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Novel splice-site and missense mutations in the ALDH1A3 gene underlying autosomal recessive anophthalmia/microphthalmia
  1. C Nur Semerci1,
  2. Ersan Kalay2,
  3. Cem Yıldırım3,
  4. Tuba Dinçer2,
  5. Akgün Ölmez4,
  6. Bayram Toraman2,
  7. Ali Koçyiğit5,
  8. Yunus Bulgu6,
  9. Volkan Okur1,
  10. Lale Şatıroğlu-Tufan1,
  11. Nurten A Akarsu7
  1. 1Department of Medical Genetics, School of Medicine, Pamukkale University, Denizli, Turkey
  2. 2Department of Medical Biology, School of Medicine, Karadeniz Technical University, Trabzon, Turkey
  3. 3Department of Ophthalmology, School of Medicine, Pamukkale University, Denizli, Turkey
  4. 4Department of Pediatric Neurology, Denizli State Hospital of Ministry of Health, Denizli, Turkey
  5. 5Department of Radiology, School of Medicine, Pamukkale University, Denizli, Turkey
  6. 6Department of Ophthalmology, State Hospital, Şuhut-Afyonkarahisar, Afyon, Turkey
  7. 7Department of Medical Genetics, Gene Mapping Laboratory, School of Medicine, Hacettepe University, Ankara, Turkey
  1. Correspondence to Dr C Nur Semerci, Department of Medical Genetics, School of Medicine, Pamukkale University, Kınıklı, Denizli 20020, Turkey; nsemerci1{at}yahoo.com

Abstract

Aim This study aimed to identify the underlying genetic defect responsible for anophthalmia/microphthalmia.

Methods In total, two Turkish families with a total of nine affected individuals were included in the study. Affymetrix 250 K single nucleotide polymorphism genotyping and homozygosity mapping were used to identify the localisation of the genetic defect in question. Coding region of the ALDH1A3 gene was screened via direct sequencing. cDNA samples were generated from primary fibroblast cell cultures for expression analysis. Reverse transcriptase PCR (RT-PCR) analysis was performed using direct sequencing of the obtained fragments.

Results The causative genetic defect was mapped to chromosome 15q26.3. A homozygous G>A substitution (c.666G>A) at the last nucleotide of exon 6 in the ALDH1A3 gene was identified in the first family. Further cDNA sequencing of ALDH1A3 showed that the c.666G>A mutation caused skipping of exon 6, which predicted in-frame loss of 43 amino acids (p.Trp180_Glu222del). A novel missense c.1398C>A mutation in exon 12 of ALDH1A3 that causes the substitution of a conserved asparagine by lysine at amino acid position 466 (p.Asn466Lys) was observed in the second family. No extraocular findings—except for nevus flammeus in one affected individual and a variant of Dandy–Walker malformation in another affected individual—were observed. Autistic-like behaviour and mental retardation were observed in three cases.

Conclusions In conclusion, novel ALDH1A3 mutations identified in the present study confirm the pivotal role of ALDH1A3 in human eye development. Autistic features, previously reported as an associated finding, were considered to be the result of social deprivation and inadequate parenting during early infancy in the presented families.

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Introduction

Anophthalmia and microphthalmia (A/M) are rare, but severe developmental disorders of the eye. True anophthalmia—in which the primary optic vesicle has stopped developing—is diagnosed histologically and is incompatible with life.1 Clinical anophthalmia, or severe microphthalmia, is characterised by a marked decrease or absence of ocular tissue within the orbit, whereas microphthalmia is a small eye (corneal diameter <10 mm and axial length of the globe <20 mm). A/M are clinically and genetically heterogeneous disorders with a combined prevalence of 3–14 per 100 000 births. Both conditions can occur as an isolated malformation or as part of a syndrome in 33% of cases.2

Autosomal recessive, autosomal dominant and X-linked patterns of inheritance have been described for both syndromic and non-syndromic A/M. Mutations in several genes including VSX2 (CHX10), RAX, PAX6, SOX2, OTX2, BCOR, CHD7, BMP4, FOXE3, STRA6, SMOC1, HCSS, SHH, SNX3, MFRP, PRSS56, GDF3 and GDF6 have been associated with A/M. Heterozygous mutations in SOX2 (MIM 184429) appear to be the most common cause of A/M malformation, as they are reported in 10–15% of cases. The remaining genes are responsible for 25% of cases, which indicates extensive genetic heterogeneity within this spectrum.3

