Application Of Preimplantation Genetic Diagnosis Research Articles

A critical view on using "life not worth living" in the bioethics of assisted reproduction.

This paper critically engages with how life not worth living (LNWL) and cognate concepts are used in the field of beginning-of-life bioethics as the basis of arguments for morally requiring the application of preimplantation genetic diagnosis (PGD) and/or germline genome editing (GGE). It is argued that an objective conceptualization of LNWL is largely too unreliable in beginning-of-life cases for deriving decisive normative reasons that would constitute a moral duty on the part of intending parents. Subjective frameworks are found to be more suitable to determine LNWL, but they are not accessible in beginning-of-life cases because there is no subject yet. Conceptual and sociopolitical problems are additionally pointed out regarding the common usage of clear case exemplars. The paper concludes that a moral requirement for the usage of PGD and GGE cannot be derived from the conceptual base of LNWL, as strong reasons that can be reliably determined are required to limit reproductive freedom on moral grounds. Educated predictions on prospective well-being might still be useful regarding the determination of moral permissibility of PGD and/or GGE. It is suggested that due to the high significance of subjective experience in the normativity of beginning-of-life bioethics, the discipline is called to more actively realize the inclusion of people with disabilities. This regards for instance research design, citation practices, and language choices to increase the accessibility of societal debates on the reproductive ethics of genetic technologies.

Assessment of attitudes towards the use of preimplantation genetic diagnosis in a single center in Riyadh, Saudi Arabia.

In this cross-sectional study, we assessed the attitudes of the general public in Saudi Arabia regarding both medical and non-medical applications of pre-implantation genetic diagnosis (PGD). The study was conducted in King Abdullah Specialist Children's Hospital (KASCH) in Riyadh with a sample size of 377. Demographic information was collected, and attitudes towards applications of PGD were assessed using a pre-validated self-administered questionnaire. Out of the total sample size, 230 (61%) were males, 258 (68%) were married, 235 (63%) had one child or more, and 255 (68%) were older than 30 years of age representing the majority of participants. Only 87 (23%) of participants reported prior experience with PGD. Personally, knowing someone who had a prior experience with PGD was associated with higher attitude scores (more favorable attitudes towards PGD) (p-value = 0.04). The findings of this study indicate that our sample of Saudi individuals generally had a positive attitude towards the use of PGD.

First Successful Application of Preimplantation Genetic Diagnosis for Lethal Neonatal Rigidity and Multifocal Seizure Syndrome in Korea: A Case Report

Lethal neonatal rigidity and multifocal seizure syndrome (RMFSL) is a severe autosomal recessive epileptic encephalopathy characterized by rigidity, intractable multifocal seizures, microcephaly, apnea, and bradycardia immediately after birth. RMFSL is related to a mutation in breast cancer 1-associated ataxia telangiectasia mutated activation-1 protein (BRAT1). We report a case of a female infant born to non-consanguineous Korean parents who developed hypertonia, dysmorphic features, progressive encephalopathy with refractory seizures at birth, and worsening intermittent apnea, leading to intubation and death at 137 days of age. The initial repeated electroencephalographic findings were normal; however, a pattern of focal seizures emerged at 35 days of life. Rapid trio whole-exome sequencing revealed heterozygous mutations c.1313_1314delAG p.(Gln438Argfs*51) and c.1276C>T p. (Gln426*) in BRAT1. After genetic counseling for pregnancy planning, a preimplantation genetic diagnosis for targeted BRAT1 mutations was successfully performed, and a healthy baby was born. To our knowledge, this is the first reported case of a Korean patient with compound heterozygous mutations in BRAT1. An early and accurate genetic diagnosis can help provide timely treatment to patients and indicate the need for reproductive counseling for parents for family planning.

