Identification of de Novo and Novel Mutations in LTBP2 in Pakistani Families with Inherited Primary Congenital Glaucoma

Rani Saira Saleem1*, Muhammad Imran Khan2, Saba Irshad1*,

Sorath Noorani Siddiqui3 and Shazia Micheal4

1School of Biochemistry and Biotechnology, University of the Punjab, 54000 Lahore, Pakistan.

2Department of Human Genetics, Radboud University Medical Centre, 6525 GA Nijmegen, The Netherlands

3Department of Pediatric Ophthalmology and Strabismus, Al-Shifa Eye Trust Hospital Jhelum Road, Rawalpindi 46000, Pakistan.

4Department of Clinical Genetics, Academic Medical Centre, Meibergdreef 9, 1105 Amsterdam, The Netherlands.

ABSTRACT

Primary congenital glaucoma (PCG) is the topmost reason for childhood blindness due to optic nerve impairment, enlarged globe, and loss of visual field. PCG is usually identified during the first year of life. The purpose of the recent research was to assess the involvement of the Latent transforming growth factor 2 (LTBP2) gene in PCG families of Pakistani origin and to find novel mutations. To extract genomic DNA from the whole blood of n=20 families were performed followed by the genotyping of the affected and unaffected persons of the families by using whole genome single nucleotide polymorphism microarray (SNP). Homozygosity mapping analysis was performed for the selected members of these families. LTBP2 gene was screened using Sanger Sequencing in n=20 consanguineous Pakistani families diagnosed with PCG by standard ophthalmological examination. Novel homozygous mutations were identified in three families with PCG in the LTBP2 gene. We found a new de novo frameshift mutation c.1762_1763del; p. (Leu588Valfs*14). In a second family, we identified a splice site mutation c.2531-2A>C, and in the third family, a splice donor site c.1686G>A; p. (Gln562Gln) mutation. In this study, we report the involvement of novel de novo frameshift mutation and two genetic variants that affect splicing in PCG families from Pakistan. The current study will help us to extend our understanding of the part of LTBP2 in PCG.

Article Information

Received 12 March 2023

Revised 20 July 2023

Accepted 11 August 2023

Available online 11 January 2024

(early access)

Published 01 May 2025

Authors’ Contribution

RSS, SM and SI conceived and designed the experiments. RSS and SM performed the experiments. RSS, SI and SM performed results and bioinformatics analysis. SNS and MIK recruited patients, collected clinical data and samples. SNS, MIK, SM and SI contributed reagents, materials, analysis tools. RSS, SI and SM wrote the manuscript. All authors have read and approved the final manuscript.

Key words

Primary congenital glaucoma, LTBP2, Homozygosity, Mutations, Microarray analysis

DOI: https://dx.doi.org/10.17582/journal.pjz/20230312090320

* Corresponding author: [email protected], [email protected]

0030-9923/2025/0003-1159 $ 9.00/00

Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Primary congenital glaucoma (PCG; OMIM 231300) is one of the types of glaucoma leading to blindness among 18% of cases in the starting years of life (DeLuise and Anderson, 1983). The disease pathology is characterized by obstruction of the aqueous humor drainage pathways owing to congenital progressive imperfections in the anterior segment of the eye (Maumenee, 1958). PCG is also commonly known as congenital buphthalmos characterized by delocalization of the sclera, damage to the optic nerve, and related structures, associated with elevated levels of intraocular pressure (IOP) (Kwitko, 1973; Shaffer, 1969). Thus, it is proposed that obstetricians should pay attention during neonatal eye development to keep a record of the corneal diameter during the growth to help with the early identification and treatment of PCG. Indeed, a strong indicator of PCG is the distensibility in the neonatal eyeball due to the excessive accumulation of elastic fibers in the sclera, optical nerve cupping, or any rise in the corneal diameter >12mm in the earlier stages of life (Hoskins, 1989; Sarfarazi et al., 1995). For this reason, a conventional triad of symptoms is also suggested for an early identification of PCG that includes tearing of eyes, oversensitive eyes to the light, and related inflammation issues (Hoskins, 1989).

