Downloaded from jmg.bmj.com on October 6, 2010 - Published by group.bmj.com Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes Alan H Handyside, Gary L Harton, Brian Mariani, et al. J Med Genet 2010 47: 651-658 originally published online October 25, 2009 doi: 10.1136/jmg.2009.069971 Updated information and services can be found at: http://img.bmj.com/content/47/10/651.full.html These include: References This article cites 28 articles, 13 of which can be accessed free at: http://img.bmj.eom/content/47/10/651.full.html#reMist-1 Article cited in: http://jmg.bmj.eom/content/47/10/651.full.html#related-urls Email alerting Receive free email alerts when new articles cite this article. Sign up in the service box at the top right corner of the online article. Notes To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://journals.bmj.com/cgi/ep Downloaded from jmg.bmj.com on October 6, 2010 - Published by group.bmj.com Original article Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes Alan H Handyside,1'2 Gary L Harton,3 Brian Mariani,3 Alan R Thornhill,1-4 Nabeel Affara,5 Marie-Anne Shaw,2 Darren K Griffin4 London Bridge Fertility, Gynaecology and Genetics Centre, London, UK 2Faculty of Biological Sciences, University of Leeds, Leeds, UK 3Genetics & IVF Institute, Fairfax, Virginia, USA department of Biosciences, University of Kent, Canterbury, UK department of Pathology, University of Cambridge, Cambridge, UK Correspondence to Professor Alan H Handyside, London Bridge Fertility, Gynaecology and Genetics Centre, One St Thomas Street, London SE1 9RY, UK; ahandyside@thebridgecentre co.uk Received 1 June 2009 Accepted 29 September 2009 Published Online First 25 October 2009 ABSTRACT The use of genome wide single nucleotide polymorphism (SNP) arrays for high resolution molecular cytogenetic analysis using a combination of quantitative and genotype analysis is well established. This study demonstrates that by Mendelian analysis of the SNP genotypes of the parents and a sibling or other appropriate family member to establish phase, it is possible to identify informative loci for each of the four parental haplotypes across each chromosome and map the inheritance of these haplotypes and the position of any crossovers in the proband. The resulting 'karyomap', unlike a karyotype, identifies the parental and grandparental origin of each chromosome and chromosome segment and is unique for every individual being defined by the independent segregation of parental chromosomes and the pattern of non-recombinant and recombinant chromosomes. Karyomapping, therefore, enables both genome wide linkage based analysis of inheritance and detection of chromosome imbalance where either both haplotypes from one parent are present (trisomy) or neither are present (monosomy/ deletion). The study also demonstrates that karyomapping is possible at the single cell level following whole genome amplification and, without any prior patient or disease specific test development, provides a universal linkage based methodology for preimplantation genetic diagnosis readily available worldwide. INTRODUCTION The use of DNA microarrays and comparative genomic hybridisation (array CGH) or single nucleotide polymorphism (SNP) arrays is now well established for high resolution molecular cytogenetic analysis of duplications, deletions and other unbalanced abnormalities1-3 and has revealed extensive copy number variation throughout the genome.4 Both of these approaches rely primarily on quantitation at known loci distributed across the genome. SNP genotyping, however, has the advantage that general features of the proband's genotype, such as loss of heterozygosity, can be analysed in conjunction with quantitation to distinguish between, for example, monosomy and uniparental isodisomy. Here we demonstrate that by Mendelian analysis of the SNP genotypes of the parents and a sibling or other appropriate