Purpose
To investigate the roles of CYP1B1 and MYOC mutations and characterize the phenotype of primary congenital glaucoma in Israeli patients from 3 different ethnic backgrounds.
Design
Interventional case series.
Methods
This institutional study included 34 Israeli primary congenital glaucoma patients (26 families) comprising 9 Jews (9 families), 17 non-Bedouin Muslim Arabs (10 families), and 8 Druze (7 families). The patients and their relatives (n = 99) were screened for CYP1B1 and MYOC mutations.
Results
Mutations in the CYP1B1 gene were detected in 12 of 26 families (46%) with primary congenital glaucoma (5 Muslim Arab, 5 Druze, and 2 Jewish). The Jewish families had compound heterozygous mutations and digenic mutations (ie, an Ashkenazi family had mutations in the CYP1B1 gene [Arg368His, R48G, A119S, and L432V haplotypes] and an Ashkenazi-Sephardic family had a mutation on the CYP1B1 gene [1908delA, Sephardic] with a second missense mutation on the MYOC gene [R76K, Ashkenazi]). The Muslim Arabs and Druze tended to have a more severe phenotype than that of the Jews.
Conclusion
The phenotype and spectrum of the CYP1B1 and MYOC mutation roles in the clinical characteristics of primary congenital glaucoma varied according to ethnicity. The rarity of mutations in the CYP1B1 gene among Ashkenazi primary congenital glaucoma patients indicates that a different locus may be involved in the phenotype.
Primary congenital glaucoma is usually present in the neonatal or infantile period and is accompanied by corneal opacity and edema, buphthalmos, increased intraocular pressure, and optic nerve cupping, occasionally followed by severe visual impairment. The incidence of the disease varies significantly in different geographic regions and is more frequently found in certain ethnic groups, especially where consanguinity is prevalent. The incidence in Western countries has been reported to range from 1 in 5000 to 1 in 22 000 births, with an average of 1 in 10 000. In populations where consanguinity is prevalent, reported incidences are higher; for example, among Slovakian Gypsies, Saudi Arabians, and Indians, incidences are 1 in 1250, 1 in 2500, and 1 in 3300 births, respectively. Primary congenital glaucoma is believed to be an autosomal-recessive transmitted disease with incomplete penetrance. Three different loci have been mapped for it: GLC3A on chromosome 2p21, GLC3B on chromosome 1p36.2, and GLC3C on chromosome 14q24.3. The major gene that has now been identified as being associated with primary congenital glaucoma is the CYP1B1 gene at locus GLC3A, which encodes a member of the cytochrome P450. The frequency of mutations in the CYP1B1 gene in primary congenital glaucoma patients varies in different geographic locations and among ethnic groups: 17% in Chinese, 18% in Germans, 20% in Japanese, ∼30% each in Indonesians, Indians, and Spanish, ∼50% each in Brazilians and French, 68% in Iranians, 90% in Saudi Arabians, and 100% in Slovakian Gypsies. Mutations in myocilin ( MYOC ) and FOXC1 24 genes have also been associated with primary congenital glaucoma.
Determining the presence of CYP1B1 mutations in primary congenital glaucoma patients will improve our ability to counsel parents regarding the cause, the pattern of inheritance, and the risk of its occurrence. Apparently healthy family members with the CYP1B1 mutation are also at risk for developing glaucoma, as recently reported by Suri and associates, and should undergo routine ophthalmic examination.
The Israeli population includes a Jewish majority (76%) and several non-Jewish communities, of which 16.5% are Muslim Arabs and 2% are Druze (a relatively small Middle Eastern monotheistic religious community in Israel living in villages on Mount Carmel and at the foot of Mount Hermon). The Israeli National Genetic Database provides knowledge on genetic disorders in the Israeli population and their distribution among the various Israeli ethnic groups. The only existing information that is relevant for primary congenital glaucoma in this database is that CYP1B1 mutations account for the majority of the cases in the Israeli Bedouin population. The Bedouins compose a fraction of the Muslim Arab population, of which they are considered a distinct group. There are no available data on the role of CYP1B1 mutations in primary congenital glaucoma among Israeli Jews, non-Bedouin Muslim Arabs (“Muslim Arabs”), and Druze. The aim of the present study was to characterize the phenotype and determine the role of CYP1B1 and MYOC mutations in primary congenital glaucoma in these 3 Israeli populations in order to provide this information.
