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Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro.

Natesan SA, Bladon AJ, Coskun S, Qubbaj W, Prates R, Munne S, Coonen E, Dreesen JC, Stevens SJ, Paulussen AD, Stock-Myer SE, Wilton LJ, Jaroudi S, Wells D, Brown AP, Handyside AH - Genet. Med. (2014)

Bottom Line: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping.Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome.Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results.

View Article: PubMed Central - PubMed

Affiliation: Illumina, Cambridge, UK.

ABSTRACT

Purpose: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization.

Methods: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping.

Results: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results.

Conclusion: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development.

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Related in: MedlinePlus

Detailed karyomaps of chromosome 19, in the terminal p13.3 region, for five embryos from a preimplantation genetic diagnosis case for Peutz–Jeghers syndrome, caused by a mutation in the STK11 gene. Also shown is the outcome of conventional testing with two proximal semi-informative STR markers (D19S565 and D19S247) and direct mutation detection. Paternal haplotypes are represented in blue/red, and maternal haplotypes are represented in orange/green. Haplotypes inherited by the reference are shown in blue/orange. The affected child has inherited the mutation in STK11 from the father. Therefore, the reference haplotype (blue) represents the affected haplotype in this case. Note that there is a common crossover on the paternal chromosome between the two STR markers, which indicates that the affected child used as a reference for linkage had a crossover in this position. Furthermore, there are three additional crossovers in the region on the paternal chromosomes in these five embryos and the maternal chromosome 19 is not present in two embryos (haploblock bar grayed out). This complex pattern of crossovers and aneuploidy detected by karyomapping is completely concordant with the STR alleles (table below) and the presence or absence of the mutation (indicated by the + or − in the STR table). However, Embryos 2, 3, and 4 have identical STR results, and only direct mutation testing identifies Embryo 3 as affected. STR, short tandem repeat.
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fig2: Detailed karyomaps of chromosome 19, in the terminal p13.3 region, for five embryos from a preimplantation genetic diagnosis case for Peutz–Jeghers syndrome, caused by a mutation in the STK11 gene. Also shown is the outcome of conventional testing with two proximal semi-informative STR markers (D19S565 and D19S247) and direct mutation detection. Paternal haplotypes are represented in blue/red, and maternal haplotypes are represented in orange/green. Haplotypes inherited by the reference are shown in blue/orange. The affected child has inherited the mutation in STK11 from the father. Therefore, the reference haplotype (blue) represents the affected haplotype in this case. Note that there is a common crossover on the paternal chromosome between the two STR markers, which indicates that the affected child used as a reference for linkage had a crossover in this position. Furthermore, there are three additional crossovers in the region on the paternal chromosomes in these five embryos and the maternal chromosome 19 is not present in two embryos (haploblock bar grayed out). This complex pattern of crossovers and aneuploidy detected by karyomapping is completely concordant with the STR alleles (table below) and the presence or absence of the mutation (indicated by the + or − in the STR table). However, Embryos 2, 3, and 4 have identical STR results, and only direct mutation testing identifies Embryo 3 as affected. STR, short tandem repeat.

Mentions: In 37 PGD cycles, all of the embryos had been unambiguously diagnosed by targeted haplotyping and direct mutation analysis, and blinded karyomap analysis was concordant with the original diagnosis in 156/156 (100%) embryos. In one PGD cycle for Peutz–Jeghers syndrome, however, a recombination between the STR markers was detected along with other presumed ADOs (Figure 2). Detailed examination of the STR marker and gene loci in this case showed that it was not possible to find informative STR markers distal to the gene, and the proximal STRs were only semi-informative. The original diagnosis was therefore mainly based on the results of the mutation analysis, which has clearly been affected by ADO in several samples. When the phase of the STR alleles was predicted blindly based on their position, taking into account all four recombinations in the parental chromosomes, there was 100% concordance at the STR level.


Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro.