family member to establish phase, it is possible to identify informative loci for each of the four parental haplotypes across each chromosome and map the inheritance of these haplotypes and the position of any crossovers in the proband. The resulting 'karyomap', unlike a karyotype, identifies the parental and grandparental origin of each chromosome and chromosome segment and is unique for every individual being defined by the independent segregation of parental chromosomes and the pattern of non-recombinant and recombinant chromosomes. Karyomapping, therefore, enables genome wide linkage based analysis of inheritance of a broad range of genetic abnormalities, identified as pre-existing in one or both parents, from structural chromosomal abnormalities and copy number variants (CNVs) to single gene defects. In addition, karyomapping identifies trisomies of meiotic origin, by the presence of both haplotypes from one parent in one or more segments of the chromosome, and monosomies and deletions, by the absence of either haplotype from that parent (figure 1), exclusively on the basis of the genotype without any need for quantitative analysis. For standard applications such as prenatal diagnosis, conventional quantitative and genotype analysis of SNP array data and karyomapping are not mutually exclusive. Together, they provide comprehensive information on the nature and parental origin of any cytogenetic abnormalities, which can be combined with linkage based analysis of any inherited condition if required. For preimplantation genetic diagnosis (PGD), which is limited to analysis of single or small numbers of cells biopsied from each embryo following in vitro fertilisation (IVF),5 karyomapping is ideal. Efficient methods for whole genome amplification by multiple displacement amplification (MDA) are now available6-8 and a single universal methodology with a broad range of applications, which is widely available, eliminates the need for labour intensive and costly test development. The only requirements are that DNA is available from both parents and an appropriate family member of known disease status and the position of the relevant gene or other abnormality is known. To demonstrate the use of karyomapping for linkage based diagnosis of a single gene defect, we have analysed two families segregating mutant alleles of the cystic fibrosis transmembrane receptor (CFTR) causing cystic fibrosis (CF) (figure 2). In the first family, the karyomaps of five children confirmed the known CF status of four of them using one of the affected children for linkage. Furthermore, using MDA to amplify the whole genome of single cells from one of the children, we J Med Genet 2010;47:B51 -658. doi:10.1136/jmg.2009.069971 651 Downloaded from jmg.bmj.com on October 6, 2010 - Published by group.bmj.com Original article b CAM D i Figure 1 Karyomapping for genome wide analysis of chromosome aneuploidy and inheritance. (A) Linkage based diagnosis of inheritance of single gene defects. The two pairs of parental chromosomes (top row) are each colour coded to represent the two parental haplotypes inherited from the grandparents; the two paternal chromosomes in blue and red (left) and the two maternal chromosomes in yellow and green (right). Using Mendelian analysis of informative single nucleotide polymorphism (SNP) genotype combinations in the parents and establishing phase by reference to the genotype of one of the children, the parental haplotypes and the position of any crossovers for each paternal (left) and maternal (right) chromosomes inherited by the four children is identified and represented as a karyomap (second row). Linkage based diagnosis of the inheritance of, for example, an autosomal recessive single gene defect is then possible by comparing the parental haplotypes flanking the position of the gene (thin yellow band) in each child with those present in a child of known