A Laboratory Study on the Molecular Basis of Primary Congenital Glaucoma

Purpose: To detect pathogenic mutations in cytochrome P450 family1 subfamily B polypeptide1 (CYP1B1) gene in nineteen sporadic Primary congenital glaucoma (PCG) cases and to identify patients lacking CYP1B1 mutations. Methods: CYP1B1 exon 2 and the coding part of exon 3 of 15 participants were amplifi ed by Polymerase chain reaction and amplicons were sequenced by Sanger sequencing. Sequencing data was analyzed to identify the gene mutations or Single Nucleotide Polymorphisms SNPs. Results: Four previously reported PCG-associated CYP1B1 mutations (c.1159G>A; p.E387K, c.230T>C; p.L77P, c.1103G>A; p.R368H and c.1568G>A; p.R523K) were found in four patients out of the 15 fully ‘sequenced’ patients. Also, ten previously reported Single Nucleotide Polymorphisms and two novel noncoding variants were identifi ed. Conclusion: The relatively low percentage of PCG patients having CYP1B1 mutations (4/15=26.6%) demonstrates that other known and unknown genes may contribute to PCG pathogenesis. Lack of CYP1B1 gene mutations in some patients stresses the need to identify other responsible candidates. Research Article A Laboratory Study on the Molecular Basis of Primary Congenital Glaucoma Grand Chikezie Ihesiulor1, Forbes Manson2 and Udo Ahanna Ubani1* 1Department of Optometry, Abia State University, Uturu, Nigeria 2Division of Genomic Studies, University of Manchester, England Received: 30 May, 2018 Accepted: 15 June, 2018 Published: 18 June, 2018 *Corresponding author: Udo Ahanna Ubani, Dept of Optometry, Abia State University, Uturu, Nigeria, Tel: +238036770850; E-mail:


Introduction
Primary congenital glaucoma (PCG) is a classical form of infant buphthalmos and a predominant type of congenital glaucoma [1,2]. Although, buphthalmos has been reported from the time of Hippocrates (460-377 BC), it was associated with high intraocular pressure (IOP) and vision loss in children in the middle of the eighteenth century [1,3,4]. In PCG, isolated maldevelopment of ocular drainage structures increases resistance to aqueous outfl ow [3,5,6]. This leads to increased IOP, optic nerve atrophy and blindness if prompt and proper diagnosis and management is not provided [4,7,8].
Infants with PCG are affected differently. However, presentation of epiphora, blepharospasm, photophobia and swollen or cloudy cornea due to high IOP, is the norm [7].
Familial, like sporadic, PCG is an autosomal recessive disease [9]. It is highly prevalent in the consanguineous and inbred Gypsy subpopulation of Slovakia. This was reported to be due to genetic drift or 'founder effect' [8,10]. In non Gypsy populations, PCG may be inherited in a multifactorial fashion [11].
Although the genetic component of PCG is debatable, 4 chromosomal loci has been mapped including, GLAUCOMA Recently, PCG phenotype was associated with mutations in cytochrome P450 family 1subfamily B polypeptide 1 (CYP1B1) gene on the 2p21 locus, gene symbol GLC3A (OMIM #231300) and Latent transforming growth factor beta-binding protein 2 (LTBP2) gene (GLC3D; OMIM 602091) [8]. The genes associated with GLC3B and GLC3C are not yet known [12].
CYP1B1 gene analysis is the focus of this study. CYP1B1gene (Ensembl transcript ESNT00000260630) is a three-exon gene in which only exons 2 and part of 3 are translated [9]. It encodes a 543-amino-acid 'drug metabolizing' protein or enzyme involved in early anterior chamber angle development [13].
Mutations in CYP1B1 have also been implicated in other anterior segment dysgenesis (ASD) syndromes [14]. The molecular role of CYP1B1 gene in the pathophysiology of PCG is not fully understood [8]. This study identifi es unrelated PCG subjects that have or lack CYP1B1 mutations by screening the coding exons of the CYP1B1gene using direct sequencing procedures.
Patients having no disease-causing CYP1B1 alleles were noted for exome sequencing to fi nd out other genetic factors involved in PCG pathogenesis [9].

