|Molecular Vision 2007;
Received 2 June 2007 | Accepted 12 August 2007 | Published 17 August 2007
Exclusion of TACSTD2 in an Iranian GDLD Pedigree
Fahimeh Asadi Amoli,4
Mehdi Hosseini Tehrani4
1National Institute of Genetic Engineering and Biotechnology, Tehran, Iran; 2School of Biology, College of Science, University of Tehran, Tehran, Iran; 3Bioinformatics Center, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; 4Farabi Eye Research Center, Tehran University of Medical Sciences, Tehran, Iran
Correspondence to: Dr. Elahe Elahi, National Institute of Genetic Engineering and Biotechnology, Medical Genetics, Tehran-Karaj Expressway Km17, Pajouhesh Boulevard, Tehran, Tehran, Iran 19857; Phone: 00989122181251; FAX: ++98 21 44580399; email: firstname.lastname@example.org
Purpose: To perform a mutation screen of TACSTD2 in an Iranian Gelatinous Drop-like Corneal Dystrophy (GDLD) pedigree.
Methods: In addition to the coding region of TACSTD2, for the first time the promoter, the entire 5'-noncoding region, and the entire 3'-untranslated region of the gene were sequenced from an affected member of the pedigree. Phenotypic features of affected individuals were assessed.
Results: The proband carried six sequence variations in TACSTD2. One of the variations was homozygous and caused a synonymous codon change. The remaining five were heterozygous variations in the 3'-untranslated region. None of the variations were assessed to be associated with GDLD. Fibroblastic scars were evident in in corneal histology sections of two affected members of the pedigree.
Conclusions: It is concluded that GDLD in the pedigree is probably not caused by mutations in TACSTD2, supporting evidence for the existence of at least one other locus for GDLD. Phenotypic features of the Iranian patients, including the existence of fibroblastic scars in the corneas, were similar to those of a previously reported GDLD patient without TACSTD2 mutations. This suggests the disease in these individuals may be due to mutations in the same gene.
Gelatinous drop-like corneal dystrophy (GDLD; OMIM 204870) is a rare inherited ocular disease reported in many countries but mostly in Japan . Clinical symptoms usually manifest within the first decade of life. It is characterized by the deposition of amyloid material in the subepithelial space of the cornea. Early stage nodular depositions later increase in number and depth and coalesce usually to form a protruding whitish-yellow mulberry appearance [2-4]. Neovascularization of the subepithelial and superficial stroma may appear as the disease progresses . Affected individuals experience lacrimation, photophobia, foreign body sensation and blurred vision. Eventually visual acuity is impaired. There is generally early recurrence after penetrating or lamellar keratoplasty, photoablation, or keratectomy .
The inheritance of GDLD is autosomal recessive [7,8]. Membrane component, chromosome 1, Surface marker 1 (M1S1), originally identified as the gene encoding gastrointestinal tumor-associated antigen and now officially named TACSTD2 (tumor associated calcium signal transducer 2), has been identified as the causative gene of GDLD [9,10]. Putative disease causing mutations in TACSTD2 were found in almost all cases where mutation screening of the gene was performed [9,11-23]. Twenty-two different disease-associated mutations have so far been reported . Screening has been done in patients from Japan, India, Tunisia, Iran, and other countries. It can be surmised that TACSTD2 is the most important gene in the etiology of GDLD. Nevertheless, three unrelated GDLD pedigrees have been identified wherein mutations in TACSTD2 were not found, suggesting genetic heterogeneity for the disease [11,22,24]. Here, we report a fourth GDLD pedigree wherein a disease associated mutation in TACSTD2 was not found. The clinical and histpathologic features of affected individuals in the pedigree are presented and compared with reported features of a previously reported GDLD patient in whom a mutation in TACSTD2 was not observed.
This research was performed in accordance with the Helsinki Declaration and with approval of the ethics board of the International Institute of Genetic Engineering and Biotechnology in Iran. A GDLD pedigree (pedigree 100-14) from a western province of Iran was identified, and the family consented to participate in the research after being informed of its nature. Family 100-14 had two affected and three unaffected siblings, born to unaffected parents who reported no consanguinity. Diagnosis of GDLD in affected individuals was made by cornea specialists at the Farabi Hospital (associated with Tehran University of Medical Sciences). Diagnosis was based on classic clinical appearance and slit lamp biomicroscopy. GDLD was confirmed with histopathology by staining for amyloid with congo red. Ocular, medical, and family histories were obtained for each available family member.
For genetic analysis, DNA was available only from the affected female sibling and the unaffected father. The coding region of TACSTD2 was amplified from these DNAs by the polymerase chain reaction (PCR) as previously reported . Additionally, sequences further upstream and downstream of the coding region were also amplified. In all 3051 nucleotides were amplified, including 212 nucleotides upstream of the transcriptional initiation site, the 5'-noncoding region (617 nucleotides), the coding region (972 nucleotides), the 3'-untranslated region (1214 nucleotides), and 36 nucleotides further downstream. The upstream regions of TACSTD2 amplified include the CAAT box, the TATA box, and a Sp1-binding site element of the gene's promoter . Sequences downstream of the coding region which were amplified include three poly-A adenylation signals . The sequences of the primers used are presented in Table 1.
The amplified products were sequenced in both forward and reverse directions with the PCR primers using the ABI BigDye terminator chemistry and an ABI Prism 3700 instrument (Applied Biosystems, Foster City, CA). Sequences were analyzed using the Sequencher software (Gene Codes Corporation, Ann Arbor, MI). Sequence variations and numbering were assessed by comparison with reference sequences available at NCBI: NT_032977, NM_002353, and NP_002344. Predicted effects of variant sequences on splicing were determined by comparison with known canonical splice site motifs.
Both affected siblings of the pedigree were diagnosed with GDLD at the age of 12 years old, albeit clinical and histopathologic findings differed somewhat from those usually associated with GDLD. They were affected bilaterally. At the time of this writing, the male sibling was 27 years old and the female sibling was 23 years old. Their visual acuity was reduced to finger counting at 0.5 m. Both patients underwent several keratectomies. Recurrence was evident in both only three months after the last keratectomy, and a penetrating keratoplasty was performed six months after recurrence.
Slit lamp photographs and histologic sections of the patients' corneas are presented in Figure 1. Band-shaped opacity is evident in the slit lamp photograph of the female sibling (Figure 1A). Opacity is more extensive in the cornea of the male sibling whose disease is more advanced and vascularization is evident (Figure 1B). A typical mulberry-like amyloid deposition was not observed at any time in the corneas of either patient. Hematoxylin-eosin staining shows amorphous eosonophilic material in subepithelial and superficial stroma (Figure 1C and Figure 1D), fibroblastic collagenized scars (Figure 1E,F), vascularization of the fibroblastic scars (Figure 1G), and disruption of Bowman's membrane (Figure 1F). Amyloid deposition was only weakly evident in congo red stained sections prepared after first surgical intervention (Figure 1H). The amyloid depositions were more disperse than is typical in GDLD. Furthermore, deposition was not limited to subepithelial and superficial stroma regions, rather extended into the central and posterior stroma. Congo red staining of sections prepared after second surgical intervention showed more intensive amyloid deposition, but the deposition remained dispersed (Figure 1I).
Six sequence variations were identified in the TACSTD2 gene of the affected female sibling (Table 2). One of the variations (c.828C>T) was in the coding region and resulted in a synonymous change of codon -GGC- coding glycine at position 276 to -GGT-. This variation was present in the homozygous state. The remaining five variations were all heterozygous and located in the 3'-untranslated region. None of the variations were predicted to affect splicing. The unaffected father was heterozygous for c.828C>T, but did not carry any of the other variations.
As a disease associated variation in TACSTD2 was not observed, exons 4, 11, 12, and 14 of TGFβI were also screened for variations by sequencing. TGFβI, encoding transforming growth factor, beta-induced protein (also named kerato-epithelin), is the causative gene for the several dominant forms of corneal dystrophies [21,22,26]. These four exons of TGFβI were selected because they include hotspots of mutations within the gene [14,18,22]. No sequence variations were found in these exons of TGFβI in the affected proband (not shown).
An association between the variations found in TACSTD2 in the affected sibling and GDLD is assessed to be unlikely. Variation c.828C>T causing G276G does not affect an amino acid change and is not predicted to affect splicing. Furthermore, the frequency of the T allele at position c.828 has been estimated at 1.3% and 3.0% (rs12121124), respectively, in African American and Caucasian cohorts. Assuming full penetrance and random mating, even these relatively low allele frequencies would predict this single mutation should cause a GDLD incidence of 1 per 10,000 individuals in the African American population and 9 per 10,000 individuals in the Caucasian population. Variation c.828C>T is therefore thought not to cause disease. Four of the variant alleles in the 3'- untranslated region have also been reported at frequencies high enough to make their role in GDLD unlikely. In populations reported, the minor allele frequency of c.1609G >A (rs7333), c.1737C>T (rs6683669), c.1773T>A (rs2268943), and c.2110A>G (rs232840) were 2.0%, 8.6%-39.7%, 11.6%, and 8.9%-55.9%, respectively. With respect to c.1878, C is the nucleotide at that position in the reference genomic sequence (NT_032977), and T is the nucleotide in the reference cDNA sequence (NM_002353), suggesting both are common alleles. None of these variations are predicted to affect splicing or biological functions known to be associated with the 3'-untranslated region of mRNAs, including polyadenyaltion or mRNA stability . Finally, as none of the 3'-untranslated region variations were observed in the DNA of the father, the affected female sibling is expected to have received from him one TACSTD2 allele with a wild type sequence. All five 3'-untranslated region variations must be linked on the allele the affected sibling received from her mother. Therefore, even the theoretical possibility that specific combinations of the variations on both copies of the gene caused disease in the patient is ruled out. All these observations support earlier evidence for the existence of at least one other GDLD locus [8,19,21]. Among reported GDLD patients in whom disease associated mutations in TACSTD2 were not found, this is the first in which, in addition to the coding region, the promoter, the entire 5'-noncoding region, and the entire 3'-untranslated region, are also analyzed. Because TACSTD2 does not contain introns, these regions constitute the entire gene. Ruling out presence of possible disease associated variations in the non-coding regions was important, as variations in those regions of other genes have been found to be the cause of some diseases [28-30].
The histological features of corneal sections of both affected individuals of pedigree 100-14 showed features distinct from those commonly reported for GDLD patients. One of these is dispersed amyloid deposition in the subepithelial, central and posterior stroma. The deposition in GDLD patients is normally limited to the subepithelial space and superficial stroma of the cornea . Although amyloid deposition in the cornea can be the consequence of various conditions such as chronic keratitis, the deposition in the two siblings is most likey related to their common disease because it is observed in both. Another feature evident in the two siblings and not generally associated with GDLD is the presence of fibroblastic scars in their corneal sections . Because of these unusual phenotypic features, it may be appropriate to define the disease observed in the siblings as a novel subtype of GDLD. It is not unreasonable to predict that defects in the gene associated with disease in these patients would produce phenotypic features somewhat different from those caused by defects in TACSTD2. Lattice corneal dystrophy has also been grouped into various subtypes on the basis of distinguishing phenotypic features .
The three previously reported GDLD patients in whom mutations in TACSTD2 were not observed were all Caucasians [11,22,24]. Photographs of the cornea and pictures of histologic sections of only one of these patients were presented . The amyloid deposits in that patient also did not present the typical mulberry appearance. Another common feature between that patient and the affected female sibling of the Iranian pedigree is the presence of fibroblastic scars in their corneal histologic sections. This suggests that these two patients may share a genetic defect in the same gene. The two patients are probably not related, as two variations found in the TACSTD2 gene of the previously reported case were not observed in the Iranian patient and the G276G variation of the Iranian patient was not observed in the previous case. In addition to GDLD, the previously reported patient also had phenotypic features of developmental delay not present in the Iranian patient. It is possible that GDLD and developmental delay in that patient were due to different genetic defects .
We thank the Iranian family for consenting to participate in this study. We also acknowledge funding by the Iranian Molecular Medicine Network, the National Institute of Genetic Engineering and Biotechnology and the University of Tehran.
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