status. In this example, from left to right, unaffected carrier (paternal mutant allele), unaffected carrier (maternal allele), unaffected homozygote, affected homozygote. (B—D) Detection of chromosome aneuploidy. (B) Monosomy is detected by the absence of either of the haplotypes from one parent (black). (C) Trisomy caused by duplication of one chromosome or mitotic non-disjunction cannot be detected by karyomapping alone since the SNP genotype of both chromosomes is identical. (D) Trisomies resulting from the inheritance of two different products of meiosis are detected by one or more regions in which both haplotypes from one parent are present at closely adjacent informative SNP loci (purple). In this illustration, the presence of both maternal haplotypes in the pericentromeric region is consistent with a maternal meiosis I type non-disjunction error. demonstrate that karyomapping is possible at the single cell level by limiting the analysis to only heterozygous informative SNP loci while retaining a sufficient density of loci for accurate linkage based testing. Similarly, in the second family, who had undergone IVF with PGD resulting in the birth of an unaffected child, the karyomaps of five embryos were consistent with the original diagnosis based on mutation detection alone, following A TO +/AF508 i a (ill Child 1 AF508/? B Child 2 +/? Child 3 AF508/+ Child 4 +/? Child 5 AF508/? -Kl +/AF508 AF508 +/AF508 <> <> <>4 Embryo 1 +/AF508 Embryo 2 Embryo 3 Embryo 4 Embryo 5 +/ AF508 AF508/ AF508 +/+ Figure 2 Pedigrees of the two families segregating mutations in CFTR. (A) Family 1. Both parents are carriers of mutations in the cystic fibrosis transmembrane receptor {CFTR) gene. The father is a carrier of the AF508 deletion and the mother is a carrier for an unknown minor mutation. Two of the five children are affected by cystic fibrosis and are compound heterozygotes for the two mutations. The other three (two sons and a daughter) are all carriers of one of the parental mutations as indicated. (B) Family 2. Both parents are carriers of the AF508 deletion and following preimplantation genetic diagnosis (PGD), five preimplan-tation embryos, which were not selected for transfer, were cryopre-served and subsequently consented for research. The PGD result based on analysis of one or two cells biopsied from each embryo is indicated. MDA of either the whole embryo or small numbers of biopsied cells. In addition, a maternal aneuploidy was detected in two of the five embryos. METHODS Two families segregating mutant alleles of the CFTR causing CP were genotyped using high density, genome wide SNP arrays for karyomap analysis. Family 1 The parents and five children of known CF status were genotyped using DNA extracted from lymphoblastoid cell lines (NIGMS Human Genetic Cell Repository, Coriell Institute, Camden, New Jersey, USA) by standard methods (figure 2A). Family 2 In this family, both parents are known carriers of the AF508 deletion (p.Phe508) and have two homozygous CF affected children. Following IVF and cleavage stage biopsy of one or two single cells for PGD using standard methods,9 two unaffected blastocyst stage embryos were transferred 5 days post-insemination resulting in the normal delivery of a healthy girl. All remaining embryos were cryopreserved and subsequently donated for research with the patients' informed consent. Three blastocyst stage and two arrested cleavage stage embryos of known CF status (figure 2B) were thawed for reanalysis by karyomapping. Three to 10 trophectoderm cells were biopsied from each blastocyst and the biopsies, one corresponding biopsied embryo and the two arrested embryos (each with B52 J Med Genet 2010;47:B51 -658. doi:10.1136/jmg.2009.069971 Downloaded from jmg.bmj.com on October 6, 2010 - Published by group.bmj.com Original article | CHROMOSOME 6 | | CHROMOSOME 7 | PATERNAL MATERNAL PATERNAL MATERNAL 12345 12345 12345 12345 