Preimplantation genetic diagnosis in the prevention of the haemoglobin disorders

Preimplantation Genetic Diagnosis (PGD) is currently an alternative for couples with high risk of pregnancies with genetic anomalies; it offers the possibility of avoiding the need to terminate affected pregnancies, since it allows the selection of unaffected embryos for transfer. PGD for inherited disorders has become extremely accurate (99.5%), and may currently be performed for any single gene disorders in which mutation is identified. PGD has been performed for more than 100 different conditions resulting in the birth of at least 1000 healthy children free of genetic disorder. PGD is presently also used together with preimplantation HLA typing for treatment of affected sibling with genetic and acquired disorders requiring HLA matched stem cell transplantation. This is not only to allow couples to have an unaffected child but also to select a potential donor progeny for stem cell transplantation. In Turkey, thalassemia is the most commonly seen genetic disorder the rate of thalassemia carriers is about 3 - 4% in Turkey. The majority of our PGD cases are thalassemia carriers. They do not only require thalassemia mutation analysis but also HLA typing for their affected child. In this study PGD results of 236 Turkish couples with or without HLA typing will be presented and discussed. A full diagnosis was achieved in 91.0% of the biopsied samples. In Group I, 17.8% of the analyzed embryos were found to be HLA compatible. HLA compatible and disease free embryos were 12.9% of all diagnosed embryos. In group II, 17.2% of embryos were found to be HLA matched and 71.4% HLA non-matched. The majority of our HLA typing combined with PGD cases were β-Thalassemia carriers (87.9%). The mutations analyzed have high heterogeneity, the most frequent mutation was IVS-I-110 G-A and comprised 46.2% of all mutations. To date, 70 healthy and HLA compatible children have been born. Twenty-five sick children have already been cured with cord blood cell and/or bone marrow transplantation. Twenty-one children are waiting for their newborn siblings to gain sufficient weight and maturity for the donation of stem cells. The successful transplantations have been performed for the following indications: β- Thalassemia (n=19), Wiskott Aldrich syndrome (n=2), Glanzmann Disease (n=1), X-Adrenoleukodystrophy (n=1) and acute myeloid leukemia (n=1) and Diamond Blackfan anemia (n=1). This data presents one of the world’s largest experiences on preimplantation HLA typing, and the outcome of stem cell transplantation is the largest number available from one center. Our results indicate HLA typing with or without mutation analysis is a promising and effective therapeutic tool for curation of an affected sibling. 如今，对于胎儿先天畸形的高风险夫妇来讲，可以选择植入前遗传学诊断（PGD），由于它能对未受地贫遗传的胎儿进行选择，因而能更有效地终止被遗传地贫的胎儿的出现几率。 遗传病植入前遗传学诊断（PGD）已日趋准确（准确率高达99.5%），如今，此方法也能用来诊断所有的确诊变异的单基因病。 植入前遗传学诊断（PGD）使至少一千多名本来可能患上遗传地贫的孩子免遭此劫难， 这些孩子中，情况不一，一百多种情况不尽相同，他们如今非常健康。现在，植入前遗传学诊断（PGD）还和胚胎植入前白细胞抗原配型一起使用，来治疗受感染的同胞种基因，以及HLA匹配的干细胞移植。 这不仅使夫妇能得到健康的孩子，还能为干细胞移植选择一个潜在的捐赠者后代。 在土耳其，地中海贫血是最常见的基因遗传病，土耳其的地贫基因携带者约为3%-4%。 我们的植入前遗传学诊断（PGD）的患者中大多数人都是地中海贫血基因携带者。 他们不仅仅被要求做地中海贫血基因突变分析，还给他们受到地贫基因遗传的孩子做HLA配型。 这份对植入前遗传学诊断（PGD）的研究结果对使用或没使用HLA配型的夫妇进行了展示和讨论。 在活体样本中，诊断的完整度达到了91.0%。 在第一组样本中，经过分析的胚胎中有17.8%与HLA匹配。 在全部诊断的胚胎中，HLA兼容和无遗传病基因的胚胎占12.9%。 在第二组样本中，有17.2%的胚胎与HLA匹配，71.4%不匹配。 我们的HLA配型与植入前遗传学诊断（PGD）中大多数案例都是β型地中海贫血基因携带者（占87.9%）。 经分析表明基因突变有高异质性，而最频繁的基因突变是IVS-I-110 G-A ，占总数46.2%。 迄今为止，70名健康的以及HLA配型成功的孩子已经降生。 25名患病儿童已在脐带血肝细胞以及/或者骨髓移植的治疗方法下得到康复。 21名患病儿童正在等待他们的同胞弟妹的出生以及成长，等他们体重达到捐献所规定的体重，以及成长到规定的年龄，就能获得他们捐出的干细胞。 移植手术已成功地在如下疾病中实施： β型地中海贫血（n=19）、德里症候群（n=2）、血小板无力症（n=1）、X-肾上腺白质营养不良症（n=1）、急性骨髓性白血病（n=1)）、先天性纯红血球再生障碍性贫血（n=1）。 这份数据展示了关于胚胎植入前HLA配型世界上最丰富的经验之一，干细胞移植的成果已达到了一个中心所能成功的最大化。 我们的研究结果表明有或者无基因突变分析的HLA配型对于治疗有遗传病的胚胎，是前景广阔并且行之有效的一种治疗手段。


