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

Primary congenital


INTRODUCTION
P rimary 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

O n l i n e F i r s t A r t i c l e
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, noncoding, 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.

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/

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

O n l i n e F i r s t A r t i c l e
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

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

O n l i n e F i r s t A r t i c l e
de Novo and Novel Mutations in LTBP2 for PCG 5 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 diseasesegregating 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 Marchesanilike 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.

Fig. 1 .
Fig. 1.Family I: LTBP2 de Novo Mutation c.1762_1763del; p. (Leu588Valfs*14).(A) A two-generation family pedigree.PB (II: 1) is indicated with an arrowhead.Affected members are indicated with black filled box.(B) sanger sequence analysis of the de novo mutation showing the chromatograms of unaffected father, Mother and the affected son.

Fig. 2 .
Fig. 2. Family II: LTBP2 canonical splice site mutation (c.2531-2A>C).(A) Homozygosity mapping in two affected and two unaffected individuals of family II.Analysis was done by using online web tool homozygosity mapper (http://www.homozygositymapper.org/).The red line indicates the homozygous regions in the SNP array analysis, one of them corresponding to 22Mb region at chromosome 14 for LTBP2.(B) Pedigree of consanguineous family II showing segregation of a novel mutation c.2531-2A>C.Proband is indicated by a black arrowhead.(C) Chromatogram showing Sanger sequencing results for the canonical splice site mutation c.2531-2A>C indicated with respect to homozygous normal (IV:1), heterozygous carrier (III:1) and homozygous mutant (IV:2).(D) In-Silico predictions for mutation (c.2531-2A>C) by ALAMUT Visual.The score for natural 3' splice site was decreased in the mutant.

Fig. 3 .
Fig. 3. Family III: LTBP2, synonymous splice site c.1686G>A; p. (Gln562Gln) mutation.(A) Homozygosity mapping in three affected and three unaffected individuals of family III.Analysis was done by using online web tool Homozygosity mapper (http://www.homozygositymapper.org/).The red line indicates the homozygous regions in the SNP array analysis, one of them corresponding to 14Mb region at chromosome 14 for LTBP2.(B) Pedigree of consanguineous family III showing segregation of a novel c.1686G>A mutation.Proband indicated by a black arrowhead.(C) Chromatogram showing Sanger sequencing results for the splice donor site mutation c.1686G>A indicated with respect to homozygous normal (IV: 6), heterozygous carrier (III: 4) and homozygous mutant (IV: 7).(D) In-Silico predictions for mutation c.1686G>A; p. (Gln562Gln) by ALAMUT Visual.The score for natural 5' splice site was decreased in the mutant.Respective HSF and SSF scores have also been listed in the table.

Fig. 4 .
Fig. 4. Structural representation of LTBP2 exons showing the N and C-Terminal of the protein.The exons with previously reported mutations have been shown in rust red color.Current study mutations and exons have been marked with red and black color, respectively.