Herein we present two autosomal recessive Turkish families with isolated A/M. Using Affymetrix 250 K single nucleotide polymorphism (SNP) array genotyping and homozygosity mapping, this malformation was mapped to chromosome 15q26.3, and a homozygous novel splice site and missense mutation in the human aldehyde dehydrogenase 1A3 (ALDH1A3) gene were identified. ALDH1A3 mutations have recently been associated with autosomal recessive A/M malformation.4–8 The present study's findings expand the mutation spectrum for this locus and confirm the critical role of ALDH1A3 in human eye development.

Materials and methods

Clinical evaluation

Family CNS_1 (figure 1A–E) and CNS_2 (figure 2A–C) were from the Aegean region of Turkey. Physical and ophthalmological evaluations were completed in three affected individuals (figure 1A; individuals VII-2, VII-4 and VII-5) in family CNS_1 and in the two affected individuals (figure 2A; individuals IV-4 and IV-6) in family CNS_2. Radiological investigation was performed using ultrasonography and MRI. After obtaining informed consent, peripheral blood samples were collected.

Figure 1

Homozygosity mapping and mutation analysis in family CNS_1. (A) Family CNS_1 pedigree (seven generations). Black squares represent affected male individuals and black circles denote affected female individuals. Empty squares and circles represent healthy male and female individuals, respectively. (B) Schematic representation of the genome-wide homozygosity mapping data. Homozygous genotypes identical to the genotype data obtained from affected individual VII-2 are shown in blue whereas contrasting homozygote genotypes are shown in white and heterozygous single nucleotide polymorphisms (SNPs) appear in orange. Non-informative SNPs appear in yellow and grey indicates no call. The single overlapping homozygous interval sharing the same haplotype in four affected individuals is about 1.02 Mb, between SNP markers rs7164647 and rs1993338 on chromosome 15. (C) The sequence chromatograms of exon 6—intron 6 boundaries showing the identified c.666G>A (p.Glu222Glu) mutation in ALDH1A3 in family CNS_1. The upper chromatogram shows the homozygous mutant sequence in affected individual VII-2, and the lower chromatogram shows the wild-type sequences in a healthy control. (D) Reverse transcriptase PCR analysis of c.666G>A transition with exon 5 sequence-specific forward primer and exon 7 sequence-specific reverse primer shows the remarkable size of the deletion in the amplicon obtained from an affected individual (line a) and a control (c). In the affected individual there is also a residual quantity of wild type amplicon. a, affected individual; c, healthy control; m, 100-bp marker; n, negative control. (E) The sequence chromatograms of cDNA shows complete deletion of exon 6 from mRNA as a result of disrupted splicing caused by c.666G>A transition.

Figure 2

Family pedigree and mutation analysis in family CNS_2. (A) Family CNS_2 pedigree (four generations). Black squares represent affected male individuals and black circles denote affected female individuals. Empty squares and circles represent healthy male and female individuals, respectively. (B) The sequence chromatograms of exon 12 showing the identified c.1389C>A (p.Asn466Lys) mutation in ALDH1A3 in family CNS_2. The upper chromatogram shows the homozygous mutant sequence in affected individual VII-6, and the lower chromatogram shows the wild-type sequences in a healthy control. (C) Multiple alignments of the partial amino acid sequences of the ALDH1A3 in a variety of species. Amino acid positions are indicated on the right side of the alignments and the missense mutation (p.Asn466Lys) is shown above the alignment.

SNP array genotyping and homozygosity mapping

DNA from affected individuals (figure 1A; individuals VII-2, VII-4, VII-5 and VI-8) and their parents (figure 1A; individuals V-1 and VI-1) were processed according to the genomic mapping 250 K NspI protocol and hybridised to 250 K NspI SNP GeneChips according to the manufacturer's instructions (Affymetrix, Santa Clara, California, USA). Genotype files (CHP files) were generated using Affymetrix GTYPE software and transferred to the VIGENOS (Visual Genome Studio) program (Hemosoft Inc, Ankara), which facilitates visualisation of a large quantity of genomic data in comprehensible visual screens.9 ,10

