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Karyomapping-a comprehensive means of simultaneous monogenic and cytogenetic PGD: comparison with standard approaches in real time for Marfan syndrome.

Thornhill AR, Handyside AH, Ottolini C, Natesan SA, Taylor J, Sage K, Harton G, Cliffe K, Affara N, Konstantinidis M, Wells D, Griffin DK - J. Assist. Reprod. Genet. (2015)

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Affiliation: School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK.

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Targeted haplotyping by multiplex fluorescent polymerase chain reaction (PCR) of closely linked or intragenic short tandem repeat (STR) markers combined with direct mutation detection improves the accuracy of single cell analysis significantly and minimizes potential errors caused by undetected allele dropout (ADO) or contamination... Mitotic chromosome duplication, which can arise through malsegregation of chromosomes in the cleavage divisions following fertilization, cannot be detected by Karyomapping per se, since the sequence of both chromosomes is identical... However, chromosome duplications may be clinically less significant, since they are often associated with poor morphology and developmental arrest... In the case of PGD for Marfan syndrome, ADO affecting the mutation site on the copy of chromosome 15 carrying the mutation, could cause an affected embryo appear normal (as only the normal allele is successfully amplified)... To reduce the risk of misdiagnosis, a strategy employing a combination of mutation detection and analysis of closely linked short tandem repeat (STRs) was used, revealing the paternal 15q21.1 haplotype associated with the mutation... Following whole genome amplification, targeted haplotyping, with all three STR markers, and direct mutation analysis was successful in 7/8 (87.5 %) of the single cells biopsied from six cleavage stage embryos (Table 2; Fig.  1)... Analysis of the STR alleles present at the FBN 1 locus were consistent with the mutation status in five embryos and identified four as unaffected (Embryos 1, 3–5) and one as affected (Embryo 2)... In the remaining single cell biopsied from Embryo 6 with a normal allele for the mutation, only one of the maternal alleles (200 bp) and neither of the paternal specific alleles (176 and 180 bp) were detected with D15S659... The remaining twin boy was healthy at 2 years... Another of the unaffected embryos (Embryo 1; Table 2), cryopreserved by vitrification at the blastocyst stage on Day 6 post ICSI, was successfully thawed 16 months later and transferred in an unstimulated cycle; no pregnancy resulted... Karyomapping however had the added advantage of not requiring the clinical work-up of a specific test beforehand (only the SNP array information from the parents and an affected child was needed)... Minisequencing in combination with the analysis of several STR markers, yielded results within 24 h for all six embryos in which whole genome amplification of the single cells was successful... In theory, screening for spontaneously arising aneuploidies should increase the likelihood that the embryo chosen for transfer will establish a viable pregnancy and ultimately a healthy child... Indeed, as a selection tool, aneuploidy screening can prioritize the embryo for transfer to achieve improved implantation rates and lower miscarriage rates in fresh transfer cycles as well as support single embryo transfer policy as part of the drive towards reducing multiple birth rates.

