomAD South Asian frequency\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
ACMG Classification\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Clinical significance\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Case 1 \& Case 2 GAPO with glaucoma\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5:135388650 C/T\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
rs201210696\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NM\_000358.3:c.968C\textgreater{}T\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
p.Ala323Val\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.001111\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
PP2, PP3, BS2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
US\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
JOAG\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5:135388650-C-A\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
rs201210696\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
NM\_000358.3:c.968C\textgreater{}A\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
p.Ala323Glu\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.00003269\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
PM1,PM2, PP2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
US with minor pathogenic evidence\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
JOAG\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5:135397226-C-\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NM\_000358.3:c.1944del\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
p.Ser649LeufsTer22\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
PVS1,PM2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Likely Pathogenic\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
JOAG with GCD\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5:135392469-C-T\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
rs121909208\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
NM\_000358.3:c.1663C\textgreater{}T\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
p.Arg555Trp\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.0002070\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
PP5,PM1, PM5,PM5, PM2, PP2,PP3\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Pathogenic\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

ACMG : American College of Medical Genetics

US: Uncertain Significance

NA: not available

GAPO: Growth retardation, alopecia, pseudoanodontia, and progressive
optic atrophy

JOAG: Juvenile onset open angle glaucoma

GCD: Granular corneal dystrophy

\textbf{References}

Abu-Amero, K. K.,Osman, E. A.,Mousa, A.,Wheeler, J.,Whigham,
B.,Allingham, R. R.,Hauser, M. A., \& Al-Obeidan, S. A. (2011).
Screening of CYP1B1 and LTBP2 genes in Saudi families with primary
congenital glaucoma: genotype-phenotype correlation. \emph{Mol Vis}
,\emph{17} , 2911-2919. https://doi.org/315

Alsaif, H. S.,Khan, A. O.,Patel, N.,Alkuraya, H.,Hashem, M.,Abdulwahab,
F.,Ibrahim, N.,Aldahmesh, M. A., \& Alkuraya, F. S. (2019). Congenital
glaucoma and CYP1B1: an old story revisited. \emph{Hum Genet}
,\emph{138} (8-9), 1043-1049. https://doi.org/10.1007/s00439-018-1878-z

Bayat, B.,Yazdani, S.,Alavi, A.,Chiani, M.,Chitsazian, F.,Tusi, B.
K.,Suri, F.,Narooie-Nejhad, M.,Sanati, M. H., \& Elahi, E. (2008).
Contributions of MYOC and CYP1B1 mutations to JOAG. \emph{Mol Vis}
,\emph{14} , 508-517.

Bayram, Y.,Pehlivan, D.,Karaca, E.,Gambin, T.,Jhangiani, S. N.,Erdin,
S.,Gonzaga-Jauregui, C.,Wiszniewski, W.,Muzny, D.,Elcioglu, N.
H.,Yildirim, M. S.,Bozkurt, B.,Zamani, A. G.,Boerwinkle, E.,Gibbs, R.
A., \& Lupski, J. R. (2014). Whole exome sequencing identifies three
novel mutations in ANTXR1 in families with GAPO syndrome. \emph{Am J Med
Genet A} , \emph{164A} (9), 2328-2334.
https://doi.org/10.1002/ajmg.a.36678

Bozkurt, B.,Yildirim, M. S.,Okka, M., \& Bitirgen, G. (2013). GAPO
syndrome: four new patients with congenital glaucoma and myelinated
retinal nerve fiber layer. \emph{Am J Med Genet A} , \emph{161A} (4),
829-834. https://doi.org/10.1002/ajmg.a.35734

Cascella, R.,Strafella, C.,Germani, C.,Novelli, G.,Ricci, F.,Zampatti,
S., \& Giardina, E. (2015). The genetics and the genomics of primary
congenital glaucoma. \emph{Biomed Res Int} .~\emph{2015} , 321291.
https://doi.org/10.1155/2015/321291

Chakrabarti, S.,Kaur, K.,Kaur, I.,Mandal, A. K.,Parikh, R. S.,Thomas,
R., \& Majumder, P. P. (2006). Globally, CYP1B1 Mutations in Primary
Congenital Glaucoma Are Strongly Structured by Geographic and Haplotype
Backgrounds. \emph{Investigative Ophthalmology \& Visual Science}
,\emph{47} (1), 43-47. https://doi.org/10.1167/iovs.05-0912

