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),
Clinical anophthalmia exhibits different patterns of genetic inheritance,
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
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.
This study was approved by the Ethics Committee of the Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Khyber Pakhtunkhwa, Pakistan. A consanguineous Pakistani family of the Pashtoon ethnic group with bilateral clinical anophthalmia, in which disease was segregating as an autosomal recessive trait, was ascertained. This family resided in the southern region of Khyber Paktunkhwa, Pakistan, known as Kohat, which is inhabited by various Pashtoon tribes (Figure 1). It was observed that ocular tissue was absent in the orbit of the eyes in affected offspring (Figure 2). On the basis of clinical features assessed by slit lamp examination and radiological assessment by CT scanning, the ophthalmologist diagnosed the anophthalmia as an isolated entity and having no syndromic presentation in the affected daughters. Written informed consent was obtained from the elders of this family to participate in the study. A pedigree of the family was created from information provided by the family using the Cyrillic (version 2.10) program (
Blood samples were collected in 10 mL vacutainer tubes (Becton Dickinson, Mountain View, CA, USA) with written informed consent from six individuals including four clinically normal (2MOP001, 2MOP002, 2MOP004, and 2MOP005), and two affected (2MOP003, and 2MOP006) daughters of this family. Genomic DNA was extracted from peripheral blood samples following the standard phenolchloroform extraction procedure [21].
Identification of the locus responsible for the isolated clinical anophthalmia phenotype in the selected family, genomic DNA from each individual was genotyped using a microsatellite short tandem repeat (STR) marker for the known clinical anophthalmia loci (Table 1). The microsatellite markers for each locus were amplified by polymerase chain reaction (PCR). Each PCR reaction was performed in a 10 μL volume, containing 1.5 mM MgCl2, 0.6 μM of each forward and reverse primer, 0.2 mM dNTPs, 1 U Taq DNA polymerase and PCR buffer [16 mM (NH4) 2SO4, 67 mM Tris-HCl (pH 8.8), and 0.01% of the nonionic detergent Tween-20] (Bioline Reagents Ltd., London, UK). Amplification was performed with an initial denaturation for 4 min. at 94 °C, followed by 35 cycles of denaturation at 94 °C for 35 seconds, annealing at 55 °C for 35 seconds, extension at 72 °C for 35 seconds, and a final extension at 72 °C for 7 min. The PCR products were separated on 10.0% nondenaturing polyacrylamide gels (Protogel; National Diagnostics, Edinburgh, Scotland, UK). The gel was stained with ethidium bromide and photographed under UV illumination. Alleles were assigned to individuals and genotypic data was used to find genotypes of all individuals of this family. The phenotype was analyzed as an autosomal recessive trait.
List of short tandem repeat markers used for genotyping in clinical anophthalmia.
Chromosome | Gene | STR Markers | Distance (cM) | Amplified Length (bp) |
---|---|---|---|---|
14q32 | – | D14S617 | 91.0 | 141-173 |
14q24.3 | D14S588 | 71.0 | 117-141 | |
18q21.3 | D18S858 | 54.9 | 193-208 | |
14q21-22 | GATA168F06 | 92.6 | 212-232 | |
3q26.3-q27 | D3S1565 | 190.3 | 239-245 |
cM: centimorgan; bp: base pair.
All individuals of this family were screened for mutations in the candidate gene. Polymerase chain reaction amplification of DNA of both normal and affected individuals was performed with forward and reverse primers sets, spanning the whole exonic region and promoter region of the candidate gene (Tables 2 and 3) that were designed using online available Primer 3 (
Primer sets used for amplification of the single exon of the
Primers | Sequences (5’>3’) | PCR Fragment Size (bp) |
---|---|---|
Sox2_1aF | CCT CTC TCT TTT TTT CCC C | 431 |
Sox2_1bF | GCG GCA ACC AGA AAA ACA | 291 |
Sox2 1cF | GCT CAT GAA GAA GGA TAA GT | 283 |
Sox2 1dF | CAT GAA CGG CTG GAG CAA | 407 |
Sox2_1eF | TTA CCT CTT CCT CCC ACT C | 286 |
PCR: polymerase chain reaction; bp: base pair; F: forward; R: reverse.
Primer sets used for amplification of the promoter sequence of the
Primers | Sequences (5’>3’) | PCR Fragment Size (bp) |
---|---|---|
5’ F2 | AGT CCC GGC CGG GCC GAG | 602 |
3’ F | GGC GTG AAC CAG CGC ATG G | 612 |
5’UTR F | CGC TGA TTG GTC GCT AGA A | 518 |
3’UTR.1F | GGG GTG CAA AAG AGG AGA GTA | 490 |
3’UTR.2F | AAC ATG GCA ATC AAA ATG TCC | 514 |
3’UTR.3F | CCC CCT TTA TTT TCC GTA GTT | 353 |
PCR: polymerase chain reaction; bp: base pair; F: forward; R: reverse; 5’UTR: 5’ untranslated region; 3’UTR: 3’ untrans-lated region.
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
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
The severity of clinical anophthalmia is variable due to mutations in various human genes that are associated with anophthalmia [4,5]. Among these, the
By sequence analysis of the coding region of the
In a 12-year-old girl with congenital bilateral clinical anophthalmia, a heterozygous nonsense mutation in the
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