CHROMOSOME 6 UNIT! 13 Mb : I - Figure 3 Karyomaps of chromosomes 6 and 7 for the five children in family 1. The consecutive series of informative single nucleotide polymorphism (SNP) loci across the length of each chromosome is represented in the karyomap by a continuous vertical column, coloured according to the parental haplotype identified. Where the SNP genotype indicates the same parental haplotype as that in the reference used to establish phase, the column is coloured blue or yellow, and where the opposite haplotype is present, the column is coloured red or green for the paternal and maternal chromosomes, respectively. The position of the centromere is indicated by the grey segment. Note that the length of the karyomap is determined by the number of informative SNPs on that chromosome and does not represent the physical distance between the SNP loci. The paternal and maternal chromosomes are grouped on the left and right, respectively, to facilitate comparison between siblings (child 1—5). Note that the grandparental origin of each chromosome can be determined by examining the parental haplotype in the pericentro-meric region. The position of the cystic fibrosis transmembrane receptor (CFTR) located on the long arm of chromosome 7 (7q31.2) is indicated by a horizontal line. The extent of the human leucocyte antigen (HLA) region (approximately 24—35.7 Mb) tested for preimplantation genetic diagnosis (PGD) with HLA matching is indicated by the box on the chromosome 6 panel. Following whole genome amplification, the call rates for biopsies from three blastocyst stage embryos (3—10 cells), one corresponding biopsied embryo and two arrested embryos in which only approximately two cells remained intact in each case, all of which had been cryopreserved subsequent to cleavage stage biopsy and PGD, were lower—averaging 92.8%. Using embryo 1, previously identified by PGD as an unaffected heterozygous carrier, as the reference genotype, the karyomaps for each of the other embryos clearly identified the parental haplotypes and positions of crossovers and consistent with the original PGD result in each case (figure 4). Furthermore, karyomapping identifies the origin of the mutant allele as maternal in both of the carriers (embryos 1 and 3). In this family, this is less definitive since both parents carry the same mutation (p.Phe508) and no linkage based testing was attempted originally. The karyomaps of the five embryos also revealed two aneuploidies of maternal origin, a monosomy for chromosome 6 (biopsy 2) and a trisomy for chromosome 9 (biopsy 3) (figure 4). With the trisomy 9, the distribution of regions in which both maternal haplotypes were detected, on both arms distal from the centromere, is consistent with non-disjunction Biopsy 3 Emb4 P M P M EmbS P M I CHROMOSOME 7 CHROMOSOME 9 Biopsy 1 Embl Biopsy2 Biopsy3 Emb4 Irin ■ PM P hi PM P M PM P M Ml ,!H' I im. I. Figure 4 Karyomaps of chromosomes 6, 7 and 9 for embryos 1—5 (family 2). Karyomaps representing the parental haplotypes identified in the biopsied cells or embryos for the paternal (left) and maternal (right) chromosomes are paired in each case. Where the SNP genotype indicates the same parental haplotype as that in the reference used to establish phase, the column is coloured blue or yellow, and where the opposite haplotype is present, the column is coloured red or green for the paternal and maternal chromosomes, respectively. The position of the centromere is indicated by the grey segment. In addition, if both or neither parental haplotypes from one parent are identified, the column is coloured purple or black, respectively. The position of the cystic fibrosis transmembrane receptor (CF77?) located on the long arm of chromosome 7 (7q31.2) is indicated by a horizontal line. in meiosis II, though other mechanisms are possible (see Discussion). DISCUSSION By Mendelian analysis of a series of closely