Advances in preimplantation genetic diagnosis

In the past 20 years, molecular genetic technology has developed rapidly. The leap forward development of single-cell genetic diagnosis technologies represented by whole genome amplification combined with microarray technology or next-generation sequencing not only increased the accuracy of preimplantation genetic diagnosis (PGD) but also greatly expanded the variety and scope of detectable diseases. This paper systematically reviews the clinical application of PGD as well as recent progress of related technologies. Key words: Preimplantation genetic diagnosis; Microarray technology; Next-generation sequencing

The first successful application of preimplantation genetic diagnosis for hearing loss in Iran

Hearing impairment (HI) caused by mutations in the connexin-26 gene (GJB2) accounts for the majority of cases with inherited, nonsyndromic sensorineural hearing loss. Due to the illegality of the abortion of deaf fetuses in Islamic countries, preimplantation genetic diagnosis (PGD) is a possible solution for afflicted families to have a healthy offspring. This study describes the first use of PGD for GJB2 associated non-syndromic deafness in Iran. GJB2 donor splicing site IVS1+1G>A mutation analysis was performed using Sanger sequencing for a total of 71 Iranian families with at least 1 deaf child diagnosed with non-syndromic deafness. In Vitro Fertilization (IVF) was performed, followed by PGD for a cousin couple with a 50% chance of having an affected child. Bi-allelic pathogenic mutations were found in a total of 12 families (~17 %); of which a couple was a PGD volunteer. The deaf woman in this family was homozygous and her husband was a carrier of the IVS1+1G>A gene mutation. Among 8 biopsied embryos, two healthy embryos were implanted which resulted in a single pregnancy and subsequent birth of a healthy baby boy. This is the first report of a successful application of PGD for hearing loss in Iran. Having a baby with a severe hearing impairment often imposes families with long-term disease burden and heavy therapy costs. Today PGD has provided an opportunity for high-risk individuals to avoid the birth of a deaf child.

Successful preimplantation genetic diagnosis by targeted next-generation sequencing on an ion torrent personal genome machine platform.

Hearing loss may place a heavy burden on the patient and patient's family. Given the high incidence of hearing loss among newborns and the huge cost of treatment and care (including cochlear implantation), prenatal diagnosis is strongly recommended. Termination of the fetus may be considered as an extreme outcome to the discovery of a potential deaf fetus, and therefore preimplantation genetic diagnosis has become an important option for avoiding the birth of affected children without facing the risk of abortion following prenatal diagnosis. In one case, a couple had a 7-year-old daughter affected by non-syndromic sensorineural hearing loss. The affected fetus carried a causative compound heterozygous mutation c.919-2 A>G (IVS7-2 A>G) and c.1707+5 G>A (IVS15+5 G>A) of the solute carrier family 26 member 4 gene inherited from maternal and paternal sides, respectively. The present study applied multiple displacement amplification for whole genome amplification of biopsied trophectoderm cells and next-generation sequencing (NGS)-based single nucleotide polymorphism haplotyping on an Ion Torrent Personal Genome Machine. One unaffected embryo was transferred in a frozen-thawed embryo transfer cycle and the patient was impregnated. To conclude, to the best of our knowledge, this may be the first report of NGS-based preimplantation genetic diagnosis (PGD) for non-syndromic hearing loss caused by a compound heterozygous mutation using an Ion Torrent Personal Genome Machine. NGS provides unprecedented high-throughput, highly parallel and base-pair resolution data for genetic analysis. The method meets the requirements of medium-sized diagnostics laboratories. With decreased costs compared with previous techniques (such as Sanger sequencing), this technique may have potential widespread clinical application in PGD of other types of monogenic disease.