Methods
This was an interventional case series study on 34 primary congenital glaucoma patients who had been referred to the Carmel Medical Center in Haifa, Israel. The patients belonged to 3 different ethnic groups: Jew (n = 9), Muslim Arab (n = 17), and Druze (n = 8). All the study patients had clinical manifestations of primary congenital glaucoma at the time of diagnosis, including at least 2 of the following clinical features: increased corneal diameter (>10 mm at birth), corneal edema, Haab’s striae, and optic disc changes. Ophthalmic examination included slit-lamp biomicroscopy, measurement of intraocular pressure, and ophthalmoscopy. The clinical diagnosis was confirmed by examination under anesthesia. Patients with other ocular or systemic diseases associated with childhood glaucoma, such as aniridia, anterior segment dysgenesis, congenital hereditary endothelial dystrophy, Peters anomaly, and Sturge Weber syndrome, were excluded.
Blood samples were taken from affected subjects and their first-degree relatives, yielding a total of 99 samples. The CYP1B1 gene coding sequences were screened in all individuals. When no mutations or only 1 heterozygous mutation was found in the CYP1B1 gene, we screened the coding region of the MYOC gene since it has been reportedly implicated in the pathogenesis of some cases of primary congenital glaucoma by digenic inheritance of the CYP1B1 gene. Since the FOXC1 gene has been suggested to be 1 of the new candidate genes for primary congenital glaucoma, the coding sequences of this gene were screened in patients from nonconsanguineous families who did not have any mutation in their CYP1B1 and MYOC gene and in patients with “milder” forms of congenital glaucoma.
For DNA extraction and polymerase chain reaction (PCR) amplification, DNA was extracted from peripheral blood leukocytes according to standard protocols. Amplifications of all coding regions of the CYP1B1 gene and intron/exon boundaries were obtained using sense and antisense oligonucleotides, designed and numbered on the basis of the known sequence of the CYP1B1 gene locus (GenBank accession no. NM_000104 ). PCR reactions were carried out in a total volume of 50 μL containing 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 50 mM KCl, 0.2 mM dNTPs, and 30 nM primers, initially at 94°C for 5 minutes, followed by 35 cycles of 94°C for 0.5 minutes, an annealing temperature for 1 minute, and 72°C for 1 minute. The reactions were completed with a final extension step at 72°C for 7 minutes. The primers, the annealing temperatures, and the sizes of the PCR products for each of the CYP1B1 exons are available upon request.
The samples underwent denaturing high-performance liquid chromatography (DHPLC) mutation screening. Scanning for DNA mutations and variants using DHPLC involves subjecting PCR products to chromatography using an ion-pair reversed-phase cartridge. PCR products are denatured and allowed to re-anneal. Under conditions of partial denaturation with a linear acetonitrile gradient, heteroduplexes from PCR samples with an internal sequence variation display a reduced column retention time relative to their homoduplex counterparts. The elution profile for heterozygous samples is typically quite distinct from that of homozygous sequences, thus making the identification of heterozygous mutations relatively straightforward. An analysis for mutations on the X chromosome in males or for homozygous autosomal mutations requires mixing the test sample with the DNA of a known sequence.
For the purpose of establishing DHPLC conditions, a mutation analysis was performed using the WAVE apparatus from Transgenomic, Inc (Omaha, Nebraska, USA). The PCR products were denatured at 95°C for 5 minutes and cooled to 65°C in a decreasing temperature gradient of 1°C/minute. The samples were kept at 4°C until 5 μL was applied to a preheated C18-reversed phase column based on nonporous (polystyrene-divinyl-benzene) particles (DNA-Sep Cartridge, cat. no. 450181; all DHPLC catalog numbers are from Transgenomic, Inc). DNA was eluted within a linear acetonitrile gradient consisting of buffer A (0.1 M triethylammonium acetate [TEAA; cat. no. SP5890])/buffer B (0.1 M TEAA, 25% acetonitrile [cat. no. 700001]). The temperature at which heteroduplex detection occurred was deduced with the Transgenomic software (Wavemaker 4.2) and the Stanford DHPLC melting program ( http://insertion.stanford.edu/meltdoc.html ), which analyzes the melting profile of each specific DNA fragment.
To identify homozygous mutations, 10 μL PCR product of wild-type DNA and 10 μL PCR product of sample DNA were mixed 1:1 and denatured at 95°C. This enabled detection of homozygous mutations by the formation of a heteroduplex. DNA sequencing was performed on the PCR products after DHPLC analysis, as previously described. Fragments showing an abnormal DHPLC pattern were investigated for the identification of sequence variants by automated sequence analysis with the direct cycle-sequence analysis, using an ABI PRISM 3100 Genetic Analyzer Sequencer (PE Biosystems, Foster City, California, USA). This was performed according to the manufacturer’s protocol, using a reamplified PCR product of the abnormal fragment (forward and reverse).
