A second locus (GLC3B) for primary congenital glaucoma (Buphthalmos) maps to the 1p36 region
A second locus ( GLC3B ) for primary congenital glaucoma (Buphthalmos) maps to the 1p36 regionA. Nurten Akarsu, M. Erol Turacli1, S. Gulderen Aktan1, Magda Barsoum-Homsy2, Line Chevrette2, B. Sitki Sayli3 and Mansoor Sarfarazi*
Surgical Research Center, Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut, USA, 1Departments of Ophthalmology and 3Medical Biology and Genetics, University of Ankara, Faculty of Medicine, Sihhiye, Ankara, Turkey, and 2Department of Ophthalmology, Sainte-Justine Hospital, University of Montreal, Montreal, Quebec, Canada
Received April 16, 1996;Revised and Accepted May 17, 1996
Primary congenital glaucoma (gene symbol: GLC3) is an ocular disorder that occurs for 0.01-0.04% of blind people. In the majority of familial cases reported so far, this condition is inherited as an autosomal recessive trait. We have recently used a group of 17 GLC3 families with a minimum of two affected offspring and consanguinity in most of the parental generation and mapped the first GLC3 locus (GLC3A) to the 2p21 region. Six families did not show any linkage to the GLC3A locus and thus provided evidence for genetic heterogeneity of this disorder. A total of eight families unlinked to the 2p21 region were used to search for the chromosomal location of the second GLC3 locus. Herein, we describe mapping of a new locus (designated GLC3B) for primary congenital glaucoma to the short arm of chromosome 1 (1p36.2-36.1) that is situated centromeric to the neuroblastoma and Charcot-Marie-Tooth type 2A (CMT2A) loci. A total of 17 DNA markers were genotyped from this region of chromosome 1. Four families showed no recombination with the two markers D1S2834 and D1S402 with a maximum lod score of 4.510 and 4.157 respectively. Pairwise and multipoint linkage analysis and inspection of the haplotypes revealed that the remaining four families are not linked to this part of chromosome 1, thus providing further evidence that at least one more locus for the autosomal recessive form of GLC3 must exist in the genome. Based on the recombination events, the overall linkage map of this region is: tel-D1S1192-D1S1635-D1S1193 - (D1S1597/- D1S489/D1S228) - [GLC3B/D1S2834/D1S402] - (D1S1176/ D1S507/D1S407) - D1S2728-(MFAP2/D1S170) - D1S1368 - D1S436-D1S1592-cen.
Primary congenital or infantile glaucoma (gene symbol: GLC3) is a rare inherited eye disorder that results from an isolated maldevelopment of the trabecular meshwork (1 ). This condition usually manifests itself within the first year of life with a typical presentation of tearing, photophobia and clouding of the cornea (2 ). Numerous research studies have been undertaken to elucidate the pathogenesis of the GLC3 phenotype, but to date the exact cause and nature of this disorder have remained unknown. However, it is certain that a genetic component plays a major role in the outcome of this developmental disease. Although it has been indicated that different modes of inheritance may be involved in the transmission of this disorder, autosomal recessive inheritance has been confirmed in a significant proportion of cases (3 -5 ). GLC3 is four times more frequent in the Middle Eastern population as compared with the Western population (6 ,7 ). Population studies, together with complex segregation analysis in the families, suggested the existence of both genetic and etiological heterogeneity of the GLC3 phenotype (8 ,9 ).
Recently, we used a group of 17 Turkish families with the autosomal recessive form of primary congenital glaucoma and mapped the first GLC3 locus to chromosome 2p21 (10 ). Haplotype and heterogeneity analyses provided evidence for genetic heterogeneity of this condition. The proportion of linked families was estimated to be 85%, thus indicating that the GLC3 locus on 2p21 (designated as GLC3A) is a major locus for this phenotype. In an earlier study, we excluded the GLC3 locus from other candidate regions of 6p21, 6p25 and the entire chromosome 11 in the same group of families (11 ). In this study, we used eight families that were unlinked to 2p21, 6p21, 6p25 and the entire chromosome 11 and searched the remaining part of the genome to identify the second GLC3 locus. Herein, we describe the localization of this locus (i.e. GLC3B) on chromosome 1p36.2-p36.1 and provide evidence for a third unmapped locus for this condition.