Mutation analysis

ENSEMBL and UCSC Human Genome databases (GRCh37/hg18) were used to identify the candidate gene in the critical interval. In all, 13 exons and adjacent intronic sequences of the largest transcript of ALDH1A3 (ENST00000329841) gene were amplified via PCR under standard conditions. Primers were designed using Primer3 web software (all primer sequences and PCR conditions are available upon request). PCR products were directly sequenced using ABI PRISM V.3130 DNA analyzer (Applied Biosystems). To test the segregation of identified sequence variations within the families, sequencing for all family members was performed. Ancestrally matched controls (n=182) were screened for c.666G>A and c.1398C>A sequence variations using the amplification refractory mutation system with allele-specific primers.11 Pathogenicity of the missense substitution of ALDH1A3 p.Asn466Lys was also analysed using PolyPhene-2 (http://genetics.bwh.harvard.edu/pph/) and SIFT (Sorting Intolerant From Tolerant) (http://sift.jcvi.org/) specialised software. Evolutionary conservation of asparagine at position 466 was evaluated via multiple sequence alignment of ALDH1A3 in various species.

RT-PCR analysis

The effects of c.666G>A substitution to the splicing of intron 6 was evaluated via RT-PCR. For total RNA we established fibroblast cell cultures from skin biopsyspecimens obtained from affected individual VII-2 from family CNS_1 and control. Total RNA was isolated from these fibroblast cells using TRI Reagent (Sigma, USA), according to the manufacturer's instructions. Subsequently, cDNA synthesis was performed using the First-Strand cDNA Synthesis Kit (MBI Fermentas, Vilnius, Lithuania), according to the manufacturer's protocol. We performed PCR reactions with gene-specific primers designed from sequences in exons 5 and 7. To evaluate splicing of exon 6 of the ALDH1A3 gene PCR fragments from agarose gel were purified and sequenced.

Results

Clinical description of the families

Clinical anophthalmia and/or microphthalmia were major findings in both families (figure 3). Ocular findings of the affected patients completed ophthalmological evaluations were summarised in table 1. Eyelids and eyelashes were intact. Large and bushy eyebrows, frequently associated with A/M malformations,12–15 existed in all affected cases. In family CNS_1 the index case (figure 1A, individual VII-2 and figure 3A) was 13 years old. Her growth parameters were normal and she had normal intelligence; no other systemic findings were observed. Ocular biomicroscopic examination showed the presence of a conjunctival sac with severe microcornea indistinguishable anterior chamber and iris structures consisting of a severe rudimentary microphthalmic globe. Ocular ultrasonography showed that the antero-posterior diameter of the right and left globes was 10 and 14 mm, respectively, and a 5 mm cyst on the left orbit was noted (figure 3B,C).

Table 1

Summaries of ocular findings of the patients in Fam CNS_1 and Fam CNS_2

Figure 3

Clinical and radiological findings in the patients. (A) Photo of eye of affected individual VII-2 in family CNS_1. (B, C) Ultrasonography scan shows bilateral rudimentary eye globes with a cyst on the left orbit. (D) Photos of the eyes of affected individual VII-4 in family CNS_1. Ocular MRI shows (E) a right remnant fibrotic globe (fat sat T2-weight axial image), (F) left hypoplastic optic nerve, but no visible right optic nerve (fat sat T2-weight axial image), indicated arrows. (G) Photo of the eyes of affected individual VII-5 in family CNS_1. Ocular MRI shows (H) bilateral cystic lesions in the intraorbital region and (I) bilateral hypoplastic optic nerves (fat sat T2-weight coronal images) indicated arrows. (J) Clinical appearance of affected individual IV-4 in family CNS_2. Ocular MRI shows (K) bilateral remnant tissue in the intraorbital region (fat sat T2-weight axial image), and (L) bilateral hypoplastic optic nerves, but no visible optic chiasm (fat sat T2-weight axial image). (M) Clinical appearance of affected individual IV-6 in family CNS_2. (N) Ultrasonography shows an anophthalmic socket. Ocular MRI shows (O) remnant fibrotic tissue in the intraorbital region and hypoplastic optic nerve bilaterally (fat sat T2-weight axial image).