The mode of inheritance for PCG is autosomal recessive with variable penetrance indicating that PCG is more prevalent in the populations with consanguinity. Genetic investigation of the families with PCG revealed four genetic loci linked to the disease; GLC3A at 2p22-p21 (Sarfarazi et al., 1995), GLC3B at 1p36.2-p36.1 (Akarsu et al., 1996), GLC3C at 14q24.3 (Stoilov and Sarfarazi, 2002) and GLC3D at 14q24 (Firasat et al., 2008). To date, disease-causing mutations have been known in three genes within these loci. Cytochrome P450 1B1 (CYP1B1) is the most common gene identified for its association with PCG and is encompassed within the GLC3A locus (Stoilov et al., 1997). LTBP2 is another important disease-causing gene localized within the GLC3D locus (Ali et al., 2009). Recently, mutations have also been known in the third PCG gene, Tunica internal endothelial cell kinase (TEK) (Souma et al., 2016).

The most frequent cause of PCG in consanguineous families are disease producing changes in the CYP1B1 gene. For example, 80%-100% of Saudi Arabian PCG families were reported to harbor CYP1B1 mutations (Abu-Amero, 2011). So far, about more than 150 different mutations including synonymous, non-synonymous, coding, non-coding, and frameshifts have been listed in CYP1B1 for causing PCG in families with different origins. However, only 37-40% of Pakistani PCG families had mutations in the CYP1B1 gene, which further suggests genetic heterogeneity of the disease (Micheal et al., 2014). Recently three novel alterations have been stated in Iranian families with PCG linked to the CYP1B1 gene (Emamalizadeh, 2021). In addition to CYP1B1, a few mutations in the LTBP2 gene have been reported for Pakistani families and other populations linked with PCG. Since the expression of LTBP2 in trabecular meshwork cell lines is 12.7 times that of CYP1B1, it is tempting to speculate on the importance of LTBP2 in the pathogenesis of PCG (Wang et al., 2001).

The purpose of the current study was to screen the probands of twenty Pakistani PCG families for mutations in the LTBP2 gene. All these families had been previously excluded for variants in the CYP1B1 gene.

Materials and Methods

Subjects

The probands were engaged by the Ophthalmology Department of Al-Shifa Eye Trust Hospital, Rawalpindi, Pakistan. The ocular investigation was executed for both affected and unaffected persons of families.

Extraction of DNA and PCR amplification of LTBP2

Extraction of DNA was done from peripheral leukocytes by using the QIAGEN DNA Blood Midi Kit (QIAGEN, Germantown, Maryland, USA) of twenty PCG families. Genotyping will be followed by whole genome SNP microarray (Illumina GSA Beadchip) analysis by using DNA samples of selected affected and unaffected individuals of these 20 families. An online mapping tool Homozygosity Mapper was deployed to evaluate the information and find the respective regions. LTBP2 gene was screened for the presence of mutations. Sanger sequencing of all 36 exons inclusive of exon borders of the gene was performed by using an automated ABI 3730 Sequencer (Applied Biosystems, Inc.) according to the provided protocol. CodonCode Aligner (version 6.1) was deployed for performing sequence alignment with the reference sequence to identify the novel variants. The primer sequences, reactions, and PCR conditions were modified from the literature.

In silico studies

In the current study, an in-silico investigation was completed for the variants found by using ALAMUT® VISUAL software to determine the functional impact. The prediction tools included to examine the pathogenicity of missense variations were SIFT, Mutation Taster, and PolyPhen-2. In the case of splice site variants, GeneSplicer, MaxEntScan, HSF, NNSPLICE, and ESE were considered for splicing site calculations. The links used to access these analytical tools were;

Human Splicing Finder (HSF) http://www.umd.be/HSF3/technicaltips.html

Maximum entropy (MaxENt) score: http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html

Sorting Intolerant from Tolerant (SIFT): http://sift.bii.a-star.edu.sg/

Polymorphism Phenotyping v2 (PolyPhen-2): http://genetics.bwh.harvard.edu/pph2/

NNSPLICE: https://omictools.com/nnsplice-tool

Exon Splicing Enhancer (ESE): http://rulai.cshl.edu/tools/ESE2/index.html

GeneSplicer: https://ccb.jhu.edu/software/genesplicer/

Results

Identification of De-Novo c.1762_1763del; p. (Leu588Valfs*14) mutation

In family 1, proband II.I was a 3-months-old boy when presented in the patient outdoor clinic. He was diagnosed with PCG. Both his parents were unaffected upon clinical examination. Both parents did not suffer from elevated IOP or glaucoma but, soon after birth, the child was reported with raised IOP > 22 mmHg. Along with this, the corneal diameter of the child was recorded to be larger than 13mm. The reported cup-to-disc ratio was >0.7.

