First systematic experience of preimplantation genetic diagnosis for de-novo mutations  Svetlana Rechitsky, Ekaterina Pomerantseva, Tatiana Pakhalchuk, Dana Pauling, Oleg Verlinsky, Anver Kuliev  Reproductive BioMedicine Online  Volume 22, Issue 4, Pages 350-361 (April 2011) DOI: 10.1016/j.rbmo.2011.01.005 Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Preimplantation genetic diagnosis (PGD) strategies for de-novo mutations (DNM) of different origin. (A) Case work-out for DNM detected in father (partner): (1) pedigree in two generations; (2) mutation verification in DNA extracted from blood and total spermatozoa; (3) amplification of partner’s single spermatozoa to establish normal and affected haplotypes required for PGD cycle preparation; (4) amplification of patient’s DNA to identify the most informative markers for PGD; and (5) PGD by blastomere or blastocyst biopsy for combined mutation and linkage analysis. (B) Case work-out for DNM detected in mother (patient): (1) pedigree in two generations; (2) mutation verification in DNA extracted from whole blood or cheek swabs and single lymphocytes (paternal haplotypes are analysed on single spermatozoa for more accurate prediction of embryos’ genotypes); (3) PGD by PB1 and PB2 analysis to identify DNM-free oocytes and establish maternal haplotypes; and (4) blastomere or blastocyst analysis to confirm the diagnosis. (C) Case work-out for DNM detected first in affected offspring: (1) pedigree in three generations; (2) verification of DNM in child’s DNA extracted from blood or cheek swabs and mutation testing of DNA extracted from parents’ whole blood and total spermatozoa; (3) mutation evaluation on single lymphocytes and single sperm testing to rule out paternal gonadal mosaicism; (4) PGD by polar body analysis to detect potential maternal gonadal mosaicism; and (5) blastomere or blastocyst analysis to confirm the absence of the mutation. Reproductive BioMedicine Online 2011 22, 350-361DOI: (10.1016/j.rbmo.2011.01.005) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Preimplantation genetic diagnosis (PGD) for de-novo mutations (DNM) in neurofibromatosis type II gene (c114+2 T–C splicing mutation) of paternal origin. (A) Family pedigree showing that that DNM in NF2 gene was first detected in father. Single sperm analysis via multiplex heminested PCR revealed gonadal mosaicism with three different haplotypes: haplotype a (normal), haplotype b (mutant containing c.114+2 T–C allele) and haplotype c (mutant without c.114+2 T–C allele in NF2). Maternal linkage was based on DNA amplification of blastomeres in PGD cycle. (B) Outcome of the first PGD cycle. Blastomeres from five embryos were subjected to combined mutation and linkage analysis by multiplex heminested PCR. Three embryos (4, 5 and 7) were predicted to be free from the paternal mutation based on the presence of normal sequence (N) in NF2 gene and confirmed by linked markers (haplotype a). Two embryos (6 and 8) were predicted to have normal sequence (N∗) on haplotype c. The accuracy of this prediction was decreased due to a potential allele drop out of the dominant mutation in single blastomere. Trophectoderm (TE) biopsy from these embryos confirmed the presence of the normal sequence of NF2 gene. Embryos 5 and 7 were transferred (ET) resulting in unaffected pregnancy and birth of a healthy boy and girl confirmed by postnatal testing. (C) Outcome of the second PGD cycle. Combined mutation and linkage analysis by multiplex heminested PCR was performed on blastomeres from 10 embryos. Mutant haplotype b was detected only in embryo 13. Embryo 6 was missing all the maternal markers suggesting monosomy of chromosome 22, in which the gene is localized. Although all the remaining embryos were predicted to be normal and free of mutation, only four (embryos 3, 4, 9 and 12) were with normal (N) paternal haplotype a, while embryos 2, 5, 7 and 11 were predicted to have normal sequence (N∗) on the mutant haplotype c. Blastocyst biopsy confirmed normal genotypes predicted on blastomeres. Two normal embryos (3 and 4) were transferred, resulting in clinical pregnancy and delivery of a healthy girl confirmed by postnatal analysis. Reproductive BioMedicine Online 2011 22, 350-361DOI: (10.1016/j.rbmo.2011.01.005) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Preimplantation