Chen, Y.,Jiang, D.,Yu, L.,Katz, B.,Zhang, K.,Wan, B., \& Sun, X. (2008).
CYP1B1 and MYOC mutations in 116 Chinese patients with primary
congenital glaucoma. \emph{Arch Ophthalmol} , \emph{126} (10),
1443-1447. https://doi.org/126/10/1443

Ferre-Fern\selectlanguage{ngerman}ández, J.-J.,Aroca-Aguilar, J.-D.,Medina-Trillo,
C.,Bonet-Fernández, J.-M.,Méndez-Hernández, C.-D.,Morales-Fernández,
L.,Corton, M.,Cabañero-Valera, M.-J.,Gut, M.,Tonda, R.,Ayuso,
C.,Coca-Prados, M.,García-Feijoo, J., \& Escribano, J. (2017).
Whole-Exome Sequencing of Congenital Glaucoma Patients Reveals
Hypermorphic Variants in GPATCH3, a New Gene Involved in Ocular and
Craniofacial Development. \emph{Scientific Reports} , \emph{7} (1),
46175. https://doi.org/10.1038/srep46175

Gagliardi, A. R.,Gonzalez, C. H., \& Pratesi, R. (1984). GAPO syndrome:
report of three affected brothers. \emph{Am J Med Genet} , \emph{19}
(2), 217-223. https://doi.org/10.1002/ajmg.1320190203

Garcia-Castellanos, R.,Nielsen, N. S.,Runager, K.,Thogersen, I.
B.,Lukassen, M. V.,Poulsen, E. T.,Goulas, T.,Enghild, J. J., \&
Gomis-Ruth, F. X. (2017). Structural and Functional Implications of
Human Transforming Growth Factor beta-Induced Protein, TGFBIp, in
Corneal Dystrophies. \emph{Structure} , \emph{25} (11), 1740-1750 e1742.
https://doi.org/S0969-2126(17)30292-7

Goloni-Bertollo, E. M.,Ruiz, M. T.,Goloni, C. B.,Muniz, M. P.,Valerio,
N. I., \& Pavarino-Bertelli, E. C. (2008). GAPO syndrome: three new
Brazilian cases, additional osseous manifestations, and review of the
literature. \emph{Am J Med Genet A} , \emph{146A} (12), 1523-1529.
https://doi.org/10.1002/ajmg.a.32157

Goyal, N.,Gurjar, H.,Sharma, B. S.,Tripathi, M., \& Chandra, P. S.
(2014). GAPO syndrome with pansutural craniosynostosis leading to
intracranial hypertension. \emph{BMJ Case Rep} , \emph{2014} .
https://doi.org/bcr-2013-201727

Gupta, V.,Somarajan, B. I.,Walia, G. K.,Kaur, J.,Kumar, S.,Gupta,
S.,Chaurasia, A. K.,Gupta, D.,Kaushik, A.,Mehta, A., \& Sharma, A.
(2018). Role of CYP1B1, p.E229K and p.R368H mutations among 120 families
with sporadic juvenile onset open-angle glaucoma. \emph{Graefes Arch
Clin Exp Ophthalmol} , \emph{256} (2), 355-362.
https://doi.org/10.1007/s00417-017-3853-0

Ilker, S. S.,Ozturk, F.,Kurt, E.,Temel, M.,Gul, D., \& Sayli, B. S.
(1999). Ophthalmic findings in GAPO syndrome. \emph{Jpn J Ophthalmol}
,\emph{43} (1), 48-52. https://doi.org/S0021515598000586

Jiang, X., \& Zhang, H. (2021). Deterioration of Avellino corneal
dystrophy in a Chinese family after LASIK. \emph{Int J Ophthalmol}
,\emph{14} (6), 795-799. https://doi.org/10.18240/ijo.2021.06.02

Kim, J. E.,Han, M. S.,Bae, Y. C.,Kim, H. K.,Kim, T. I.,Kim, E. K., \&
Kim, I. S. (2007). Anterior segment dysgenesis after overexpression of
transforming growth factor-beta-induced gene, beta igh3, in the mouse
eye. \emph{Mol Vis} , \emph{13} , 1942-1952. https://doi.org/v13/a219