spaced informative SNP loci across each chromosome within a family, karyomapping enables highly accurate, genome wide linkage based analysis of inheritance of a broad range of genetic abnormalities from structural chromosomal abnormalities and CNVs to single gene defects identified as pre-existing in one or both parents. All that is required is that the exact position of the genetic abnormality B54 J Med Genet 2010;47:B51 -658. doi:10.1136/jmg.2009.069971 Downloaded from jmg.bmj.com on October 6, 2010 - Published by group.bmj.com is known. With genomic DNA (family 1), karyomapping unambiguously confirmed the known status of each of the four siblings for mutations in the CFTR causing CF, using one of the affected children to establish phase and linkage to the mutations in both parents. Also, crossovers between parental haplotypes were sharply defined (figure 3) and the number and distribution of crossovers in the five children from family 1 (table 2) was broadly consistent with previous studies.10-12 There were a few isolated contradictory SNP genotype calls at informative loci (table 1). Most of these are likely to be genotyping errors in either the reference genotype or the proband, since a double recombination between closely spaced flanking SNP loci is extremely unlikely,13 although rare mutations or gene conversion cannot be excluded in some cases. In principle, therefore, for single gene defects, there may be no need for direct mutation detection. In practice, however, many if not most families do not fulfil the criteria for linkage testing even after extensive investigation to understand the mode of inheritance and identify the causative mutation. For example, many families present singleton cases, so that one cannot easily determine whether or not it is a new mutation or an inherited case without mutation testing. In these families, karyomapping may only be useful as an aid to investigate possible linkage. For PGD of single gene defects, following IVF and cleavage stage embryo biopsy, knowledge of the causative mutation and/ or established linkage to one or more family members are essential prerequisites because of the relatively high incidence of errors with single cell genetic analysis. For example, ADO, in which one of the parental alleles randomly fails to amplify by PCR or following whole genome amplification by MDA, is a major potential source of errors.7 14 Tests for single gene defects, therefore, typically include one or more closely spaced flanking markers in addition to direct mutation detection and informative, highly polymorphic short tandem repeat (STR) markers would normally be preferred to biallelic SNPs.15 Indeed, karyomap analysis of single cells genotyped following MDA, at all informative SNP loci, resulted in an unacceptably high incidence of contradictory calls, presumably caused by ADO, which often made it impossible to define the position of crossovers accurately. By limiting analysis to only those informative loci that are heterozygous, this problem is virtually eliminated (table 1). Although the average interval between these heterozygous informative SNP loci is increased (64.8—106.4 Kb for the autosomes), with few exceptions, the spacing is more than adequate for accurate linkage based diagnosis with or without direct mutation detection. This was demonstrated by karyomap analysis of samples with small numbers of cells following MDA from a PGD cycle for CF (family 2), which was consistent with the original conventional single cell analysis (figure 4). Thus a single universal linkage based methodology providing genome wide linkage markers, which is widely available, eliminates the need for labour intensive and costly patient or disease specific test development. Another feature of karyomapping is that analysis of informative loci across each chromosome not only provides genome wide linkage markers but also enables detection