Ana Peciña

Experience of Preimplantation Genetic Diagnosis with HLA Matching at the University Hospital Virgen del Rocío in Spain: Technical and Clinical Overview

Preimplantation genetic diagnosis (PGD) of genetic diseases, combined with HLA matching (PGD-HLA), is an option for couples at risk of transmitting a genetic disease to select unaffected embryos of an HLA tissue type compatible with that of an existing affected child. Here we present the results of our PGD-HLA program at the Department of Genetics, Reproduction and Fetal Medicine of the University Hospital Virgen del Rocío in Seville. Seven couples have participated in our program because of different indications. Overall, 26 cycles were performed, providing a total of 202 embryos. A conclusive molecular diagnosis and HLA-typing could be assured in 96% of the embryos. The percentage of transfers per cycle was 26.9% and the birth rate per cycle was 7.7% per transfer. Our PGD-HLA program resulted in the birth of 2 healthy babies, HLA-identical to their affected siblings, with successful subsequent haematopoietic stem cell (HSC) transplantations. Both HSC-transplanted children are currently doing well 48 and 21 months following transplantation, respectively. All the procedures, including HSCs umbilical cord transplantation, were performed in our hospital.

One-step multiplex polymerase chain reaction for preimplantation genetic diagnosis of Huntington disease.

OBJECTIVE To develop a multiplex polymerase chain reaction (PCR) method for Huntington disease (HD) preimplantation genetic diagnosis (PGD) based on the coamplification of CAG repeats and three different polymorphic microsatellites in a single step of PCR. DESIGN Techniques and instrumentation. SETTING Tertiary clinical and academic medical center. PATIENT(S) Thirty-six embryos from seven clinical PGD cycles. INTERVENTION(S) Patients underwent a PGD cycle with transfer of two unaffected embryos on day 5. MAIN OUTCOME MEASURE(S) PGD based on mutation identification or exclusion testing for at-risk HD carriers. RESULT(S) Thirty-six embryos from seven clinical PGD cycles were analyzed with the new method here developed, and results were obtained for 34 of them. Two embryos were transferred on day 5, resulting in two singleton pregnancies. CONCLUSION(S) An interesting application of this approach can be considered for PGD cycles in which numerous markers must be used. We have also used this one-step multiplex method for PGD for other pathological conditions.

Novel One-Step Multiplex PCR-Based Method for HLA Typing and Preimplantational Genetic Diagnosis of -Thalassemia

Preimplantation genetic diagnosis (PGD) of single gene disorders, combined with HLA matching (PGD-HLA), has emerged as a tool for couples at risk of transmitting a genetic disease to select unaffected embryos of an HLA tissue type compatible with that of an existing affected child. Here, we present a novel one-step multiplex PCR to genotype a spectrum of STRs to simultaneously perform HLA typing and PGD for β-thalassemia. This method is being routinely used for PGD-HLA cycles in our department, with a genotyping success rate of 100%. As an example, we present the first successful PGD-HLA typing in Spain, which resulted in the birth of a boy and subsequent successful HSC transplantation to his affected brother, who is doing well 4 years following transplantation. The advantage of our method is that it involves only a round of single PCR for multiple markers amplification (up to 10 markers within the HLA and 6 markers at the β-globin loci). This strategy has allowed us to considerably reduce the optimization of the PCR method for each specific PGD-HLA family as well as the time to obtain molecular results in each cycle.

Clinical and Technical Overview of Preimplantation Genetic Diagnosis for Fragile X Syndrome: Experience at the University Hospital Virgen del Rocio in Spain.

Fragile X syndrome (FXS) accounts for about one-half of cases of X-linked intellectual disability and is the most common monogenic cause of mental impairment. Reproductive options for the FXS carriers include preimplantation genetic diagnosis (PGD). However, this strategy is considered by some centers as wasteful owing to the high prevalence of premature ovarian failure in FXS carriers and the difficulties in genetic diagnosis of the embryos. Here we present the results of our PGD Program applied to FXS, at the Department of Genetics, Reproduction and Fetal Medicine of the University Hospital Virgen del Rocío in Seville. A total of 11 couples have participated in our PGD Program for FXS since 2010. Overall, 15 cycles were performed, providing a total of 43 embryos. The overall percentage of transfers per cycle was 46.67% and the live birth rate per cycle was 13.33%. As expected, these percentages are considerably lower than the ones obtained in PGD for other pathologies. Our program resulted in the birth of 3 unaffected babies of FXS for 2 of the 11 couples (18.2%) supporting that, despite the important drawbacks of PGD for FXS, efforts should be devoted in offering this reproductive option to the affected families.

Co‐segregation of a homozygous SMN1 deletion and a heterozygous PMP22 duplication in a patient

Despite co‐segregation of two different genetic neurological disorders within a family is rare, clinicians should take into consideration this possibility in patients presenting with unusual complex phenotypes or with unexpected electrophysiological findings. Here, we report a Spanish 11‐month‐old patient with spinal muscular atrophy type 2 and Charcot‐Marie‐Tooth 1A.

Atypical Association of Angelman Syndrome and Klinefelter Syndrome in a Boy with 47,XXY Karyotype and Deletion 15q11.2-q13

Angelman syndrome (AS, OMIM 105830) is a neurogenetic disorder with firm clinical diagnostic guidelines, characterized by severe developmental delay and speech impairment, balanced and behavioral disturbance as well as microcephaly, seizures, and a characteristic electroencephalogram (EEG). The majority of AS cases (70%) are caused by a 15q11.2-q13 deletion on the maternally derived chromosome. The frequency of AS has been estimated to be between 1/10000 and 1/20000. Klinefelter syndrome (KS) occurs due to the presence of an extra X chromosome (karyotype 47,XXY). The main features in KS are small testes, hypergonadotropic hypogonadism, gynecomastia, learning difficulties, and infertility. We present what is, to our knowledge, the first case of a patient with both KS and AS due to a 15q11.2-q13 deletion on the maternally derived chromosome and an extra X chromosome of paternal origin. He showed dysmorphic features, axial hypotonia, and delayed acquisition of motor skills. Early diagnosis is essential for optimal treatment of AS children; this is one of the earliest diagnosed cases of AS probably due to the presence of two syndromes. Clinical findings in this patient here described may be helpful to identify any other cases and to evaluate recurrence risks in these families.

Preimplantation Genetic Diagnosis for Myotonic Dystrophy Type 1 and Analysis of the Effect of the Disease on the Reproductive Outcome of the Affected Female Patients

Myotonic dystrophy type 1 (DM1) is the most common adult muscular dystrophy and presents an autosomal dominant inheritance. A reproductive option for the families affected is preimplantation genetic diagnosis (PGD). One limitation of this option is the nonoptimal response to ovarian stimulation of the women with DM1, although controversial results exist regarding this subject. In this study, we have analyzed the results of the PGD program applied to DM1 at our institution. A total of 35 couples have been included in our program since 2010, and 59 cycles have been performed. The percentage of transfers per cycle was 64.4% and the live birth rate per cycle was 18.6%. Interestingly, statistically significant differences were observed for the clinical results in the group of couples with an affected female versus the group with an affected male or versus a group of couples with different referral reasons. Specifically, both the percentage of mature oocytes out of the total oocytes retrieved and the percentage of fertilization were considerably lower in the group of DM1 females. Our findings would suggest the possibility of achieving less favourable PGD outcomes in women with DM1 in comparison with other pathologies, although the underlying mechanism remains unknown.