Parti c ipants
Informed consent was obtained from the parents or responsible guardians of the subjects. The probands and their immediate families were questioned on their past medical history and examined at the hospital. Reviewing the eye charts from their respective eye physicians helped confi rm their visual function. The inclusion criteria were intraocular pressure ≥21mmHg in the fi rst years of life, the presence of cornea oedema, scar, or Haab striae or an ocular history consistent with infantile glaucoma. The presence of enlarged globe in their teens or adulthood suggests a history of congenital glaucoma in the fi rst few years of life. Other clinical signs were examined using portable slit lamp and direct ophthalmoscope. (Table 1) The study did not report the individual visual acuity, visual fi eld and intraocular pressure of the patients.

DNA qu a ntifi cation and quality assessment
In order to determine the concentration and quality of extracted DNA (patient samples), the Nanodrop 8000 (Thermo Scientifi c) was used to obtain the absorbance of the DNA samples at 260nm; A 260 =1 was noted as the equivalent to 50ng/ml for double stranded DNA. The Nanodrop machine was 'blanked' initially by adding of nuclease-free water1μl (dH2O ) onto the tip at the base of the spectrophotometer. The concentration and 260/280 values were noted (not shown).
A 260/280 absorbance ratio of ≥ 1.8 was considered a better amount of purity for DNA samples. Samples with concentration of more than 100ng/μl were diluted properly with dH2O to make 100ng/μl concentration.
The DNA samples (in 100ng/ul concentrations) were spun down for 3-5 secs using Minispin at a speed of 2000rpm to prevent DNA from being shattered.

Designing primers
In order to design primers of the CYP1B1 gene (DNA) target, the following databases were used; ensembl; a massive database/genome browser, primer3plus and reverse complement.
Human CYP1B1 (ENST00000260630) gene sequence was obtained from ensembl. Oligonucleotide sequences were synthesized by Eurofi ns MWG Operon.
Primers were designed to include exons and a minimum of 50 base pairs (bp) of introns for desired sequence to be completely amplifi ed.

Polymerase chain reaction (PCR) optimization
The best conditions for PCR were determined using gradient PCR. Primers were hydrated with a given volume of dH 2 O (Table   2) [15,16]    SafeView dye is safer (less harmful) and stains DNA effectively to allow its visualization within the Agarose gel. After swirling gently and pouring the gel into the mold, a comb was placed in the second groove of the mold. After 20-30 minutes of gel setting, the combs were removed and the tray and gel were introduced into the electrophoresis tank containing suffi cient 1 x TAE buffer. 5μl appropriately sized DNA ladder (hyperladder1 or 100bp #3231S) was loaded with pipette into the fi rst well.
5μl of each amplicon was then loaded into the remaining wells carefully. Gels were electrophoresed at 100V voltage, 400Amps for 20 -60 mins.
After electrophoresis, the gel was visualized to show the size (length) of the amplicon. This was carried out by UVtransillumination (Fluorchem imager) and an integrated camera was used to capture the images ( Figure 1).

PCR amplifi c ation
In order to effi ciently amplify the desired target DNA, a

Purifi cation of sequencing reactions
In order the remove the unincorporated dye terminator, Agencourt CleanSEQ paramagnetic bead solution (Beckman Coulter) and Beckman Coulter Biomek NX robotics were used.
Multichannel pipette was used to introduce 5μl of CleanSEQ beads into skirted well (PCR tubes containing the sequencing product). The purifi cation procedure was performed according to manufacturer's guideline.

