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Biallelic mutation of protocadherin-21 (PCDH21) causes retinal degeneration in humans.

Henderson RH, Li Z, Abd El Aziz MM, Mackay DS, Eljinini MA, Zeidan M, Moore AT, Bhattacharya SS, Webster AR - Mol. Vis. (2010)

Bottom Line: No color vision was detected in any proband.The fundus appearance included the later development of characteristic circular patches of pigment epithelial atrophy at the macula and in the peripheral retina.Biallelic mutations in the photoreceptor-specific gene PCDH21 cause recessive retinal degeneration in humans.

View Article: PubMed Central - PubMed

Affiliation: Moorfields Eye Hospital, London, UK.

ABSTRACT

Purpose: To describe the clinical findings and mutations in affected members of two families with an autosomal recessive retinal dystrophy associated with mutations in the protocadherin-21 (PCDH21) gene.

Methods: A full genome scan of members of two consanguineous families segregating an autosomal recessive retinal dystrophy was performed and regions identical by descent identified. Positional candidate genes were identified and sequenced. All patients had a detailed ophthalmic examination, including electroretinography and retinal imaging.

Results: Affected members of both families showed identical homozygosity for an overlapping region of chromosome 10q. Sequencing of a candidate gene, PCDH21, showed two separate homozygous single-base deletions, c.337delG (p.G113AfsX1) and c.1459delG (p.G487GfsX20), which were not detected in 282 control chromosomes. Affected members of the two families first reported nyctalopia in late teenage years and retained good central vision until their late 30s. No color vision was detected in any proband. The fundus appearance included the later development of characteristic circular patches of pigment epithelial atrophy at the macula and in the peripheral retina.

Conclusions: Biallelic mutations in the photoreceptor-specific gene PCDH21 cause recessive retinal degeneration in humans.

Show MeSH

Related in: MedlinePlus

Electropherograms of the mutations detected in Protocadherin-21 (PCDH21). Panel A illustrates the index c.338delG mutation in family 1; the wild-type (WT) sequence is displayed on the top row. The middle row shows the proband in family 1 (IV-2) with a homozygous (HM) c.338delG change, illustrated with a dash for the missing nucleotide when aligned with the wildtype sequence. The bottom row displays an unaffected parent of family 1 (III-3) with the c.338delG change in the heterozygous state (Het): the latter section of the heterozygous electropherogram shows two superimposed sequences due to the synchronous addition of nucleotides due to two distinct DNA templates derived from the wild type and the shorter mutant alleles of the heterozygote. Panel B illustrates the second mutation that was identified in PCDH21, in family 2. The top row displays the control individual with the wildtype (WT) allele; while the bottom row displays the affected proband (II-1) with a homozygous (HM) c.1463delG variant.
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f2: Electropherograms of the mutations detected in Protocadherin-21 (PCDH21). Panel A illustrates the index c.338delG mutation in family 1; the wild-type (WT) sequence is displayed on the top row. The middle row shows the proband in family 1 (IV-2) with a homozygous (HM) c.338delG change, illustrated with a dash for the missing nucleotide when aligned with the wildtype sequence. The bottom row displays an unaffected parent of family 1 (III-3) with the c.338delG change in the heterozygous state (Het): the latter section of the heterozygous electropherogram shows two superimposed sequences due to the synchronous addition of nucleotides due to two distinct DNA templates derived from the wild type and the shorter mutant alleles of the heterozygote. Panel B illustrates the second mutation that was identified in PCDH21, in family 2. The top row displays the control individual with the wildtype (WT) allele; while the bottom row displays the affected proband (II-1) with a homozygous (HM) c.1463delG variant.

