Novel mutations in LTBP2 identified in familial cases of primary congenital glaucoma

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: Primary congenital glaucoma (PCG) is a genetically heterogeneous disorder caused by developmental defects in the anterior chamber and trabecular meshwork. This disease is an important cause of childhood blindness. In this study, we aim to identify the genetic determinants of PCG in three consanguineous families of Pakistani descent.

Methods: Affected members of all three families underwent detailed ophthalmological examination including slit-lamp biomicroscopy. Blood samples were collected from affected and healthy members of all three families, and genomic DNA was extracted. Linkage analysis was performed for the known or reported loci of PCG to localize the disease interval, and logarithm of odds (LOD) scores were calculated. All protein-coding exons of the candidate gene, latent transforming growth factor-beta binding protein 2 (LTBP2), were bidirectionally sequenced to identify the disease-causing mutation.

Results: Short tandem repeat (STR) marker-based linkage analysis localized the critical interval to chromosome 14q with a maximum two-point LOD score of 2.86 (PKGL076), 2.8 (PKGL015), and 2.92 (PKGL042). Bidirectional Sanger sequencing of LTBP2 revealed three novel pathogenic variants, i.e., c.3028G>A (p.Asp1010Asn), c.3427delC (p.Gln1143Argfs*35), and c.5270G>A (p.Cys1757Tyr) in PKGL076, PKGL015, and PKGL042, respectively. All three mutations segregated with the disease phenotype in their respective families and were absent in 200 ethnically matched normal chromosomes.

Conclusions: We identified three novel mutations, p.D1010N, p.Q1143Rfs*35, and p.C1757Y, in LTBP2 responsible for PCG.

The term glaucoma represents a heterogeneous group of neurodegenerative disorders that have a specific type of optic neuropathy, causing irreversible defects in the visual field. The disease is deceptive, and sometimes, individuals remain undertreated or undiagnosed because central vision is usually not lost at an early stage of the disease [1,2]. Glaucoma is the second leading cause of blindness accounting for nearly 12.3% cases of visual loss worldwide [1,2].

Primary congenital glaucoma (PCG; OMIM 231300), a severe type of glaucoma, is characterized by developmental defects in the trabecular meshwork (trabeculodysgenesis) with neonatal or infantile onset [3]. Genetic defects in the trabecular meshwork are responsible for various forms of glaucoma [4]. The classical triad of symptoms presented by PCG is photophobia (hypersensitivity to light), epiphora (excessive tearing), and blepharospasm (inflammation of eyelids) [5]. Other clinical findings include Haab’s striae (rupture of Descemet’s membrane), elevated intraocular pressure (IOP), buphthalmos, conjunctival erythema, corneal edema, and optic atrophy in later stages of the disease [6-9]. It is a rare form of glaucoma representing 1% to 5% of all cases of glaucoma [10]. Although sporadic cases of PCG are more common, familial cases have also been documented. In familial cases, PCG is normally inherited in an autosomal recessive pattern with incomplete penetrance, but recently, an autosomal dominant inheritance case was reported [3,11,12].

As the molecular etiology of PCG is not fully understood, only a few genes responsible for the disease are known [13-18]. Four chromosomal locations, GLC3A (2p22-p21), GLC3B (1p36.2–36.1), GLC3C (14q24.3), and GLC3D (14q24.2–24.3), and two causative genes, cytochrome P450 family 1 subfamily B member 1 (CYP1B1; OMIM 601771) and latent transforming growth factor-beta binding protein 2 (LTBP2; OMIM 602091), have been identified for PCG thus far [19-25]. Pathogenic mutations in CYP1B1 are the most common cause of PCG and are responsible for 27% of sporadic and 87% of familial PCG cases worldwide [26]. To date, a total of nine mutations, including missense and frameshift, have been identified in LTBP2 responsible for PCG [20,22,27-29]. Recently, an autosomal dominant model of PCG with variable expression was identified in a fifth locus, GLC3E (9p21.2) harboring mutations in the tunica interna endothelial cell kinase (TEK; OMIM 600221) gene [12]. These findings were consistent with previous findings in terms of high penetrance as well as variable expression of the paired-like homeodomain transcription factor 2 (PITX2; OMIM 601542), forkhead box C1 (FOXC1; OMIM 601090), paired box gene 6 (PAX6; OMIM 607108), and optic atrophy 1 (OPA1; OMIM 605290) genes responsible for developmental glaucoma in an autosomal dominant inheritance pattern [30-36].

