Gene mapping in an anophthalmic pedigree of a consanguineous Pakistani family opened new horizons for research

Clinical anophthalmia is a rare genetic disease of the eye and phenotype refers to the absence of ocular tissue in the orbit of eye [1-3]. Congenital clinical anophthalmia is commonly bilateral [4] but it may also be unilateral [5]. Often anophthalmia is part of a syndrome and is accompanied by other brain anomalies [6,7]. Clinically, in the absence of apparent ocular tissue, congenital anophthalmia and extreme microphthalmia (A/M), i.e., the presence of a small eye, are considered the same, because computerized tomography (CT) scan investigations indicated residual neuroectoderm in the orbit of the eye in some cases [8].

Clinical anophthalmia exhibits different patterns of genetic inheritance, i.e. autosomal dominant, autosomal recessive and X-linked recessive [4,9]. Studies on autosomal recessive mode of inheritance of anophthalmia have rarely been reported [1,10]. Similarly, previous research studies have also provided rare evidence about consanguinity association with congenital anophthalmia [8,11].

Anophthalmia/microphthalmia cause a considerable percentage of congenital visual impairments in children [12]. Epidemiological data has reported that the prevalence of congenital anophthalmia is three in 100,000. However, other evidence estimated the combined prevalence of congenital anophthalmia and microphthalmia up to three in 100,000 [13,14]. In developed countries, a prevalence of 0.2-0.4 per 10,000 births has been reported [15-17]. Epidemiological studies have also investigated some risk factors for anophthalmia including late maternal age, multiple births [15,16], low birth weight and premature birth complications [18]. Epidemiological studies have reported that both genetic and environmental factors cause anophthalmia and microphthalmia, however, environmental factors account for a lesser number of cases [4].

Genetic linkage analysis studies have identified the same loci and mutations in the same genes for both clinical anophthalmia and extreme microphthalmia [4]. It was considered that congenital A/M showed genetic heterogeneity due to linkage of a large number of loci with them. Similarly, mutations in several well-defined human genes such as CHX10, RAX, SOX2 and OTX2, are also associated with heritable forms of clinical anophthalmia and severe microphthalmia [9].

In the Khyber Pakhtunkhwa region of Pakistan, different Pashtoon tribes are prominent ethnic groups and due to cultural impacts, they have very strong reservations regarding marriages outside their tribal boundaries, as they believe that dilution of tribal blood may result in losing their specific tribal characteristics. Moreover, they practice consanguineous marriages to strengthen family ties and to maintain the family structure and property [19]. The causes of congenital inherited diseases in Khyber Pakhtunkhwa are maternal illiteracy, mother’s age to be less than 20 years at the birth of first child, birth interval of less than 18 months [20], and the influencing culture factor to have more children, particularly sons, until menopause. The lack of public awareness toward prenatal diagnosis or prevention of inherited disease and health risks associated with consanguineous unions, is limited. Many people do not agree with medical explanations of a genetic mode of disease inheritance, even in case where there is an affected child. Because of this, inherited diseases are frequently observed in Khyber Pakhtunkhwa that follow Mendelian patterns of inheritance, and the molecular bases are not known. In present study, a consanguineous Pakistani family of the Pashtoon ethnic group with isolated clinical anophthalmia in Khyber Pakhtunkhwa was investigated.

In an ascertained consanguineous family with isolated clinical anophthalmia, the phenotypically normal parents with pedigree ID 2MOP001and 2MOP002, produced two affected daughters with pedigree ID 2MOP003 and 2MOP 006 (Figure 3). The proband, (2MOP003), was the first daughter identified with bilateral clinical anophthalmia, and she helped in tracing the disease in this family. Bilateral clinical anophthalmia was present at birth in both the affected daughters and the ages of these affected daughters were between 4-13 years.

In this study, no evidence of linkage was observed with any of the STR markers for the 14q32, 14q24.3, 18q 21.3 and 14q21-22 loci and were therefore excluded. However, in view of the obtained results, this family with clinical anophthalmia was mapped to a locus on chromosome 3q26.3-q27, where the SOX2 gene resides, as affected daughters showed homozygosity for this locus within a 3 cM (centimorgan) in this region for STR markers D3S 2427, D3S1262, D1S2436 and D3S1580 (Figure 4). In the pedigree under study, the parents of the affected daughters were first cousins, and both carried the same disease chromosome in a heterozygous state (Figure 3). However, the mutations were not identified in the single exonic sequence and regulatory element of the SOX2 gene by comprehensive mutational analysis of both normal and affected individuals. Only two individuals were found to be affected, thus, the Lod score genes could not be calculated to examine the combined effects of the genes.

The term clinical anophthalmia was first used by Duke-Elder [8], and is a rare disease. The reported average prevalence of congenital anophthalmia is three in 100,000 [14]. Clinical anophthalmia is the absence of the eye and diagnosed without histological examination [22]. The most common phenotype in affected individuals is bilateral anophthalmia [4], and unilateral anophthalmia may rarely be seen [5].