Mentions: Genome-wide linkage scans were performed to identify regions of autozygosity (where both alleles are identical by descent and are copies of a common ancestral gene). DNA samples from all affected family members were genotyped using the Affymetrix human GeneChip® Mapping Array (version Xba142 2.0) for family 1 and the GeneChip® Human Mapping 100 K set array for family 2 (Affymetrix, Santa Clara, CA). The detailed methodology for genotyping using the GeneChip® array has been previously described [4]. Briefly, 250 ng of genomic DNA was digested with XbaI (New England Biolabs, Ipswich, MA) for 2 h at 37 °C, ligated with XbaI adaptors using T4 DNA ligase (New England Biolabs). The ligation reaction was diluted in 1:4 (vol/vol) with molecular grade water (Sigma-Aldrich, St. Louis, MO) to 100 µl. Ten µl of the diluted ligation mix was used to setup selection by PCR (Fragment Selection by PCR) in triplicates. The pooled PCR products were purified using QIAGEN MinElute 96 UF plate (Qiagen, Duesseldorf, Germany). The concentration of PCR products was quantified using ND-1000 Nanodrop spectrometry (Thermo Fisher Scientific, Waltham, MA). Purified PCR product (90 ng) was fragmented with 0.25 units DNaseI ('Fragmentation Reagent'; Affymetrix), labeled with 'Labeling Regent' (Affymetrix). The labeling reaction (70 µl) was mixed with 190 μl hybridization reagent and denatured at 99 °C for 10 min (all as detailed in the Affymetrix GeneChip Mapping 100K Assay Manual). Finally the denatured hybridization mixture was injected into Affymetrix Human Mapping XbaI chips and incubated at 48 °C for 16 h, followed by automatic washing and staining in a Fluidics Station 450, and scanned by using the GeneChip® Scanner 3000 7G (Affymetrix). Primers for direct sequencing were as described by Bolz et al. [5]. PCR amplification was performed using a 20-µl reaction mix, including Abgene Reddymix with or without dye (AB795, AB793; Abgene, Epsom, UK). Ten microliters of Reddymix was combined with 5 µl of deionized water, 2 µl (10 pmol) of forward and reverse primer, and 1 µl (50–100 ng) of genomic DNA. All exons were optimized and amplified at 55 °C. DNA fragments were purified using Montage PCR cleanup plates, according to the standard protocol (Millipore, Watford, UK). PCR cleanup (purple plates) was performed using 15 μl of PCR product made up to 100 μl with deionized water (ddH2O) and transferred to the purple plates. Ten min of vacuum was applied to the plates until the wells were empty. Twenty five μl of ddH2O was added to each well and a further 3 min vacuum was applied. Twenty μl of ddH2O was added to each well; the plate covered and vortexed at 1,000 rpm for 10 min. The remaining contents of each well (clean PCR product) was then transferred for storage. The Big Dye terminator cycle sequencing reaction was then performed using a 10-µl reaction mixture containing 0.5 µl of BigDye v3.1 Applied Biosystems™ (ABI Ltd, Warrington, Cheshire. UK), 2 µl of PCR product, 1.5 µl of 5 picomolar (pM) forward or reverse primer, 3.5 µl of ddH2O, and 2.5 µl of sequencing buffer (ABI Ltd) containing 5x Tris-HCl and MgCl2. The sequencing reaction mixture was purified using the Montage sequence cleanup plate (Millipore, Watford, UK): 25 μl of injection solution (Millipore,) were added to each well, and the total 35 μl was transferred to the blue plate and vacuumed for 3 min. A further 25 μl of injection solution was added to each well and vacuumed again for 4 min. Twenty five μl of injection solution was again added to each well, the plate covered and vortexed for 10 min. The remaining volume was transferred to the sequencing plate and run on the ABI 3700 sequencer. The DNA sequence was analyzed using DNAStar® Inc. (Madison, WI). To check segregation of the mutant alleles with disease, restriction digests were performed. Family 1 was investigated using HaeIII enzyme (Figure 2; New England Biolabs Ltd, NEB, Hitchin, Herts, UK), while in family 2 a digest using an E. coli strain that carries the BglI gene from Bacillus globigii (BglI [NEB]) was performed. One unit (0.2 μl) of enzyme was combined with 2 μl of 1x NEBuffer 3 (NEB, Herts, UK), 10 μl of PCR product and brought to a 20 μl reaction volume with deionized H2O (ddH2O) and digested for 16 h at 37 °C. Heat inactivation was performed at 80 °C for 20 min and the product run out on a 5% agarose gel.