The clinical features at the onset of disease were ascertained in all patients by examining the clinical records of those who were treated by us as well as the patients who were treated elsewhere. First, we compared the clinical characteristics among patients from the Muslim Arab, Druze, and Jewish origins. We then separated the patients into groups according to whether they had a CYP1B1 or MYOC mutation (“mutation group”) or did not (“no mutation group”). The clinical phenotype was compared among the 2 groups.
Patients were also compared for consanguinity, sex, age at onset of disease, appearance of the cornea (corneal edema, Haab’s striae) at disease onset, and the number of eyes needing more than 1 surgical procedure. For the purposes of this analysis, the onset of disease was defined as the time of the initial diagnosis. Onset within the first month of life was considered as onset at birth. Degrees of severity were assigned on the basis of the corneal presentation and response to therapy, reflecting the number of eyes needing more than 1 operation. Eyes that were in phthisis upon arrival of the patient to our department were counted as having had more than 1 surgical procedure. All parents and siblings that participated in the study were examined for evidence of mild forms of anterior segment dysgenesis and late-onset glaucoma.
Mutation analyses were performed with the EpiInfo 2000 software ( http://www.cdc.gov/epiinfo ). The odds ratio (OR) and confidence interval (CI) were calculated as an estimation of risk among mutation carriers. The χ 2 test was used to determine the statistical significance of the different genetic variation frequencies between patients and healthy subjects (controls). Analyses of the clinical features were performed with the SPSS Statistical Package, version 15. The chi-square test was used to determine the statistical significance among the different frequencies of the clinical signs in Muslim Arab, Druze, and Jewish primary congenital glaucoma patients and in patients with and without mutations. The Fisher exact test was used when the sample size was small. The Kruskal-Wallis test was employed for comparing the age at onset between ethnic groups. All P values were 2-sided and significance was set at P < .05.
Results
The clinical phenotype of the study patients was identified by gathering clinical information on 34 primary congenital glaucoma patients (26 families) ( Tables 1 and 2 ). The Muslim Arab group included 17 patients of 8 unrelated families and 2 related families, of whom 5 patients (29%) had relatives (siblings) with primary congenital glaucoma. The Druze group included 8 patients of 7 unrelated families, and the Jewish group included 9 patients of 9 unrelated families. There was no difference in the male-to-female ratio or the frequency of bilateral cases between the 3 ethnic groups. There was, however, a significant difference among them in the frequency of consanguinity: 65% (11/17) of the Muslim Arabs and 75% (6/8) of the Druze were offspring of consanguineous marriages, while consanguinity was not reported in any of the Jewish patients. There was a significant group difference in the mean age at the onset of disease (1.8 months in the Muslim Arab group, at birth in the Druze group, and 6.1 months in the Jewish group). The disease was diagnosed at birth in 82% (14/17) of the Muslim Arab patients, 100% (8/8) of the Druze patients, and 11% of the Jewish patients. Severe corneal edema and buphthalmos was found in at least 1 eye when the patient was first examined in 41% (7/17) of the Muslim Arab patients, 50% (4/8) of the Druze patients, and only 11% (1/9) of the Jewish patients ( P = nonsignificant). Similarly, there was no significant group difference with respect to the number of eyes that needed more than 1 operation. There was no evidence of mild forms of anterior segment dysgenesis and later-onset glaucoma in parents or older siblings of the patients with no mutations.
