Volume 14 Supplement 1
Preimplantation genetic diagnosis of hemophilia A
- Ming Chen†1, 2, 3, 4, 5, 6Email author,
- Shun-Ping Chang†1, 2,
- Gwo-Chin Ma†1, 2, 7, 8,
- Wen-Hsian Lin1, 2,
- Hsin-Fu Chen3,
- Shee-Uan Chen3,
- Horng-Der Tsai7,
- Feng-Po Tsai9 and
- Ming-Ching Shen10
© The Author(s). 2016
Published: 4 October 2016
Preimplantation genetic diagnosis (PGD) is a powerful tool to tackle the transmission of monogenic inherited disorders in families carrying the diseases from generation to generation. It currently remains a challenging task, despite PGD having been developed over 25 years ago. The major difficulty is it does not have an easy and general formula for all mutations. Different gene locus needs individualized, customized design to make the diagnosis accurate enough to be applied on PGD, in which the quantity of DNA is scanty, whereas timely laboratory diagnosis is mandatory if fresh embryo transfer is desired occasionally. Indicators for outcome assessment of a successful PGD program include the successful diagnosis rate on blastomeres (Day 3 cleavage-stage embryo biopsy) or trophectoderm cells (Day 5/6 blastocyst biopsy), the implantation rate per embryo transferred, and the livebirth rate per oocyte retrieval cycle. Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by various types of pathological defects in the factor VIII gene (F8). The mutation spectrum of the F8 is complex, according to our previous report, including large segmental intra-gene inversions, large segmental deletions spanning a few exons, point mutations, and total deletion caused by chromosomal structural rearrangements. In this review, the molecular methodologies used to tackle different mutants of the F8 in the PGD of HA are to be explained, and the experiences of successful use of amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR) and linkage analysis for PGD of HA in our laboratory are also provided.
KeywordsPGD ARMS-qPCR Linkage analysis STR marker Polymorphism
Preimplantation genetic diagnosis (PGD) had become a standard of care when dealing with stopping the transmission of the heritable disease from generation to generation since it was firstly introduced in 1990 [1, 2]. The gold standard of molecular technology used for PGD nowadays is the coamplification of the polymorphic microsatellite linkage markers [3, 4]. However, such techniques cannot avoid the possibility of recombination occurred within the segment which separated the linked polymorphic markers and the disease loci, and it is advised to combine more informative linkage markers to reduce the chance of misdiagnosis. On the other hand, direct mutation detection assay, either rapid PCR-based or the more time-consuming sequencing-based genotyping platforms, is prone to allele dropout (ADO), which may ensue a catastrophic false-negative misdiagnosis in PGD of autosomal dominant monogenic disorder [4, 5].
Hemophilia A (HA) (OMIM 306700), a bleeding disorder which causes long-term disability, is a X-linked recessive disorder and its causative gene is situated at Xq28, the factor VIII (F8) gene, is a serious threat for public health in Taiwan, and we had first published its mutation spectrum in the Taiwanese population in 2008 . The mutation spectrum included rearrangements such as intron 1 inversions (INV1) and intron 22 inversions (INV22), large deletions spanning for consecutive exons, small deletions involving only a few base pairs, and point mutations . The broad spectra of F8 mutations have also been reported in several other studies [7–9]. The genotyping itself for the F8 is already a daunting task given its complicated existing mutations patterns, let alone the PGD. In spite of the challenges, we managed to tackle these difficulties in a few families who came to our hospital seeking for PGD. Meanwhile, a few similar efforts had been reported from other laboratories [10, 11], which indicates PGD for HA is feasible, at least in those families the mutation has been confirmed. Here we will give a concise review of PGD for HA, including the different molecular technologies used to tackle different mutation patterns, and also to cite a few of our experience with successful outcome, that is, to give birth to normal unaffected babies in families suffered from HA.
Mutation spectrum of the F8 gene, genotyping strategies, and possible PGD approaches
Since the publication of the sequence of the F8 in 1984, more than 2000 gene mutations causing HA have been described and these are catalogued in the Human Gene Mutation Database (HGMD; http://www.hgmd.cf.ac.uk/ac/index.php) and Factor VIII Variant Database (http://www.factorviii-db.org/). In 2008, we had first published the mutation spectrum in the Taiwanese population . Of 31 unrelated HA patients (19 severe and 10 moderate/mild males, and 2 severe females), 12 (38.7 %) and 1 (3.2 %) severe males were genotyped with INV22 and INV1 respectively. The F8 defects in the remaining 18 inversion-negative patients cover a wide spectrum, in which 17 different mutations were identified (10 missense and 3 nonsense mutations, and 2 small and 2 large deletions). Eleven of these mutations are novel and unique, confirming a high diversity of molecular defects in HA . A systematic review for data from 30 studies on 5383 patients had been reported and showed 45 % of HA had INV22, 2 % INV1, 3 % large deletions, 16 % small deletions or insertions, and 28 % point mutations (15 % missense mutations, 10 % nonsense mutations, and 3 % splicing site mutations). In 4.6 % of patients, the mutation was unknown . Overall, with the exceptions of recurrent INV22 and INV1, no mutation hot spots have been identified.