A total of 30 familial cases affected with PCG were identified and recruited from different cities in Pakistan. Of these cases, 23 families were linked to CYP1B1 with a total of 11 mutations reported previously, and three families showed linkage to LTBP2 [37]. In the present study, we report three novel mutations, including two missense and a frameshift in the LTBP2 gene, in three consanguineous families with PCG who were negative for mutations in CYP1B1.

In this study, we report three familial cases, PKGL076, PKGL015, and PKGL042, recruited from different cities in Punjab, Pakistan. A detailed history was obtained for affected individuals in each family through family interviews and available medical records. Pedigree and haplotype analysis revealed the disease phenotype segregates with an autosomal recessive pattern. All affected individuals developed the disease symptoms within the first 3 years of life. Affected individuals from these familial cases underwent a detailed ophthalmic examination including slit-lamp biomicroscopy to confirm the diagnosis.

Ophthalmic examination of PKGL076 showed bilateral (B/L) buphthalmos, corneal edema, B/L corneal haze, and an increased corneal diameter (>13 mm) in individual 7. The maximum IOP recorded for this individual was 36/26 mmHg (oculus dexter/oculus sinister [OD/OS]) and visual acuity reduced to the perception of light in both eyes. Individual 8 developed PCG symptoms at 1.5 years of age. Medical records showed B/L buphthalmos, B/L nystagmus, elevated IOP of 28/24 mmHg (OD/OS), and an increased corneal diameter (>13 mm). Detailed medical examination of individual 9 showed B/L buphthalmos and B/L myopic fundus. Fundoscopy of this affected individual revealed a cup to disc ratio of 0.6/0.4 (OD/OS). Visual acuity was reduced to the perception of light in the left eye whereas the right eye had no perception of light (Table 1).

Detailed medical examination of individual 12 in PKGL015 showed B/L buphthalmos, corneal edema, maximum IOP of 40/21 mmHg (OD/OS), and a C/D ratio of 1.0/ no view (NV; OD/OS). Visual acuity of this affected member was reduced to counting fingers and no perception of light in the right and left eyes, respectively. Medical records of individual 15 showed a maximum IOP of NV/36 mmHg (OD/OS), a C/D ratio of NV/0.9 (OD/OS) and visual acuity reduced to counting fingers. Clinical data of individual 17 showed aphakia, peripheral iridectomy, a C/D ratio of 1.0/NV, and visual acuity reduced to counting fingers only (Table 1).

All affected individuals of PKGL042 exhibited a severe PCG phenotype. Clinical data of affected individual 9 showed B/L buphthalmos and corneal opacity. The maximum IOP recorded was 36/NV mmHg (OD/OS), and the C/D ratio was 1.0/NV (OD/OS). Visual acuity was reduced to hand movement only. Medical records of individual 10 showed B/L buphthalmos, B/L corneal opacity, and an increased corneal diameter (>13 mm). The ophthalmic examination of affected individual 11 revealed a maximum IOP of 24/22 mmHg (OD/OS), a C/D ratio of 0.7/0.4 (OD/OS), and a corneal diameter of 13/14 mm (OD/0S). Additional clinical findings included B/L buphthalmos, corneal opacification, and B/L nystagmus (Table 1).

Initially, linkage analysis was performed on all families for previously reported genes and loci responsible for PCG, including the GLC3D locus on chromosome 14q24.2–24.3. All available members of the families (PKGL076, PKGL015, and PKGL042) were genotyped for microsatellite markers (D14S63, D14S258, D14S43, D14S1036, D14S61, D14S59, and D14S74) flanking the GLC3D locus. The haplotype analysis of chromosome 14 markers in all families revealed the linkage of the PCG phenotype to the GLC3D locus (Figure 1). Maximum two-point LOD scores of 2.86, 2.8 and 2.92, at recombination fraction θ=0, were obtained for the D14S1036, D14S59, and D14S59 markers, for PKGL076, PKGL015, and PKGL042, respectively (Table 2).

