Molecular Vision 1999; 5:38 <http://www.molvis.org/molvis/v5/p38/> Received 26 February 1999 | Accepted 27 December 1999 | Published 29 December 1999

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North Carolina macular dystrophy (MCDR1) locus: A fine resolution genetic map and haplotype analysis

Kent W. Small,¹ Nitin Udar,¹ Svetlana Yelchits,¹ Ronald Klein,² Charlie Garcia,³ Guillermo Gallardo,³ Bernard Puech,⁴ Virginie Puech,⁴ David Saperstein,⁵ Jennifer Lim,⁵ Julia Haller,⁶ Christina Flaxel,⁷ Rosemary Kelsell,⁸ David Hunt,⁸ Kevin Evans,⁸ Felicia Lennon,⁹ Margaret Pericak-Vance⁹
 
 

¹Department of Ophthalmology, the Jules Stein Eye Institute, University of California, Los Angeles, CA; ²University of Wisconsin; ³University of Texas, Houston, TX; ⁴Centre Hospitalier Regional Universitaire de Lille, France; ⁵Emory Eye Center, Emory University, Atlanta, GA; ⁶Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD; ⁷University of West Virginia, Morgantown, WV; ⁸Department of Molecular Genetics, Institute of Ophthalmology, University College London, London, UK; ⁹Section of Medical Genetics, Duke University Medical Center, Durham, NC

Correspondence to: Kent W. Small, M.D., 200 Stein Plaza, UCLA, Los Angeles, CA, 90095; Phone: (310) 206-7475; FAX: (310) 794-7904; email: small@jsei.ucla.edu

Abstract

Purpose: We previously reported linkage of North Carolina macular dystrophy in a single isolated family to a broad region on chromosome 6q16. In order to refine the localization of the MCDR1 gene (North Carolina macular dystrophy), additional families with this disease and new markers were studied.

Methods: We ascertained 10 families with the North Carolina macular dystrophy phenotype (MCDR1). These families were of various ethnic and geographic origins such as Caucasian, Mayan Indian, African-American, French, British, German, and American of European decent. Two hundred thirty-two individuals in these families underwent comprehensive ophthalmic examinations and blood was collected for genotyping. One hundred seventeen were found to be affected. Linkage simulation studies were performed. Two-point linkage, haplotype analysis, and multipoint linkage was performed using VITESSE and FASTLINK. HOMOG was used to test for genetic heterogeneity.

Results: The clinical features were consistent with the diagnosis of North Carolina macular dystrophy in all families. Multipoint linkage analysis indicates that the MCDR1 gene is in the interval between D6D249 and D6S1671 with a maximum LOD score of 41.52. There was no evidence of genetic heterogeneity among the families studied. Families 765, 768, 772, 1193, and 1292 shared the same chromosomal haplotype in this region.

Conclusions: This is the largest single data set of families with the MCDR1 phenotype. The single large family from North Carolina continues to be informative for the closest flanking markers and alone supports the minimal candidate region as suggested by previous studies. There remains no evidence of genetic heterogeneity in this disease. Most of the American families appear to have descended from the same ancestral mutation. The remaining families could each represent independent origins of the mutation in the MCDR1 gene.

Introduction

North Carolina macular dystrophy is an autosomal dominant inherited trait consisting of congenital or infantile onset of a macular degeneration that tends to not progress [1-11]. The fundus appearance can vary considerably between individuals even within the same family. Approximately a third of affected individuals have normal vision with only drusen centrally (20/20 - 20/25). Approximately a third have the appearance of confluent drusen moderate impairment of central vision (20/20-20/40) and a third have colobomatous-appearing or disciform-appearing lesions with moderate impairment if central vision (20/40-20/800). The original report of this disease by Lefler, Wadsworth, and Sidbury described the descendants of a large family that had settled in the mountains of North Carolina in the 1800s [2]. This report called the disease "dominant macular degeneration and aminoaciduria." A subsequent report of the same family by Frank and associates revealed that the aminoaciduria was in fact unrelated to the macular degeneration and renamed the disease "dominant progressive foveal dystrophy" [3]. Subsequently, families with "central areolar pigment epithelial dystrophy" and "autosomal dominant central pigment epithelial and choroidal degeneration" were shown to be descendents of the original North Carolina family [5,6,8-10]. These early studies were limited by the size of the ascertained families. The diverse names given this one disease reflect in part, the wide phenotypic variability.

