RESULTS
We found a missense homozygous ANTXR1 gene mutation in all three
children affected with GAPO (NM_032208.2:c.572T>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>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 TEKgene (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 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,MYOC (GLC1A), CYP1B1 (GLC3A), WDR36 (GLC1G),ASB10 (GLC1F), OPTN (GLC1E), NTF4 (GLC1O),TBK1 (GLC1P), LTBP2 (GLC3C), FOXC1, PITX2 ,TEK, and PAX6. Exonic variants specific to the two sisters
with congenital glaucoma and the variants they inherited from either of
the parents are listed in Suppl Table 1.
Four missense variants exclusive to the two sisters with GAPO and
congenital glaucoma (case 1 and case 2); PTPN4 , PCDHB4,
GGT2 and TGFBI, were observed. The variants on PTPN4 andPCDHB4 were rejected as they were found to be benign. TheGGT2 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 inTGFBI , NM_000358.3:c.968C>T
NP_000349.1:p.Ala323Val (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 Clinvarstar 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 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 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, aTGFBI 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>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>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
(Fig 3 ). WES analysis of this patient revealed a heterozygousTGFBI mutation NM_000358.3:c.1663C>T,
NP_000349.1:p.Arg555Trp which is reported to be pathogenic inClinvar with 3 submissions associated with granular corneal
dystrophies (GCD). (Table 2 )
A comparative modeling of the 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 α-helical region in the C-terminal, as shown inFigure 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 ofTGFBI 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
(Supplementary Figure 1 ). MD simulations followed by Principal
Component Analysis (PCA) were performed to study the overall motions of
the 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 Figure 5 . We found that TGFBIfluctuated 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) 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 TGFBI (Figure 6 ).