In the DNA of the proband (II-1), we found a homozygous de novo mutation c.1762_1763del by Sanger sequencing in the LTBP2 gene. The absence of this somatic, non-hereditary variant in both parents suggests this particular variant is a novel cause of PCG. This 2bp deletion in Exon 8 (Fig. 1B) causes a frameshift at codon Leu588 and, subsequently, a stop codon 13 positions downstream. This may result in the non-sense-mediated decay of the mRNA.

LTBP2 canonical splice site mutation (c.2531-2A>C)

In family 2, two affected individuals were diagnosed with bilateral PCG. Male Proband IV: 2 and his affected sister (IV: 3) were, respectively, 6 and 3 months old at the time of diagnosis. All the affected (IV:2, IV:3) and unaffected (III:1, III:2, IV:1, IV:4) members of the family were checked for the presence of any other related abnormalities. After the Whole genome analysis, the homozygosity mapper yielded two relevant regions: one

 

 

confined to chromosome 14 and the other referred to chromosome 20; as revealed in Figure 2A. LTBP2 gene spans at the 22Mb localized homozygous region of chromosome 14. Sanger sequencing of the entire coding sequence and intronic boundaries identified a novel homozygous acceptor splice site mutation c.2531-2A>C neighboring exon 16. The mutation was found cosegregating with the disease in the family. In the affected patients, the mutation is existing in homozygous form, as shown in Figure 2C. The prediction analysis of human splicing finder (HSF) and other tools, such as MaxEntScore and NNSPLICE, and ExoSKIP, all predicted a pathogenic modification of the wild acceptor site. Most likely, the consequence of this change is of the skipping of LTBP2 exon 16, as indicated by ExoSKIP in Figure 2D. This change has not been stated in any of the databases including genomAD.

LTBP2, synonymous splice site c.1686G>A; p. (Gln562Gln) mutation

In family 3 (Fig. 3B), proband IV: 7 was identified with PCG when he was just 9 months old. In addition to this, members IV: 2, IV: 5, and IV: 7 were also diagnosed with bilateral PCG with corneal diameters >13mm and IOP > 23 mmHg. No secondary ocular anomalies were observed in these individuals. Whole genome homozygosity mapping analysis revealed multiple homozygous regions. Among them was a 14Mb region on chromosome 14 encompassing for LTBP2 gene, found to be homozygous (Fig. 3A). Sanger Sequencing of the LTBP2 gene in the affected members of the family caused the recognition of a c.1686G>A; p. (Gln562Gln) mutation at the splice donor site. The genetic variant was found to be cosegregated with the disease in the family (Fig. 3C). The consensus nucleotide positions flanking this canonical splice donor

 

site are highly conserved indicating its highly pathogenic potential (Ma et al., 2015). The pathogenicity of the synonymous change c.1686G>A is further supported in silico by the computer program scores, SSF, MaxEnt, and HSF (Fig. 3D). These scores indicate that as a result of the G>A change at position c.1686 the splice donor site will not be recognized by the splicing machinery anymore, and most likely will lead to intron retention. As introns frequently have stop codons, so this synonymous modification in the open reading frame will likely be introducing a premature stop codon.