Genotype-phenotype relationship, genetic testing and preimplantation genetic diagnosis (PGD) in hemophilia A
Frequency of occurrancea
• 45 %
• 2 %
• I-PCR (for INV22)
• Long-distance PCR (for INV22)
• Southern blotting (for INV22)
• Multiplex PCR (for INV1)
• Linkage analysis
• Splicing site
• 15 %
• 10 %
• 3 %
Mild, Moderate, Severe
• Mild, Moderate (majority)
• Severe (majority)
• Severe (majority)
Direct DNA sequencing
• Linkage analysis
Small deletion/insertion (<1 exon)
Direct DNA sequencing
• Linkage analysis
Large deletion (≥1 exon)
• Linkage analysis
Others (e.g., Complex rearrangement)
Depending on mutation entities
• Linkage analysis
Given the marked morbidity associated with severe HA, PGD has become a feasible option for couples at risk of having a child with HA since it reduces the risk of termination of affected pregnancies. Gender selection by fluorescence in situ hybridization (FISH) and transfer of only female embryos is a simple strategy for X-linked recessive disorders, such as HA, and has been adopted in many clinics . However, in practice, gender selection is illegal in some countries (e.g., Taiwan) and methods allowing the correct and more definitive diagnosis of the HA status of every embryo are more desirable because the number of embryos available for transfer is increased. PGD involving whole genome amplification (WGA) step was broadly applied to mutation detection strategies, but the high rate of amplification bias renders WGA an imperfect option . Recently, co-amplification of polymorphic microsatellite markers, linked with the targeted mutation, had been the gold-standard genotyping strategy for PGD [4, 23–26] (Table 1). A linkage approach using polymorphic markers located near the mutation allows monitoring the occurrence of allele dropout, a known problem associated with PCR amplification bias in PGD. Below, we describe our experience with two HA families seeking for PGD.
Experience of PGD of hemophilia A in our laboratory
The INV22 of F8 is one of the most frequent cause of severe HA, known as a result of homologous recombination between the int22h-1 region within the F8 locus and either int22h-2 (Inv22 type II) or int22h-3 (Inv22 type I), which lie nearly 400 kb distal to F8 . The gene rearrangements tend to increase the difficulty of PGD experimental design performed in the affected families. In the second PGD family with F8 INV22 mutation, the couple (2–1 and 2–2) has had a healthy boy and they would like to get more babies without HA. We performed 3 PGD cycles of HA during the period of Sep. 2014 to Dec. 2015 using short tandem repeat (STR) markers and capillary electrophoresis for direct linkage analysis. Five informative STR markers distributed within or near the flanking region of the F8 were selected for PGD performing (see family 2 in Fig. 3). Among a total of 19 examined embryos, 5 wild types and 4 INV22 carriers were selected and transferred during the period. Unfortunately, pregnancy outcome did not occur as expected, possibly due to ad maternal age, embryo morphology, development and other abnormalities. Despite the fact that no pregnancy was achieved in the PGD experience so far, they are still willing to keep trying.
The outcome indicators of PGD can be classified as successful diagnosis rate (the number of embryos which diagnosis was made/the total number of embryos being biopsied), implantation rate (the number of embryos implanted/the total number of embryos being transferred), and the live-birth rate (the rate of liveborn pregnancy per transferred cycle or the rate of liveborn pregnancy per oocyte-retrieval). It is now still under debate whether frozen or fresh embryo transfer can achieve a better outcome against the other. However, it is vitally important that PGD laboratories developed a timely genotyping platform to cope with the need of fresh embryo transfer, especially when Day 5/6 blastocyst biopsy is undertaken. For rapid PGD of HA, the direct mutation detection, e.g., ARMS-qPCR, can greatly increase the reliability of mutation detection in embryos with small insertions/deletion and point mutations (see the exemplified couple 1). However, for large and complex F8 defects, e.g., INV1 and INV22, PGD by direct genotyping is not easily feasible and indirect linkage analysis with informative markers may be considered (see the exemplified couple 2). Of noted, the chance of recombination between the markers and mutation can lead to small diagnostic error and some families may not be informative for any of the available markers.