| Pedigree ID | Patient Serial Number | Ethnicity | Consanguinity | Gender | Age at Diagnosis | Affected Eye | Corneal Characteristics at Diagnosis/Affected Eye | Mutations Identified |
|---|---|---|---|---|---|---|---|---|
| 1 | PCG-1 | Muslim Arab | Yes | F | Newborn | OU |
| Arg469Trp |
| 1 | PCG-2 | Muslim Arab | Yes | M | Newborn | OU |
| Arg469Trp |
| 2 | PCG-3 | Muslim Arab | Yes | M | Newborn | OU | Buphthalmos, severe edema OU |
|
| 2 | PCG-4 | Muslim Arab | Yes | F | Newborn | OU | Buphthalmos, severe edema OU |
|
| 3 | PCG-5 | Muslim Arab | No | M | 2 mos. | OU | Moderate edema OU | – |
| 3 | PCG-6 | Muslim Arab | No | M | Newborn | OU | Moderate edema OU | – |
| 4 | PCG-7 | Muslim Arab | Yes | M | Newborn | OU | Buphthalmos, severe edema, scarring OU | – |
| 5 | PCG-8 | Muslim Arab | No | F | 18 mos. | OD | Moderate edema OD |
|
| 5 | PCG-9 | Muslim Arab | No | F | Newborn | OU | Moderate edema OU |
|
| 6 | PCG-10 | Muslim Arab | No | F | Newborn | OU | Buphthalmos, severe edema OU |
|
| 7 | PCG-11 | Muslim Arab | Yes | F | Newborn | OU | Buphthalmos, severe edema, scar OU | Arg469Trp |
| 7 | PCG-12 | Muslim Arab | Yes | M | Newborn | OU | Moderate edema OU | Arg469Trp |
| 7 | PCG-13 | Muslim Arab | Yes | F | Newborn | OU | Mild edema OU | Arg469Trp |
| 8 | PCG-14 | Muslim Arab | Yes | F | Newborn | OU | Moderate edema OU | – |
| 8 | PCG-15 | Muslim Arab | Yes | F | Newborn | OU | Moderate edema OU | – |
| 9 | PCG-16 | Muslim Arab | No | F | 10 mos. | OU | Mild edema OU | – |
| 10 | PCG-17 | Muslim Arab | Yes | M | Newborn | OU | Moderate edema OU | Arg469Trp |
| 11 | PCG-18 | Druze | No | M | Newborn | OU | Buphthalmos, severe edema OU |
|
| 12 | PCG-19 | Druze | No | F | Newborn | OD | Moderate edema OD | Arg469Trp |
| 13 | PCG-20 | Druze | Yes | M | Newborn | OU | Moderate edema OU | Arg469Trp |
| 14 | PCG-21 | Druze | Yes | F | Newborn | OU | Buphthalmos, severe edema, scarring OU | Met1Thr |
| 14 | PCG-22 | Druze | Yes | F | Newborn | OS | Moderate edema OS |
|
| 15 | PCG-23 | Druze | Yes | F | Newborn | OU | Buphthalmos, severe edema, scarring OU | Arg469Trp |
| 16 | PCG-24 | Druze | Yes | M | Newborn | OU | Buphthalmos, severe edema, scarring OU | – |
| 17 | PCG-25 | Druze | Yes | F | Newborn | OD | Moderate edema OD | – |
| 18 | PCG-26 | Jewish | No | F | 3 mos. | OS | Mild edema OS | – |
| 19 | PCG-27 | Jewish | No | M | 2 mos. | OU | Moderate edema OU | – |
| 20 | PCG-28 | Jewish | No | M | 3 mos. | OD | Mild edema OD | – |
| 21 | PCG-29 | Jewish | No | F | Newborn | OU |
|
|
| 22 | PCG-30 | Jewish | No | M | 3 mos. | OU | Moderate edema OU | – |
| 23 | PCG-31 | Jewish | No | F | 6 mos. | OU | Moderate edema OU | – |
| 24 | PCG-32 | Jewish | No | F | 30 mos. | OU | Moderate edema OS | – |
| 25 | PCG-33 | Jewish | No | M | 6 mos. | OU | Haab’s striae OU |
|
| 26 | PCG-34 | Jewish | No | M | 2 mos. | OU | Haab’s striae OU | – |
| Muslim Arabs | Druze | Jews | P Value | |
|---|---|---|---|---|
| Patients, n | 17 | 8 | 9 | |
| Male/female | 7/10 | 3/5 | 5/4 | .74 |
| Consanguinity, n (%) | 11 (65) | 6 (75) | – | .002 |
| Bilateral disease, n (%) | 16 (94) | 5 (62) | 7 (78) | .087 |
| Mean age at onset, months (range) | 1.8 (0–18) | 0 | 6.1 (0–30) | <.0001 |
| Diagnosed at birth, n (%) | 14 (82) | 8 (100) | 1 (11) | <.0001 |
| Patients with severe corneal edema and buphthalmus in at least 1 eye, n (%) | 7 (41) | 4 (50) | 1 (11) | .03 a |
| Eyes needing >1 operation/total of eyes with PCG, n/n (%) | 18/33 (54) | 9/13 (69) | 8/16 (50) | .5 |
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