In our previous study, we genotyped a total of 126 markers from 17 different chromosomes before a significant linkage was found with the GLC3A locus on 2p21 (10 ). Once linkage was found and genetic heterogeneity established, we re-analyzed the two-point linkage data and tested for haplotype transmission in each of the six original unlinked families (10 ). When a given chromosomal region was identified for which a group of loosely linked DNA markers showed a small positive lod score, saturation mapping of that region was achieved using all known flanking PCRable markers. The first two candidate regions from chromosome 5 and 2failed to detect any linkage using this approach. However, other markers from the 1p36 region (D1S1635, D1S228, D1S507, D1S407 and D1S1368) were found to segregate conspicuously with the disease phenotype in four of our families (Fam 1, 4, 14 and 28). Figure 1 1 illustrates the segregation of the disease with DNA markers from the 1p36 region in a typical three generations of an autosomal recessive GLC3 pedigree (Fam 4). The remaining four families (Fam 6, 7, 20 and 501) showed no linkage to this region of chromosome 1 by both pairwise and multipoint linkage analyses. Haplotype analysis also revealed that the affected individuals in these pedigrees received completely different haplotypes from their respective parents and hence provided further evidence for genetic heterogeneity of this disorder. Therefore, we excluded the four unlinked families from further analysis and designated the locus on the 1p36 region as `GLC3B' (new OMIM # 600975). Thereafter, a total of 17 markers from 1p36.2-1p36.1 were used to saturate this region. For the linked families, positive lod scores were obtained with all the DNA markers studied. The two markers D1S2834 and D1S402 showed no recombination in the linked families and provided the maximum lod score values of 4.510 and 4.157 respectively. The result of pairwise linkage analysis for markers that provided the maximum linkage information is presented in Table 1 .
To localize further the position of the disease gene, haplotype analysis was achieved according to the observed recombination events. The information on the order of DNA markers was obtained from the Genome Data Base (12 ) or previously published genetic linkage maps of Genethon (13 ,14 ), Utah (15 ) and Cooperative Human Linkage Center (CHLC; 16 -18 ). In order to combine the marker information from different maps, D1S228 and D1S507 were used as anchor markers. By using the recombination events in our family panel, we positioned D1S170 centromeric to D1S407. In one of our families (Fam 28), null alleles were observed with markers D1S402 and D1S1176, as previously reported by the Utah group (15 ).
With regard to the affection status, one single recombination in an affected individual (Fam 1, person II-3) positioned the disease locus telomeric to D1S2728 and centromeric to D1S1193. Further recombinations in two normal individuals (Fam 4, IV-2 and Fam 28, II-1) localized the GLC3B locus between the (D1S1597/ D1S489/D1S228) and (D1S1176/D1S507/D1S407) markers and within an ~3 cM distance (Fig. 2 ). The status of one of these normal individuals (Fig. 1 1; Fam 4, person IV-2) is confirmed as a proven gene carrier since she has already produced an affected offspring. The other individual has been clinically examined and proven to be normal for primary congenital glaucoma. In order to obtain maximum linkage information, the following group of markers were consolidated and used as a haplotype to calculate lod score: (D1S1597/D1S489/D1S228), (D1S2834/D1S402) and (D1S1176/D1S507/D1S407). Three-point linkage analyses were performed using this haplotype conformation in both linked and unlinked families (Fig. 3 ). The lod score values obtained from all the eight families were used with the HOMOG program and tested for genetic heterogeneity. As predicted by the two-point linkage and inspection of the constructed haplotypes, highly significant evidence ([chi]2 = 11.5, P = 0.0003, likelihood ratio = 314.66) for the existence of genetic heterogeneity was obtained. Furthermore, multipoint linkage analysis using LINKMAP in the linked families confirmed that the GLC3B locus is centromeric to DNA markers (D1S1597/D1S489/D1S228) and telomeric to DNA markers (D1S1176/D1S507/ D1S407), within an estimated region of ~3 cM (Fig. 3 ). Since no recombination has been observed with DNA markers D1S2834 and D1S402, the GLC3B locus may be located on either side of these two markers.
We have identified the chromosomal localization of a second locus for primary congenital glaucoma (GLC3B) in the 1p36.2-1p36.1 region. This GLC3B locus is confined within a 3 cM interval that is flanked by two groups of tightly linked markers of (D1S1597/D1S489/D1S228) and (D1S1176/D1S507/ D1S407). The maximum lod score values were obtained with two closely linked markers, D1S2834 (Z = 4.510) and D1S402 (Z = 4.157) that showed no recombination with the disease condition in four families (Table 1 ). The haplotype and heterogeneity analyses revealed that the remaining four families are not linked to this region of chromosome 1 and thus implied that at least one more locus for the autosomal recessive form of primary congenital glaucoma must exist in the genome. Overall analysis of the recombination events in our group of families suggested the most likely order of markers to be: tel-D1S1192-D1S1635- D1S1193- (D1S1597/D1S489/D1S228)-[GLC3B/D1S2834/D1S402](D1S116/D1S507/D1S407)-D1S2728-(MFAP2/D1S170)-D1S1368-D1S436-D1S1592-cen. The exact position of D1S1193 in relation to the above mentioned map was not known. However, four different recombinants in three of our families indicated that the position of this marker is most likely to be between D1S1635 and (D1S1597/D1S489/D1S228).