Discussion

The LTBP2 gene lies near in to the GLC3C locus that was reported for the first time in a large Turkish PCG family (Firasat et al., 2008; Stoilov and Sarfarazi, 2002). In the recent study, three novel mutations in LTBP2 are reported which includes a canonical splice site mutation c.2531-2A>C, a de novo mutation c.1762_1763del; p. (Leu588Valfs*14), and a synonymous missense c.1686G>A; p. (Gln562Gln) in 3 out of 20 Pakistani PCG families. Previously, more than 28 different alterations in the LTBP2 gene have been implicated in various forms of glaucoma, especially PCG (Fig. 4). The previously reported mutations for PCG in Pakistani families include a c.412delG; p. (Ala138Prfs*278) mutation in exon 1; a nonsense variant in exon 4, c.895C>T; p. (Arg299X); a c.1243-1256del; p. (Glu415Argfs*596) homozygous removal of 14 bp in exon 6; and c.331C>T; p. (Gln111X) in exon 1 (Ali et al., 2009). One of these mutations (c.895C>T; p. (Arg299X); exon 4) has also been found in a European gypsy family supporting the idea that this mutation may have a common origin. Interestingly, another recent study described null variants in LTBP2 in 2 Pakistani families characterized by secondary glaucoma along with ectopia lentis, myopia, microspherophakia, megalocornea and Merfanoid structures in elder children. These null mutations included novel homozygous mutations consisting of exons 4 and 9 (Désir et al., 2010). Whole genome homozygosity mapping for two additional Iranian PCG families also revealed two disease-segregating mutations in LTBP2, p.(Ser472fsX3) and p.(Tyr1793fsX55) resulting in a nonfunctional protein. These mutations were suggested to affect significantly the protein assembly and its role thereby interfering with both fibrillin 5 and fibrillin 1 interaction which is critical for the role of LTBP2 (Narooie-Nejad et al., 2009). WES in the two PCG families with Pakistani origin led to the identification of two additional new missense p. (Arg1645Glu) and a new variant p. (Asp1345Glyfs*6) resulting in a frameshift in LTBP2 found segregating with the diseased phenotype (Micheal et al., 2016).

 

In yet another family of south Indian origin, diagnosed with microspherophakia (a rare eye disorder characterized by spherical lens), in exon 36 a variant c.5446dupC has been stated. Thus, an elongation of 21 residues was resulted due to this variant in LTBP2 protein which replaced the preceding 6 with an additional 27 residues (Kumar et al., 2010). Finally, three novel changes have also been described segregating in three consanguineous families with Saudi Arabian origin, one in exon 4; p. (Ser338PfsX4), in exon 33, p. (Gln1619X) and exon 29, p. (Cys1438Tyr). The medical aspects of the disease carrying individuals in these families consisted of an ocular syndrome of congenital megalocornea and associated secondary glaucoma (Khan et al., 2011). Recently, another study reported three new pathogenic LTBP2 variants p. (Gln1143Argfs*35), p. (Cys1757Tyr), and p. (Asp1010Asn) in Pakistani PCG families (Rauf et al., 2020). LTBP2 changes have also been associated with Weill Marchesani-like syndrome, primary open-angle glaucoma, pseudo exfoliative glaucoma, and microspherophakia in addition to PCG (Alías et al., 2018; Jelodari-Mamaghani et al., 2013; Javadi et al., 2012). The LTBP2 gene has also been investigated in northern India, the United States, the United Kingdom, and Chinese PCG cohorts but, in these countries, no mutations were found associated with PCG or related disease (Wang et al., 2018; Lim et al., 2013). A recent study regarding phenotype-genotype association for childhood glaucoma revealed a strong association between Weill Marchesani-like syndrome, childhood glaucoma, and LTBP2 mutations by presenting a ratio of about 12.5% among all the cases studied for childhood glaucoma (Stingl et al., 2022).

The LTBP2 protein relates to the transforming growth factor superfamily that performs a significant part in the differentiation of organs and tissues. The gene codes for 1821 amino acids long protein molecule containing (Ali et al., 2009). LTBP2 is known as an extracellular matrix microfibril protein. The actual mechanism of LTBP2 mutations causing PCG is still not clear. Interestingly, however, its high expression has been profiled in Trabecular Meshwork (TM), in the frontal chamber of the eye, and in the ciliary zonule microfibrils. At the cellular level, various causative mutations have been reported for PCG along with its role in ECM maintenance (Lim et al., 2013; Horiguchi et al., 2012). The protein is involved in the regulation of TGFβ1 signaling. It is important to regulate the downstream signaling and initiation of transcription factors by phosphorylation and activation of TGFBR1, upon the binding of TGFβ1 to TGFBR2 (Kielty et al., 2005). While interacting, the C-terminal of LTBP2 come in close association with the N-terminus of fibrillin 1, a critical component of the microfibrils (Verstraeten et al., 2016). The participation of LTBP2 in the development of elastic fibers during elastic tissues development has also been shown (Hirani et al., 2007). It is hypothesized that due to the presence of mutations in LTBP2, there results in changes in the structure of the protein that cease it to bind with FBN1 (Hirani et al., 2007). Furthermore, lens dislocation has also been reported in LTBP2 null mice, this may be the result of the loss of arrangement of ciliary zonules, consisting of microfibrils (Inoue et al., 2014). Thus, the loss of ciliary organization and associated structures and tissues may result in high IOP and apoptosis of RGCs thus contributing towards glaucoma.

Conclusions

In conclusion, we report here a de novo pathogenic variant c.1762_1763del; p. (Leu588Valfs*14) in a family for PCG. We also report two novel splice site mutations c.2531-2A>C and c.1686G>A identified in LTBP2 for two consanguineous families of Pakistan. This study along with the previous studies on the roles of LTBP2 predicts that there is a lot more to explore for elucidating the exact mechanisms involved in the pathogenesis of PCG.

Acknowledgment

The authors are thankful to the affected and unaffected participants and their families for their cooperation. The authors also acknowledge the Department of Clinical Genetics, Academic Medical Centre, Amsterdam, The Netherlands and Higher Education Commission, Pakistan and its International Research Initiative Program for providing this Research Study opportunity.

Availability of data

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

IRB approval

The consent for the research was obtained from the Institutional Review Board of the Al-Shifa Eye Trust Hospital (Rawalpindi, Pakistan).

Ethical statement

Ethical Committee of the University of Punjab Lahore, approved the study following the tenets of the Declaration of Helsinki. For inclusion in research, written well-versed consensus was gained either from the members and/or their parents, as a pre-requisite for participation in the study.

Statement of conflict of interest

The authors have declared no conflict of interest.

References

Abu-Amero, K.K., Osman, E.A., Mousa, A., Wheeler, J., Whigham, B., Allingham, R.R., Hauser, M.A. and Al-Obeidan, S.A., 2011. Screening of CYP1B1 and LTBP2 genes in Saudi families with primary congenital glaucoma: Genotype-phenotype correlation. Mol. Vis., 17: 2911-2919.

Akarsu, A.N., Turacli, M.E., Aktan, S.G., Barsoum-Homsy, M., Chevrette, L., Sayli, B.S. and Sarfarazi, A., 1996. Second locus (GLC3B) for primary congenital glaucoma (Buphthalmos) maps to the 1p36 region. Hum. mol. Genet., 5: 1199-1203. https://doi.org/10.1093/hmg/5.8.1199

Ali, M., McKibbin, M., Booth, A., Parry, D.A., Jain, P., Riazuddin, S.A., Hejtmancik, J.F., Khan, S.N., Firasat, S., Shires, M., Gilmour, D.F., Towns, K., Murphy, A.L., Azmanov, D., Tournev, I., Cherninkova, S., Jafri, H., Raashid, Y., Toomes, C., Craig, J., Mackey, D.A., Kalaydjieva, L., Riazuddin, S. and Inglehearn, C.F., 2009. Null mutations in LTBP2 cause primary congenital glaucoma. Am. J. Hum. Genet., 84: 664-671. https://doi.org/10.1016/j.ajhg.2009.03.017

Alías, L., Crespi, J., González-Quereda, L., Téllez, J., Martínez, E., Bernal, S. and Gallano, M.P., 2018. Next-generation sequencing reveals a new mutation in the LTBP2 gene associated with microspherophakia in a Spanish family. BMC med. Genet., 19: 77. https://doi.org/10.1186/s12881-018-0590-0

DeLuise, V.P. and Anderson, D.R., 1983. Primary infantile glaucoma (Congenital glaucoma). Surv. Ophthalmol., 28: 1–19. https://doi.org/10.1016/0039-6257(83)90174-1

Désir, J., Sznajer, Y., Depasse, F., Roulez, F., Schrooyen, M., Meire, F. and Abramowicz, M., 2010. LTBP2 null mutations in an autosomal recessive ocular syndrome with megalocornea, spherophakia, and secondary glaucoma. Eur. J. Hum. Genet., 18: 761–767. https://doi.org/10.1038/ejhg.2010.11

Emamalizadeh, B., Daneshmandpour, Y., Kazeminasb, S., Aghaei, Moghadam, E., Bahmanpour, Z., Alehabib, E., Alinaghi, S., Doozandeh, A., Atakhorrami, M. and Darvish, H., 2021. Mutational analysis of CYP1B1 gene in Iranian pedigrees with glaucoma reveals known and novel mutations. Int. Ophthalmol., 41: 3269–3276. https://doi.org/10.1007/s10792-021-01888-w

Firasat, S., Riazuddin, S.A., Hejtmancik, J.F. and Riazuddin, S., 2008. Primary congenital glaucoma localizes to chromosome 14q24.2-24.3 in two consanguineous Pakistani families. Mol. Vis., 14: 1659–1665.