Introduction
Preimplantation Genetic Diagnosis (PGD) is currently an alternative for couples with high risk of pregnancies with genetic anomalies.PGD offers to these couples the possibility of avoiding the need to terminate affected pregnancies, since it allows the selection of unaffected embryos for transfer.PGD for inherited disorders has become extremely accurate (99.5%), and may currently be performed for any single gene disorders in which mutation is identified.
PGD has been performed for more than 100 different conditions resulting in the birth of at least 1000 healthy children free of genetic disorder.PGD is presently also used together with preimplantation HLA typing for treatment of affected sibling with genetic and acquired disorders requiring HLA matched stem cell transplantation (Fiorentino et al., 2006;Kahraman et al., 2004Kahraman et al., , 2007;;Verlinsky et al., 2001Verlinsky et al., , 2004;;Van de Velde et al., 2004, 2009).This is not only to allow couples to have an unaffected child but also to select a potential donor progeny for stem cell transplantation.
Due to a small number of children per family, only one third of patients are able to find an HLA identical sibling.This may further be improved by 3% using an extended family research for a matched related donor.In the remaining patients the only resort is the identification of a matched unrelated donor, which may be maximized by establishing national registries.
It is therefore of a great value for hematopoietic and other life threatening diseases as stem cells in the cord blood and bone marrow from an HLA compatible newborn can be used for transplantation without graft rejection, thus saving an affected children's life.PGD for HLA matching has also been used as a primary indication in cases not requiring mutation testing (Verlinsky et al., 2004) (i.e. leukemia).Considering that the theoretical probability of finding HLA compatible and mutation free embryos is about 18%, obtaining a sufficient number of suitable embryos as well as good quality necessitates a higher number of oocytes to be fertilized and embryos to be biopsied for a given cycle.Apart from being a valuable treatment approach, there may exist several patients or cycle-specific limitations and it seems that not all couples can benefit from the present procedures.
Correspondence: S. Kahraman, Istanbul Memorial Hospital ART and Reproductive Genetics Center, Istanbul, Turkey.
Key words: hemoglobin disorders, genetic diagnosis.©Copyright S. Kahraman et al., 2011Licensee PAGEPress, Italy Thalassemia Reports 2011;1(s2):e5 doi:10.4081/thal.2011.s2.e5This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.In Turkey, thalassemia is the most commonly seen genetic disorder the rate of thalassemia carriers is about 3 -4% in Turkey (Basak et al., 2007).The majority of our PGD cases are thalassemia carriers.They do not only require thalassemia mutation analysis but also HLA typing for their affected child.In this chapter, PGD results of 236 Turkish couples with or without HLA typing will be presented and discussed.

