LTBP2 gene analysis in the GLC3C -linked family and 94 CYP1B1 -negative cases with primary congenital glaucoma

14

INTRODUCTION

Primary congenital glaucoma (PCG) is an isolated tra-beculodysgenesis ocular disorder with a broad clini-cal spectrum presented from birth to the age of 3 years or more. This condition represents between 1–5% of all glaucoma cases1 and is relatively rare in Western populations. The incidence of PCG mainly depends on ethnicity and prevalence of parental consanguinity in the individual geographical region; it ranges from 1 in 1,250 in the Gypsies2 to 1 in 30,200 in the Irish.3 Approximately, 10% of the familial forms of PCG are inherited in an autosomal recessive mode.2,4–9 Clinical

and genetic data suggest that PCG is a genetically het-erogeneous group of conditions.8,10–12

The exact nature of PCG is still unknown. However, abnormal differentiation of the trabecu-lar meshwork during the early stages of develop-ment and the presence of anterior chamber angle anomalies at birth are consistent findings in this condition.1,13–16 Reduction in the outflow facility and consequential increase of intraocular pressure (IOP) ultimately lead to buphthalmos,9 optic nerve damage, corneal opacities, cataracts and amblyopia, in the absence of effective treatment, may lead to eventual vision loss.17

Ophthalmic Genetics, 34(1-2), 14–20, 2013© 2013 Informa Healthcare USA, Inc.ISSN: 1381-6810 print/1744-5094 onlineDOI: 10.3109/13816810.2012.716486

Received 27 December 2011; revised 25 July 2012; accepted 25 July 2012Correspondence: Mansoor Sarfarazi, PhD, Molecular Ophthalmic Genetics Laboratory, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA. Tel: +1 860 679 3629 (Office). Fax: +1 860 679 7524. E-mail: [email protected]

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RESEARCH REpoRt

LTBP2 gene analysis in the GLC3C-linked family and 94 CYP1B1-negative cases with primary

congenital glaucomaRoshanak Sharafieh1,2, Anne H. Child2, Peng T. Khaw3, Brian Fleck4, and

Mansoor Sarfarazi1

1Molecular Ophthalmic Genetics Laboratory, University of Connecticut Health Center, Farmington, CT, USA, 2Department of Cardiac and Vascular Sciences, St George’s Hospital, University of London, UK, 3NIHR BRC

Moorfields Eye Hospital & UCL Institute of Ophthalmology, London, UK, and 4Department of Ophthalmology, Royal Infirmary of Edinburgh, Scotland, UK

ABSTRACT

Purpose: Primary congenital glaucoma (isolated trabeculodysgensis, PCG) generally presents between birth and 3 years of age. Recently, mutations in Latent Transforming Growth Factor (TGF)-beta Binding Protein 2 (LTBP2) have been reported in several families that were diagnosed with PCG, who actually had a more complex ocular phenotype with ectopia lentis and Marfanoid features. We screened this gene for mutations in the original Turkish GLC3C-linked PCG family and in a group of CYP1B1-negative British PCG cases and their matched normal control subjects.

Methods: The 36-coding exons of the LTBP2 gene were sequenced in 94 familial or sporadic CYP1B1-negative PCG cases and 96 matched normal control subjects.

Results: No disease-causing mutations were identified in the original GLC3C-linked family. Screening of LTBP2 in 94 PCG and 96 control subjects identified three novel synonymous variations (L429L, P680P, S1031S) in 12 PCG and seven control subjects. A novel heterozygous missense mutation (R538W) was also identified in 1 of 90 PCG cases that is unlikely to be disease-causative.

Conclusions: LTBP2 mutations were not found in the Turkish GLC3C-linked PCG family or in 94 British CYP1B1-negative PCG cases. Our data suggest that LTBP2 mutations are not a significant cause for isolated trabeculodysgenesis.