In the present study, we reported a consanguineous family with two affected daughters of isolated clinical anophthalmia from the Kohat region of Khyber Pakhtunkhwa, Pakistan. Affected daughters do not have any congenital malformations except for bilateral clinical anophthalmia. In addition, the family history showed that there was no other member with anophthalmia. In the pedigree under study, the affected daughters have unaffected parents, who are first cousins, thus inheritance is undoubtedly autosomal recessive. Moreover, members of this family practiced consanguineous marriages to follow the family tradition of marriages between cousins. Consanguinity in a family as a risk factor and consequently autosomal recessive mode of inheritance for clinical anophhalmia, has rarely been reported [1,8,10,11]. However, X-linked inher-itance has been described for clinical anophthalmia [4,23]. Epidemiological studies have also reported other risk factors including late maternal age, multiple births, low birth weight, premature birth complications, mechanical abortion and severe vitamin A deficiency [4,15,16,18]. These risk factors were not identified in this family as a cause of clinical anophthalmia.

In the present study, linkage analysis of family was performed with STR markers corresponding to the candidate genes involved in clinical anophthalmia phenotypes. This Pakistani family was linked to a locus at chromosome 3q26.3-q27, which carries the SOX2 gene. The critical disease region was flanked by STR markers D3S1565 and D3S1311 in the affected daughters; therefore, it is probable that the disease gene lies between these two markers within a region of approximately 23 cM on chromosome 3. However, the affected daughters showed homozygosity in the disease region of approximately 3 cM for markers D3S 1262, D1S2436 and D3S1580. The linkage data presented in this study suggested that a gene for clinical anophthalmia was present within the region of homozygosity at chromosome 3. However, mutation screening did not reveal any mutation in the exonic sequence and regulatory element of the SOX2 gene in the parents and offspring of this family. This indicates that another gene might possibly be present in the mapped region for disease phenotype and needs to be identified and screened to identify the disease-associated mutation in this family. The Lod score calculation in linkage analysis is very successful in mapping Mendelian disease genes or to examine combined effects of genes. However, the Lod score could not be calculated, as there were only two affected daughters, and that is the limitation of our study.

The severity of clinical anophthalmia is variable due to mutations in various human genes that are associated with anophthalmia [4,5]. Among these, the SOX2 has been reported as a major causative gene for clinical anophthalmia [4]. By genetic analysis, the single-exon SOX2 gene was identified in an intron of a noncoding SOX2OT (SOX2 overlapping transcript) gene [24]. By using the fluorescent in situ hybridization (FISH) approach, the SOX2 gene was mapped to chromosome 3q26.3-q27 [25]. The SOX2 gene is universally expressed in neural stem and neural precursor cells throughout the central nervous system including the neural retina [26-28], and mutations in this gene are common causes of retinal and ocular malformations in humans.

By sequence analysis of the coding region of the SOX2 gene, a heterozygous loss-of-function mutation was identified in individuals with unilateral and bilateral anophthalmia in various research studies. By SOX2 mutation analysis in four unrelated individuals with unilateral or bilateral clinical anophthalmia, Fantes et al. [24] identified heterozygous de novo truncating mutations in the SOX2 gene. Similarly, in an 11-month-old Mexican female infant with bilateral clinical anophthalmia and brain malformations, Zenteno et al. [29] identified heterozygosity for a 20 bp deletion in the SOX2 gene. De novo missense mutations and frameshift mutations in the heterozygous state in the coding region of the SOX2 gene in patients with bilateral anophthalmia/ microphthalmia were also reported [30].

In a 12-year-old girl with congenital bilateral clinical anophthalmia, a heterozygous nonsense mutation in the SOX2 gene was found [31]. Similarly, a heterozygous missense mutation was found in the SOX2 gene in a girl with bilateral clinical anophthalmia. However, the clinically normal mother was found to be heterozygous for this mutation [32]. The SOX2 gene was analyzed in two female siblings with clinical bilateral anophthalmia and found heterozygosity for a 17 bp deletion on this gene [6,33]. Similarly, in an Italian male with clinical bilateral anophthalmia and micropenis, a heterozygous insertion mutation was reported in the SOX2 gene responsible for such phenotypes [34].

In conclusion, the phenotypically normal parents of the affected daughters were first cousins, and both carried disease chromosome in the heterozygous state, and their affected daughters were homozygotes. Therefore, the present study strongly supports the fact on the basis of pedigree and linkage analysis that the consanguineous marriage contributed to the congenital isolated clinical anophthalmia that is inherited in an autosomal recessive manner. Moreover, this study could not reveal a molecular basis for congenital clinical anophthalmia in this family, as a consequence of mutations in the SOX2 gene. If more samples of congenital clinical anophthalmia subjects would be investigated in families of the same ethnic group, a better estimation of disease-related genes other than SOX2, but not yet identified in congenital isolated clinical anophthalmia, could be made in future.