A brother and sister of the index case (figure 1A, individuals VII-1 and VII-3) died due to unknown causes at age 1.5 years. Her two other affected brothers (figure 1A individuals VII-4 and VII-5 and figure 3D,G) were aged 6 and 7 years, respectively. Their growth parameters were normal, but their all developmental mental and motor milestones like walking, speech care, self care, toilet use, feeding were delayed according to the history given by parents. They had behavioural and cognitive problems, and autistic-like features, such as stereotypies, intense interest in music, and aggressive behaviour especially towards strangers. Since no psychometric test ‘(Wechsler Intelligence Scale for Children Revised (WISC-R))’ could be performed they were clinically estimated as moderate mental retardation. The parents’ parenting skills were inadequate during early infancy; however, the index case was raised by her grandmother under better environmental and social conditions. Ocular biometric examinations could not be performed in these affected individuals. In individual VII-4 ocular MRI showed a 6×7 mm irregular fibrotic remnant tissue in the left intraorbital area (figure 3E) and three cystic lesions in the right intraorbital area. Extraocular muscles were hypoplastic. His optic nerves were hypoplastic on the left side and were not visible in the optic canal on right side (figure 3F). The optic chiasm was not visible. Cranial MRI showed mild hypoplasia of the vermis (variant of Dandy–Walker malformation) (data not shown). Individual VII-5 had bilateral intraorbital cystic lesions and hypoplastic extraocular muscles, based on ocular MRI (figure 3H).

His optic nerves were hypoplastic and the optic chiasm was not visible (figure 3I). Individuals VII-4 and VII-5 had no other dysmorphic or systemic findings except nevus flammeus in individual VII-5.

In family CNS_2 individual IV-4 was a 17-year-old female with normal intelligence who was attending high school (figures 2A and 3J). Ocular biomicroscopic examination showed the presence of a conjunctival sac with indistinguishable anterior chamber and iris structures. Ultrasonography showed anophthalmic sockets bilaterally. Ocular MRI showed remnant fibrotic tissue bilaterally (9×8 mm on the right side and 7×6 mm on the left side) (figure 3K) and a 9×4×6 mm cystic lesion in the left intraorbital region. Bilateral extraocular muscles and optic nerves were hypoplastic (figure 3L). The optic chiasm was not visible. She had no other systemic or dysmorphic findings. Her brother (figure 2A, individual IV-6 and figure 3M) was aged 12 years. Ocular biomicroscopic examination showed the presence of a conjunctival sac with indistinguishable anterior chamber and iris structures. Ultrasonography showed anophthalmic sockets on the right side (data not shown) and left side (figure 3N). Ocular MRI showed remnant fibrotic tissue and hypoplastic optic nerve bilaterally (figure 3O). Since he could not undergo the WISC-R test he was clinically estimated as borderline mental retardation according to his present skills. He had poor anger management and self-control, and some autistic like features as stereotypies and ecolalia. As with individuals VII-4 and VII-5 in family CNS_1, IV-6's parenting skills during early infancy were inadequate. He had no other systemic or dysmorphic findings.

Homozygosity mapping

In all, four affected individuals and two parents from family CNS_1 were typed using the 250 K SNP mapping array (figure 1B). Affected individual VII-2 was chosen to construct genome-wide haplotypes. Haplotypes indicating homozygosity by descent were compared with the identical homozygous haplotypes of affected individuals VII-4, VII-5 and VI-8. A single homozygote segment on chromosome 15q26.3 was identified (figure 1B); within this region haplotypes residing between SNP markers rs7164647 and rs1993338 were identical in the four affected individuals. The critical interval was approximately 1.02 Mb (figure 1B), which harbours seven protein-coding genes (ADAMTS17, HSD47, LASS3, LINS1, ASB7, ALDH1A3, LRRK1 and CHSY). As Aldh1a3 was shown to be highly expressed in the developing sensory neuroepithelia of the eye in chicken and mouse embryos, and Aldh1a3 deficiency causes eye and nasal defects, the ALDH1A3 gene was selected as the most promising candidate gene from the critical region for mutation analysis.16 ,17

Mutation results and RT-PCR analysis

Sequencing of the ALDH1A3 gene from genomic DNA of affected individual VII-2 showed a homozygous c.666G>A substitution leading to a silent mutation (p.Glu222Glu) (figure 1C). Although c.666G>A does not cause an amino acid substitution, it resides at the 3′ end of exon 6, where it is highly conserved and essential for proper intron splicing.18 In silico splice site analysis predicted that c.666G>A substitution destroyed the exon–intron junction (splice site prediction by Neural Network)19; therefore, the cDNA of ALDH1A3 obtained from cell culture of the skin biopsy specimen from patient VII-2 was evaluated (figure 1D,E). Skipping of exon 6 that leads to in-frame deletion of 43 amino acids encompassing Trp180 to Glu222 was observed (p.Trp180_Glu222del).