CYP1B1 mutational analysis
Exons 2 and 3 were (Polymerase chain reaction) PCramplifi ed and sequenced for each patient as described in the Materials and Methods. Sequence fi les were read with either the GeneScreen or Staden programs. One patient DNA (sample AK1) failed to give readable sequence (in both exons) despite multiple attempts. 15 out of 19 patients had both exons fully sequenced (Figure 1).
In tables 1,3 non-synonymous mutations were identifi ed in fi ve probands. In one patient only a single mutation was found meaning that the pick-up rate for CYP1B1 mutation being pathogenic in this panel of 15 'fully sequenced' patients was 4/15 (26.67 %). All 5 mutations had been previously reported. Four (75%) of these mutations were found in exon 3 while one (25%) was identifi ed in exon2. Exon 3 was fully sequenced in all 19 patients, while exon 2 was only fully sequenced in 15.
Out of the fi ve probands that had two mutations, three were homozygous for a CYP1B1 mutation and one was compound heterozygous. The mutant alleles segregate with the disease in an autosomal recessive manner of inheritance [9,18]. Mutation c.1159G>A, a nucleotide change at cDNA position 1159 of the CYP1B1 gene sequence is the most prevalent homozygous mutation found in two patients (

Single nucleotide polymorphisms (SNP) analyses
Sequencing of 19 unrelated probands with PCG detected 12 SNPs which included four intronic variants (two in intron 1 and two in intron 2), fi ve variants in exon 2, and 3 variants in exon 3 (Table 2) (Figure 2).    Sequencing analysis shows previously reported homozygous missense change c.1159G>A p.E387K in CYP1B1 exon 3. Also, results show three coding SNPs and 2 noncoding variants (Table 4). P.E387K mutation is a founder PCG mutation in the Gypsy population as previously described in patient JH5.

Variants found i n fi ve PCG patients
Patient BY17: c.1568G>A p.R523K Brief clinical details and family history: Patient BY17 is 9 years old that presented with congenital glaucoma, heart murmur delay and Rieger phenotype. This patient has terminal deletion of a gene on Chromosome 6 (result not shown).
Sequencing analysis showed a single heterozygous CYP1B1 mutation on exon 3. In addition, two coding and one noncoding variant were found in this patient (Figure 7).
Congenital glaucoma in this patient was not due to CYP1B1 gene mutation. As stated earlier, glaucoma-associated Axenfi eld Rieger syndrome is due to deletion of gene located in chromosome 6p25 (forkhead Box C1 gene). This is responsible for the glaucoma in this patient.