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Detailed Karyomaps for chromosome 15q21.1 in single blastomeres biopsied from each cleavage stage embryo. Consecutive informative single nucleotide polymorphism (SNP) loci for the four parental chromosomes are represented by two pairs of columns in each case (paternal, left and maternal, right) in which each segment is an informative SNP. Single cell genotypes identifying the presence of one of the four parental chromosomes at informative SNP loci are coloured (paternal chromosomes P1 and P2 are indicated in blue and red respectively; maternal chromosomes M1 and M2 in yellow and green respectively). The Karyomaps of a 5–6 Mb region of chromosome 15q21.1 of the affected child, known to be a carrier of both paternal mutations (P1—blue) and used as a reference for phase, and seven single blastomeres biopsied from six cleavage stage embryos are presented (M1—yellow chromosome also assigned). Otherwise, informative SNP genotypes, which indicate the absence of that chromosome or are not called, are coloured grey. The position of fibrillin-1 (FBN1) relative to the SNP loci is indicated by the light blue bars. The positions of the three short tandem repeat (STR) markers, D15S143, D15S196 and D15S659 used for conventional analysis are indicated on the left. Three embryos are identified as having the unaffected (red) paternal chromosome (Embryos 1, 4 and 5); one embryo has the affected paternal chromosome (blue) also present in the affected child (Embryo 2), and two embryos are missing the paternal chromosomes either because of the complete absence of the paternal genome in a parthenogenetically activated embryo (Embryo 3) or paternal monosomy 15 (Embryo 6). (See “Results” and “Discussion” Sections for more detail). The genomic DNA from the affected child, the first single blastomere from Embryo 1 and the two abnormal embryos, Embryos 3 and 6, were genotyped on the normal 3 day protocol. For comparison, all of the other cells, including the second blastomere from Embryo 1, were genotyped using the 24 h protocol. Abbreviations used: Emb embryo, Bm blastomere (cell), X crossover, asterix* miscall
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Fig2: Detailed Karyomaps for chromosome 15q21.1 in single blastomeres biopsied from each cleavage stage embryo. Consecutive informative single nucleotide polymorphism (SNP) loci for the four parental chromosomes are represented by two pairs of columns in each case (paternal, left and maternal, right) in which each segment is an informative SNP. Single cell genotypes identifying the presence of one of the four parental chromosomes at informative SNP loci are coloured (paternal chromosomes P1 and P2 are indicated in blue and red respectively; maternal chromosomes M1 and M2 in yellow and green respectively). The Karyomaps of a 5–6 Mb region of chromosome 15q21.1 of the affected child, known to be a carrier of both paternal mutations (P1—blue) and used as a reference for phase, and seven single blastomeres biopsied from six cleavage stage embryos are presented (M1—yellow chromosome also assigned). Otherwise, informative SNP genotypes, which indicate the absence of that chromosome or are not called, are coloured grey. The position of fibrillin-1 (FBN1) relative to the SNP loci is indicated by the light blue bars. The positions of the three short tandem repeat (STR) markers, D15S143, D15S196 and D15S659 used for conventional analysis are indicated on the left. Three embryos are identified as having the unaffected (red) paternal chromosome (Embryos 1, 4 and 5); one embryo has the affected paternal chromosome (blue) also present in the affected child (Embryo 2), and two embryos are missing the paternal chromosomes either because of the complete absence of the paternal genome in a parthenogenetically activated embryo (Embryo 3) or paternal monosomy 15 (Embryo 6). (See “Results” and “Discussion” Sections for more detail). The genomic DNA from the affected child, the first single blastomere from Embryo 1 and the two abnormal embryos, Embryos 3 and 6, were genotyped on the normal 3 day protocol. For comparison, all of the other cells, including the second blastomere from Embryo 1, were genotyped using the 24 h protocol. Abbreviations used: Emb embryo, Bm blastomere (cell), X crossover, asterix* miscall

Mentions: SNP genotyping and Karyomaping analysis was successful with all seven single cells from which MDA products were available. Genomic DNA samples from the parents and affected child gave call rates of about 97 % and heterozygous call rates of 28–29 %. Overall SNP call rates were lower in si ngle cells, ranging from 78 to 82 % with a significant rate of ADO (approximately 15 %) and heterozygous call rates ranging from 14 to 20 % (excluding the cell from the parthenogenetic haploid embryo). Karyomapping identified the parental chromosomes present at the FBN1 locus on chromosome 15q21.1 in 11/12 (92 %) of the chromosomes present in the seven cells analyzed (Fig. 2, Table 2). The only chromosome which could not be haplotyped confidently at the FBN1 locus was the maternal chromosome 15 in Embryo 3. In that case, there was a crossover immediately distal to FBN1 and without any intragenic informative SNP loci, the exact location of the recombination event could not be identified unequivocally. Otherwise, the paternal and maternal haplotypes identified (where present) were concordant with the targeted haplotyping and direct mutation analysis, including the proximal region of the maternal chromosome 15 in Embryo 3 (Table 2).Fig. 2


Karyomapping-a comprehensive means of simultaneous monogenic and cytogenetic PGD: comparison with standard approaches in real time for Marfan syndrome.