Natesan SA, Bladon AJ, Coskun S, Qubbaj W, Prates R, Munne S, Coonen E, Dreesen JC, Stevens SJ, Paulussen AD, Stock-Myer SE, Wilton LJ, Jaroudi S, Wells D, Brown AP, Handyside AH - Genet. Med. (2014)

Detailed karyomaps of chromosome 19, in the terminal p13.3 region, for five embryos from a preimplantation genetic diagnosis case for Peutz–Jeghers syndrome, caused by a mutation in the STK11 gene. Also shown is the outcome of conventional testing with two proximal semi-informative STR markers (D19S565 and D19S247) and direct mutation detection. Paternal haplotypes are represented in blue/red, and maternal haplotypes are represented in orange/green. Haplotypes inherited by the reference are shown in blue/orange. The affected child has inherited the mutation in STK11 from the father. Therefore, the reference haplotype (blue) represents the affected haplotype in this case. Note that there is a common crossover on the paternal chromosome between the two STR markers, which indicates that the affected child used as a reference for linkage had a crossover in this position. Furthermore, there are three additional crossovers in the region on the paternal chromosomes in these five embryos and the maternal chromosome 19 is not present in two embryos (haploblock bar grayed out). This complex pattern of crossovers and aneuploidy detected by karyomapping is completely concordant with the STR alleles (table below) and the presence or absence of the mutation (indicated by the + or − in the STR table). However, Embryos 2, 3, and 4 have identical STR results, and only direct mutation testing identifies Embryo 3 as affected. STR, short tandem repeat.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225458&req=5

fig2: Detailed karyomaps of chromosome 19, in the terminal p13.3 region, for five embryos from a preimplantation genetic diagnosis case for Peutz–Jeghers syndrome, caused by a mutation in the STK11 gene. Also shown is the outcome of conventional testing with two proximal semi-informative STR markers (D19S565 and D19S247) and direct mutation detection. Paternal haplotypes are represented in blue/red, and maternal haplotypes are represented in orange/green. Haplotypes inherited by the reference are shown in blue/orange. The affected child has inherited the mutation in STK11 from the father. Therefore, the reference haplotype (blue) represents the affected haplotype in this case. Note that there is a common crossover on the paternal chromosome between the two STR markers, which indicates that the affected child used as a reference for linkage had a crossover in this position. Furthermore, there are three additional crossovers in the region on the paternal chromosomes in these five embryos and the maternal chromosome 19 is not present in two embryos (haploblock bar grayed out). This complex pattern of crossovers and aneuploidy detected by karyomapping is completely concordant with the STR alleles (table below) and the presence or absence of the mutation (indicated by the + or − in the STR table). However, Embryos 2, 3, and 4 have identical STR results, and only direct mutation testing identifies Embryo 3 as affected. STR, short tandem repeat.
Mentions: In 37 PGD cycles, all of the embryos had been unambiguously diagnosed by targeted haplotyping and direct mutation analysis, and blinded karyomap analysis was concordant with the original diagnosis in 156/156 (100%) embryos. In one PGD cycle for Peutz–Jeghers syndrome, however, a recombination between the STR markers was detected along with other presumed ADOs (Figure 2). Detailed examination of the STR marker and gene loci in this case showed that it was not possible to find informative STR markers distal to the gene, and the proximal STRs were only semi-informative. The original diagnosis was therefore mainly based on the results of the mutation analysis, which has clearly been affected by ADO in several samples. When the phase of the STR alleles was predicted blindly based on their position, taking into account all four recombinations in the parental chromosomes, there was 100% concordance at the STR level.

Bottom Line: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping.Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome.Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results.

View Article: PubMed Central - PubMed

Affiliation: Illumina, Cambridge, UK.

ABSTRACT

Purpose: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization.

Methods: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping.

Results: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results.

Conclusion: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development.

Show MeSH
Related in: MedlinePlus