Discussion
CYP1B1 gene mutat ions are the major molecular cause of PCG [20]. PCG is a genetically heterogeneous phenotype [13]. In addition, one patient (BY17) had a single heterozygous mutation in CYP1B1 and a terminal deletion in forkhead transcription factor gene (FKHL 7). The deletion in the short arm of chromosome 6 (6p25) is responsible for Axenfeld-Rieger Syndrome (ARS) and glaucoma [21]. Patient (TC7) was born prematurely. And it is known that anterior segment anomalies can be present in premature babies [22,23].
The pick-up rate (26.67%) may be because sequencing was limited to CYP1B1 coding exons leaving the promoter or non-coding regions [8]. Other reasons could be that the PCG phenotype may be caused by mutation in other PCG-associated loci (such as GLC3B, GLC3C or GLC3D (LTBP2) [8]. Pathogenic mutation in the LTBP2 gene associated with PCG has been reported in Pakistani, European Gypsy and Iranian probands [24,25]. Moreover, it has been suggested that a pathogenic mutation in MYOC gene may cause PCG with a CYP1B1 mutation via a digenic mode [26]. However, this fi nding is not established.
Mutation in other unknown genes may be responsible for PCG in the CYP1B1 negative patients [8].
The rest fourteen patients were CYP1B1 negative. BY17 has ARS, so can be discounted. Although exons 2 in four probands (JH5, MF10, CS15 and JL21) was not fully sequenced, homozygous pathogenic mutations were found in two (JH5 and MF 10) (Table 2)      From our results, PCG showed allelic heterogeneity in the patients (JH5 and MF10) associated with homozygosity for 2 different CYP1B1 mutations and on patient (TC7) showing compound heterozygosity in two distinct CYP1B1 mutations. Allelic heterogeneity explains the molecular contribution for a uniform clinical manifestation of probands, and homozygous and compound heterozygous explains the autosomal recessive nature of PCG [15,27].
p.R368H (found in TC7 and HZ9) is a hypomorphic mutation located in the conserved core structure (CCS), J helix region of the CYP1B1 protein [16]. This causes a decline in CYP1B1 enzymatic activity (~ 20% of wild type protein) or decrease in protein stability [27]. In a study by Li et al. [20], p.R368H was mostly found in white Europeans [20].
Mutation p.E387K (found in JH5 and MF10) is most common among Slovakian Gypsies, less in caucasians (4.90%), present in Amish population and absent in Asians and middle easterners [18,20]. For effective genetic screening in PCG patients, it is necessary to fi nd out the common and founder mutations in a given population [21].
E387K is the founder mutation that accounts for 79.63% of CYP1B1 gene mutations [15]. The high rate of consanguinity (especially cousin to cousin marriages) and high coeffi cient of inbreeding in the Middle Eastern and Gypsy populations explains the high occurrence of the E387K mutation [20].
The amino acid position (Glu387) in which p.E387K mutation changes glutamic acid to lysine, is a highly conserved position in all documented species and P450 enzymes [28]. Also it's a core element located in helix K, a region suspected to be essential for proper folding and active haem binding of the CYP1B1 enzyme [19,29].
The mutation found in patient BY17 (c.1568G>A) was previously identifi ed in Israeli Bedouin kindred and it was stated that it obliterates the DdeI restriction site and disrupts the CYP enzyme active site found in the C-terminal region of the CYP1B1 enzyme [27]. Bar-yoseph et al. [27], reports this variant that changes arginine to lysine in amino acid position 523 (p.R523K), was not found in 100 healthy individuals [27].
In our study, fourteen patients had no disease-associated mutations in the fully sequenced regions of the CYP1B1 gene. This supports the report that some unidentifi ed molecular etiology is behind the PCG in some patients [27].
Residue change from leucine (L) to Proline (P) at position 77 (p.L77P), results from c.230T>C missense mutation. This previously reported mutation (found in patient TC7) occurs in conserved position. It has been reported to be associated with PCG in Saudi families [16].
Information from this study shows that early genetic testing is pertinent to determine the carrier status of individuals and their phenotype [20]. Late presentation of PCG is associated with profound visual impairment in children [21].
Having identifi ed CYP1B1 negative patients, future studies can be undertaken to determine other genetic causes of PCG by whole exome sequencing of these subjects. Furthermore, the absence of CYP1B1 mutations in some PCG patients supports that another unidentifi ed gene is mutated.
The residual level of CYP1B1 activity is modulated by the presence of modifi er genes [9].

Conclusions
The number of CYP1B1 negative (11/15) probands was higher than the CYP1B1 positive (4/15) patients. Some mutations may have been missed due to the genetic screening strategy applied. This is the fi rst drawback of this study. Screening was not extended up to the regulatory sequences in the upstream or downstream regions in the introns although bit of introns were sequenced due to primer design [9].
The noncoding regions (including exon 1) may have the disease-related variants whereas this study screened only protein coding exons 2 and 3 of the gene [8]. For accurate genetic diagnosis of PCG, all three exons need to be sequenced [9].
Secondly, the small sample size (19) prevents conclusions to be confi dently made; confi rmation of result is therefore needed with larger number of subjects [20].
In conclusion, the analysis of CYP1B1 genotypes of 19 unrelated patients is reported in this study. A relatively low proportion of our subjects (~ 27%) tested positive for PCGrelated CYP1B1 mutations demonstrate the need to identify other PCG-causing genes.
Exome sequencing of CYP1B1 negative patients to detect new genes mutated in PCG.
Co-segregation analysis of other unaffected and affected family members may be carried in the future.