Biallelic mutation of protocadherin-21 (PCDH21) causes retinal degeneration in humans.

Henderson RH, Li Z, Abd El Aziz MM, Mackay DS, Eljinini MA, Zeidan M, Moore AT, Bhattacharya SS, Webster AR - Mol. Vis. (2010)

Electropherograms of the mutations detected in Protocadherin-21 (PCDH21). Panel A illustrates the index c.338delG mutation in family 1; the wild-type (WT) sequence is displayed on the top row. The middle row shows the proband in family 1 (IV-2) with a homozygous (HM) c.338delG change, illustrated with a dash for the missing nucleotide when aligned with the wildtype sequence. The bottom row displays an unaffected parent of family 1 (III-3) with the c.338delG change in the heterozygous state (Het): the latter section of the heterozygous electropherogram shows two superimposed sequences due to the synchronous addition of nucleotides due to two distinct DNA templates derived from the wild type and the shorter mutant alleles of the heterozygote. Panel B illustrates the second mutation that was identified in PCDH21, in family 2. The top row displays the control individual with the wildtype (WT) allele; while the bottom row displays the affected proband (II-1) with a homozygous (HM) c.1463delG variant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f2: Electropherograms of the mutations detected in Protocadherin-21 (PCDH21). Panel A illustrates the index c.338delG mutation in family 1; the wild-type (WT) sequence is displayed on the top row. The middle row shows the proband in family 1 (IV-2) with a homozygous (HM) c.338delG change, illustrated with a dash for the missing nucleotide when aligned with the wildtype sequence. The bottom row displays an unaffected parent of family 1 (III-3) with the c.338delG change in the heterozygous state (Het): the latter section of the heterozygous electropherogram shows two superimposed sequences due to the synchronous addition of nucleotides due to two distinct DNA templates derived from the wild type and the shorter mutant alleles of the heterozygote. Panel B illustrates the second mutation that was identified in PCDH21, in family 2. The top row displays the control individual with the wildtype (WT) allele; while the bottom row displays the affected proband (II-1) with a homozygous (HM) c.1463delG variant.
Mentions: Genome-wide linkage scans were performed to identify regions of autozygosity (where both alleles are identical by descent and are copies of a common ancestral gene). DNA samples from all affected family members were genotyped using the Affymetrix human GeneChip® Mapping Array (version Xba142 2.0) for family 1 and the GeneChip® Human Mapping 100 K set array for family 2 (Affymetrix, Santa Clara, CA). The detailed methodology for genotyping using the GeneChip® array has been previously described [4]. Briefly, 250 ng of genomic DNA was digested with XbaI (New England Biolabs, Ipswich, MA) for 2 h at 37 °C, ligated with XbaI adaptors using T4 DNA ligase (New England Biolabs). The ligation reaction was diluted in 1:4 (vol/vol) with molecular grade water (Sigma-Aldrich, St. Louis, MO) to 100 µl. Ten µl of the diluted ligation mix was used to setup selection by PCR (Fragment Selection by PCR) in triplicates. The pooled PCR products were purified using QIAGEN MinElute 96 UF plate (Qiagen, Duesseldorf, Germany). The concentration of PCR products was quantified using ND-1000 Nanodrop spectrometry (Thermo Fisher Scientific, Waltham, MA). Purified PCR product (90 ng) was fragmented with 0.25 units DNaseI ('Fragmentation Reagent'; Affymetrix), labeled with 'Labeling Regent' (Affymetrix). The labeling reaction (70 µl) was mixed with 190 μl hybridization reagent and denatured at 99 °C for 10 min (all as detailed in the Affymetrix GeneChip Mapping 100K Assay Manual). Finally the denatured hybridization mixture was injected into Affymetrix Human Mapping XbaI chips and incubated at 48 °C for 16 h, followed by automatic washing and staining in a Fluidics Station 450, and scanned by using the GeneChip® Scanner 3000 7G (Affymetrix). Primers for direct sequencing were as described by Bolz et al. [5]. PCR amplification was performed using a 20-µl reaction mix, including Abgene Reddymix with or without dye (AB795, AB793; Abgene, Epsom, UK). Ten microliters of Reddymix was combined with 5 µl of deionized water, 2 µl (10 pmol) of forward and reverse primer, and 1 µl (50–100 ng) of genomic DNA. All exons were optimized and amplified at 55 °C. DNA fragments were purified using Montage PCR cleanup plates, according to the standard protocol (Millipore, Watford, UK). PCR cleanup (purple plates) was performed using 15 μl of PCR product made up to 100 μl with deionized water (ddH2O) and transferred to the purple plates. Ten min of vacuum was applied to the plates until the wells were empty. Twenty five μl of ddH2O was added to each well and a further 3 min vacuum was applied. Twenty μl of ddH2O was added to each well; the plate covered and vortexed at 1,000 rpm for 10 min. The remaining contents of each well (clean PCR product) was then transferred for storage. The Big Dye terminator cycle sequencing reaction was then performed using a 10-µl reaction mixture containing 0.5 µl of BigDye v3.1 Applied Biosystems™ (ABI Ltd, Warrington, Cheshire. UK), 2 µl of PCR product, 1.5 µl of 5 picomolar (pM) forward or reverse primer, 3.5 µl of ddH2O, and 2.5 µl of sequencing buffer (ABI Ltd) containing 5x Tris-HCl and MgCl2. The sequencing reaction mixture was purified using the Montage sequence cleanup plate (Millipore, Watford, UK): 25 μl of injection solution (Millipore,) were added to each well, and the total 35 μl was transferred to the blue plate and vacuumed for 3 min. A further 25 μl of injection solution was added to each well and vacuumed again for 4 min. Twenty five μl of injection solution was again added to each well, the plate covered and vortexed for 10 min. The remaining volume was transferred to the sequencing plate and run on the ABI 3700 sequencer. The DNA sequence was analyzed using DNAStar® Inc. (Madison, WI). To check segregation of the mutant alleles with disease, restriction digests were performed. Family 1 was investigated using HaeIII enzyme (Figure 2; New England Biolabs Ltd, NEB, Hitchin, Herts, UK), while in family 2 a digest using an E. coli strain that carries the BglI gene from Bacillus globigii (BglI [NEB]) was performed. One unit (0.2 μl) of enzyme was combined with 2 μl of 1x NEBuffer 3 (NEB, Herts, UK), 10 μl of PCR product and brought to a 20 μl reaction volume with deionized H2O (ddH2O) and digested for 16 h at 37 °C. Heat inactivation was performed at 80 °C for 20 min and the product run out on a 5% agarose gel.