The disease-causing gene was linked to chromosome 6q16 by Small and associates [12]. The Human Genome Organization named this disease locus MCDR1 (MC = macular, D = dystrophy, R = retinal, 1 = first macular degeneration genetically mapped). Originally, the markers linked to MCDR1 were crudely localized, were all centromeric to the gene and were in an area of the human genome where no other markers were available for 20 cM. The only subsequent report of linkage studies in the original North Carolina family defined the telomeric cross at marker D6S283 [13].

Linkage studies in newly reported families have been reported by Small et al. and others and have narrowed the interval [14-18]. A family (1463) studied by Rabb and Small et al. from Belize, Central America, showed linkage to the region flanked by D6S501 centromerically and D6S475 telomerically [16]. Studies by Small et al. in a Texan family (768) found linkage to the region flanked by D6S492 and D6S468 [14]. A family from the United Kingdom with the MCDR1 phenotype defined the critical region flanked by D6S251 and D6S468 [18]. In Germany 3 families were reported with the MCDR1 phenotype and were found to share a common haplotype flanked by the markers D6S249 and D6S475 [17]. Small et al. studied a family with the MCDR1 phenotype from France (769) that defined the minimal candidate region to D6S424-D6S1671 [15]. The marker D6S1671 remains the closest telomeric cross. No subsequent studies of the original North Carolina family (765) have been reported. A graphical summation of all previous and current MCDR1 reports is shown in Figure 1.

Herein, we report the accumulation of the genetic linkage data obtained from 10 families with this disorder. Five of these families have not been previously reported and in the remaining 5 families, new genetic markers were studied.

Methods

We ascertained and examined 232 individuals from ten families with the MCDR1 phenotype (Table 1). Seven of these families were American (families 765, 768, 1193, 1292, 771, 770, 772) and primarily located in North Carolina, Texas, Illinois, Washington DC, West Virginia, Wisconsin, and South Carolina respectively. One family was of Mayan Indian decent living in Belize, Central America. Another family was from northern France in the Flanders region. One family was from London. All were Caucasian except for the family from Chicago, Illinois (1193) that was African-American and the Mayan Indian family in Belize (1463). All of these families were thought to be genealogically independent at the time of ascertainment. Institutional review board approved signed consents were obtained from all subjects. Blood samples were collected in EDTA tubes. The DNA was extracted using a Purgene kit (Gentra systems, Minneapolis, MN).

Genotyping was performed with modifications to the method described by Weber and May [19]. Primers were end labeled with [gamma]³³P-dATP (NEN, Boston, MA). The DNA samples were subjected to PCR for 18 to 20 cycles. The alleles were separated on a denaturing 6% polyacrylamide gel (USB, Cleveland, OH), 7M Urea (USB) and electrophoresed at constant power. The standard sequence ladder (plasmid-PUC18) was prepared as recommended by the manufacturer using the Thermo Sequenase Kit (Amersham, Piscataway, NJ). The PCR conditions were as follows. Denaturation was at 94 °C for 2 min, followed by 18 cycles of 94 °C for 30 min, 57 °C for 30 min, 72 °C for 30 min. The reaction was allowed to extend for 72 °C for 10 min and then maintained at 4 °C. Polymorphic markers in the chromosome 6q16.3 region were tested. The fragments were then separated on a polyacrylamide gel by electrophoresis. Five microliters of formamide dye solution (formamide 10 ml, xylene cyanol FF 10 mg, bromophenol blue 10 mg, 0.5 M EDTA (pH 8.0) 200 µl) were added to the PCR mix and denatured at 94 °C for 4 min and immediately put on ice. Five microliters of this were then loaded onto a 6% polyacrylamide (29:1 acrylamide:bisacrylamide) gel containing 7M Urea on a 38 x 50 cm sequencing gel apparatus (BioRad, Hercules, CA) and run at 80W constant power. A pUC18 plasmid and -40 M13 forward (23-mer) primer were used in a separate sequencing reaction (Thermo Sequenase, USB) and run on the same gel as a standard ladder. The gel was then transferred to a blotting paper backing, dried, and exposed to autoradiograph film. Allele sizes were determined by comparing it to the standard ladder.