Subsequently, all 36 coding exons, exon–intron boundaries along with 5′ and 3′ untranslated regions (UTRs) of LTBP2 were sequenced in all available family members of PKGL076, PKGL015, and PKGL042. In PKGL076, a novel homozygous missense mutation (c.3028G>A) was identified in exon 19 of LTBP2 that results in the substitution of asparagine for aspartic acid at position 1010 (p.Asp1010Asn; Figure 2A,B). In PKGL015, bidirectional sequencing of the LTBP2 gene identified a novel single-base deletion at the coding nucleotide position: c.3427delC (p.Q1143Rfs*35; Figure 2C,D). In PKGL042, a novel guanine to adenine transition mutation (c.5270G>A) was identified in exon 35 which results in the substitution of tyrosine for cysteine at position 1757 (p.Cys1757Tyr; Figure 2E,F). All three mutations showed complete segregation with the PCG phenotype in the respective families and were absent in 200 ethnically matched control chromosomes. Moreover, both missense mutations (c.3028G>A and c.5270G>A) were absent from the Exome Variant Server (EVS), Genome Aggregation Database (gnomAD), dbSNP database, and 1000 Genomes database whereas c.3427delC has been reported in ExAC and gnomAD with a minor allele frequency (MAF) of 0.000008258 and 0.000003981, respectively.

Evolutionary conservation analysis showed high conservation of both residues (Asp1010 and Cys1757) among primates, placental mammals, and vertebrates (Figure 3). The PolyPhen-2, SIFT, LoFtool, and Condel analyses predicted the p.D1010N mutation to be deleterious to the functional and enzymatic activity of the LTBP2 protein with scores of 1.0000, 0.0000, 0.0599, and 0.9450, respectively. MutationTaster also predicted this mutation to be disease-causing affecting the protein features. In silico analysis predicted that the p.C1757Y mutation would be damaging to the protein function by providing maximum scores of 0.0000, 1.0000, 0.9450, 0.0599, and 194.00 for SIFT, PolyPhen-2, Condel, LoFtool, and MutationTaster, respectively. MutationTaster also provided the conservation of the wild-type nucleotide (g.111113G) based on multiple alignments of genome sequences of 46 different species. MutationTaster predicted g.111113G to be highly conserved with maximum PhastCons and phyloP scores of 1.000 and 5.924, respectively.

In the present study, we identified three novel pathogenic mutations in the LTBP2 gene in three consanguineous families with PCG (PKGL076, PKGL015, and PKGL042) from Punjab, Pakistan. The homozygous substitution (c.3028G>A; p. Asp1010Asn) identified in PKGL076 occurs within the splice donor site of intron 19, and most likely will perturb the normal splicing pattern of the LTBP2 protein. We used HSF, an online bioinformatics tool, to predict the effect of c.3028G>A on LTBP2 mRNA splicing. The HSF analysis generated consensus values of 74.68 and 45.73 for the wild-type (c.3028G) and mutant (c.3028A) nucleotides, respectively. The predicted consensus deviation value of −38.77% for c.3028G>A suggests the loss of the wild-type donor splice site. In parallel, HSF also detected the activation of an exonic cryptic splice acceptor site using the adenine nucleotide (c.3028G>A) in exon 19. The algorithm predicted consensus values of 37.26 and 66.20 for the wild-type (c.3028G) and the new cryptic splice site (c.3028A), respectively. The predicted consensus value deviation of +77.67% for the new exonic splice acceptor site suggests the loss of the wild-type splice donor site that would result in a frameshift and eventually, would lead to a premature stop codon in the mutant protein. Sanger sequencing of the LTBP2 gene identified a novel homozygous single-base deletion at c.3427delC in all affected members of PKGL015. This single nucleotide deletion in exon 23 substitutes the amino acid glutamine with arginine at codon 1143 (p.Gln1143Argfs*35), causing a frameshift mutation. This frameshift resulted in truncating the open reading frame of the LTBP2 protein by creating a premature stop codon, 35 amino acids downstream after the last original amino acid P1142. As a result of this frameshift mutation, a truncated protein of 1,176 amino acids was produced of which only the first 1,142 amino acids were similar to the wild-type LTBP2 protein. These frameshift mutations change the amino acid composition of the LTBP2 enzyme which, as a result, renders the functional inactivity of the protein by changing its primary, secondary, and tertiary structures.

