|Molecular Vision 2000;
Received 2 May 2000 | Accepted 13 June 2000 | Published 19 June 2000
Physical and genetic mapping of the macular corneal dystrophy locus on chromosome 16q and exclusion of TAT and LCAT as candidate genes
Ning-Pu Liu,1,2 Susan
Dew-Knight,2 Fridbert Jonasson,3 John R. Gilbert,2
Gordon K. Klintworth,1,4
Jeffery M. Vance2
1Department of Ophthalmology, Duke University Medical Center, Durham, NC; 2Center for Human Genetics, Duke University Medical Center, Durham, NC; 3University Department of Ophthalmology, Landspitalinn, Reykjavik, Iceland; 4Department of Pathology, Duke University Medical Center, Durham, NC
Correspondence to: Gordon K. Klintworth, Department of Ophthalmology, Box 3802, Duke University Medical Center, Durham, NC, 27710; Phone: (919) 684-3550; FAX: (919) 684-9225; email: firstname.lastname@example.org
Purpose: Macular corneal dystrophy (MCD) is an inherited autosomal recessive disorder that has been subdivided into three immunophenotypes, MCD types I, IA and II. We previously mapped the MCD type I gene to chromosome 16q22 and suggested that the MCD type II gene was linked to the same region. The purpose of this study was to construct a genomic contig spanning the MCD region and to narrow the MCD critical interval by haplotype analysis. The TAT and LCAT genes were mapped to determine if they might be the MCD gene.
Methods: The MCD contig was constructed by screening YAC, PAC, and BAC libraries with microsatellite, STS and EST markers, employing a systematic "DNA walking" technique. Polymorphic markers mapped and ordered on the contig were used to screen the MCD affected individuals and their family members for haplotype analysis.
Results: Twenty-two YAC, 30 PAC, and 17 BAC clones were mapped to form the MCD contig. Markers mapped on the contig include 19 microsatellite, 14 STS, and 15 EST markers. Moreover, 18 novel STS markers were generated. Using the mapped and ordered microsatellite markers, haplotype analysis on 21 individuals with MCD type I or type II and their family members from Iceland narrowed the MCD interval to 3 overlapping PAC clones. In addition, the TAT and LCAT genes were mapped outside the MCD region.
Conclusions: We established a genomic contig for the MCD region and dramatically narrowed the MCD critical interval. Mapping data show that the TAT and LCAT genes are not the cause of MCD.
Macular corneal dystrophy (MCD) is an inherited autosomal recessive bilateral corneal disorder with characteristic clinical and histopathologic features [1,2]. Irregular, cloudy opacifications begin axially in both corneas and these cloudy regions progressively merge until eventually the entire corneal stroma becomes opaque. MCD is rare in most parts of the United States and in other countries, but it is more frequent in populations in which consanguineous marriages are common. In Iceland, for example, MCD is the most frequent condition necessitating penetrating keratoplasty and accounts for one-third of all corneal grafts performed there .
Based on whether antigenic keratan sulfate (aKS) is identified in the corneal stroma and serum, cases of MCD have been subdivided into three immunophenotypes: MCD types I, IA and II [3-5]. In MCD type I, neither the cornea nor the serum contain appreciable levels of aKS, while in MCD type II aKS is present in the cornea and serum [3-5]. MCD type IA has no aKS immunoreactivity in the corneal stroma and serum, but the keratocytes react with the anti-keratan sulfate antibody . In a previous pedigree linkage study, we localized the MCD type I gene to an interval of approximately 7 cM on the long arm of chromosome 16 (16q22), flanked by markers D16S512 and D16S518 . Moreover, we provided strong evidence suggesting that the MCD type II was linked to the same region as MCD type I, with a LOD score of 2.5 in MCD type II families for markers in the MCD type I region . We also found that both MCD type I and type II coexisted in a single sibship  and shared the same disease haplotype [7,8], suggesting that different immunophenotypes of MCD are likely to be allelic manifestations of the same abnormal gene.
Here we report the formation of a genomic contig across the MCD interval between D16S512 and D16S518. With three newly mapped microsatellite polymorphic markers (NP4, AFMB317WF9, and D16S395), we subsequently reduced the MCD critical region to 3 overlapping PAC (P1 artificial chromosome) clones. Moreover, because the tyrosine aminotransferase (TAT) and lecithin-cholesterol acyltransferase (LCAT) genes both cause corneal lesions and are located in the same region of chromosome 16q [9-12], we investigated if these two genes could lead to MCD. However, physical mapping demonstrated that both TAT and LCAT genes are outside the MCD interval, and therefore, are not the cause of MCD.
The contig formation
The order of framework microsatellite markers used in this study was originally initiated based on the Genéthon genetic map  and the Whitehead physical map. The final order was determined by the genomic contig that was constructed to span the entire MCD region between D16S512 and D16S518 (Figure 1). This contig was constructed by screening the CEPH Yeast Artificial Chromosome (YAC) megabase library, PAC library and human bacterial artificial chromosome (BAC) library [14-16]. Briefly, DNAs from the YAC plates were pooled based on a two-dimensional polymerase chain reaction (PCR) screening strategy [17,18]. Microsatellite markers were then screened by PCR as previously described . PAC and BAC clones were isolated by either screening the library filters or PCR screening of "Down to the Well Release I and II DNA" pools (Genome Systems Inc., St. Louis, MO), according to the manufacturer's recommendations. PAC and BAC clones described in the present study were obtained from Genome Systems Inc. The PCR reaction was performed as previously described .