KEYWORDS: LTBP2, PCG, GLC3C, CYP1B1-Negative

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LTBP2 screening in PCG cases

R. Sharafieh et al.

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LTBP2 screening in PCG cases 15


Early genetic and segregation analysis of PCG families suggested an autosomal recessive mode of inheritance in certain populations. The presence of pseudo-dominance has also been reported in a few families.18 However, presentation of isolated cases and co-existence of PCG with a broad spectrum of other clinical phenotypes signifies other genetic contributions to this phenotype. It is generally agreed that co-existence of multiple affected subjects within a given family is evidence of autosomal recessive inheritance. Genetic studies of PCG families has so far identified 3 loci on chromosomes 2p22 (GLC3A)8, 1p36 (GLC3B)4 and 14q24.3-q31.1 (GLC3C).19,20 We originally reported the first series of truncating mutations in the Cytochrome P4501B1 (CYP1B1) gene as a major contributor to the etiology of familial PCG cases.21 This has subsequently been confirmed worldwide.22 Our study showed that approximately 80% of familial and 33% of sporadic PCG cases are caused by CYP1B1 gene mutations.23 However, depending on the ethnic and geographic distribution of PCG subjects, between 17%24,25 and 100%26,27 of cases are caused by mutations in this gene.21 As CYP1B1 mutations do not account for all PCG cases, it is important to identify other genes that contribute to the etiology of this phenotype.

Recently, null mutations in the Latent Transforming Growth Factor (TGF)-β Binding Protein 2 (LTBP2) gene on chromosome 14q24.3 were reported in a group of Pakistani, Gypsy and Iranian families who were diag-nosed with PCG.28,29 Although LTBP2 mutations were described as a cause for PCG in four Pakistani families, eight European Gypsies, and three Iranian families,28,29 in retrospect those families likely actually had a second-ary glaucoma with primary megalocornea.30–32 The first study identified mutations in a group of consanguine-ous Punjab Pakistani PCG families initially linked to the GLC3C region.28 Four LTBP2 mutations (p.Q111X; p.A138PfsX278; p.R299X; p.E415RfsX596) were reported in these families. Thereafter, two homozygous deletions (p.S472fsX3; p.Y1793fsX55), a synonymous altera-tion (p.L429L), and four polymorphisms (rs2304707; rs862031; rs862031; rs61505039) were also reported in consanguineous Iranian PCG families.29

Since the LTBP2 gene is located near the GLC3C locus (approximately 1.16-Mb above D14S61), we hypothesized that it might also be involved in the PCG Turkish family we mapped to this locus.19,20 Therefore, we screened this GLC3C-linked Turkish family for possible LTBP2 gene mutations. We also screened 94 familial and sporadic PCG subjects of British descent, who did not have muta-tions in CYP1B1 or any other glaucoma causing genes.

PATIENTS AND METHODS

Ascertainment of PCG Subjects

In addition to a large consanguineous Turkish fam-ily that was originally used to map the GLC3C locus

on the 14q24.3-q31.1 region,19,20 we used a group of British (from Moorfields Eye Hospital) and Scottish (Edinburgh Royal Infirmary) familial and isolated cases that were clinically examined and diagnosed by the two contributing ophthalmologists (PTK and BF). The clinical diagnosis of PCG was based on enlarge-ment of the anterior segment (buphthalmos), photo-phobia, excessive tearing, epiphora, opacification of the cornea, abnormal anterior chamber, rupture of Descemet’s membrane, progressive optic damage, blepharospasm and elevated intraocular pressure (IOP). We only included subjects with “primary” forms of congenital glaucoma and excluded all other cases that were part of a more complex clinical syndrome, including those resulting from different chromosomal abnormalities. For each PCG case, a complete family history was obtained and blood samples were drawn from the immediate nuclear pedigree, including both parents and all siblings of the affected subjects. We also ascertained a group of matched normal control subjects that were clinically examined and determined to be free of any glaucoma subtypes. Most of these were spouses married into the PCG families, with no history of any type of glaucoma.

Genome-Wide Scan and Mutation Screening of the GLC3C Family

We used a five-generation consanguineous Turkish family with 46 members, including 12 obligate gene carriers and four affected PCG subjects.21 We had not identified mutations in CYP1B1 in this family, and its gene locus did not map to the GLC3B locus on 1p36.4 Homozygosity mapping localized the gene in this fam-ily to 14q24.3-q31.1, a region of ~5.77-Mb that contains over 40 genes.19,20 This locus was named GLC3C and because of the proximity of the LTBP2 gene location, this family was screened using direct sequencing of this gene.