of chromosome imbalance, including identifying the parent of origin, where either both (trisomy) or no (monosomy or deletion) parental haplotypes from one parent are present, exclusively on the basis of the SNP genotype without any need for quantitative analysis (figure 1). Thus, any trisomy of meiotic origin, in which the whole of the chromosomes or one or more segments of the two chromosomes are derived from the two parental haplotypes, will be detected. Furthermore, the pericentromeric or distal position of Original article these dual haplotype segments may identify their origin as resulting from non-disjunction in meiosis I or II, respectively, although other mechanisms have been proposed.16 Without quantitation, however, it is not possible to detect duplication of chromosomes or smaller segments such as de novo CNVs since the genotype is identical. For example, uniparental isodisomy cannot be distinguished from monosomy by karyomapping alone. By contrast, whole chromosome monosomies and segmental or even smaller deletions should be detected with a resolution only limited by the spacing between informative loci. This ability of karyomapping to combine detection of chromosome imbalance with genome wide linkage analysis is particularly important for PGD since there is a relatively high incidence of chromosome aneuploidy and other complex structural abnormalities of meiotic and postzygotic origin in human preimplantation embryos following IVF.17 18 This was demonstrated here, where reanalysis by karyomapping, following a conventional PGD cycle for CF, revealed that two out of five embryos had maternal aneuploidies, one monosomy 6 and one trisomy 9 (meiosis II type), and would not have established a viable pregnancy if transferred (figure 4). Recently, several randomised clinical trials of the use of PGD and fluorescent in situ hybridisation (FISH) analysis of 5—7 chromosomes have shown that live birth rates are not increased in women of advanced maternal age in which the incidence of aneuploidy is significantly increased.19 This may be partly explained by the limited number of chromosomes tested and errors caused by chromosomal mosaicism which arises through postzygotic malsegregation or loss of chromosomes,20 so that the biopsied single cell is not representative of the remaining cells in the embryo. Amplification bias during whole genome amplification from single cells by either PCR or MDA methods has, until recently, made the use of DNA microarray based CGH for comprehensive aneuploidy testing unreliable.21-23 However, several groups have now reported accurate detection of aneuploidies by conventional quantitative analysis of SNP array data.24 Karyomapping is exclusively based on genotype analysis and has several advantages. These include genome wide analysis for all chromosomes, identifying the parent of origin, which may be clinically significant, and, for trisomies, providing direct evidence for the inheritance of two chromosomes from one parent even if only a single cell is analysed. In this event, although trisomy rescue is a theoretical possibility, there is a risk of uniparental disomy, and unlike monosomies, which are generally not viable, some trisomies result in viable but abnormal pregnancy. A major application of karyomapping for PGD is for carriers of balanced structural chromosome abnormalities, mainly reciprocal and Robertsonian translocations, which can cause infertility or repeated miscarriage. Karyomapping should in these cases detect both the duplicated and deleted segments of any unbalanced combination in the embryo resulting from fertilisation with an unbalanced gamete. Furthermore, unlike FISH based methods with combinations of chromosome specific centromeric and telomeric probes,25 karyomapping should also distinguish between normal and balanced embryos by identifying the parental haplotypes across the breakpoints. Another