We identified the second novel substitution (c.5270G>A) resulting in the substitution of tyrosine for cysteine at position 1757 (p.Cys1757Tyr) in epidermal growth factor (EGF)-like domain 19 of LTBP2. The high conservation of cysteine and the flanking amino acids indicates an important role in the structural and functional stability of the LTBP2 protein. Cysteine, a nonpolar hydrophobic amino acid, involves the formation of intrachain disulfide bonds. MutationTaster-based in silico analysis ranked p.Cys1757Tyr as disease-causing and predicted the potential loss of EGF-like domain 19 and EGF-like domain 20 and subsequent downstream disulfide bond formations in the mutant LTBP2 protein. In conclusion, the substitution of cysteine with tyrosine will result in the loss of intrachain disulfide bonds and ultimately, disrupts the proper folding and the normal functioning of the LTBP2 protein.

LTBP2 has 36 exons and encodes for an 1,821 amino acid protein [43,44], that belongs to a superfamily of extracellular matrix LTBP proteins and fibrillins comprised of EGF-like domains, transforming growth factor β (TGF-β) binding protein (TB)-like motifs [22,45-47]. LTBP2 protein is an extracellular matrix protein and has a presumed role in cell adhesion and elastin microfibril assembly [48-52]. The association of LTBP2 with microfibrils and its noticeable presence in elastic tissues has been reported [53].

The LTBP2 protein is expressed in the trabecular meshwork, and is important in the regulation of IOP and for the development of ciliary zonules and the anterior chamber, as well as the production of aqueous humor [54]. Furthermore, LTBP2 is highly expressed in tissues rich in elastic fibers, i.e., arteries, and lungs [55]. Mutations in LTBP2 were identified to cause PCG in families and patients from Pakistani, Gypsy, Iranian, and Indian populations [20,22,27-29].

PCG is a genetically heterogeneous disease with variable patterns and expressivity. The most common clinical features persistent with PCG are elevated IOP, buphthalmos, increased corneal diameter, corneal edema or haze, and reduced visual acuity. The clinical presentation of PCG cases harboring mutations in CYP1B1 or LTBP2 is similar with the exception of a few subtle phenotypic differences. For instance, in a family reported from Pakistani population, one of the patients had a persistent pupillary membrane and had a bilateral subluxated lens. The other patient in the same family had microspherophakia and iridogoniodysgenesis in addition to the cardinal symptoms of PCG, whereas the second family had typical symptoms [28]. Furthermore, in a recent study of 36 large consanguineous Pakistani families, several patients affected by PCG had classical or typical symptoms (reduced vision and corneal haze) in only one eye which is in direct contrast with the present study in which all the individuals were affected bilaterally in the first 3 years of life. Similarly, one affected member of the family had high IOP but no corneal haze or vision impairment, which is also in slight contrast to the present study in which all affected individuals have much reduced visual acuity along with corneal haze, corneal edema, or megalocornea [56].

In summary, we have identified three novel LTBP2 mutations, c.3028G>A (p. Asp1010Asn), c.3427delC (p.Gln1143Argfs*35), and c.5270G>A (p.Cys1757Tyr), in three Pakistani familial cases with PCG. Thus far, only nine mutations in LTBP2 have been reported in patients with PCG of Pakistani, Gypsy, Iranian, and Indian descent which suggests that these mutations are a relatively rare cause of PCG (Table 3). The present study results along with those of previous studies suggest that mutations in LTBP2 are a plausible cause of ocular anomalies that may lead to elevated IOP and eventually cause PCG.