DNA extraction and sequencing were performed using standard methods.33 The 36 exons of LTBP2 were sequenced with primers designed to include the intron-exon boundaries. One member in each British PCG family and two members (one carrier and one affected) of the GLC3C-linked Turkish family were selected for sequencing.

RESULTS

Table 1 summarizes all the DNA variations that were observed in this gene. Screening of the LTBP2 gene in the GLC3C-linked Turkish family identified only one synonymous change in exon 6 (L429L) that was also observed in the normal control subjects. Six known DNA polymorphisms (rs2304707, rs4899522, rs34755459, rs862031, rs699371 and rs3815329) were also observed in

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16 R. Sharafieh et al.

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this family. Therefore, the role of LTBP2 gene mutations in this original GLC3C-linked is now excluded.

Direct sequencing of 94 familial and sporadic British PCG subjects identified a total of 18 DNA variations in the LTBP2 gene, 14 known SNPs and 4 novel alterations (Table 1). One of the novel changes observed in 4 of 92 (4.35%) affected subjects (3 British and 1 Turkish) was a heterozygous DNA variation in exon 6 (CTG>CTA). This synonymous change (L429L) was also identified in 5 out of 96 (5.21%) normal control subjects and, there-fore, was classified as a common polymorphism. The second novel variation (exon 7; CGG>TGG; R538W) was observed in 1 of 90 (1.11%) PCG probands and none of the 96 normal control subjects screened. When the co-segregation of this non-synonymous change (R538W)

was investigated in all the available family members with two consanguineous obligate-gene carrier parents (Figure 1), the same change was observed in the father and not in the mother, who had the wild-type (C/C) alleles. Also, of the five available normal siblings, four had the same R538W change, possibly suggesting that this could be a private polymorphism in this family and unlikely to be involved in the etiology of PCG. This is further substantiated by the fact that for this autoso-mal recessive condition, one would expect to observe a homozygous DNA alteration that is contributed by the two consanguineous obligate gene carrier parents to their affected offspring. The DNA from the second affected subject of this family was not available for testing. The next novel variation (exon 11; CCC>CCT;

TABLE 1 Observed coding variations in the LTBP2 gene.SNP # Exon no. DNA variation Amino acid change PCG subjects Normal subjectsrs3742793 2 C → G N/A: Intronic 53/95 Het* = 49.98%rs60337900 4 ACG → ACA T305T 1/77 1/96 & Het* = 50%rs2304707 4 CCA → CAA P319Q 8/77 8/96 & Het* = 4.9%Novel 6 CTG → CTA L429L 4/92 5/96Novel 7 CGG → TGG R538W 1/90 0/96rs4899522 9 A → G N/A: Intronic 29/95 Het* = 50%rs3742794 9 A → C N/A: Intronic 31/93 Het* = 34%rs862025 10 G → A N/A: Intronic 28/88 Het* = 31.8%Novel 11 CCC → CCT P680P 1/90 0/96rs34755459 13 C → A N/A: Intronic 6/61 Het* = 37.5%rs699374 14 ACT → ACC T802T 42/91 Het* = 41.3%rs862030 15 A → G N/A: Intronic 15/72 Het* = 40%rs862031 15 ACC → ACT T834T 18/56 Het* = 46.2%rs862032 15 A → T N/A: Intronic 5/75 Het* = 7.7%rs699371 16 G → A N/A: Intronic 23/78 Het* = 46.9%Novel 20 TCC → TCT S1031S 7/80 2/96rs3815329 26 T → C N/A: Intronic 26/68 Het* = 42.4%rs2286411 28 C → T N/A: Intronic 19/76 Het* = 28.7%Het* = Percentage of Heterozygosity from the HapMap Project.

FIGURE 1 Partial sequence of LTBP2 in Exon-7. A heterozygous alteration was identified at amino acid 538 (R538W). The wild-type codon CGG was observed as TGG, changing the Arginine to Tryptophan. As only one PCG subject and five normal family members showed the same non-synonymous change, it is unlikely that this is PCG-causing.