The clinical application of preimplantation genetic diagnosis to prevent spinal muscular atrophy in a Chinese family: A case report

The clinical application of preimplantation genetic diagnosis to prevent spinal muscular atrophy in a Chinese family: A case report

P100 A novel sequence enrichment protocol allowing HLA-typing on 5-cells

Aim Limited genomic DNA template in a sample can make accurate HLA-typing difficult. We therefore aimed to develop a method that allows HLA-typing using next generation sequencing (NGS) up to p-group resolution with starting DNA template of only 5 cells. Methods Aliquots of 5 cells were sorted from aneuploid and euploid cell lines (Coriell Institute of Medical Research, USA). Whole genome amplification was performed using the DOPlify™ TSE (Targeted Sequence Enrichment) protocol (RHS Ltd, Australia) with the inclusion of SBTessenz® primers (GenDx, the Netherlands) to increase amplification of exon 2 to 3 of the HLA-A locus. Enrichment was determined using semi-quantitative PCR for HLA-A. The HLA-A amplicon was subsequently pooled with the original DOPlify™ TSE WGA DNA in a 1:10 and 1:20 ratio before library preparation. After indexing and multiplexing of 40 samples for sequencing on a MiSeq platform according to standard 2 × 75 bp protocol (Illumina), the NGS data was analyzed and typed by the HLA-typing software NGSengine®. Results Targeted sequence enrichment increased the breadth and depth of coverage of exon 2, intron 2 and exon 3 for the HLA-A locus, with concurrent detection of aneuploidy. When the HLA-A amplicon was pooled with the DOPlify™ TSE WGA DNA, coverage of 100% was achieved and the average read depth following NGS was greater than 200. Amplification of both alleles was confirmed with a ratio of approximately 30:70% which enabled clear detection and analysis of the heterozygous positions. The quality of the sequence data allowed resolution of all ambiguities that are present in exon 2, intron 2 and exon 3; including the sequence up front of exon 2 required to exclude several null alleles. Conclusions We developed a novel sequence enrichment protocol to amplify and type HLA-A from a 5 cell sample. The data quality meets general laboratory standards for HLA-typing and allows typing up to p-group resolution. This protocol has application in preimplantation genetic diagnosis and low template input HLA typing such as buccal swabs.

What is Karyomapping and where does it fit in the world of preimplantation genetic diagnosis (PGD)?

The first application of preimplantation genetic diagnosis (PGD) recently celebrated its 25th birthday. Aside from the very early days when chromosomal diagnoses were used (by sexing) for the selective implantation of embryos unaffected by sex linked disorders, the paths of chromosomal and monogenic PGD have diverged. For monogenic disorders, progress has been impeded by the need to tailor each diagnosis to the mutation in question. For chromosomal diagnoses, fluorescent in situ hybridization (FISH) technology was replaced by array comparative genomic hybridization (aCGH), and then next generation sequencing (NGS). Karyomapping is a novel approach that allows the detection of the inheritance of (grand) parental haploblocks through the identification of inherited chromosomal segments. It involves genome-wide single nucleotide polymorphism (SNP) analysis of parental DNA, a reference from a related individual of known disease status (typically an affected child) and amplified DNA form biopsied cells of the (usually blastocyst) embryos in question. Identification of informative loci for each of four parental haplotypes is followed by direct comparison to the reference, ultimately creating a Karyomap. The Karyomapping programme (Illumina) displays homologous chromosomes, points of crossing over and the haplotype of each of the embryos. It also detects meiotic trisomy, monosomy, triploidy and uniparental heterodisomy (some of which NGS and aCGH will not). Inherent in the design is the analysis of “key SNPs” (heterozygous informative calls) thereby avoiding the risk of misdiagnoses caused by the phenomenon of allele drop out (ADO). Karyomapping is currently in use for the detection of monogenic disorders and around 1000 clinics offer it worldwide making use of about 20 diagnostic laboratories. At the time of writing, over two and a half thousand clinical cases have been performed. Because of the limited detection of some post-zygotic errors such as post-zygotic trisomy which can also lead to mosaicism, Karyomapping has not yet been fully applied clinically for aneuploidy screening. The diagnostic potential of the technique will be fully recognised with the application of this technology on clinical cases.