genetic diagnosis (PGD) for de-novo mutations (DNM) in NF1 gene (intron 27–38 deletion) of maternal origin. (A) Family pedigree of a couple with affected son carrying deletion of intron 27–38 in the NF1 gene. This deletion was not detected in maternal DNA from whole blood, although two haplotypes (c and d) were present, the latter corresponding to mutant haplotype corresponding to affected son’s haplotype, but with no deletion. The expected deleted area on this ‘benign’ chromosome (same haplotype as affected son received from the mother but without deletion) is framed. The actual mutant haplotype (e) with deletion was detected on maternal single lymphocytes. Paternal normal haplotypes (a) and (b) were established based on markers detected on the son’s normal chromosome. (B) Outcome of PGD cycle, performed by multiplex heminested PCR on blastomeres from 11 embryos. Three embryos (3, 4 and 5) were predicted normal (N) based on the presence of maternal normal haplotype (c) and suitable for embryo transfer (ET). Four embryos (1, 2, 10 and 12) inherited the ‘benign’ mutant maternal haplotype (d) and were also predicted normal (N∗) and suitable for ET. Of the remaining four embryos, embryos 7 and 11 were predicted to have monosomy of chromosome 17, based to the absence of maternal alleles, while the other two (embryos 6 and 8) were predicted to be affected, based on the absence of maternal markers in deleted area (DEL). Two embryos (2 and 3) were transferred and resulted in a biochemical pregnancy. Reproductive BioMedicine Online 2011 22, 350-361DOI: (10.1016/j.rbmo.2011.01.005) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Preimplantation genetic diagnosis (PGD) for autosomal recessive de-novo mutations (DNM) detected first in an affected child who was compound heterozygous for C750G/E837X mutations in the FANCI gene, combined with human leukocyte antigen (HLA) genotyping. (A) Family pedigree, showing HLA and mutation haplotypes, based on parental and affected child’s genomic DNA testing. E837X mutation was detected in the carrier mother, but C750G mutation was absent in DNA extracted from paternal blood or whole sperm samples. However, both normal and mutant haplotypes were detected in testing of single spermatozoa. (B) PGD cycle combined with HLA testing: (1b) Multiplex heminested and fully nested amplification performed on blastomeres from six embryos did not reveal the paternal mutation. Four embryos (1, 2, 4 and 5) were predicted normal based on the absence of both mutations, of which embryos 1, 4 and 5 inherited ‘benign’ paternal haplotype a, similar to one of the mutant haplotypes in the affected child, and embryo 2 inherited the normal haplotype b. The remaining embryos (3 and 6) were predicted to be carriers of the maternal mutation E837X, but inherited the paternal haplotype a; and (2b) HLA marker analysis demonstrated the presence of two HLA-matched embryos (3 and 6), which were transferred but did not achieve pregnancy. Normal∗ = benign paternal haplotype a similar to the mutant haplotype of the affected child; white bars = HLA-match genotypes; grey bars = non-HLA-match haplotypes. Reproductive BioMedicine Online 2011 22, 350-361DOI: (10.1016/j.rbmo.2011.01.005) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Preimplantation genetic diagnosis (PGD) for chronic granulomatous disease, determined by X-linked DNM IVS9+5 G–A, combined with human leukocyte antigen (HLA) genotyping and aneuploidy testing. (A) Family pedigree showing the mutation and closely linked to CYBB gene markers. (B) Sequential PB1 and PB2 analysis, showing that all the tested oocytes are normal, despite four of them containing the ‘benign’ mutant haplotype without IVS9+5 mutation (N∗). (C) (1c) multiplex heminested PCR for combined mutation analysis; (2c) HLA genotyping; and (3c) karyotyping, for six chromosomes by PCR on blastomeres. Two of four embryos (8 and 9), predicted to be HLA matched and free of mutation and aneuploidy, were transferred, resulting in a clinical pregnancy that spontaneously aborted at 9weeks. White bars = HLA-match genotypes; grey bars = non-HLA-match haplotypes. Reproductive BioMedicine Online 2011 22, 350-361DOI: (10.1016/j.rbmo.2011.01.005) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions