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Fine mapping of the GLC1K juvenile primary open-angle glaucoma locus and exclusion of candidate genes.

Sud A, Del Bono EA, Haines JL, Wiggs JL - Mol. Vis. (2008)

Bottom Line: Four candidate genes within the refined region were screened for biologically significant mutations using direct genomic sequencing: bone morphogenetic protein 2 (BMP2); phospholipase C beta 1 (PLCB1); phospholipase C beta 4 (PLCB4); and BTB POZ domain containing 3 (BTBD3).Biologically significant DNA sequence variants were not identified in the BMP2, PLCB1, PLCB4, or BTBD3 genes in these families.Four genes that are located within the refined region with attractive ocular expression and function have been excluded as causative genes for JOAG.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA, USA.

ABSTRACT

Purpose: Primary open-angle glaucoma is a leading cause of blindness worldwide. We previously identified a region on chromosome 20p12 associated with juvenile-onset primary open-angle glaucoma (JOAG) that was designated GLC1K. The aim of this study is to refine the boundaries of the GLC1K region and to screen selected candidate genes located within the refined region for biologically significant mutations.

Methods: Four JOAG families (44 individuals) with linkage to GLC1K were used for this study. Informative single nucleotide polymorphism (SNP) markers located throughout the previously defined region were used for haplotype analysis. Four candidate genes within the refined region were screened for biologically significant mutations using direct genomic sequencing: bone morphogenetic protein 2 (BMP2); phospholipase C beta 1 (PLCB1); phospholipase C beta 4 (PLCB4); and BTB POZ domain containing 3 (BTBD3).

Results: Haplotype analysis identified a new critical interval of 12.7 Mb using a combination of SNPs and microsatellite markers. This analysis extended the region of GLC1K from D20S846 to rs6081603 in affected individuals, and the region was further reduced to 9 Mb if unaffected recombinant individuals were included in the analysis. Biologically significant DNA sequence variants were not identified in the BMP2, PLCB1, PLCB4, or BTBD3 genes in these families.

Conclusions: Using recombinant breakpoint mapping and haplotypes based on a combination of SNP and microsatellite markers, the GLC1K region has been reduced to a maximum of 12.7 Mb and a minimum of 9 Mb. Four genes that are located within the refined region with attractive ocular expression and function have been excluded as causative genes for JOAG.

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Related in: MedlinePlus

GLC1K haplotype analysis in JOAG families. Haplotypes consisting of alleles for microsatellite repeat markers and SNPs are shown under each individual in the pedigrees. The boxed regions indicate the haplotype that segregates with the affected status. The small arrow identifies the location of a recombination event on the non-disease chromosome. The UCSC genome browser [27] was used to determine the location of the markers.
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f1: GLC1K haplotype analysis in JOAG families. Haplotypes consisting of alleles for microsatellite repeat markers and SNPs are shown under each individual in the pedigrees. The boxed regions indicate the haplotype that segregates with the affected status. The small arrow identifies the location of a recombination event on the non-disease chromosome. The UCSC genome browser [27] was used to determine the location of the markers.

Mentions: The GLC1K region was previously identified as a glaucoma gene locus by a microsatellite-based genome scan using a population of 25 multi-generational JOAG pedigrees (overall multipoint LOD score of 4.0) [26]. Haplotypes that were based on the microsatellite repeat markers used in the scan identified key recombination events in affected members in pedigree JOAG-52, a three-generation family with sufficient size and structure to establish independent linkage to the GLC1K region (maximum LOD score=3.2). These recombination breakpoints defined a 46 cM region extending from marker D20S846 to marker D20S891 in affected individuals and a 23 cM region extending from marker D20894 to marker D20878 if unaffected individuals were included in the analysis. To reduce the size of the critical region, 40 SNPs (single nucleotide polymorphisms) with minor allele frequencies of at least 40% were selected at approximately 100 Kb intervals throughout the previously defined GLC1K region [29]. Fifteen SNPs were informative for haplotype analysis, and alleles from these SNPs were evaluated for segregation in all members of pedigree JOAG-52 (Figure 1A). Haplotype analysis using the previous microsatellite alleles as well as the added SNP alleles identified recombination breakpoints that defined a new critical interval of 12.7 Mb, extending from marker D20S846 to marker rs6081603, a reduction of approximately 26 Mb. The critical recombination events defining the 12.7 Mb region occurred in individuals II-1 and II-3, both affected. A third recombination event occurred in individual III-5 who is unaffected at age 45. This recombination event would reduce the size of the region to 9 Mb, extending from marker rs1232605 to marker rs6081603. Because of the unknown penetrance of JOAG, we are using the most conservative measure for the size of the region of 12.7 Mb, which is based on the recombination events in individuals known to be affected.


Fine mapping of the GLC1K juvenile primary open-angle glaucoma locus and exclusion of candidate genes.

Sud A, Del Bono EA, Haines JL, Wiggs JL - Mol. Vis. (2008)

GLC1K haplotype analysis in JOAG families. Haplotypes consisting of alleles for microsatellite repeat markers and SNPs are shown under each individual in the pedigrees. The boxed regions indicate the haplotype that segregates with the affected status. The small arrow identifies the location of a recombination event on the non-disease chromosome. The UCSC genome browser [27] was used to determine the location of the markers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2480480&req=5

f1: GLC1K haplotype analysis in JOAG families. Haplotypes consisting of alleles for microsatellite repeat markers and SNPs are shown under each individual in the pedigrees. The boxed regions indicate the haplotype that segregates with the affected status. The small arrow identifies the location of a recombination event on the non-disease chromosome. The UCSC genome browser [27] was used to determine the location of the markers.
Mentions: The GLC1K region was previously identified as a glaucoma gene locus by a microsatellite-based genome scan using a population of 25 multi-generational JOAG pedigrees (overall multipoint LOD score of 4.0) [26]. Haplotypes that were based on the microsatellite repeat markers used in the scan identified key recombination events in affected members in pedigree JOAG-52, a three-generation family with sufficient size and structure to establish independent linkage to the GLC1K region (maximum LOD score=3.2). These recombination breakpoints defined a 46 cM region extending from marker D20S846 to marker D20S891 in affected individuals and a 23 cM region extending from marker D20894 to marker D20878 if unaffected individuals were included in the analysis. To reduce the size of the critical region, 40 SNPs (single nucleotide polymorphisms) with minor allele frequencies of at least 40% were selected at approximately 100 Kb intervals throughout the previously defined GLC1K region [29]. Fifteen SNPs were informative for haplotype analysis, and alleles from these SNPs were evaluated for segregation in all members of pedigree JOAG-52 (Figure 1A). Haplotype analysis using the previous microsatellite alleles as well as the added SNP alleles identified recombination breakpoints that defined a new critical interval of 12.7 Mb, extending from marker D20S846 to marker rs6081603, a reduction of approximately 26 Mb. The critical recombination events defining the 12.7 Mb region occurred in individuals II-1 and II-3, both affected. A third recombination event occurred in individual III-5 who is unaffected at age 45. This recombination event would reduce the size of the region to 9 Mb, extending from marker rs1232605 to marker rs6081603. Because of the unknown penetrance of JOAG, we are using the most conservative measure for the size of the region of 12.7 Mb, which is based on the recombination events in individuals known to be affected.

Bottom Line: Four candidate genes within the refined region were screened for biologically significant mutations using direct genomic sequencing: bone morphogenetic protein 2 (BMP2); phospholipase C beta 1 (PLCB1); phospholipase C beta 4 (PLCB4); and BTB POZ domain containing 3 (BTBD3).Biologically significant DNA sequence variants were not identified in the BMP2, PLCB1, PLCB4, or BTBD3 genes in these families.Four genes that are located within the refined region with attractive ocular expression and function have been excluded as causative genes for JOAG.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA, USA.

ABSTRACT

Purpose: Primary open-angle glaucoma is a leading cause of blindness worldwide. We previously identified a region on chromosome 20p12 associated with juvenile-onset primary open-angle glaucoma (JOAG) that was designated GLC1K. The aim of this study is to refine the boundaries of the GLC1K region and to screen selected candidate genes located within the refined region for biologically significant mutations.

Methods: Four JOAG families (44 individuals) with linkage to GLC1K were used for this study. Informative single nucleotide polymorphism (SNP) markers located throughout the previously defined region were used for haplotype analysis. Four candidate genes within the refined region were screened for biologically significant mutations using direct genomic sequencing: bone morphogenetic protein 2 (BMP2); phospholipase C beta 1 (PLCB1); phospholipase C beta 4 (PLCB4); and BTB POZ domain containing 3 (BTBD3).

Results: Haplotype analysis identified a new critical interval of 12.7 Mb using a combination of SNPs and microsatellite markers. This analysis extended the region of GLC1K from D20S846 to rs6081603 in affected individuals, and the region was further reduced to 9 Mb if unaffected recombinant individuals were included in the analysis. Biologically significant DNA sequence variants were not identified in the BMP2, PLCB1, PLCB4, or BTBD3 genes in these families.

Conclusions: Using recombinant breakpoint mapping and haplotypes based on a combination of SNP and microsatellite markers, the GLC1K region has been reduced to a maximum of 12.7 Mb and a minimum of 9 Mb. Four genes that are located within the refined region with attractive ocular expression and function have been excluded as causative genes for JOAG.

Show MeSH
Related in: MedlinePlus