Sequencing of the ALDH1A3 gene in two additional individuals from family CNS_2 showed a homozygous c.1398C>A transversion in exon 12 that leads to substitution of asparagine with lysine at evolutionary conserved position 466 (p.Asn466Lys) (figure 2A–C). Evaluation of p.Asn466Lys using specialised prediction software PolyPhene-2 and SIFT also classified this substitution as very likely pathological for protein function, with a score of 1.00 and 0.97, respectively. c.666G>A substitution and c.1398C>A mutations were segregating with disease in both families, whereas neither was detected in 182 ancestrally matched controls.

Discussion

In the present study genome-wide homozygosity mapping followed by the candidate gene approach was used to identify novel mutations—a 43-amino-acid in-frame deletion (p.Trp180_Glu222del) and p.Asn466Lys substitution—in the ALDH1A3 gene in two autosomal recessive isolated A/M families. The following points support the disease-causing nature of these mutations: p.Trp180_Glu222del mutation deletes a critical portion of nicotinamide adenine dinucleotide (NAD)-binding pocket for which another in-frame deletion has previously been reported in isolated A/M;4 the substitution of asparagine with lysine at evolutionary conserved position 466 (p.Asn466Lys) is clearly deleterious based on analysis by PolyPhene-2 and SIFT mutation prediction programs; both mutations cosegregated with the disease in the families; and they are absent in the dbSNP132 database and 182 Turkish controls.

ALDH1A3 encoding of the A3 isoform of cytosolic class 1 aldehyde dehydrogenase plays a critical function via conversion of retinol (vitamin A) to the signalling molecule retinoic acid (RA). RA is an important molecule that mediates transcriptional activation of different sets of genes that play a crucial role in early embryonic development, including the eyes.20 The association between severe developmental eye defects, such as microphthalmia, coloboma, abnormal folding of the retina, anterior chamber defect, gaps between the neural and pigmented retina, thickened retrolenticular membrane filling the vitreous cavity, delayed lens differentiation, and vitamin A and/or RA deficiency, indicates the importance of RA and RA-triggered pathways in eye development.17 ,21–23 Consistent with these earlier reports, loss of function mutations in STRA6, the gene encoding a cell membrane protein that triggers RA uptake from retinol-binding protein, have been associated with isolated anophthalmia with coloboma and Matthew–Wood syndrome (MIM 601186), also known as pulmonary agenesis, microphthalmia and diaphragmatic defect syndrome.24–26

The mutations in the ALDH1A3 gene reported in the present study provide another direct link between the RA signalling pathway and severe eye anomalies in humans. Isolated A/M, optic nerve hypoplasia/aplasia, and optic chiasm hypoplasia/aplasia were major findings in the presented families with distinct ALDH1A3 mutations. Recently, six missense, two nonsense and one splicing site ALDH1A3 mutations were reported.4–8 Table 2 summarises the clinical findings of all reported cases with ALDH1A3 mutations and provides a comparison between previously reported cases and those presented herein. Approximately 50% of the mutations accumulate in exon 5 and exon 6, which encode the NAD-binding pocket at the protein level.4 In addition to a previously reported in-frame deletion containing exon 5, the present study reports skipping of exon 6 that leads to a 43-amino-acid in-frame deletion at the protein level. The deletion encompassing Trp180 to Glu222 contains critical amino acid residues, such as p.W180, p.N181, p.W189, p.K204, that are directly involved in NAD binding or catalytic (p.N181) activity.4 ,27–29 The phenotype expands from anophthalmia to severe bilateral microphthalmia with cysts, including dysplastic globes, and a hypoplastic/aplastic optic chiasm and optic nerves in both splice variants in exon 5 and exon 6; however, phenotype evaluation of families with various mutations, that is, splice, nonsense and missense variations, showed that there is not a genotype–phenotype correlation between the nature of mutation and a particular phenotype (table 2). Although isolated A/M is the consistent outcome of the ALDH1A3 mutations, associated ocular findings such as retinal detachment, iris and chorioretinal coloboma, cysts, displaced pupils, hiperopia, optic chiasm and optic nerve abnormalities are variable within and between families.4–8