Klintworth, G. K.,Bao, W., \& Afshari, N. A. (2004). Two mutations in
the TGFBI (BIGH3) gene associated with lattice corneal dystrophy in an
extensively studied family. \emph{Invest Ophthalmol Vis Sci} ,\emph{45}
(5), 1382-1388. https://doi.org/10.1167/iovs.03-1228

Knight, L. S. W.,Ruddle, J. B.,Taranath, D. A.,Goldberg, I.,Smith, J. E.
H.,Gole, G.,Chiang, M. Y.,Willett, F.,D'Mellow, G.,Breen, J.,Qassim,
A.,Mullany, S.,Elder, J. E.,Vincent, A. L.,Staffieri, S. E.,Kearns, L.
S.,Mackey, D. A.,Luu, S.,Siggs, O. M.,Souzeau, E., \& Craig, J. E.
(2021). Childhood and Early Onset Glaucoma Classification and Genetic
Profile in a Large Australasian Disease Registry. \emph{Ophthalmology}
,\emph{128} (11), 1549-1560. https://doi.org/S0161-6420(21)00288-8

Kocabay, G., \& Mert, M. (2009). GAPO syndrome associated with dilated
cardiomyopathy: an unreported association. \emph{Am J Med Genet A}
,\emph{149A} (3), 415-416. https://doi.org/10.1002/ajmg.a.32638

Lakshminarayanan, R.,Chaurasia, S. S.,Anandalakshmi, V.,Chai, S.
M.,Murugan, E.,Vithana, E. N.,Beuerman, R. W., \& Mehta, J. S. (2014).
Clinical and genetic aspects of the TGFBI-associated corneal
dystrophies. \emph{Ocul Surf} , \emph{12} (4), 234-251.
https://doi.org/S1542-0124(14)00125-6

Liu, Y.,Munro, D.,Layfield, D.,Dellinger, A.,Walter, J.,Peterson,
K.,Rickman, C. B.,Allingham, R. R., \& Hauser, M. A. (2011). Serial
analysis of gene expression (SAGE) in normal human trabecular
meshwork.\emph{Mol Vis} , \emph{17} , 885-893. https://doi.org/99

Mackay, D. S.,Bennett, T. M., \& Shiels, A. (2015). Exome Sequencing
Identifies a Missense Variant in EFEMP1 Co-Segregating in a Family with
Autosomal Dominant Primary Open-Angle Glaucoma. \emph{PLoS One}
,\emph{10} (7), e0132529. https://doi.org/10.1371/journal.pone.0132529

Manouvrier-Hanu, S.,Largilliere, C.,Benalioua, M.,Farriaux, J. P., \&
Fontaine, G. (1987). The GAPO syndrome. \emph{Am J Med Genet} ,\emph{26}
(3), 683-688. https://doi.org/10.1002/ajmg.1320260323

Mullaney, P. B.,Jacquemin, C.,al-Rashed, W., \& Smith, W. (1997). Growth
retardation, alopecia, pseudoanodontia, and optic atrophy (GAPO
syndrome) with congenital glaucoma. \emph{Arch Ophthalmol} ,\emph{115}
(7), 940-941. https://doi.org/10.1001/archopht.1997.01100160110028

Pang, C. P.,Fan, B. J.,Canlas, O.,Wang, D. Y.,Dubois, S.,Tam, P. O.,Lam,
D. S.,Raymond, V., \& Ritch, R. (2006). A genome-wide scan maps a novel
juvenile-onset primary open angle glaucoma locus to chromosome
5q.\emph{Mol Vis} , \emph{12} , 85-92. https://doi.org/v12/a9

Quinodoz, M.,Peter, V. G.,Bedoni, N.,Royer Bertrand, B.,Cisarova,
K.,Salmaninejad, A.,Sepahi, N.,Rodrigues, R.,Piran, M.,Mojarrad,
M.,Pasdar, A.,Ghanbari Asad, A.,Sousa, A. B.,Coutinho Santos,
L.,Superti-Furga, A., \& Rivolta, C. (2021). AutoMap is a high
performance homozygosity mapping tool using next-generation sequencing
data. \emph{Nat Commun} , \emph{12} (1), 518.
https://doi.org/10.1038/s41467-020-20584-4

Rim, P. H., \& Marques-de-Faria, A. P. (2005). Ophthalmic aspects of
GAPO syndrome: case report and review. \emph{Ophthalmic Genet}
,\emph{26} (3), 143-147. https://doi.org/LQ8L22N53744224K