Recently, it is noteworthy that because of the popularity of preimplantation genetic screening (PGS), there is a growing need of concurrent PGD/PGS. At the moment the strategies used in PGS, if we exclude the outdated FISH-based diagnostics , include array-based (either array comparative genomic hybridization or single nucleotide polymorphism chromosomal microarray) techniques [29–31], q-PCR based techniques [32, 33], and next generation sequencing (NGS)-based techniques [34, 35]. Some of the techniques had been reported to successfully being applied in PGD combined with PGS [36–39]. It is inevitable that in the near future, women will opt for select the unaffected embryos with certain heritable monogenic disorders, such as HA, as well as the euploid embryos which will reduce the chance of abortion due to aneuploidy in the later gestational period or improved the implantation rate as many researchers advocated . However, it will be undisputable only after more convincing randomized trials to prove the efficacy of PGS, the combination of PGD and PGS should be offered to all women underwent PGD . Those couple who opted for PGD combined with PGS should be counseled that double selection will inevitable reduce the number of embryos which are classified as “suitable” for transfer, thereby reducing all the outcome indicators of PGD, the most important live-birth rate is certainly included.
PGD of HA by direct mutation analysis or indirect linkage analysis has become a feasible option for couples at risk of having an affected child. However, given the broad spectra of the F8 mutations, genetic counseling along with the technical aspects of the accuracy and limitations of tests should be provided for couples who request PGD.
Amplification refractory mutation system-quantitative polymerase chain reaction
Fluorescence in situ hybridization
Human gene mutation database
Intron 1 inversion
Intron 22 inversion
Inverse polymerase chain reaction
- long-distance PCR:
Long-distance polymerase chain reaction
Multiplex ligation-dependent probe amplification
Next generation sequencing
Preimplantation genetic diagnosis
Preimplantation genetic screening
Short tandem repeat
Whole genome amplification
This study was partially supported by grants from Changhua Christian Hospital to M. Chen and to M-C. Shen.
Publication fees for this article have been funded by APSTH 2016.
This article has been published as part of Thrombosis Journal Volume 14 Supplement 1, 2016. The full contents of the supplement are available at https://thrombosisjournal.biomedcentral.com/articles/supplements/volume-14-supplement-1.
Availability of data and material
The unidentified dataset and material information of this article are available upon request by contacting the corresponding author.
MC, SPC, GCM designed the study. MCS recruited the patients. MCS, HFC, SUC, HDT, FPT collected the clinical data. MC, SPC, GCM, WHL did the experiments and performed the analyses. MC, SPC, GCM wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Handyside A, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature. 1990;344:768–70.View ArticlePubMedGoogle Scholar
- Brezina PR, Brezina DS, Kearns WG. Preimplantation genetic testing. BMJ. 2012;345:e5908.View ArticlePubMedGoogle Scholar
- Harton GL, De Rycke M, Fiorentino F, Moutou C, SenGupta S, Traeger-Synodinos J, et al. European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium: ESHRE PGD consortium best practice guidelines for amplification-based PGD. Human Reprod. 2011;26:33–40.View ArticleGoogle Scholar
- Chen HF, Chang SP, Wu SH, Lin WH, Lee YC, Ni YH, et al. Validating a rapid, real-time, PCR-based direct mutation detection assay for preimplantation genetic diagnosis. Gene. 2014;548:299–305.View ArticlePubMedGoogle Scholar
- Kuo SJ, Ma GC, Chang SP, Wu HH, Chen CP, Chang TM, et al. Preimplantation and prenatal genetic diagnosis of aromatic L-amino acid decarboxylase deficiency with an amplification-refractory mutation system-quantitative polymerase chain reaction. Taiwan J Obstet Gynecol. 2011;50:468–73.View ArticlePubMedGoogle Scholar
- Ma GC, Chang SP, Chen M, Kuo SJ, Chang CS, Shen MC. The mutation spectrum of the factor 8 (F8) defects in Taiwanese patients with severe hemophilia A. Haemophilia. 2008;14:787–95.View ArticlePubMedGoogle Scholar