Table 2

Phenotypic features of the patients with various ALDH1A3 mutations

However, Fares-Taie et al4 reported a patient who had pulmonary stenosis and atrial septal defect, and another patient who had autism as extraocular findings. The presented patients did not have any severe extraocular systemic findings, except for nevus flammeus on the face in individual VII-5 and a variant of Dandy–Walker malformation in individual VII-4; however, we also observed autistic-like behaviours in family CNS_1 individuals VII-4, VII-5, and in family CNS_2 individual IV-6. Recently two other studies also reported intellectual disability and/or autism in anophthalmia/microphthalmia cases with ALDH1A3 mutations.6 ,8 However, these phenotypes show intrafamilial and interfamilial variation.

The aetiology of autism in individuals who are blind—especially those with congenital blindness—is not well known.30–32 Early social and environmental deprivation are well known causes of mental retardation and delayed speech in children, and such deprivation has also been shown to cause autism.33 The frequency of autism and autistic-like features are rather high, and may be reversible in children in whom early social and environmental deprivation are aetiological.30 So the families’ knowledge of the possibility of autism in A/M can provide better educational and environmental conditions from the beginning of early infancy. Family CNS_1 provided a good history of insufficient maternal care during the early infancy of two individuals, as did family CNS_2 in the case of individual IV-6. As older sisters in the two families had normal intelligence and social development, we think that the cause of autistic-like behaviours and mental retardation in the two sibs in family CNS_1 and poor anger management and borderline mental retardation in family CNS_2 might not be directly related to ALDH1A3 mutations. However, it is known that multiple regions of homozygosity in each child of consangineous families can cause different phenotypes occurring in the same patient coincidentally.

The clinical molecular approach to diagnosis of individuals is complicated due to extensive genetic heterogeneity in isolated and syndromic forms of A/M malformation. It was suggested that a screen for heterozygous SOX2 and OTX2 mutations should be the first-line test in isolated cases with A/M.34 Regarding the autosomal recessive form of this malformation, a total of five genes (MFRP [MIM 606227], PRSS56 [MIM 613858], RAX [MIM 601881], CHX10 [MIM 142993], STRA6 [MIM 610745]) have been associated with this malformation, to which ALDH1A3 (MIM 600463) is added. Among this group, MFRP, PRSS56 and RAX mutations are extremely rare and almost specific to individual families; however, biallelic ALDH1A3 and CHX10 mutations manifesting isolated A/M malformation associated with coloboma were reported in families of varying ethnic origin. Homozygous STRA6 mutations usually manifest as severe extraocular malformations, although variable expressivity has been documented; therefore, for diagnostic purposes biallelic mutation screening of ALDH1A3 and CHX10 genes should be the first-line test in isolated cases of A/M malformation in recessive populations.

Prenatal diagnosis of facial abnormalities and A/M can be made by transvaginal ultrasonography, three-dimensional (3D) ultrasonography and MRI usually after the first trimester.35 ,36 The identification of causative mutations in A/M provides the opportunity of a precise preimplantation and/or early prenatal molecular diagnosis for the affected families.

In conclusion, novel ALDH1A3 mutations identified in the present study confirm the pivotal role of ALDH1A3 in human eye development. Autistic features, previously reported as an associated finding, were considered to be the result of social deprivation and inadequate parenting during early infancy in the presented families.

References

Footnotes

  • Contributors The conception and design of the project were planned by CNS and NAA. The review of phenotypes and the sample collection were performed by CNS, CY, AÖ, AK and YB. Data analysis and interpretation of data were performed by NAA, CNS, EK, TD, BT, VO and LŞT. The manuscript was written by NAA, CNS and EK. Revising the draft critically for important intellectual content were done by NAA. Final approval of the version to be published was performed by CNS, EK and NAA. CNS and EK contributed equally.

  • Funding This study was supported by the Pamukkale University Scientific Research Grants Fund, grant number 2012ARŞ001.

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval The study protocol was approved by the Pamukkale University Ethics Committee (approval number: 2011/14).

  • Provenance and peer review Not commissioned; externally peer reviewed.