Saeedi, O.,Yousaf, S.,Tsai, J.,Palmer, K.,Riazuddin, S., \& Ahmed, Z. M.
(2018). Delineation of Novel Compound Heterozygous Variants in LTBP2
Associated with Juvenile Open Angle Glaucoma. \emph{Genes (Basel)}
,\emph{9} (11). https://doi.org/genes9110527

Sayli, B. S., \& Gul, D. (1993). GAPO syndrome in three relatives in a
Turkish kindred. \emph{Am J Med Genet} , \emph{47} (3), 342-345.
https://doi.org/10.1002/ajmg.1320470309

Siggs, O. M.,Souzeau, E.,Taranath, D. A.,Dubowsky, A.,Chappell, A.,Zhou,
T.,Javadiyan, S.,Nicholl, J.,Kearns, L. S., \& Staffieri, S. E. (2020).
Biallelic CPAMD8 variants are a frequent cause of childhood and juvenile
open-angle glaucoma. \emph{Ophthalmology} , \emph{127} (6), 758-766.

Song, Y.,DiMaio, F.,Wang, R. Y.,Kim, D.,Miles, C.,Brunette, T.,Thompson,
J., \& Baker, D. (2013). High-resolution comparative modeling with
RosettaCM. \emph{Structure} , \emph{21} (10), 1735-1742.
https://doi.org/S0969-2126(13)00297-9

Stranecky, V.,Hoischen, A.,Hartmannova, H.,Zaki, M. S.,Chaudhary,
A.,Zudaire, E.,Noskova, L.,Baresova, V.,Pristoupilova, A.,Hodanova,
K.,Sovova, J.,Hulkova, H.,Piherova, L.,Hehir-Kwa, J. Y.,de Silva,
D.,Senanayake, M. P.,Farrag, S.,Zeman, J.,Martasek, P.,Baxova, A.,Afifi,
H. H.,St Croix, B.,Brunner, H. G.,Temtamy, S., \& Kmoch, S. (2013).
Mutations in ANTXR1 cause GAPO syndrome. \emph{Am J Hum Genet}
,\emph{92} (5), 792-799. https://doi.org/S0002-9297(13)00160-2

Van Der Spoel, D.,Lindahl, E.,Hess, B.,Groenhof, G.,Mark, A. E., \&
Berendsen, H. J. (2005). GROMACS: fast, flexible, and free. \emph{J
Comput Chem} , \emph{26} (16), 1701-1718.
https://doi.org/10.1002/jcc.20291

Vanommeslaeghe, K.,Hatcher, E.,Acharya, C.,Kundu, S.,Zhong, S.,Shim,
J.,Darian, E.,Guvench, O.,Lopes, P.,Vorobyov, I., \& Mackerell, A. D.,
Jr. (2010). CHARMM general force field: A force field for drug-like
molecules compatible with the CHARMM all-atom additive biological force
fields. \emph{J Comput Chem} , \emph{31} (4),
671-690.https://doi.org/10.1002/jcc.21367

Wajntal, A.,Koiffmann, C. P.,Mendonca, B. B.,Epps-Quaglia, D.,Sotto, M.
N.,Rati, P. B., \& Opitz, J. M. (1990). GAPO syndrome (McKusick
23074)--a connective tissue disorder: report on two affected sibs and on
the pathologic findings in the older. \emph{Am J Med Genet} ,\emph{37}
(2), 213-223. https://doi.org/10.1002/ajmg.1320370210

Williams, C. J.,Headd, J. J.,Moriarty, N. W.,Prisant, M. G.,Videau, L.
L.,Deis, L. N.,Verma, V.,Keedy, D. A.,Hintze, B. J.,Chen, V. B.,Jain,
S.,Lewis, S. M.,Arendall, W. B., 3rd,Snoeyink, J.,Adams, P. D.,Lovell,
S. C.,Richardson, J. S., \& Richardson, D. C. (2018). MolProbity: More
and better reference data for improved all-atom structure
validation.\emph{Protein Sci} , \emph{27} (1), 293-315.
https://doi.org/10.1002/pro.3330

\textbf{Funding:~} This study was supported by funding from the Indian
Council of Medical Research, New Delhi, India (Grant no.
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