Application of preimplantation genetic diagnosis in equine blastocysts

Application of preimplantation genetic diagnosis in equine blastocysts

The clinical application of preimplantation genetic diagnosis for the patient affected by congenital contractural arachnodactyly and spinal and bulbar muscular atrophy.

To investigate the usefulness of preimplantation genetic diagnosis (PGD) for the patient affected by congenital contractural arachnodactyly (CCA) and spinal and bulbar muscular atrophy (SBMA). Multiple displacement amplification (MDA) was performed for whole genome amplification (WGA) of biopsied trophectoderm (TE) cells. Direct mutation detection by sequencing and next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping were used for CCA diagnosis. Direct sequencing of the PCR products and sex determination by amplification of sex-determining region Y (SRY) gene were used for SBMA diagnosis. After PGD, the unaffected blastocyst (B4) was transferred in the following frozen embryo transfer (FET). In this PGD cycle, sixteen MII oocytes were inseminated by ICSI with testicular spermatozoa. Four blastocysts (B4, B5, B10, B13) were utilized for TE cell biopsy on day 5 after ICSI. After PGD, B4 was unaffected by CCA and SBMA. B5 was affected by CCA and carried SBMA. B10 was unaffected by CCA and carried SBMA. B13 was affected by CCA and unaffected by SBMA. B4 was the only unaffected blastocyst and transferred into the uterus for the subsequent FET cycle. The accuracy of PGD was confirmed by amniocentesis at 21weeks of gestation. A healthy boy weighing 2850g was born by cesarean section at the 38th week of gestation. PGD is a valid screening tool for patienst affected of CCA and SBMA to prevent transmission of these genetic diseases from parents to children.

Validation of a semiconductor next-generation sequencing-based protocol for preimplantation genetic diagnosis of reciprocal translocations.

We aim to validate a semiconductor next-generation sequencing (NGS)-based method to detect unbalanced chromosome translocation in preimplantation embryos. The study consisted of a blinded retrospective evaluation with NGS of 145 whole-genome amplification products obtained from biopsy of cleavage-stage embryos or blastocysts, derived from 33 couples carrying different balanced translocations. Consistency of NGS-based copy number assignments was evaluated and compared with the results obtained by array-comparative genomic hybridization. Reliably identified with the NGS-based protocol were 162 segmental imbalances derived from 33 different chromosomal translocations, with the smallest detectable chromosomal segment being 5 Mb in size. Of the 145 embryos analysed, 20 (13.8%) were balanced, 43 (29.6%) were unbalanced, 53 (36.5%) were unbalanced and aneuploid, and 29 (20%) were balanced but aneuploid. NGS sensitivity for unbalanced/aneuploid chromosomal call (consistency of chromosome copy number assignment) was 99.75% (402/403), with a specificity of 100% (3077/3077). NGS specificity and sensitivity for unbalanced/aneuploid embryo call were 100%. Next-generation sequencing can detect chromosome imbalances in embryos with the added benefit of simultaneous comprehensive aneuploidy screening. Given the high level of consistency with array-comparative genomic hybridization, NGS has been demonstrated to be a robust high-throughput technique ready for clinical application in preimplantation genetic diagnosis for chromosomal translocations, with potential advantages of automation, increased throughput and reduced cost.

Preimplantation genetic diagnosis of infantile malignant osteopetrosis in a Chinese family

To explore the application of preimplantation genetic diagnosis (PGD) for infantile malignant osteopetrosis (IMO). For a family affected with IMO, PGD was provided using combined parental mutation detection and haplotype constructions with microsatellite markers spanning the TCIRG1 gene. Prenatal diagnosis was performed on the chorionic villus and amniocentesis samples by direct sequencing. Prenatal diagnosis showed that the fetus by the third pregnancy has carried the parental mutations [c.242delC (p.Pro81Argfs*85) and c.1114C>T (p.Gln372*)], and the pregnancy was terminated. PGD was subsequently performed through mutations detection and haplotype analyses following whole genome amplification (WGA) of each of 13 cells. The results showed that 6 of the 13 embryos were unaffected, 3 were carriers and 4 were affected. Well developed unaffected/carrier embryos were selected and transferred into the uterus. A single pregnancy was confirmed. Subsequently pre- and post-natal diagnoses have confirmed development of a healthy child. The study demonstrated the advantage of PGD over prenatal diagnosis when natural pregnancies have repeatedly produced IMO children/fetuses.