important application is for human leucocyte antigen (HIA) matching, with or without single gene defect testing if required, with the aim of isolating cord blood stem cells at birth for transplantation to an existing child with a serious blood related illness.26 27 Here the provision of genome wide linkage markers for the HLA region on chromosome 6 as well as any other locus for single gene defect analysis is a significant advantage and J Med Genet 2010;47:B51 -658. doi:10.1136/jmg.2009.069971 655 Downloaded from jmg.bmj.com on October 6, 2010 - Published by group.bmj.com Table 2 The number and location of crossovers detected in the autosomes and X chromosomes of child 1—5 (family 1) Paternal Maternal Chromosome 1 2 3 4 5 Average ±SD Range 1 2 3 4 5 Average ±SD Range 1 P 2 2 0 1 2 1.4 0.89 0-2 3 0 3 2 0 1.6 1.52 0-3 q 2 1 1 1 1 1.2 0.45 1-2 3 2 2 2 2 2.2 0.45 2-3 All 4 3 1 2 3 2.6 1.14 1-4 6 2 5 4 2 3.8 1.79 2-6 2 p 0 1 1 0 2 0.8 0.84 0-2 1 1 2 0 0 0.8 0.84 0-2 q 0 0 1 4 0 1 1.73 0-4 1 0 1 2 1 1 0.71 0-2 All 0 1 2 4 2 1.8 1.48 0-4 2 1 3 2 1 1.8 0.84 1-3 3 p 1 0 1 1 0 0.6 0.55 0-1 1 3 1 1 1 1.4 0.89 1-3 q 1 0 1 0 1 0.6 0.55 0-1 1 1 2 2 1 1.4 0.55 1-2 All 2 0 2 1 1 1.2 0.84 0-2 2 4 3 3 2 2.8 0.84 2-4 4 p 0 1 2 0 1 0.8 0.84 0-2 0 0 1 0 2 0.6 0.89 0-2 q 0 1 2 1 1 1 0.71 0-2 3 2 2 0 0 1.4 1.34 0-3 All 0 2 4 1 2 1.8 1.48 0-4 3 2 3 0 2 2 1.22 0-3 5 p 1 0 0 1 1 0.6 0.55 0-1 0 1 1 1 2 1 0.71 0-2 q 1 1 1 0 1 0.8 0.45 0-1 1 2 1 2 2 1.6 0.55 1-2 All 2 1 1 1 2 1.4 0.55 1-2 1 3 2 3 4 2.6 1.14 1-4 6 p 0 1 0 2 0 0.6 0.89 0-2 1 1 0 1 2 1 0.71 0-2 q 1 0 0 0 0 0.2 0.45 0-1 1 1 2 1 1 1.2 0.45 1-2 All 1 1 0 2 0 0.8 0.84 0-2 2 2 2 2 3 2.2 0.45 2-3 7 p 1 0 1 0 0 0.4 0.55 0-1 2 0 0 1 1 0.8 0.84 0-2 q 1 0 2 0 1 0.8 0.84 0-2 3 0 2 2 1 1.6 1.14 0-3 All 2 0 3 0 1 1.2 1.30 0-3 5 0 2 3 2 2.4 1.82 0-5 B p 0 1 1 0 1 0.6 0.55 0-1 0 1 1 2 1 1 0.71 0-2 q 1 0 0 2 1 0.8 0.84 0-2 2 0 0 1 2 1 1.00 0-2 All 1 1 1 2 2 1.4 0.55 1-2 2 1 1 3 3 2 1.00 1-3 9 p 1 0 0 1 0 0.4 0.55 0-1 1 1 2 1 2 1.4 0.55 1-2 q 1 0 1 0 1 0.6 0.55 0-1 1 2 2 1 2 1.6 0.55 1-2 All 2 0 1 1 1 1 0.71 0-2 2 3 4 2 4 3 1.00 2-4 10 p 0 1 0 0 1 0.4 0.55 0-1 2 1 1 2 2 1.6 0.55 1-2 q 0 0 0 1 0 0.2 0.45 0-1 0 2 2 3 2 1.8 1.10 0-3 All 0 1 0 1 1 0.6 0.55 0-1 2 3 3 5 4 3.4 1.14 2-5 11 p 1 1 1 0 1 0.8 0.45 0-1 1 1 1 0 1 0.8 0.45 0-1 q 0 0 1 1 1 0.6 0.55 0-1 1 1 1 0 1 0.8 0.45 0-1 All 1 1 2 1 2 1.4 0.55 1-2 2 2 2 0 2 1.6 0.89 0-2 12 p 0 1 0 0 0 0.2 0.45 0-1 1 1 0 0 0 0.4 0.55 0-1 q 1 1 2 2 1 1.4 0.55 1-2 2 1 2 3 0 1.6 1.14 0-3 All 1 2 2 2 1 1.6 0.55 1-2 3 2 2 3 0 2 1.22 0-3 13 p Not analysed Not analysed q 1 1 2 3 2 1.8 0.84 1-3 0 2 3 2 0 1.4 1.34 0-3 All 1 1 2 3 2 1.8 0.84 1-3 0 2 3 2 0 1.4 1.34 0-3 14 p Not analysed Not analysed q 1 1 1 2 1 1.2 0.45 1-2 2 1 2 1 2 1.6 0.55 1-2 All 1 1 1 2 1 1.2 0.45 1-2 2 1 2 1 2 1.6 0.55 1-2 15 p Not analysed Not analysed q 1 1 1 2 2 1.4 0.55 1-2 1 2 2 1 0 1.2 0.84 0-2 All 1 1 1 2 2 1.4 0.55 1-2 1 2 2 1 0 1.2 0.84 0-2 16 p 0 1 0 1 0 0.4 0.55 0-1 0 0 2 1 2 1 1.00 0-2 q 0 1 0 0 0 0.2 0.45 0-1 1 1 1 2 1 1.2 0.45 1-2 All 0 2 0 1 0 0.6 0.89 0-2 1 1 3 3 3 2.2 1.10 1-3 17 p 1 0 0 0 0 0.2 0.45 0-1 2 1 1 0 1 1 0.71 0-2 q 0 0 1 3 2 1.2 1.30 0-3 2 1 2 2 1 1.6 0.55 1-2 All 1 0 1 3 2 1.4 1.14 0-3 4 2 3 2 2 2.6 0.89 2-4 18 p 0 1 1 1 0.6 0.55 0-1 1 0 0 0 1 0.4 0.55 0-1 q 0 0 1 0 0 0.2 0.45 0-1 1 1 2 1 1 1.2 0.45 1-2 All 0 1 1 1 1 0.8 0.45 0-1 2 1 2 1 2 1.6 0.55 1-2 19 p q 0 1 1 1 ! 