Javadi, H.S.R., Jelodari-Mamaghani, S., Paylakhi, S.H., Yazdani, S., Nilforushan, N., Fan, J.B., Klotzle, B., Mahmoudi, M.J., Ebrahimian, M.J., Chelich, N., Taghiabadi, E., Kamyab, K., Boileau, C., Paison-Ruiz, C., Ronaghi, M. and Elahi, E., 2012. LTBP2 mutations cause Weill-Marchesani and Weill-Marchesani-like syndrome and affect disruptions in the extracellular matrix. Hum. Mutat., 33: 1182–1187. https://doi.org/10.1002/humu.22105

Hirani, R., Hanssen, E. and Gibson, M.A., 2007. LTBP2 specifically interacts with the amino-terminal region of fibrillin-1 and competes with LTBP1 for binding to this microfibrillar protein. Matrix Biol., 26: 213-223. https://doi.org/10.1016/j.matbio.2006.12.006

Horiguchi, M., Ota, M. and Rifkin, D.B., 2012. Matrix control of transforming growth factor-beta function. J. Biochem., 152: 321–329. https://doi.org/10.1093/jb/mvs089

Hoskins, H.D. Jr., 1989. Angle closure glaucoma with pupillary block. In: Becker-shaffer’s Diagnosis and Therapy of the Glaucomas. 6th ed. CV Mosby, St Louis.

Hoskins, H.D.Jr. and Kass, M.A., 1989. Becker-Shaffer’s diagnosis and therapy of the glaucoma. 6th ed. CV Mosby, St Louis. pp. 356.

Inoue, T., Ohbayashi, T., Fujikawa, Y., Yoshida, H., Akama, T.O., Noda, K., Horiguchi, M., Kameyama, K., Hata, Y., Takahashi, K., Kusumoto, K. and Nakamura, T., 2014. Latent TGF-beta binding protein-2 is essential for the development of ciliary zonule microfibrils. Hum. mol. Genet., 23: 5672-5682. https://doi.org/10.1093/hmg/ddu283

Jelodari-Mamaghani, S., Haji-Seyed-Javadi, R., Suri, F., Nilforushan, N., Yazdani, S., Kamyab, K. and Elahi, E., 2013. Contribution of the latent transforming growth factor-β binding protein 2 gene to etiology of primary open angle glaucoma and pseudoexfoliation syndrome. Mol. Vis., 19: 333-347.

Khan, A.O., Aldahmesh, M.A. and Alkuraya, F.S., 2011. Congenital megalocornea with zonular weakness and child-hood lens related secondary glaucoma-a distinct phenotype caused by recessive LTBP2 mutations. Mol. Vis., 17: 2570-2579.

Kielty, C.M., Sherratt, M.J., Marson, A. and Baldock, C., 2005. Fibrillin microfibrils. Adv. Protein Chem., 70: 405–436. https://doi.org/10.1016/S0065-3233(05)70012-7

Kumar, A., Duvvari, M.R., Praphakaran, V.C., Shetty, J.S., Murthy, G.J. and Blanton, S.H., 2010. A homozygous mutation in LTBP2 causes isolated micrspherophakia. Hum. Genet., 128: 365-371. https://doi.org/10.1007/s00439-010-0858-8

Kwitko, M.L., 1973. Glaucoma in infants and children. Appleton-Century-Crofts.

Lim, S.H., Tran-Viet, K.N., Yanovitch, T.L., Freedman, S.F., Klemm, T., Call, W., Powell, C., Ravichandran, A., Metlapally, R., Nading, E.B., Rozen, S. and Young, T.L., 2013. CYP1B1, MYOC and LTBP2 mutations in primary congenital glaucoma patients in United States. Am. J. Opthalmol., 155: 508-517. https://doi.org/10.1016/j.ajo.2012.09.012

Ma, S.L., Vega-Warner, V., Gillies, C., Sampson, M.G., Kher, V., Sethi, S.K. and Otto, E.A., 2015. Whole exome sequencing reveals novel PHEX splice site mutations in patients with hypophosphatemic rickets. PLoS One, 10: e0130729. https://doi.org/10.1371/journal.pone.0130729

Maumenee, E.A., 1958. The pathogenesis of congenital glaucoma: A new theory. Trans. Am. Ophthalmol. Soc., 56: 507–570.

Micheal, S., Ayub, H., Zafar, S.N., Bakker, B., Ali, M., Akhtar, F., Islam, F., Khan, M.I., Qamar, R. and den Hollander, A.I. 2014. Identification of novel CYP1B1 gene mutations in patients with primary congenital and primary open angle glaucoma from Pakistan. Clin. exp. Ophthalmol., 43: 31-39. https://doi.org/10.1111/ceo.12369

Micheal, S., Siddiqui, S.N., Zafar, S.N., Iqbal, A., Khan, M.I. and den Hollander, A.I., 2016. Identification of novel variants in LTBP2 and PXDN using whole-exome sequencing in developmental and congenital glaucoma. PLoS One, 11: e0159259. https://doi.org/10.1371/journal.pone.0159259

Narooie-Nejad, M., Paylakhi, S.H., Shojaee, S., Fazlali, Z., Kanavi, M.R., Nilforushan, N., Yazdani, S., Babrzadeh, F., Suri, F., Ronaghi, M., Elahi, E. and Paisa´n-Ruiz C., 2009. Loss of function mutations in the gene encoding latent transforming growth factor beta binding protein 2, LTBP2, cause primary congenital glaucoma. Hum. mol. Genet., 18: 3969–3977. https://doi.org/10.1093/hmg/ddp338

Rauf, B., Irum, B., Khan, S.Y., Kabir, F., Naeem, M.A., Riazuddin, S., Ayyagari, R. and Riazuddin, S.A., 2020. Novel mutations in LTBP2 identified in familial cases of primary congenital glaucoma. Mol. Vis., 26: 14-25.

Sarfarazi, M., Akarsu, A.N., Hossain, A., Turacli, M.E., Aktan, S.G., Barsoum-Homsy, M., Chevrette, L. and Sayli, B.S., 1995. Assignment of a locus (GLC3A) for primary congenital glaucoma (Buphthalmos) to 2p21 and evidence for genetic heterogeneity. Genomics, 30: 171–177. https://doi.org/10.1006/geno.1995.9888

Shaffer, R.N.,1969. The glaucomatous disc in infants. A suggested hypothesis for disc cupping. Trans. Am. Acad. Ophthalmol. Otolaryngol., 73: 929-935.

Souma, T., Tompson, S.W., Thomson, B.R., Siggs, O.M., Kizhatil, K., Yamaguchi, S. and Yanovitch, T.L., 2016. Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity. J. clin. Invest., 126: 2575-2587. https://doi.org/10.1172/JCI85830

Stingl, J.V., Diederich, S., Diel, H., Schuster, A.K., Wagner, F.M., Chronopoulos, P., Aghayeva, F., Grehn, F., Winter, J., Schweiger, S. and Hoffmann, E.M., 2022. First results from the prospective german registry for childhood glaucoma: Phenotype genotype association. J. clin. Med., 11: 16. https://doi.org/10.3390/jcm11010016

Stoilov, I., Akarsu, A.N. and Sarfarazi, M., 1997. Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21. Hum. mol. Genet., 6: 641–647. https://doi.org/10.1093/hmg/6.4.641

Stoilov, I.R. and Sarfarazi, M., 2002. The third genetic locus (GLC3C) for primary congenital glaucoma (PCG) maps to chromosome 14q24.3. Invest. Ophthalmol. Vis. Sci., 43: 3015.

Verstraeten, A., Alaerts, M., Van-Laer, L. and Loeys, B., 2016. Marfan syndrome and related disorders: 25 years of gene discovery. Hum. Mutat., 37: 524-531. https://doi.org/10.1002/humu.22977

Wang, H., Sun, P., Chen, Y., Jiang, L., Wu, H., Zhang, W. and Gao, F., 2018. Research progress on human genes involved in the pathogenesis of glaucoma (Review). Mol. Med. Rep., 18: 656-674. https://doi.org/10.3892/mmr.2018.9071

Wang, W.H., McNatt, L.G., Shepard, A.R., Jacobson, N., Nishimura, D.Y., Stone, E.M., Sheffield, V.C. and Clark, A.F., 2001. Optimal procedure for extracting RNA from human ocular tissues and expression profiling of the congenital glaucoma gene FOXC1 using quantitative RT-PCR. Mol. Vis., 7: 89–94.