H.A. Tac

Successful haematopoietic stem cell transplantation in 44 children from healthy siblings conceived after preimplantation HLA matching

Haematopoietic stem cell transplantation (HSCT) remains the best therapeutic option for many acquired and inherited paediatric haematological disorders. Unfortunately, the probability of finding an HLA matched donor is limited. An alternative technique is PGD combined with HLA matching, which offers the possibility of selecting unaffected embryos that are HLA compatible with the sick child, with the aim of possible use of stem cells from the resulting baby in future. Since the first successful report for Fanconi anaemia a decade ago, the therapeutic success of this technique was reported in a few cases and for a limited number of disorders. Here, we report full recovery of 44 sick children who received HSCT from healthy infants conceived after pre-implantation HLA matching for the following 10 indications; beta-thalassaemia, Wiskott-Aldrich syndrome, Fanconi anaemia, sickle cell anaemia, acute myeloid leukaemia, acute lymphoblastic leukaemia, Glanzmanns thrombasthaenia, Diamond-Blackfan anaemia, X-linked adrenoleukodystrophy and mucopolysaccharidosis type I. No serious complications were observed among recipients and donors. Graft failure occurred in four children with beta-thalassaemia where a second HSCT was planned. Preimplantation HLA matching is a reliable technique and provides a realistic option for couples seeking treatment for an affected child when no HLA-matched donor is available.

Birth of a healthy infant after preimplantation genetic diagnosis by sequential blastomere and trophectoderm biopsy for β-thalassemia and HLA genotyping

BACKGROUND Preimplantation genetic diagnosis (PGD) is a widely used technique for couples at genetic risk and involves the diagnosis and transfer of unaffected embryos generated through in vitro fertilization (IVF) techniques. STUDY DESIGN For those couples who are at risk of transmitting a genetic disease to their offspring, preimplantation embryos can be selected according to their genetic status as well as human leukocyte antigen (HLA) compatibility with the affected child. Stem cells from the resulting babys umbilical cord blood can be used for transplantation to the affected sibling without graft rejection. RESULTS Here we report successful hematopoietic stem cell transplantation (HSCT) after the birth of a healthy infant, who was born after successful PGD testing with both cleavage stage and blastocyst stage biopsy for the purpose of diagnosis of β-thalassemia and HLA compatibility. CONCLUSION The specific feature of this work is not only to have the first successful HSCT achieved in Bulgaria after using preimplantation HLA typing technique, it also demonstrates how to accomplish this success via cross-border collaboration of different units, which makes the application of these sophisticated methods possible in hospitals not having the necessary equipments and expertise.

O2 Outcomes of 303 cycles on preimplantation Human Leukocyte Antigen typing with or without mutation analysis

Aims: Single cell diagnosis for PGD requires simultaneous analysis of multiple linked polymorphic markers in addition to mutation analysis in order to reduce misdiagnosis. This type of analysis requires building family haplotypes spanning at least two generations. In cases of germ line mosaicism the wild type haplotype can be seen in both affected and unaffected children making the diagnosis more complex. We present a family with two children affected with Tuberous Sclerosis (TSC1 C1327T) and two healthy children in which both parents tested negative for the mutation. Material and Methods: Twelve informative markers flanking the TSC1 gene were used to construct haplotypes. Genomic DNA isolated from blood and buccal cells was used to analyze the 1327C>T mutation. Single sperm analysis was performed using a multiplex assay that included the 12 markers and the 1327C>T mutation. Results: The identified 1327C>T mutation was not detected in genomic DNA derived from blood or buccal cells from either parent. Both affected children shared the same paternal allele and different maternal alleles. However, one of the two healthy children also shared this same paternal allele, and in order to confirm paternal transmission we performed single sperm analysis for the mutation and 12 markers. Of 44 single sperm analyzed 4 bared the T allele allowing linkage between the mutation and markers. Conclusions: Germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm or polar body analysis are useful tools for verifying allelic linkage.

Recent advances in preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) gives couples who are at risk of having a child with an inherited genetic disorder or chromosome abnormality, a chance to have an unaffected child without undergoing termination or miscarriage of an affected pregnancy. Embryos obtained from in-vitro fertilisation (IVF) with or without intracytoplasmic sperm injection are tested genetically prior to selective transfer of unaffected ones into the uterus. The physical and psychological complications of a termination or miscarriage, especially in repeated situations, should not be underestimated. In PGD, DNA can be obtained by blastomere biopsy at the cleavage stage, trophectoderm cell biopsy when an embryo has developed to the blastocyst stage or biopsy of one or both polar bodies. Compared with cleavage stage biopsy, trophectoderm biopsy does not adversely impact the embryo, although vitrification and cryopreservation of the embryo may be required to allow time for genetic analysis.1 Although polar body biopsy is less invasive, it is less predictive of actual clinical outcome than direct embryo assessment.2 Genetic laboratories have developed their own protocols to perform different molecular tests on the limited amount of DNA obtained from biopsy. Traditionally, fluorescent in-situ hybridisation is used for cytogenetic diagnosis, and polymerase chain reaction for molecular diagnosis. New technologies, including array comparative genomic hybridisation (CGH) and single nucleotide polymorphism (SNP) microarrays, can improve diagnostic accuracy.3,4 The single-cell whole genome amplification (WGA) method allows subsequent mutation study, directly by minisequencing and/or indirectly by linkage analysis alongside the mutation test. It also allows simultaneous PGD for more than one indication.5 The indications for PGD are increasing. Common ones include single-gene disorders, X-linked diseases, and inherited chromosome abnormalities. Preimplantation genetic diagnosis of predisposition to inherited cancer such as breast cancer (BRCA mutation) is also emerging.6 Nonetheless, social sexing is prohibited in Hong Kong and Europe. Legislation and regulation of PGD also vary among different countries. Aneuploidy is the most common cause of repeated implantation failure and recurrent Hong Kong Med J 2015;21:296–7 DOI: 10.12809/hkmj154638 KY Leung*, MD, FHKAM (Obstetrics and Gynaecology)