- Bogdanova N, Markoff A, Pollmann H, Nowak-Göttl U, Eisert R, Wermes C, et al. Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A. Hum Mutat. 2005;26:249–54.View ArticlePubMedGoogle Scholar
- Guillet B, Lambert T, d’Oiron R, Proulle V, Plantier JL, Rafowicz A, Peynet J, et al. Detection of 95 novel mutations in coagulation factor VIII gene F8 responsible for hemophilia A: results from a single institution. Hum Mutat. 2006;27:676–85.View ArticlePubMedGoogle Scholar
- Bogdanova N, Markoff A, Eisert R, Wermes C, Pollmann H, Todorova A, et al. Spectrum of molecular defects and mutation detection rate in patients with mild and moderate hemophilia A. Hum Mutat. 2007;28:54–60.View ArticlePubMedGoogle Scholar
- Laurie AD, Hill AM, Harraway JR, Phillipson GT, Benny PS, Smith MP, et al. Preimplantation genetic diagnosis of hemophilia A using indirect linkage analysis and direct genotyping approaches. J Thrombo Haemost. 2010;8:783–9.View ArticleGoogle Scholar
- Fernández RM, Peciña A, Sánchez B, Lozano-Arana MD, García-Lozano JC, Pérez-Garrido R, et al. Experience of preimplantation genetic diagnosis for hemophilia at the University Hospital Virgen Del Rocio in Spain: Technical and clinical overview. Biomed Res Int. 2015;2015:406096.PubMedPubMed CentralGoogle Scholar
- Gouw SC, van den Berg HM, Oldenburg J, Astermark J, de Groot PG, Margaglione M, et al. F8 gene mutation type and inhibitor development in patients with severe hemophilia A: systematic review and meta-analysis. Blood. 2012;119:2922–34.View ArticlePubMedGoogle Scholar
- Liu Q, Nozari G, Sommer SS. Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in haemophilia A. Blood. 1998;92:1458–9.PubMedGoogle Scholar
- Rossetti LC, Radic CP, Larripa IB, De Brasi CD. Genotyping the hemophilia inversion hotspot by use of inverse PCR. Clin Chem. 2005;51:1154–8.View ArticlePubMedGoogle Scholar
- Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood. 2002;99:168–74.View ArticlePubMedGoogle Scholar
- Tantawy AAG. Molecular genetics of hemophilia A: clinical perspectives. Egypt J Med Hum Genet. 2010;11:105–14.View ArticleGoogle Scholar
- Shetty S, Ghosh K, Mohanty D. Alternate strategies for carrier detection and antenatal diagnosis in haemophilias in developing countries. Indian J Hum Genet. 2003;9:5–9.Google Scholar
- Mitchell M, Keeney S, Goodeve A. UK Haemophilia Centre Doctors’ Organization Haemophilia Genetics Laboratory Network. The molecular analysis of haemophilia A: a guideline from the UK haemophilia centre doctors’ organization haemophilia genetics laboratory network. Haemophilia. 2005;11:387–97.View ArticlePubMedGoogle Scholar
- Peyvandi F, Jayandharan G, Chandy M, Srivastava A, Nakaya SM, Johnson MJ, et al. Genetic diagnosis of haemophilia and other inherited bleeding disorders. Haemophilia. 2006;12 Suppl 3:82–9.View ArticlePubMedGoogle Scholar
- Castaldo G, D’Argenio V, Nardiello P, Zarrilli F, Sanna V, Rocino A, et al. Haemophilia A: molecular insights. Clin Chem Lab Med. 2007;45:450–61.View ArticlePubMedGoogle Scholar
- Goodeve A. Molecular genetic testing of hemophilia A. Semin Thromb Hemost. 2008;34:491–501.View ArticlePubMedGoogle Scholar
- Renwick PJ, Lewis CM, Abbs S, Ogilvie CM. Determination of the genetic status of cleavage-stage human embryos by microsatellite marker analysis following multiple displacement amplification. Prenat Diagn. 2007;27:206–15.View ArticlePubMedGoogle Scholar
- Laurie AD, Hill AM, Harraway JR, Fellowes AP, Phillipson GT, Benny PS, et al. Preimplantation genetic diagnosis for hemophilia A using indirect linkage analysis and direct genotyping approaches. J Thromb Haemost. 2010;8:783–9.View ArticlePubMedGoogle Scholar
- Chang LJ, Huang CC, Tsai YY, Hung CC, Fang MY, Lin YC, et al. Blastocyst biopsy and vitrification are effective for preimplantation genetic diagnosis of monogenic diseases. Hum Reprod. 2013;28:1435–44.View ArticlePubMedGoogle Scholar
- De Rycke M, Georgiou I, Sermon K, Lissens W, Henderix P, Joris H, et al. PGD for autosomal dominant polycystic kidney disease type 1. Mol Hum Reprod. 2005;11:65–71.View ArticlePubMedGoogle Scholar