Pre-clinical work up
First, a haplotype analysis of mother, father and child, and when available of other family members, was performed for each family prior to preimplantation HLA typing.For this, genomic DNA is isolated from peripheral blood samples of father, mother and the affected child.A panel of 50 different short tandem repeat (STR) markers (Figure 1) were tested on genomic DNAs to ensure the presence of enough informative markers (Figure 2) to aid the identification of monosomy, trisomy, recombination, allele-drop out (ADO) and uniparental disomy (UPD) of the analyzed chromosomes and regions.For each family at least 12 heterozygous markers spanning the HLA-A, HLA-B, HLA-C, HLA-DR,HLA-DQ regions (HLA Classes I, II, and III) were selected for PGD Study.

Preimplantation genetic diagnosis study
DNA testing was performed by two rounds of PCR (polymerase chain reactions): in the first round, using multiplex PCR which allows simultaneous amplification of HLA regions and mutation-linked markers and in the second round, using singleplex PCR which is a fluorescent PCR with semi or heminested primers.Primer sequences and polymerase chain reaction conditions used in this study have been reported previously (Verlinsky et al., 2001;Fiorentino et al., 2004;2005;Rechitsky et al., 2004;Verlinsky et al., 2004).

IVF and embryo biopsy procedure
The stimulation protocols were as outlined previously (Kahraman et al., 2004).Oocyte retrievals were performed 36 h after the injection of rhCG (ovitrel) by transvaginal-ultrasound-guidance.Approximately 2-3 h after oocyte retrieval, cumulus cells were enzymatically removed.Intracytoplasmic sperm injection (ICSI) was applied to metaphase II oocytes.One blastomere was removed from cleavage stage embryos (Figure 3a) from an opening made using laser (IodoLaser, Research Instruments).Subsequently, embryo transfer was performed usually on day-4 but rarely on day-5.Recently, since 2009, trophectoderm tissue biopsies have also been performed.

Results
In HLA+mutation testing group (Group I), and HLA-only group (Group II), 62.2% and 72.4% of the initiated cycles reached the stage of embryo transfer, respectively.The detailed distribution of indications and overall results for each group was shown in Table 1 and in Table 2.
A full diagnosis was achieved in 91.0% of the biopsied samples.In Group I, 17.8 % of the analyzed embryos were found to be HLA compatible.HLA compatible and disease free embryos were 12.9 % of all diagnosed embryos.In group II, 17.2% of embryos were found to be HLA matched and 71.4% HLA non-matched.
The majority of our HLA typing combined with PGD cases were β-Thalassemia carriers (87.9%).The mutations analyzed have high heterogeneity, the most frequent mutation was IVS-I-110 G-A and comprised 46.2% of all mutations.The total frequency of the most frequent 6 mutations were 74,0% (Table 3).
A total of 85 clinical pregnancies (36.5%) were achieved from 233 ET cycles.5 pregnancies are ongoing.To date, 70 healthy and HLA compatible children have been born.25 sick children have already been cured with cord blood cell and/or bone marrow transplantation.21 children are waiting for their newborn siblings to gain sufficient weight and maturity for the donation of stem cells (Table 2).The successful transplantations have been performed for the following indications: β-Thalassemia (n=19), Wiskott Aldrich syndrome (n=2), Glanzmann Disease (n=1), X-Adrenoleukodystrophy (n=1) and acute myeloid leukemia (n=1) and Diamond Blackfan anemia (n=1) (Table 4).

Conclusion
This data presents one of the world's largest experiences on preimplantation HLA typing, and the outcome of stem cell transplantation is the largest number available from one center.To date 25 children have been cured with this approach and 21 children are awaiting appropriate time for transplantation.Our results indicate HLA typing with or without mutation analysis is a promising and effective therapeutic tool for curation of an affected sibling.

Figure 1 .
Figure 1.Polymorphic STR markers used in HLA typing of the embryos.

Figure
Figure 2. Identification of informative markers during HLA set up study.

Table 2 . Overall clinical results of HLA typing.
* 21 children are awaiting an appropriate time for HSC transplantation.