MCDR1 was analyzed as a completely penetrant, autosomal dominant trait with a disease allele frequency of 0.00001. The maximum possible LOD score for each pedigree was obtained via computer simulation with the SIMLINK program [20]. Simulation studies were performed using 5000 replicates. Allele frequencies for the polymorphic markers were determined using a minimum of 70 unrelated spouses from the MCDR1 families (Duke Center for Human Genetics). This genetic map information was based on current genetic maps in the internet databases (The Sanger Centre, Human Chromosome 6 Home and Marshfield Center for Medical Genetics) as well as from our unpublished data from our physical map. The physical map data consists of information from YACs, BACs, PACs and, Radiation Hybrid maps (data not shown). VITESSE and FASTLINK computer packages were used assuming equal recombination rates in males and females [21,22]. For two-point LOD scores greater than 3.0, a 1-LOD unit down support interval was calculated as an approximation to a 95% confidence limit [20]. The family from the United Kingdom was not used in the multipoint analysis but was used in the haplotype analysis. The entire family's DNA was unobtainable for this study [18]. The reason for using the UK family in this study was to search for shared haplotypes.

Results

The original description of the disease phenotype was in a single family from North Carolina. With the additional families described herein and by others, it is apparent that the disease is present in many different locations throughout the world and ethnic groups. Table 1 shows the ethnicity and the location of the various families that have been ascertained.

The clinical findings were identical to that previously reported [1-11]. Affected individuals in these families ranged in age from 2 months to 48 years. The family from Wisconsin was not large enough to observe the most severe forms of the disease such as the grade 3 macular coloboma-like lesion [8]. Therefore, there is a possibility that this family represents a phenocopy such as dominant drusen.

Most of the subjects had good visual acuity, the range being from 20/20 to 20/100. All individuals with grade I findings had visual acuities ranging from 20/20 to 20/25. Only those with grade 2-3 findings had visual acuities of 20/40 or worse.

The order of the markers and the genetic distances between them are shown in Figure 1. A summary of the actual two-point LOD scores of the analyzed markers in these families is shown in Table 2. The maximum two-point LOD scores actually achieved were for markers D6S249 and D6S1671 with LOD scores of 32.00 and 35.71 respectively. Multi-point analysis in families 765, 768, 1463, 771, 769, 772, 1193, 1292 generated a maximum LOD score of 41.52 when MCDR1 was placed between D6S249 and D6S6171 (Figure 2). There were no recombinations observed with marker D6S1717.

Analysis for genetic heterogeneity in the multipoint analysis using HOMOG (Copyright (C) Jurg Ott 1999, available at Genetic Linkage Analysis at Rockefeller University) showed that there was no evidence of genetic heterogeneity (Chi square = 28.68, p<0.0001, likelihood odds = 14.33, square difference = 1,700,000). Odds in favor of homogeneity versus heterogeneity were 1689258:1.

Several recombination events in the families support the closest flanking markers as shown with multipoint analysis. (Table 3). The closest telomeric crosses were with marker D6S1671 seen in family 765, individual 8125, and family 1463, individual 9034. The closest centromeric cross is observed in family 765, individual 8151, who crosses with D6S249. Thus, both the closest centromeric and telomeric crosses were observed in the original North Carolina family 765.

Haplotype analysis of the data (Figure 3) suggests that families 765, 768, 772, 1292, and 1193 share the same haplotype. The affected haplotype (1-1-1-4-1) was not observed in any unaffected individuals. Each of the other families is most likely a separate haplotype and therefore represents separate independent mutations.