Thornhill AR, Handyside AH, Ottolini C, Natesan SA, Taylor J, Sage K, Harton G, Cliffe K, Affara N, Konstantinidis M, Wells D, Griffin DK - J. Assist. Reprod. Genet. (2015)

Detailed Karyomaps for chromosome 15q21.1 in single blastomeres biopsied from each cleavage stage embryo. Consecutive informative single nucleotide polymorphism (SNP) loci for the four parental chromosomes are represented by two pairs of columns in each case (paternal, left and maternal, right) in which each segment is an informative SNP. Single cell genotypes identifying the presence of one of the four parental chromosomes at informative SNP loci are coloured (paternal chromosomes P1 and P2 are indicated in blue and red respectively; maternal chromosomes M1 and M2 in yellow and green respectively). The Karyomaps of a 5–6 Mb region of chromosome 15q21.1 of the affected child, known to be a carrier of both paternal mutations (P1—blue) and used as a reference for phase, and seven single blastomeres biopsied from six cleavage stage embryos are presented (M1—yellow chromosome also assigned). Otherwise, informative SNP genotypes, which indicate the absence of that chromosome or are not called, are coloured grey. The position of fibrillin-1 (FBN1) relative to the SNP loci is indicated by the light blue bars. The positions of the three short tandem repeat (STR) markers, D15S143, D15S196 and D15S659 used for conventional analysis are indicated on the left. Three embryos are identified as having the unaffected (red) paternal chromosome (Embryos 1, 4 and 5); one embryo has the affected paternal chromosome (blue) also present in the affected child (Embryo 2), and two embryos are missing the paternal chromosomes either because of the complete absence of the paternal genome in a parthenogenetically activated embryo (Embryo 3) or paternal monosomy 15 (Embryo 6). (See “Results” and “Discussion” Sections for more detail). The genomic DNA from the affected child, the first single blastomere from Embryo 1 and the two abnormal embryos, Embryos 3 and 6, were genotyped on the normal 3 day protocol. For comparison, all of the other cells, including the second blastomere from Embryo 1, were genotyped using the 24 h protocol. Abbreviations used: Emb embryo, Bm blastomere (cell), X crossover, asterix* miscall
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Related In: Results  -  Collection

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Fig2: Detailed Karyomaps for chromosome 15q21.1 in single blastomeres biopsied from each cleavage stage embryo. Consecutive informative single nucleotide polymorphism (SNP) loci for the four parental chromosomes are represented by two pairs of columns in each case (paternal, left and maternal, right) in which each segment is an informative SNP. Single cell genotypes identifying the presence of one of the four parental chromosomes at informative SNP loci are coloured (paternal chromosomes P1 and P2 are indicated in blue and red respectively; maternal chromosomes M1 and M2 in yellow and green respectively). The Karyomaps of a 5–6 Mb region of chromosome 15q21.1 of the affected child, known to be a carrier of both paternal mutations (P1—blue) and used as a reference for phase, and seven single blastomeres biopsied from six cleavage stage embryos are presented (M1—yellow chromosome also assigned). Otherwise, informative SNP genotypes, which indicate the absence of that chromosome or are not called, are coloured grey. The position of fibrillin-1 (FBN1) relative to the SNP loci is indicated by the light blue bars. The positions of the three short tandem repeat (STR) markers, D15S143, D15S196 and D15S659 used for conventional analysis are indicated on the left. Three embryos are identified as having the unaffected (red) paternal chromosome (Embryos 1, 4 and 5); one embryo has the affected paternal chromosome (blue) also present in the affected child (Embryo 2), and two embryos are missing the paternal chromosomes either because of the complete absence of the paternal genome in a parthenogenetically activated embryo (Embryo 3) or paternal monosomy 15 (Embryo 6). (See “Results” and “Discussion” Sections for more detail). The genomic DNA from the affected child, the first single blastomere from Embryo 1 and the two abnormal embryos, Embryos 3 and 6, were genotyped on the normal 3 day protocol. For comparison, all of the other cells, including the second blastomere from Embryo 1, were genotyped using the 24 h protocol. Abbreviations used: Emb embryo, Bm blastomere (cell), X crossover, asterix* miscall
Mentions: SNP genotyping and Karyomaping analysis was successful with all seven single cells from which MDA products were available. Genomic DNA samples from the parents and affected child gave call rates of about 97 % and heterozygous call rates of 28–29 %. Overall SNP call rates were lower in si ngle cells, ranging from 78 to 82 % with a significant rate of ADO (approximately 15 %) and heterozygous call rates ranging from 14 to 20 % (excluding the cell from the parthenogenetic haploid embryo). Karyomapping identified the parental chromosomes present at the FBN1 locus on chromosome 15q21.1 in 11/12 (92 %) of the chromosomes present in the seven cells analyzed (Fig. 2, Table 2). The only chromosome which could not be haplotyped confidently at the FBN1 locus was the maternal chromosome 15 in Embryo 3. In that case, there was a crossover immediately distal to FBN1 and without any intragenic informative SNP loci, the exact location of the recombination event could not be identified unequivocally. Otherwise, the paternal and maternal haplotypes identified (where present) were concordant with the targeted haplotyping and direct mutation analysis, including the proximal region of the maternal chromosome 15 in Embryo 3 (Table 2).Fig. 2