Sequencing and generation of new STS markers
Direct cycle sequencing with T7 and SP6 primers was performed on the PAC and BAC clones using a Thermo Sequenase fluorescence-labeled primer kit (Amersham Pharmacia Biotech, Piscataway, NJ) or Beckman CEQ DTCS Cycle Sequencing Kit (Beckman Instruments Inc., Fullerton, CA), according to the manufacturer's recommendations. PCR primers were then designed from the newly-generated sequences and were subsequently used to screen the libraries for additional clones for the MCD candidate gene region.
Family data and diagnostic criteria
With approved consent under the Duke University Institutional Review Board, twenty-one affected individuals with MCD (16 type I, 5 type II) and their family members from ten Icelandic families were included in this study, of whom 12 individuals with MCD have been included in a previous publication . The diagnosis of MCD was based on a combination of the clinical presentations and the typical histopathologic features of the dystrophy in corneal tissue obtained after penetrating keratoplasty , and clinicopathologic data on these cases has been included in a previous publication . Serum levels of aKS were determined using a well-established enzyme-linked immuno-sorbent assay (ELISA) and an anti-keratan sulfate monoclonal antibody (5-D-4, ICN Biomedical, Costa Mesa, CA) directed against a highly sulfated epitope present on keratan sulfate chains [20,21]. The immunohistochemical evaluation of the excised corneal tissue used the same antibody on pathologic corneal tissue [3-5].
Genomic DNA from each individual was extracted as previously described  and genotyped with polymorphic makers located around the MCD region. Except for two dinucleotide repeat markers generated by ourselves (NP4, forward primer 5-TTTTGAAAATTGAGTCATGAAAC-3, reverse primer 5-AATAGTATCAAAATAAGGAGCCAG-3, product size 91-97 base pair; JSB16A, forward primer 5-CCATAGAAGACTAGTAGGCA-3, reverse primer 5-TCAATAGCTTGGAGGTTAG-3, product size 204-230 base pair), the primer sequences for all other polymorphic markers were obtained through the Genome Database. NP4 and JSB16A were isolated from PAC clones p81E7 and p134D23, respectively, using an indirect sequencing method as previously described .
Microsatellite repeats were amplified by PCR . PCR products were then electrophoresed on 6.5% polyacrylamide gels. Gels were stained with SyberGreen (Molecular Probes, Eugene, OR) and detected by a FluorImager SI (Molecular Dynamics, Sunnyvale, CA) or Hitachi FMBIO II (Hitachi Software Engineering America Ltd., San Bruno, CA). To ensure accuracy of allele analysis, PCR products from all affected individuals was amplified and analyzed side-by-side on the same gel as DNA from control individuals (CEPH 952645 and 952646) at the same time for comparison of different gels. Haplotype analysis was performed as previously described . All marker data were entered into a database and managed using the PEDIGENE system .
Radiation hybrid mapping
The Stanford G3 Radiation hybrid panel (Research Genetics, Huntsville, AL) was used to map marker stSG1784 by PCR. The primer sequence of stSG1784 was obtained through the Genome Database. The data was then submitted to the Stanford RH Server for two-point statistical analysis with all assayed G3 markers to determine which marker, if any, links closely with stSG1784.
Allele frequencies in Icelandic controls
DNAs extracted from the blood of 50 Icelandic, unrelated controls were used to determine the allele frequencies.
The contig formation and marker order
A genomic contig spanning the MCD region was generated as a resource for the identification of the MCD gene, by screening the YAC, PAC, and BAC libraries (Figure 1). Twenty-two YAC, 30 PAC, and 17 BAC clones were identified and mapped to form this contig. A gap in the middle of the YAC map was closed by PAC clones (Figure 1).
Nineteen polymorphic markers, 14 sequence tagged sites (STS) markers, and 15 expressed sequence tags (EST) markers were mapped and ordered on the contig. Information for all of these STS and EST markers can be obtained from the Genome Database, except for one marker, stSG5082, that was generated by the Sanger Center and information on this marker can be found at the Sanger web site. Moreover, 18 novel STS markers (Table 1) were generated by sequencing the ends of PAC and BAC clones, which were once used to screen the genomic libraries further, employing a systematic "DNA walking" technique across gap regions to complete the contig.