Discussion

Our earlier reports had localized the MCDR1 gene to a broad area on chromosome 6q16 [12,13]. Herein, we show the accumulation of the largest single data set from multiple families with the MCDR1 phenotype and have confirmed the genetic interval to be between D6S249 and D6S1671. Previously, the closest telomeric cross was defined by the French family (769) and the closest centromeric cross defined by the North Carolina family [15]. Herein we show that the original North Carolina family remained the most informative and provided the key recombinant individuals that narrowed the genetic interval both centromerically and telomerically. This is the only single MCDR1 family to achieve this. All of the other families, in our data set as well as in other's, provided no additional information that was useful for narrowing the interval.

Haplotype analysis showed interesting potential ancestral relationships among the different families (Figure 3). Of the 10 families reported herein, 5 have not been previously reported. Most of the American families appeared to have a single ancestral mutation in their MCDR1 gene. These American families shared the rare disease-associated allele #1 for D6S249 on the centromeric end and the rarer allele #1 for D6S1671 on the telomeric end as well as the same alleles of all markers in between. This suggests a common founder of the disease causing gene and thus likely harbor the same mutation. Initially, there was no known genealogical relationship between the North Carolina, Texas, South Carolina, Washington D.C., and Chicago families after obtaining extensive family history information. All of these families were Caucasian except for the family from Chicago which was African-American. Because racial admixture occurred in the Southern United States before and after the Civil War, this racial difference should not deter one from considering that these five families have a common ancestor. Additionally, there was a period after the Civil War when immigration did occur from the mountains of North Carolina to Texas. This may account for the common ancestor between the North Carolina and the Texas families. Family 772 lived in the foothills of South Carolina about 100 miles from the North Carolina family. Additionally, they shared the same last name as the original founding brothers in the North Carolina family. While there was no direct genealogical tie made between the South Carolina and the North Carolina families, it was not surprising to find that they shared the common disease associated haplotype.

The remaining families could each potentially represent independent origins of mutations in the MCDR1 gene. The haplotype sharing in these families was not so clear because in most instances the shared alleles were commonly occurring in the general population. The potential relationship of the Belize family with the British and West Virginia families was obscure. It appeared that part of the disease associated haplotype was shared among these three families. Because Belize was a British colony from the mid-1800s until 1973, it is conceivable that this shared haplotype could be due to a common founder. However, the affected family in Belize is of Mayan Indian ancestry. The West Virginia family had possible partial sharing of the disease associated haplotype but this was more difficult to explain unless there was a common British founder for both families from Belize and West Virginia. While this is possible, the assumptions may be too excessive to rely on this apparent haplotype sharing to base positional cloning strategies.

North Carolina macular dystrophy (MCDR1) was originally named because of the founder effect that concentrated the mutated gene in the mountains of North Carolina [4]. There was very little consanguinity in the family. In the 1800s, families in this agricultural region typically had many children as did the founders of our North Carolina family (765). Therefore, the increased frequency of the disease allele in this population is primarily because of the large and stable families there. Because of the disease's name, many clinicians and researchers have assumed that this was an extremely rare entity present in only one family in the world. Small et al. demonstrated that the previously published North American individuals affected with North Carolina macular dystrophy did indeed emanate from the same family in North Carolina [10]. Recently, Small et al. reported unrelated families in France and Belize with the MCDR1 phenotype [15,16]. Others have reported families in the UK and in Germany [11,17,18]. The name, MCDR1, as established by the Human Genome Organization, seems more appropriate than North Carolina macular dystrophy [12].

The MCDR1 gene is important to understand for several reasons. This disease seems to be macula-specific, as evidenced by the funduscopic examination as well as the lack of electroretinogram and electro-oculograms abnormalities. Therefore, understanding this gene's function will give insight into specific features of macular function and dysfunction. Additionally, the MCDR1 phenotype appears in some aspects similar to age-related macular degeneration, the most common cause of blindness in the elderly American population. Therefore, understanding the MCDR1 gene may shed light on our understanding and possible management of age-related macular degeneration.

Acknowledgements

This work was supported in part by NIH/NEI RO1-EY12039 (Dr. Small), and The McCone Endowment (Dr. Small). Parts of this manuscript were used by Dr. Small in a thesis requisite for gaining membership into the American Ophthalmological Society.

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