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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

Comparison of targeted haplotyping and direct mutation detection with karyomapping in a nonconcordant single-blastomere sample (Embryo 6) from a preimplantation genetic diagnosis cycle for Crigler–Najjar syndrome, type 1, caused by a mutation in UGT1A1. 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 father and mother are carriers for this autosomal recessive disorder, and the reference child is affected. This means that both the blue and the orange haplotypes carry the mutation. Embryo 6 has the blue paternal and green maternal haplotypes, indicating a paternal carrier. On the basis of the four flanking STR markers and two intragenic markers, along with the mutation analysis (lower panel), this embryo was diagnosed as an unaffected maternal carrier. In the mutation analysis, + indicates the presence of the mutation, and − indicates its absence. Only the intragenic SNP marker identifies the presence of the normal paternal allele. All other markers are ambiguous and were labeled as suspected ADOs. Phasing of the UGT1A1 locus by karyomapping is unequivocal (upper panel) and indicates that the embryo is an unaffected paternal carrier. Note that the terminal regions of both chromosome arms have the characteristic karyomapping patterns of key and non-key informative SNPs, indicating regions (marked by square braces) in which both parents have a chromosome with an identical SNP genotype, i.e., identical by descent. ADO, allele dropout; SNP, single-nucleotide polymorphism; STR, short tandem repeat.
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fig3: Comparison of targeted haplotyping and direct mutation detection with karyomapping in a nonconcordant single-blastomere sample (Embryo 6) from a preimplantation genetic diagnosis cycle for Crigler–Najjar syndrome, type 1, caused by a mutation in UGT1A1. 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 father and mother are carriers for this autosomal recessive disorder, and the reference child is affected. This means that both the blue and the orange haplotypes carry the mutation. Embryo 6 has the blue paternal and green maternal haplotypes, indicating a paternal carrier. On the basis of the four flanking STR markers and two intragenic markers, along with the mutation analysis (lower panel), this embryo was diagnosed as an unaffected maternal carrier. In the mutation analysis, + indicates the presence of the mutation, and − indicates its absence. Only the intragenic SNP marker identifies the presence of the normal paternal allele. All other markers are ambiguous and were labeled as suspected ADOs. Phasing of the UGT1A1 locus by karyomapping is unequivocal (upper panel) and indicates that the embryo is an unaffected paternal carrier. Note that the terminal regions of both chromosome arms have the characteristic karyomapping patterns of key and non-key informative SNPs, indicating regions (marked by square braces) in which both parents have a chromosome with an identical SNP genotype, i.e., identical by descent. ADO, allele dropout; SNP, single-nucleotide polymorphism; STR, short tandem repeat.

Mentions: In the remaining seven cycles, which were identified by karyomapping as having extensive regions of consanguinity (e.g., Figure 3 and Supplementary Figure S2c online), some or all of the STR markers were only semi-informative, and the results frequently deviated from the expected parental haplotypes. Hence, the original diagnosis was often based mainly on mutation detection. In these cases, karyomapping was concordant in 52/57 (91.2%) embryo samples. However, in five (8.8%) of the single-blastomere samples, the results were nonconcordant. To investigate these discrepancies, the expected STR and mutation site alleles were predicted in a blinded manner, based on the karyomap of each of the embryos. This showed that in two of these nonconcordant samples, there was only a single STR or marker allele that had led to the original diagnosis and that the karyomapping evidence from the many key or non-key SNPs in the region of interest strongly supported the contrary diagnosis (Figure 3). Furthermore, because the STR markers were in general only semi-informative, the strength of evidence in the other samples was similarly weak. The reasons for these nonconcordances therefore need further investigation. Overall, 208/213 (97.7%) embryo samples were completely concordant with the original analysis based on targeted haplotyping and mutation detection.


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)

Comparison of targeted haplotyping and direct mutation detection with karyomapping in a nonconcordant single-blastomere sample (Embryo 6) from a preimplantation genetic diagnosis cycle for Crigler–Najjar syndrome, type 1, caused by a mutation in UGT1A1. 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 father and mother are carriers for this autosomal recessive disorder, and the reference child is affected. This means that both the blue and the orange haplotypes carry the mutation. Embryo 6 has the blue paternal and green maternal haplotypes, indicating a paternal carrier. On the basis of the four flanking STR markers and two intragenic markers, along with the mutation analysis (lower panel), this embryo was diagnosed as an unaffected maternal carrier. In the mutation analysis, + indicates the presence of the mutation, and − indicates its absence. Only the intragenic SNP marker identifies the presence of the normal paternal allele. All other markers are ambiguous and were labeled as suspected ADOs. Phasing of the UGT1A1 locus by karyomapping is unequivocal (upper panel) and indicates that the embryo is an unaffected paternal carrier. Note that the terminal regions of both chromosome arms have the characteristic karyomapping patterns of key and non-key informative SNPs, indicating regions (marked by square braces) in which both parents have a chromosome with an identical SNP genotype, i.e., identical by descent. ADO, allele dropout; SNP, single-nucleotide polymorphism; STR, short tandem repeat.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Comparison of targeted haplotyping and direct mutation detection with karyomapping in a nonconcordant single-blastomere sample (Embryo 6) from a preimplantation genetic diagnosis cycle for Crigler–Najjar syndrome, type 1, caused by a mutation in UGT1A1. 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 father and mother are carriers for this autosomal recessive disorder, and the reference child is affected. This means that both the blue and the orange haplotypes carry the mutation. Embryo 6 has the blue paternal and green maternal haplotypes, indicating a paternal carrier. On the basis of the four flanking STR markers and two intragenic markers, along with the mutation analysis (lower panel), this embryo was diagnosed as an unaffected maternal carrier. In the mutation analysis, + indicates the presence of the mutation, and − indicates its absence. Only the intragenic SNP marker identifies the presence of the normal paternal allele. All other markers are ambiguous and were labeled as suspected ADOs. Phasing of the UGT1A1 locus by karyomapping is unequivocal (upper panel) and indicates that the embryo is an unaffected paternal carrier. Note that the terminal regions of both chromosome arms have the characteristic karyomapping patterns of key and non-key informative SNPs, indicating regions (marked by square braces) in which both parents have a chromosome with an identical SNP genotype, i.e., identical by descent. ADO, allele dropout; SNP, single-nucleotide polymorphism; STR, short tandem repeat.
Mentions: In the remaining seven cycles, which were identified by karyomapping as having extensive regions of consanguinity (e.g., Figure 3 and Supplementary Figure S2c online), some or all of the STR markers were only semi-informative, and the results frequently deviated from the expected parental haplotypes. Hence, the original diagnosis was often based mainly on mutation detection. In these cases, karyomapping was concordant in 52/57 (91.2%) embryo samples. However, in five (8.8%) of the single-blastomere samples, the results were nonconcordant. To investigate these discrepancies, the expected STR and mutation site alleles were predicted in a blinded manner, based on the karyomap of each of the embryos. This showed that in two of these nonconcordant samples, there was only a single STR or marker allele that had led to the original diagnosis and that the karyomapping evidence from the many key or non-key SNPs in the region of interest strongly supported the contrary diagnosis (Figure 3). Furthermore, because the STR markers were in general only semi-informative, the strength of evidence in the other samples was similarly weak. The reasons for these nonconcordances therefore need further investigation. Overall, 208/213 (97.7%) embryo samples were completely concordant with the original analysis based on targeted haplotyping and mutation detection.

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