Single-tube nonaplex microsatellite PCR panel for preimplantation genetic diagnosis of Hb Bart's hydrops fetalis syndrome.

To develop a single-tube multi-marker assay for improved preimplantation genetic diagnosis (PGD) of deletional and/or non-deletional Hb Bart's hydrops fetalis syndrome, providing haplotype confirmation of deletional status, and maximization of linkage informativity. We performed in silico mining to identify novel microsatellites within 1 Mb flanking the alpha-globin gene cluster, and optimized a single-tube assay combining detection of α(0) -thalassemia deletions with multi-marker linkage analysis. We performed validation on 100 single cells prior to clinical PGD application. Of 42 markers encompassing the α-globin gene cluster that were identified in silico, 9 were highly polymorphic (0.68 ≤ polymorphism information content ≤ 0.92; 0.66 ≤ Ho ≤ 0.90; 10 ≤ alleles ≤ 35) and optimized to co-amplify directly from a single cell. A validation analysis of 100 single lymphoblasts yielded 100% amplification success for all markers, and individual marker allele drop-out (ADO) rates of 0-5%. Clinical application of the assay in PGD for Hb Bart's (2 cases/cycles) resulted in a twin pregnancy and healthy live birth of two baby girls. This single-tube nonaplex microsatellite PCR panel can be applied directly to PGD of most deletional Hb Bart's without the need for deletion-specific customization, and to linkage-based PGD of non-deletional Hb Bart's.

Update on preimplantation genetic diagnosis and screening

Recent advancement in both human embryology and genomics has created a completely new situation for practical and widespread application of preimplantation genetic diagnosis and screening with a dramatic effect on assisted reproduction. The mapping of the first human genome and the advancement in sequencing technology and bioinformatics has led to the discovery of the exact genetic background of exponentially increasing number of diseases. In parallel, methods for culturing human embryos have also radically improved, enabling the late transfer, and the procedure of vitrification the safe cryopreservation. In consequence, refined genetic analyses have become available from blastocyst biopsy followed by the application of novel genomic methods. Furthermore, some studies suggest that by the selection of aneuploid embryos the pregnancy- and birth-rates can be increased. The amount and the depth of information obtainable from the embryos raise several technical and ethical questions that can be answered by further prospective randomized trials.

Birth of a healthy boy after PGD for X-linked heterotaxy syndrome

Heterotaxy syndrome is a clinically and genetically heterogeneous rare disorder included in the group of congenital heart diseases (CHD). The word heterotaxy comes from the Greek “heteros”, meaning “different”; and “taxis”, meaning “arrangement”. The thoracic and visceral organs (including the heart, liver, lungs, spleen, and stomach) have an abnormal placement, as they are situated randomly with respect to the left-right axis and with respect to one another [20]. The inheritance pattern of the disease is heterogeneous, as it could be autosomal recessive, autosomal dominant or X-linked. At least six different disease-causing genes have been identified [3, 13, 20] and one novel candidate has been determined [15]. Mutations in the zinc finger transcription factor gene (ZIC3, MIM *300265) cause the X-linked heterotaxy syndrome (OMIM #306955) [7], which accounts for less than 5 % of sporadic heterotaxy cases. Female carriers in the reported familial cases are usually asymptomatic, although in one case they presented phenotype [20]. The estimated prevalence is unclear, ranging from 2.5/10000 to 1/15000 (from Orphanet reports series, November 2012) [14]. Affected patients present high morbidity and mortality resulting from a wide variety of congenital defects, as they typically present complex cardiovascular malformations. Clinical diagnosis and surgical treatment for patients have improved recently. However, survival rates of patients undergoing surgery are not satisfactory. In this context, preimplantation genetic diagnosis (PGD) has been proved over the years to be a valid reproductive option for couples carrying a genetic disorder who wish to have offspring free of the disease, avoiding the traumatic termination of pregnancy (TOP). Since its first application in 1990 [8], PGD has been made available for a large number of rare genetic disorders [16] and the number of cycles continuously increases year by year [9]. The aim of this paper is to show, for the first time, the successful application of PGD for X-linked heterotaxy, resulting in the birth of a healthy boy.