- Korzebor A, Derakhshandeh-Peykar P, Meshkani M, Hoseini A, Rafati M, Purhoseini M, et al. Heterozygosity assessment of five STR loci located at 5q13 region for preimplantation genetic diagnosis ofspinal muscular atrophy. Mol Biol Rep. 2013;40:67–72.View ArticlePubMedGoogle Scholar
- Naylor JA, Buck D, Green P, Williamson H, Bentley D, Gianneill F. Investigation of the factor VIII intron 22 repeated region (int22h) and the associated inversion junctions. Hum Mol Genet. 1995;4:1217–24.View ArticlePubMedGoogle Scholar
- Mastenbroek S, Twisk M, van der Veen F, Repping S. Preimplantation genetic screening: a systematic review and meta-analysis of RCTs. Hum Reprod Update. 2011;17:454–66.View ArticlePubMedGoogle Scholar
- Schoolcraft WB, Treff NR, Stevens JM, Ferry K, Katz-Jaffe M, Scott Jr RT. Live birth outcome with trophectoderm biopsy, blastocyst vitrification, and single-nucleotide polymorphism microarray-based comprehensive chromosome screening in infertile patients. Fertil Steril. 2011;96:638–40.View ArticlePubMedGoogle Scholar
- Treff NR, Levy B, Su J, Taylor D, Scott Jr RT. Development and validation of an accurate quantitative real-time polymerase chain reaction- based assay for human blastocyst comprehensive chromosomal aneuploidy screening. Fertil Steril. 2012;97:819–24.View ArticlePubMedGoogle Scholar
- Rubio C, Rodrigo L, Mir P, Mateu E, Peinado V, Milán M, et al. Use of array comparative genomic hybridization (array-CGH) for embryo assessment: clinical results. Fertil Steril. 2013;99:1044–8.View ArticlePubMedGoogle Scholar
- Treff NR, Tao X, Ferry KM, Su J, Taylor D, Scott Jr RT. Development and validation of an accurate quantitative real-time polymerase chain reaction- based assay for human blastocyst comprehensive chromosomal aneuploidy screening. Fertil Steril. 2012;97:819–24.View ArticlePubMedGoogle Scholar
- Yang YS, Chang SP, Chen HF, Ma GC, Lin WH, Lin CF, et al. Preimplantation genetic screening of blastocysts by multiplex qPCR followed by fresh embryo transfer: validation and verification. Mol Cytogenet. 2015;8:49.View ArticlePubMedPubMed CentralGoogle Scholar
- Wells D, Kaur K, Grifo J, Glassner M, Taylor JC, Fragouli E, et al. Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. J Med Genet. 2014;51:553–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Ma GC, Chen HF, Yang YS, Lin WH, Tsai FP, Lin CF, et al. A pilot proof-of-principle study to compare fresh and vitrified cycle preimplantation genetic screening by chromosome microarray and next generation sequencing. Mol Cytogenet. 2016;9:25.View ArticlePubMedPubMed CentralGoogle Scholar
- Giménez C, Sarasa J, Arjona C, Vilamajó E, Martínez-Pasarell O, Wheeler K, et al. Karyomapping allows preimplantation genetic diagnosis of a de-novo deletion undetectable using conventional PGD technology. Reprod Biomed Online. 2015;31:770–5.View ArticlePubMedGoogle Scholar
- Zimmerman RS, Jalas C, Tao X, Fedick AM, Kim JG, Northrop LE, et al. Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders and comprehensive chromosomal aneuploidy screening without whole genome amplification. Fertil Steril. 2016;105:286–94.View ArticlePubMedGoogle Scholar
- Gui B, Yang P, Yao Z, Li Y, Liu D, Liu N, et al. A new next-generation sequencing-based assay for concurrent preimplantation genetic diagnosis of Charcot-Marie-Tooth disease yype 1A and aneuploidy screening. J Genet Genomics. 2016;43:155–9.View ArticlePubMedGoogle Scholar
- Goldman KN, Nazem T, Berkeley A, Palter S, Grifo JA. Preimplantation genetic diagnosis (PGD) for monogenic disorders: the value of concurrent aneuploidy screening. J Genet Couns. 2016. [Epub ahead of print]
- Forman EJ, Hong KH, Franasiak JM, Scott Jr RT. Obstetrical and neonatal outcomes from the BEST Trial: single embryo transfer with aneuploidy screening improves outcomes after in vitro fertilization without compromising delivery rates. Am J Obstet Gynecol. 2014;210:157.e1-6.View ArticlePubMedGoogle Scholar
- Yang Z, Lin J, Zhang J, Fong WI, Li P, Zhao R, et al. Randomized comparison of next-generation sequencing and array comparative genomic hybridization forpreimplantation genetic screening: a pilot study. BMC Med Genomics. 2015;8:30.View ArticlePubMedPubMed CentralGoogle Scholar