1 1 1 1 0.6 1 0.55 0.00 0-1 1 0 1 1 1 0 1 0 1 0 0 0.2 0.8 0.45 0.45 0-1 0-1 All 1 2 1 2 2 1.6 0.55 1-2 1 2 1 1 0 1 0.71 0-2 20 p 0 0 0 1 0 0.2 0.45 0-1 1 0 1 1 0 0.6 0.55 0-1 q 1 1 0 0 0 0.4 0.55 0-1 1 1 2 0 0 0.8 0.84 0-2 All 1 1 0 1 0 0.6 0.55 0-1 2 1 3 1 0 1.4 1.14 0-3 Continued B5B J Med Genet 2010;47:B51 -B58. doi:10.1136/jmg.2009.069971 Downloaded from jmg.bmj.com on October 6, 2010 - Published by group.bmj.com Original article Table 2 Continued Paternal Maternal Chromosome 1 2 3 4 5 Average ±SD Range 1 2 3 4 5 Average ±SD Range 21 P 0 0 0 0 0 0 0.00 q 1 0 1 1 0 0.6 0.55 All 1 0 1 1 0 0.6 0.55 22 p Not analysed q 0 1 1 2 2 1.2 0.84 All 0 1 1 2 2 1.2 0.84 X (non- PAR) p 0 0 0 q 0 0 0 All 0 0 0 Total 23 23 28 36 30 28 5.43 avoids the need for developing a patient specific test with multiple markers in both these regions. Using family 1 as an example, in the 11.7 Mb interval between D6S299 and D6S1645, two of the most distal and proximal STR markers currently used for PGD analysis,28 for the five single cells from child 3, there were an average of 79 (range 66—98) and 119 (range 86—115) heterozygous informative SNP loci for the paternal and maternal chromosomes, respectively, far exceeding the 6—10 STR markers normally used. For PGD, there is only limited time available for genetic analysis between embryo biopsy, normally at cleavage stages early on day 3 following insemination, and transfer on days 4—6. Preliminary results with shortened protocols indicate that whole genome amplification, SNP array processing and data analysis for karyomapping can be achieved within 24—48 h, which would avoid the need to cryopreserve biopsied embryos (unpublished observations). Compared with standard methods, the use of arrays is relatively expensive, particularly as multiple embryos may need to be tested. However, expensive labour intensive test development is eliminated. Also, with improvements in embryo culture and cryopreservation by vitrification, which involves ultra rapid cooling, it is now possible to biopsy only those viable embryos able to develop to the later blastocyst stage, cryopreserve them pending genetic analysis, and then thaw selected embryos for transfer in a later reproductive cycle. Using this strategy and testing for aneuploidy by conventional CGH, survival of vitrified blastocysts following thawing was close to 100% and clinical pregnancy rates were 69% per embryo transfer.25 Use of genome wide SNP genotyping or other arrays for clinical applications raises challenging clinical and ethical issues. Because karyomapping analysis is genome wide, there is a strong likelihood of its use in postnatal or adult counselling for multifactorial disorders. While our knowledge of the genes contributing to common complex disorders is incomplete, knowledge of the major loci contributing to a disorder will inform our views on risk. Karyomapping, however, simply uses SNP genotype information anonymously to map inheritance from one generation to the next across the genome. It cannot per se identify de novo changes at individual SNP loci and, if deemed necessary, it would be straightforward to blind or remove some or all of the genotype data from clinical records. With rapid advances in new sequencing technologies, including quantitative single molecule methods, the cost of sequencing targeted regions or the whole genome of individuals is steadily decreasing.30 If this becomes possible for routine clinical application, complete data on inherited and de novo alterations will be available. The challenge will then be to interpret and counsel patients on the significance of a multitude of rare 0 0 0 0 0 0 0.00 0-1 0 0 2 2 0 0.8 1.10 0-2 0-1 0 0 2 2 0 0.8 1.10 0-2 Not analysed 0-2 1 0 1 1 1 0.8 0.45 0-1 0-2 1 0 1 1 1 0.8 0.45 0-1 0 2 1 1 1 1 1.2 0.45 0-2 0 0 2 4 0 3 1.8 1.79 0-4 0 2 3 5 1 4 3 1.58 1-5 23-36 48 40 59 46 43 47.2 7.26 40-! 59 variants. For single cell analysis, however, the extent and accuracy of sequence information may be limited without further improvements in whole genome amplification. Acknowledgements AHH is grateful to Paul Williams, The Bridge Centre Ltd, for encouraging and supporting this work. GLH wishes to thank the staff of the Genetics & IVF Institute, especially Kasie Heeres and Khoa Tran, for their hard work, dedication and very gifted hands in the laboratory. DKG is supported by a BBSRC Career Development Fellowship BB/E024211/1. 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