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P680P) was observed in 1 of 90 (1.11%) PCG and was absent in 96 normal control subjects screened. Since this is a synonymous change (P680P) observed as heterozy-gote and other family members of this affected subject were unavailable for screening, it was unlikely to be involved in the etiology of PCG and most probably is an unknown rare polymorphism. However, still fur-ther screening of additional PCGs and normal control subjects are needed to resolve the potential disease-causative nature of such DNA variation. The last novel heterozygous variation was identified in exon 20 of the LTBP2 gene. This was also a synonymous change (TCC>TCT; S1031S) that was observed in 7 of 80 (8.75%) unrelated affected subjects. This alteration was also present in 2 out of 96 (2.08%) normal control subjects thus, indicating that this is an unknown polymorphism rather than a PCG-causing mutation. Contrary to nor-mal expectation for an autosomal recessive condition, we did not observe any PCG subjects with a homozy-gous DNA alteration in the LTBP2 gene. Screening of these 94 PCG cases also identified another 10 intronic DNA variations in this gene (Table 1), and all of these have already been established as known SNPs by pub-lic databases and, therefore, were not investigated any further in our analysis. No other changes were noted within the LTBP2 gene in the PCG subjects screened.

DISCUSSION

Since LTBP2 maps in close proximity to the GLC3C locus that we originally identified in a large Turkish family,19,20 we screened this family as well as a representative group of British PCG cases for possible mutations in this gene. To the best of our knowledge, this is the first population study of familial and sporadic British PCG subjects aim-ing to investigate the contributions of LTBP2 mutations to PCG in a Western population. Our Turkish family had six known polymorphisms and one synonymous change (L429L). Of the seven alterations, four were in the introns and three were within the coding exons. The two subjects selected for screening, were shown to be heterozygous for rs2304707 (CCA>CAA; P319Q). This known polymorphism has a low heterozygosity (4.9%) in the European population and was also observed in 8/77 (10.39%) of our British PCG and 8/96 (8.33%) of normal control subjects. Therefore, it is unlikely that this SNP is associated with PCG. Next, the previously reported L429L synonymous change (CTG>CTA) in the Iranian PCG subjects29 was also found in our GLC3C-linked family, 4/92 (4.35%) British PCG and 5/96 (5.21%) control subjects. Therefore, L429L appears to be an unreported polymorphism. Likewise, SNP rs862031 (ACC>ACT; T834T) observed in the GLC3C-linked family and in British PCG subjects (18 C/T, 12 T/T and 26 C/C out of 56) is a known polymorphism in the European population (49.1%), previously enlisted

in the public databases and, therefore, is unlikely to be associated with the PCG phenotype.

A total of seven LTBP2 sequence alterations were observed in familial and sporadic British PCG subjects, three of which were known polymorphisms and four were not present in any public databases. One non-syn-onymous change (R538W) was of particular interest and was present in one of 90 PCG subjects in a heterozygous state (CGG>TGG), and was absent in 96 matched control individuals. The segregation of this change was further investigated in the family (Figure 1). The two parents of the PCG proband are first-cousins, and both would be obligate mutation carriers if this were a pathogenic mutation. However, only one (father) had the same heterozygous change, while the other (mother) was homozygous for the normal allele. Of the six normal siblings of the proband, five were heterozygous and one was homozygous for the normal allele. Unfortunately, the other affected sibling of the proband was not avail-able for testing. The R538W variant may be a private polymorphism in this family, and does not appear to be related to the PCG phenotype. We conclude that in our cohort of PCG subjects, mutations in the LTBP2 gene do not play a role and, therefore, other gene(s) must be involved in the etiology of the glaucoma. Although close to the GLC3C locus on chromosome 14q24.3-q31.1, LTBP2 is not the gene involved in this particular family with PCG. Our findings confirm those of another study of 36 US families (20 Caucasian, 14 African American, and 2 Hispanic American) for mutations in the LTBP2 gene, that did not find any mutations in affected PCG subjects,34 further confirming the limited role that this gene plays in Caucasian patients with PCG.

In 2008, two independent studies of consanguineous Pakistani PCG families defined an overlapping region with the previously identified GLC3C locus;19 the locus in two Pakistani families was flanked by markers D14S289 and D14S74335; three other families mapped between D14S258 and D14S983.36 Subsequently, the families in these two studies were reported to have mutations in LTBP228 with a total of four homozygous mutations in the Pakistani families and several Gypsy samples (p.Q111X, p.A138PfsX278, p.R299X and p.E415RfsX596). Interestingly, in addition to the PCG phenotype, affected subjects had several other notable clinical features. Members of one family (MEP47) showed clinical characteristics similar to Marfan syn-drome including joint hypermobility, tall stature and arachnodactyly. Other PCG families had members with high arched palate, osteopenia, ectopia lentis and an abnormal aortic valve; traits that are usually seen in Marfan syndrome.28

Soon after, two Iranian PCG families were also reported to have other novel changes in the LTBP2 gene.29 This study identified two homozygous deletions (p.S472fsX3 and p.Y1793fsX55), a synonymous change (L429L) and three known polymorphisms (rs862031,

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rs2304707 and rs61505039). In addition to congenital glaucoma, members of one family had ectopia lentis. No further information was provided on other clinical features in these PCG subjects.

Studies in the last few years have called into ques-tion whether or not LTPB2 gene mutations are a cause for simple PCG.30–32 A recent paper reported null muta-tions in LTBP2 in two families with an autosomal recessive ocular syndrome characterized by ectopia lentis with secondary glaucoma, myopia, megalocornea, microspherophakia and Marfanoid features in older children.30 One Moroccan family with three affected subjects had an alteration in exon 9 (p.Val600GlyfsX2), while a second Macedonian Gypsy family with one affected subject had a nonsense mutation in exon 4 (p.R299X). Characteristics of the affected members

included impaired vision, spherophakia, abnormally large cornea, ocular hypertension in older children, anterior dislocation of lens causing secondary glau-coma, tall stature, arachnodactyly, high arched palate, below average IQ and ectopia lentis. Coincidentally, the same p.R299X mutation previously described by Ali28 in eight of their European PCG Gypsies and a Pakistani family (PKGL005), also presented with a high arched palate and osteopenia.28 This mutation may possibly correspond to an ancient founder effect linking the above mentioned affected members to the same region30 and may not necessarily be involved in primary forms of congenital glaucoma.

Likewise, another study screened a South Indian-Karnataka family with microspherophakia, an autosomal recessive congenital eye disorder that is

TABLE 2 Summary of five published LTBP2 gene mutation studies.Patient origin Phenotype Mutations identified ReferencePakistan, Punjab province – Khokhar clan, city of Khushab

PCG; Marfanoid features Homozygous- (Ex1) c.142delG (p.A138PfsX278)

28

Pakistan, Punjab province – Jatt clan, city of Muzaffargarh

PCG; osteopenia; high arched palate Homozygous- (Ex4) c.895C>T (p.R299X)

Pakistan, Punjab province – Lohar clan, city of Mianwali

PCG; ectopia lentis Homozygous- (Ex6) c.1243–1256del (p.E415RfsX596)

Pakistan, Punjab province – Macchi clan, city of Gujranwala

PCG; osteopenia; high arched palate Homozygous- (Ex1) c.331C>T (p.Q111X)

European Gypsies PCG Homozygous- (Ex4) c.895C>T (p.R299X)

Iran, Azari PCG; ectopia lentis Homozygous- (Ex36) c.5376delC (p.Y1793fsX55)

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Iran, Varamin PCG Homozygous- (Ex7) c.1415delC (p.S472fsX3)

Iran, Varamin PCG Homozygous- (Ex6) c.1287G>A (p.L429L)

Moroccan Ocular syndrome w/megalocornea, spherophakia and secondary glaucoma; Marfanoid features; high arched palate; below average IQ; ectopia lentis

Homozygous- (Ex9) c.1796dupC (p.V600GlyfsX2)

30

Macedonian Gypsies Ocular syndrome w/megalocornea, spherophakia and secondary glaucoma; Marfanoid features; high arched palate; ectopia lentis

Homozygous- (Ex4) c.895C>T (p.R299X)

South Indian-Karnataka Microspherophakia; lens dislocation Homozygous- (Ex36) c.5446dupC (p.H1816PfsX28)

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South Indian-Karnataka Microspherophakia; lens dislocation Homozygous- (Ex21) c.3262G>A (p.G1088S)

Saudi Arabia Megalocornea; secondary glaucoma from spherophakia and/or ectopia lentis;Some also presented with complete crystalline lens dislocation, tall stature and high arched palate

Homozygous- (Ex4)c.1012delT (p.S338PfsX4)

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Homozygous- (Ex33) c.4855C>T (p.Q1619X)


Homozygous- (Ex29) c.4313G>A (p.C1438Y)

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characterized by small spherical lenses. This pheno-type is observed individually or in combination with other syndromic disorders such as Marfan syndrome; microspherophakia with hernia; glaucoma-lens ecto-pia-microspherophakia-stiffness-shortness syndrome; microspherophakia-metaphyseal dysplasia or Weill-Marchesani syndrome. A homozygous duplication (c.5446dupC) in exon 36 of LTBP2 was reported in this consanguineous family.37 This duplication elongated the LTBP2 protein by 21 amino acids and substituted the last six with another 27 new amino acids. Additionally, the authors reported a missense alteration (p.G1088S) in exon 21 that is unlikely to be pathogenic in their family, as another family unlinked to the same region also had this alteration.37

The latest study reported three novel homozygous LTBP2 mutations in three consanguineous Saudi Arabian families. Each family had a different mutation in the gene: exon 4 (p.S338PfsX4), exon 33 (p.Q1619X) and exon 29 (p.C1438Y). Patients in these families pre-sented with a prominent ocular syndrome distinguished by congenital megalocornea leading to childhood-onset secondary glaucoma resulting from spherophakia and/or ectopia lentis. In addition to these features, some patients also presented with complete crystal-line lens dislocation, tall stature and a high arched palate.32 Previously, three other consanguineous fami-lies had been observed to have this ocular syndrome (Macedonian, Moroccan and Turkish), two of which now have been identified as having LTBP2 mutations.30

As shown in Table 2, reported mutations in the LTBP2 gene are present in a variety of ocular conditions such as abnormal shape of the lens (increased spherical cur-vature), and reduced zonule tension leading to ectopia lentis. These abnormalities could partly be caused by abnormal fibrillin structure of the zonules. Lens disloca-tion can lead to pupillary block and secondary angle-closure glaucoma, which can be mistaken for primary congenital glaucoma in infants and young children. In order to fully understand the role of LTBP2 in PCG, one should revisit the diagnosis of the studied patients. Clinically, a possibility exists that recurrent pupillary block of the normal flow of aqueous humor along the pupil boundary and anterior capsule of the lens (pupil-lary block), ultimately leads to secondary glaucoma, which in turn is mistaken for PCG.30 Since publication of the original four Pakistani families, eight European Gypsies, and three Iranian families,28,29 no other authors have found LTBP2 mutations in their PCG subjects, including patients from Saudi Arabia.38

PCG patients from the Middle East are at high risk for CYP1B1 mutations and it is doubtful that LTBP2 mutations cause PCG. The overall impact of LTBP2 in the etiology of PCG remains to be determined, and it will be necessary to screen other PCG populations, as well as assess the biological mechanisms and other protein-protein interactions that are involved in the etiology of PCG and other associated syndromes.

ACKNOWLEDGMENTS

We particularly want to thank the patients and families who have generously volunteered their time and pro-vided samples for this study. We are also indebted to Mr Glen Brice and clinical colleagues for family ascertain-ment, clinical information and initial blood sampling of the individual subjects in this study.

Declaration of interest: This work was supported in part by The International Glaucoma Association (UK), The Glaucoma Foundation (New York), The US National Institutes of Health (National Eye Institute), Rosetrees Trust (UK), The Bluff Field Charitable Trust, St. George’s NHS Hospital Trust, St. George’s, University of London (UK), the NIHR Biomedical Research Center at Moorfields Eye Hospital and UCL Institute of Ophthalmology. The authors report no con-flicts of interest. The authors alone are responsible for the content and writing of the paper.

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LTBP2 gene analysis in the GLC3C -linked family and 94 CYP1B1 -negative cases with primary congenital glaucoma