KRT9 gene mutation as a reliable indicator in the prenatal molecular diagnosis of epidermolytic palmoplantar keratoderma.

Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries.

Preimplantation Diagnosis for Single Gene Disorders

Preimplantation genetic diagnosis (PGD) allows patients who are carriers or who are affected by genetic diseases to select unaffected embryos for transfer before becoming pregnant. The practice of PGD is evolving with rapid advances in technology and biopsy methods. Testing for a specific gene mutation can be performed in combination with 24-chromosome aneuploidy screening. Several unique applications of PGD are reviewed, including exclusion diagnosis for couples from Huntington disease families, testing for fragile X premutations, and human leukocyte antigen matching for stem cell donor siblings. Although PGD for single gene mutations allows patients to gain information about their embryos and perhaps avoid a difficult decision about whether or not to terminate an ongoing pregnancy, this technique also provides for much ethical debate encompassing the well-being of the prospective couple, embryo, child, and people in the community affected by the diseases being screened.

Atelosteogenesis Type 2/Diastrophic Dysplasia Phenotypic Spectrum: From Prenatal to Preimplantation Genetic Diagnosis

Atelosteogenesis type II (AO2) and diastrophic dysplasia (DTD) are two recessively inherited, severe skeletal dysplasias caused by mutations in the SLC26A2 gene. AO2 is an invariably lethal condition, while DTD patients may reach adult life, although both diseases have overlapping diagnostic features. Here we report a patient with an intermediate phenotype between AO2 and DTD and present the successful application of preimplantation genetic diagnosis (PGD) in this situation. Sequencing of SLC26A2 alleles in the infant identified two compound heterozygous mutations, p.Arg178Ter and p.Arg279Trp, of paternal and maternal origin, respectively. At request from the parents, PGD was developed by haplotype mapping of parental SLC26A2 alleles in eleven five-day embryos. Transference to the mother was attempted twice, finally resulting in pregnancy and delivery of a healthy baby. This exemplifies the utility of PGD for inherited lethal conditions with a significant risk of recurrence, and highlights the importance of accurate diagnosis of skeletal dysplasias with prenatal manifestation.

The Ethics of Pre-Implantation Genetic Diagnosis in Practice: An Analysis of the Feasibility and Ethical Considerations of Applying and Regulating Genetic Enhancement

<span>Pre-Implantation genetic diagnosis (PGD) has many therapeutic and enhancement ap- plications. In a previous work, I presented arguments in favour of all types of PGD, whether for medical therapies or human enhancement. These arguments were based on the absence of moral distinctions between genetic therapy and genetic enhancement. The implication of these arguments is that, if one cannot distinguish between therapy and enhancement on moral grounds, then all PGD applications must be either moral or immoral. Although logically speaking this argument may be true, in practice I believe that it is possible and necessary to draw a line between what is morally permissible and what is not with respect to applications of PGD for genetic enhancement. In order to draw this line, I move away from analyzing the moral substance of PGD as a technology and focus instead on the moral agents that will employ PGD. As humans, I believe we are both morally accountable and mora</span>