The 1p36 region has been subjected to an intensive investigation and cloning bias, as many tumor suppresser genes are known to be located in this region (19 ). This part of the genome has also merited attention because it contains a high concentration of G + C-rich DNA, suggesting an accumulation of genes in this portion of the chromosome (20 ). A large number of genes have been reported in the 1p36.2-36.1 band, but none of these can be attributed directly as a possible cause of congenital glaucoma. Moreover, the 1p36 region has frequently been involved in chromosomal aberrations that resulted in various malignancies, but none of these abnormalities have been reported to segregate with the congenital glaucoma phenotype. The GLC3B locus identified in this study maps centromeric to Charcot-Marie-Tooth Type 2a (CMT2A) and neuroblastoma loci (21 ,22 ). The information obtained from the CMT2A study suggested that this region is a hot spot for recombination, and the actual physical distances in this region of chromosome 1 may be much less than the estimated distances from the genetic linkage maps (~10 cM of genetic linkage map corresponds to 1 Mb of DNA in physical distance) (21 ). Therefore, the physical location of the GLC3B locus is expected to be considerably less than the estimated genetic distance of ~3 cM. Using the existing STS and contig maps of this region (23 ), work is currently in progress to search for a candidate gene that may be involved in the etiology of this condition.
The kindreds included in this study are a subgroup of 86 primary congenital glaucoma families that originated from Turkey and Canada (5 ,24 ). Their clinical description (5 ,24 ), pedigree structure and their linkage inclusion criteria have been published previously (10 ,11 ). The family panel used in this study consisted of seven Turkish and one Canadian kindred. Six of the Turkish families (i.e. Fam 1, 4, 6, 7, 14 and 20) were used in our previous study and proved to be unlinked to the GLC3A locus on 2p21 (10 ). A new Turkish family (Fam 28 with two affected and one normal sib) and a Canadian family (Fam 501 with two affected sibs) were tested for a possible linkage to the first locus on 2p21. However, both linkage information and haplotype analysis with the flanking markers of D2S1788/D2S1325 and D2S1356 proved that these two families are also not linked to the GLC3A locus. Taken together, this panel of eight families provides a total of 37 offspring, 17 of whom are affected with the GLC3 phenotype.
Genomic DNA was amplified with sequence-specific primers using the polymerase chain reaction (PCR) technique. Information on primer sequences, number of alleles, band sizes and type of polymorphisms was obtained from the Genome Data Base (GDB, 12 ), Genethon (13 ,14 ), Utah Marker Development Group (15 ) and Cooperative Human Linkage Center (CHLC; 16 -18 ). A total of 25 [mu]l was used per reaction, which included 1* PCR buffer (10 mM Tris-HCl with various concentrations of MgCl2, pH 8.4, 50 mM KCl, 0.01% gelatin and 0.1% Triton), 0.25 [mu]M of each primers, 100 ng of template DNA, 0.2 mM of each dNTP and 0.25 U of Taq polymerase (Amplitaq-Perkin Elmer). Although the amplification conditions varied with different markers, we constantly used an initial denaturation at 94oC for 2 min which was followed by a two-cycle PCR amplification of 94oC for 10 s denaturation and 54-60oC annealing temperature for a total of 30-32 cycles. The amplified products were separated by electrophoresis on 6-7% denaturing polyacrylamide gels. The gels were silver stained (25 ), genotyped and thereafter photographed manually.
A dedicated computer program (D.M.S., unpublished) was used for data entry, error checking and preparation of input files for the LINKAGE package (26 ,27 ). Two-point linkage analysis was calculated using MLINK component (FASTLINK version 2.20) of the LINKAGE package. Lod scores were obtained under the assumption of an autosomal recessive disorder with full penetrance. Multipoint linkage and heterogeneity analyses were performed using the LINKMAP module of LINKAGE and HOMOG (28 ) programs respectively.
We would like to convey special thanks to the members of our families for their participation in this study. We thank Dr Engin Yilmaz for his help in extracting DNA and Dr Altaf Hossain for his initial genotyping. This work was funded in part (to M.S.) by the `NATO Collaborative Research Grant', `National Glaucoma Research, The American Health Assistance Foundation', `Knights Templar Eye Foundation', `National Institutes of Health (National Eye Institute) grant #EY11095' and by the University of Connecticut General Clinical Research Center (M01-RR-06192). M.S. would also like to thank the International Glaucoma Association for their continuous help and financial support. A.N.A. is a recipient of a postdoctoral research fellowship supported by `The Theodore N. Garbis Research Fund of the Fight For Sight research division of Prevent Blindness America'.
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