Twenty-one individuals with MCD (16 type I, 5 type II) and their family members were genotyped for haplotype analysis, using the mapped and ordered polymorphic markers on the MCD contig. In addition to sixteen previously reported markers in the region , the haplotype analysis included three newly mapped markers (NP4, AFMB317WF9, and D16S395). Five disease haplotypes were found that spanned the MCD region (haplotypes 1-5) as shown in Figure 2. With the three new markers, ancestral recombination analysis refined the MCD critical interval between markers NP4 and AFMB317WF9 (Figure 2, see discussion), an interval covered by three overlapping PAC clones (p36D12, p69E21, and p262N10, Figure 1). Of the EST markers mapped on the contig, 5 EST markers (SHGC-33475, stSG13096, stSG8571, SHGC-60690, and stSG3997) were mapped between NP4 and AFMB317WF9 (Figure 1).
Mapping of TAT and LCAT genes against the contig
Markers SHGC-11957 and stSG1784, which were derived from TAT and LCAT gene sequences, respectively, were used to map these two genes against the MCD contig. The TAT gene was mapped on the contig between markers D16S3033 and D16S2624, just outside the MCD interval (Figure 1). The LCAT gene was not mapped on the contig. Using the Stanford radiation hybrid G3 panel, the LCAT gene was linked to SHGC-35392 (LOD score greater than 6), a marker that was outside the MCD region based on the Stanford radiation hybrid G3 map.
In this study, a complete genomic contig for the MCD region on chromosome 16 was constructed using a combination of YAC, PAC, and BAC clones. Nineteen polymorphic markers, 14 STS markers, and 15 EST markers have been mapped on the contig. Moreover, 18 novel STS markers were generated from the end sequences of the PAC and BAC clones (Table 1).
Using the mapped and ordered polymorphic markers, twenty-one affected individuals with MCD (16 type I, 5 type II) and their family members were genotyped for haplotype analysis. Of the twenty-one individuals with MCD, 12 have been included in a previous publication . In addition to sixteen previously reported markers in the region , the polymorphisms analyzed include two previously unmapped microsatellite markers (AFMB317WF9 and D16S395) that we recently mapped to our contig and one polymorphic CA repeat marker (NP4) developed in our laboratory by PAC sequencing. As shown in Figure 2, among 42 haplotypes of the disease-bearing chromosomes, five haplotypes were found that spanned the MCD region (haplotypes 1-5). The haplotypes in Figure 2 were named in the same way as in a previous publication . The haplotype 1 was the major haplotype presenting in its complete form in fourteen haplotypes, of which thirteen were from MCD type I patients and one was from a person with MCD type II. Two individuals with MCD type I were homozygotes for haplotype 1. The disease haplotypes 1a-1g, 1i and 1j could be distinguished from the putative haplotype 1 by one or two recombination events, among which haplotypes 1c and 1e were from the MCD type II patients. The haplotype 1 is therefore entirely or partly present in 29 disease chromosomes from both MCD types I and II patients. Assuming an independent segregation of the markers studied, the calculated frequencies of haplotypes 1 in the control population is only 3 x 10-11, indicating that this haplotype derives from a common ancestral founder chromosome along with the MCD mutation, with haplotypes 1a-1g, 1i and 1j being its resulting traces after ancestral recombination events. The haplotype 1f shows an ancestral recombination to NP4, and thus NP4 is the new centromeric boundary for the MCD type I locus. If one uses only MCD type I patients, the D16S3083 is still the telomeric marker as shown by haplotype 1d . However, if both MCD type I and type II are used, the haplotype 1e from the MCD type II patients would put AFMB317WF9 as the new telomeric marker for MCD gene. Thus, the use of the three newly mapped polymorphic markers in the current study enabled us to narrow the MCD critical region between markers NP4 and AFMB317WF9, an interval that is covered by only three overlapping PAC clones (p36D12, pp69E21, and p262N10). Five EST markers (SHGC-33475, stSG13096, stSG8571, SHGC-60690, and stSG3997) were mapped to this refined MCD critical interval (Figure 1), therefore, they would be good candidates for the MCD gene.
The finding that MCD type I and type II individuals share the same disease haplotype 1 in this study further support our previous observations [3,5-8], suggesting that MCD types I and II are unlikely to be independent entities and are probably phenotypic variations in the expression of the same gene. Currently, however, no allele sharing has been observed among haplotypes 1 to 5.
In the region of the MCD gene on the long arm of chromosome 16 are located the TAT and LCAT genes, which cause corneal lesions when mutated [9-12]. The TAT gene is responsible for tyrosinemia that is characterized by herpetiform corneal ulcers, and the LCAT gene causes fish-eye disease [11,12]. Since different phenotypes can be due to the same genetic entity, we evaluated these two genes as potential candidates for the MCD gene. However, the TAT and LCAT genes cannot cause MCD because both were mapped outside the MCD interval. It is noteworthy that three genes in a small region of chromosome 16q cause different corneal diseases. Efforts are currently being conducted to find new candidate genes in the region.
In summary, we have established a genomic contig for the region of the MCD gene. Based on the observation that MCD types I and II are likely to be allelic, haplotype analysis with new polymorphic markers has narrowed the gene to three overlapping PAC clones. This represents a significant refinement of the MCD candidate interval and should facilitate the ultimate cloning of the MCD gene.
We would like to thank the families who participated in this study and acknowledge the assistance of the personnel of the Center for Human Genetics at Duke University. This study was supported by NIH Grant R01-EY08249.
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