Bottom Line: No color vision was detected in any proband.The fundus appearance included the later development of characteristic circular patches of pigment epithelial atrophy at the macula and in the peripheral retina.Biallelic mutations in the photoreceptor-specific gene PCDH21 cause recessive retinal degeneration in humans.

View Article: PubMed Central - PubMed

Affiliation: Moorfields Eye Hospital, London, UK.

ABSTRACT

Purpose: To describe the clinical findings and mutations in affected members of two families with an autosomal recessive retinal dystrophy associated with mutations in the protocadherin-21 (PCDH21) gene.

Methods: A full genome scan of members of two consanguineous families segregating an autosomal recessive retinal dystrophy was performed and regions identical by descent identified. Positional candidate genes were identified and sequenced. All patients had a detailed ophthalmic examination, including electroretinography and retinal imaging.

Results: Affected members of both families showed identical homozygosity for an overlapping region of chromosome 10q. Sequencing of a candidate gene, PCDH21, showed two separate homozygous single-base deletions, c.337delG (p.G113AfsX1) and c.1459delG (p.G487GfsX20), which were not detected in 282 control chromosomes. Affected members of the two families first reported nyctalopia in late teenage years and retained good central vision until their late 30s. No color vision was detected in any proband. The fundus appearance included the later development of characteristic circular patches of pigment epithelial atrophy at the macula and in the peripheral retina.

Conclusions: Biallelic mutations in the photoreceptor-specific gene PCDH21 cause recessive retinal degeneration in humans.

Show MeSH
Related in: MedlinePlus