Primary congenital glaucoma (PCG) refers to a glaucoma occurring within the first 3 years of life, due to isolated trabeculodysgenesis [1]. It is relatively common in Saudi Arabia (SA), with an estimated incidence of 1: 2500 live births, secondary to a high prevalence of consanguinity in Saudi families [2]. It is the second most common cause of blindness in schools of the blind in SA [3]. The congenital glaucoma registry at King Khaled Eye Specialist Hospital showed that PCG geographic distributions in SA is: south region (27.8%), western province (23.6%), central region (22.2%), eastern province (11.1%), and north province (9%) [4].

The link between PCG and gene abnormalities is the initial step in the establishment of the pathophysiology of the disease. Three previous major studies [5]-[7] were conducted in SA, to identify the abnormal genes associated with PCG. So far CYP1B1 was the only gene found to be associated with bilateral PCG in consanguineous Saudi Arabian families. CYP1B1 mutations were identified in 96% of cases of the study described by Bejjani et al. [6] and in 75.9% of cases of the study described by Abu-Amero et al. [7]. Unilateral PCG were studied by Khan et al. [8] but was not associated with CYP1B1 mutations. Expatriates were excluded from all previous gene studies [4]-[8].

This study was conducted to identify CYP1B1 mutations in Saudi PCG patients with various ethnic backgrounds, and to identify those patients who carry novel mutations. Patients were collected at King Abdulaziz University Hospital, one of the major hospitals in the Western province of Saudi Arabia and a main referral centre for congenital glaucoma in the province.

The populations of the Arabian Peninsula descend from a small number of founders. Their descendants established various tribes that dispersed across the kingdom. Therefore, the majority of native Saudi Arabians living today have common ancestors [5]. This is may be true in all parts of SA except the Western region, where the population consists of a different ethnic groups due to the presence of the two holy cities, Makkah and Madinah, and immigration of people from Arabic and Islamic countries to this region over the last 14 centuries. Jeddah is the sea port of the two holy cities, and thus also has a population with a high cultural and ethnical diversity. Non-native Saudi PCG patients were excluded from all previously published gene studies, and only conducted on native patients from various tribes and regions of SA [4]-[8]. To the best of our knowledge this is the only gene study in SA, which addresses the effect of ethnicity on gene anomalies in PCG.

The CYP1B1 gene was selected to screen for mutations in our PCG patients, since it was considered the main candidate gene for PCG in Saudi Arabia. CYP1B1 mutations were found in 79.4% of our patients, which is higher than that reported by Abu-Amero et al. [7] and lower than that reported by Bejjani et al. [6].

We detected CYP1B1 mutations in 91% and 54.5% of native and non-native Saudi Arabian PCG patients, respectively. This difference in rate of mutations could be secondary to the lower consanguinity rate in the non-native Saudi population (54.6%) compared to the native population (91.3%). The rate of CYP1B1 mutations in our non-native Saudi cases is low and comparable to populations with low consanguinity rates like India (44%) [10] and Brazil (50%) [11]. In addition, the high rate of CYP1B1 mutations in the native Saudi PCG cases may be explained by the population structure. Since the native Saudi population descended from a limited number of founders, it is likely that one of a small number of founder mutations have arisen that explain the majority of the cases among the native Saudi populations. Two of the pathogenic variants (p.Gly61Glu, p.Arg469Trp) identified in our cases were previously reported in the literature in PCG patients of various populations (ethnic groups), including native Saudi Arabians [5]-[7]. The Gly61Glu mutation is the most frequent mutation in the Saudi patients of our current study as well as in patients from Kuwait. Both SA and Kuwait share a common genetic background because of the neighborhood and frequent migration routes between the two populations [12]. We identified a missense mutation (p.Glu229Lys) in one of our non-native Saudi PCG patients. This patient's family is originally from Burma. This mutation has not been reported previously in native Saudi Arabian PCG patients [5]-[7], but it has been reported in Turkish [13], Indian [10], Iranian [14] patients, and in an index patient of family from Oman in heterozygous manner [15].

In a previous study of CYP1B1 analysis by Bejjani et al. [6] in PCG patients from SA a haplotype was constructed for the six SNPs. We also constructed haplotypes ie C/C/G/G/T/A using the same SNPs which includes one intronic c.-1-12C > T and five exonic SNPs i.e. c.142C > G; R48G, c.355G > T; A119S, c.1294C > G; V432L, c.1347T > C; D449D and c.1358A > G; N453S. In their study they had observed that this distinctive haplotype is associated with the five different mutations and we also saw a same haplotype C/G/G/T/A in case of the two mutations Gly61Glu and Arg469Trp in 27 patients. This same haplotype C/C/G/G/T/A was associated with at least seven separate mutations and appears to be common in all the individuals carrying the most frequent mutation, 4340delG in CYP1B1 in Brazilian PCG patients [11],[16]. However in the 7 patients which remained unsolved with CYP1B1 in the current study, 4 patients had this haplotype T/G/T/C/C/A while three individuals were heterozygous for all these five SNPs. Bejjani et al. [6] also found that the three families unsolved with CYP1B1 mutations has a heterozygous haplotype for these five SNPs. It seems likely, that individuals lacking haplotype C/C/G/G/T/A have another genetic loci involved in the pathogenesis of PCG. We are currently exploring this possibility.

In the literature, there has not been a consistent correlation between the severity of PCG and presence and types of CYP1B1 mutations [17]. Hollander et al. [18] found a correlation between the type of CYP1B1 mutations and the severity of glaucoma on the basis of anterior chamber angle histological changes and difficulty in controlling IOP. Panicker et al. [19] found that severe cases (using severity index) of PCG associated with ins376A (100%), E229K (80%), E368H (72%), G61E (66.7%), and P193L (62.5%). The severity of PCG in our patients was assessed on the basis of clinical assessment of IOP level, corneal diameter, and corneal clarity at the time of presentation. We did find severe cases of PCG associated with two mutations, G61E (50%), and R469W (30%), in ten of our patients.

This study confirms that CYP1B1 mutations are the most frequent cause of PCG in the Saudi Arabian population, with p.Gly61Glu being the major disease-associated allele. The p.Glu229Lys is a new mutation in Saudi Arabian PCG patients. Genetic studies of recessive diseases such as PCG is important in consanguineous populations, since it will increase awareness and allows genetic counseling to be offered to patients and their relatives. This will not only reduce the disease to be inherited to future generations, but will also reduce the disease burden in the community.

OB is the principal investigator of this research project. He wrote the proposal and obtained the grant from King Abdulaziz University. He enrolled patients in the study at King Abdulaziz university hospital. He analyzed the clinical data and wrote the manuscript. SM and AH carried the Gene studies at the Human Genetic lab at Radboud University Medical Centre. They analyzed and wrote the gene results and revised the manuscript. RK assisted in writing the proposal, analyzed the results, and revising the manuscript. MH enrolled patients in the study from King Fahd Armed Forces Hospital. All authors read and approved the final manuscript.