The clinical applications of preimplantation genetic diagnosis, carrier screening for inherited genetic disorder and newborn genetic screening
Date Issued
2015
Date
2015
Author(s)
Hung, Chia-Cheng
Abstract
Introduction With the rapid development of genetic diagnostic technologies in recent years, there are more and more inherited diseases have been linked to single gene variation. Hence, the genetic testing is very important that can provide information about an individual’s genes or chromosomes. Recently, the common genetic tests include prenatal screening, carrier screening, preimplantation testing, forensic testing, newborn screening, and so on. However, because establishment of a standard protocol for genetic testing is not easy, the current genetic testing in Taiwan, still limited to traditional platforms, such as prenatal diagnosis by karyotyping and newborn screening for congenital metabolic and endocrine diseases. Some widespread genetic tests are still not available now. So, how to create the latest genetic screening protocol for clinical use has become an important issue without delay. Materials and Methods PART 1 Preimplantation genetic diagnosis for chromosomal translocation The study included 46 couples with recurrent first-trimester or second-trimester spontaneous miscarriage, who visited the fertility centers in Taiwan from 2011 to 2014, and one of the partners, carries a balanced structural chromosomal anomaly. After discussing with the doctor and genetics counselor, couples underwent in vitro fertilization (IVF), and a total of 365 embryos in 76 IVF cycles were biopsied for preimplantation genetic diagnosis (PGD) by array comparative genome hybridization (array CGH) screening. PART 2 Carrier screening for spinal muscular atrophy A prospective large population-based cohort study of 139,404 pregnancies was investigated in 25 counties of Taiwan during a 10 year period, 2005 through 2014. Two different validated platforms were used for parallel SMN1/SMN2 gene quantification: denaturing high-performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA). A three-stage screening program was used: (1) pregnancies were tested for SMA heterozygosity, (2) if a pregnancy was heterozygous for SMA (carrier status), the paternal partner was then tested, and (3) if both partners were SMA carriers, a prenatal diagnosis was offered. PART 3 Preimplantation genetic diagnosis for spinal muscular atrophy In this part, we report data to identify the SMN1 gene deletion on eighteen clinical embryos obtained from one participating couple, where both partners are heterozygous SMA carriers with 1-SMN1/3-SMN2 genotype. We validated and applied protocol clinically for PGD through the use of blastocyst biopsy, whole genome amplification, mini-sequencing genotype coupling with genetic linkage of SMN gene involving three informative microsatellite markers, and thawed embryo transfer. PART 4 Newborn genetic screening for hereditary hearing impairment This is a prospective population-based cohort study of 26,764 newborns in Taiwan conducted during the period 2011 to 2014. Based on Taiwanese genetic database of hereditary hearing impairment, the newborn genetic screening targeted four deafness-associated mutations commonly found in the Taiwanese population, including c.109G>A of the GJB2 gene, c.235delC of the GJB2 gene, c.919-2A>G of the SLC26A4 gene, and mitochondrial m.1555A>G of the 12S rRNA gene. The newborn genetic screening were performed using polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridization probes in a real-time PCR detection system. Results PART 1 Preimplantation genetic diagnosis for chromosomal translocation A total of 365 embryos from 76 cycles were analyzed, and the overall diagnostic efficiency was 89.04% (325/365). Then 315 embryos were analyzed by aCGH, as well as the euploidy rate and aneuploidy rate were 26.67% (84/315) and 73.33% (231/315), respectively. 49 cycles of thawed embryo transfer (ET) were carried out, and the average transfer embryo was 1.11 pieces. According our record for 52 IVF cycles of 32 couples, there were 15 women got pregnant and pregnancy rate per cycle was 28.85% (15/52). PART 2 Carrier screening for spinal muscular atrophy We found 2,859 individuals with one copy of the SMN1 genotype, recognized to be SMA carriers, among the 139,404 pregnancies screened. The carrier rate in our population was approximately 1 in 49 (2.05%). Of these individuals, 58 couples were at high risk for having offspring with SMA after testing of partners or spouses 2,504 who were also determined to be SMA carriers. Prenatal diagnoses were determined for 49 pregnancies (84.48%), of which 13 (26.53%) fetuses were diagnosed with SMA; the prevalence of SMA in our population was 1 in 10,723. PART 3 Preimplantation genetic diagnosis for spinal muscular atrophy Approximately 77.78% (14/18) of blastocysts were successfully amplified in a single PGD cycle. Among these embryos, ten (72%, 10/14) were diagnosed as unaffected, two (14%, 2/14) as affected, and two embryos (14%, 2/14) had no conclusive diagnosis due to allele drop-out (ADO). Two unaffected embryos were thawed and transferred in the next cycle resulting in a singleton pregnancy, and the birth of a healthy girl who carries the 1-SMN1/3-SMN2 genotype. PART 4 Newborn genetic screening for hereditary hearing impairment Of the 26,764 newborns, a total of 5,253 (19.63%) babies were found to have at least 1 mutated allele on the newborn genetic screening for hearing impairment. A total of 399 newborns (1.49%) carrier either homozygous, compound heterozygous or homoplasmic mutations in targeted gene, who may potentially have hearing loss. 293 (1.11%) of whom were homozygous for GJB2 c.109 G>A, 2 (0.01%) were homozygous for GJB2 c.235delG, 5 (0.02%) were homozygous for SLC26A4 c.919-2A>G, 28 (0.11%) compound heterozygous for GJB2 c.109 G>A and c.235delC, and 66 (0.25%) homoplasmic or heteroplasmic for m.1555A>G in 12SrRNA gene. Conclusion Here we established four novel genetic testing platforms, including PGD for chromosomal translocation, carrier screening for SMA, PGD for single gene disorder and newborn genetic screening for hereditary hearing impairment. With constantly improved technology and drastically reduced cost, genetic testing can be a powerful tool for achieving molecular diagnosis. We hope the application to clinical can help more and more patients and families.
Subjects
preimplantation genetic diagnosis
inherited genetic disorder
carrier testing
newborn genetic screening
clinical application
Type
thesis