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Targeted haplotyping by multiplex fluorescent polymerase chain reaction (PCR) of closely linked or intragenic short tandem repeat (STR) markers combined with direct mutation detection improves the accuracy of single cell analysis significantly and minimizes potential errors caused by undetected allele dropout (ADO) or contamination... Mitotic chromosome duplication, which can arise through malsegregation of chromosomes in the cleavage divisions following fertilization, cannot be detected by Karyomapping per se, since the sequence of both chromosomes is identical... However, chromosome duplications may be clinically less significant, since they are often associated with poor morphology and developmental arrest... In the case of PGD for Marfan syndrome, ADO affecting the mutation site on the copy of chromosome 15 carrying the mutation, could cause an affected embryo appear normal (as only the normal allele is successfully amplified)... To reduce the risk of misdiagnosis, a strategy employing a combination of mutation detection and analysis of closely linked short tandem repeat (STRs) was used, revealing the paternal 15q21.1 haplotype associated with the mutation... Following whole genome amplification, targeted haplotyping, with all three STR markers, and direct mutation analysis was successful in 7/8 (87.5 %) of the single cells biopsied from six cleavage stage embryos (Table 2; Fig.  1)... Analysis of the STR alleles present at the FBN 1 locus were consistent with the mutation status in five embryos and identified four as unaffected (Embryos 1, 3–5) and one as affected (Embryo 2)... In the remaining single cell biopsied from Embryo 6 with a normal allele for the mutation, only one of the maternal alleles (200 bp) and neither of the paternal specific alleles (176 and 180 bp) were detected with D15S659... The remaining twin boy was healthy at 2 years... Another of the unaffected embryos (Embryo 1; Table 2), cryopreserved by vitrification at the blastocyst stage on Day 6 post ICSI, was successfully thawed 16 months later and transferred in an unstimulated cycle; no pregnancy resulted... Karyomapping however had the added advantage of not requiring the clinical work-up of a specific test beforehand (only the SNP array information from the parents and an affected child was needed)... Minisequencing in combination with the analysis of several STR markers, yielded results within 24 h for all six embryos in which whole genome amplification of the single cells was successful... In theory, screening for spontaneously arising aneuploidies should increase the likelihood that the embryo chosen for transfer will establish a viable pregnancy and ultimately a healthy child... Indeed, as a selection tool, aneuploidy screening can prioritize the embryo for transfer to achieve improved implantation rates and lower miscarriage rates in fresh transfer cycles as well as support single embryo transfer policy as part of the drive towards reducing multiple birth rates.

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Related in: MedlinePlus