The Human Genome Project took almost 13 years to complete and some 3 billion dollars in order to generate the first draft of the human genome, which contains 3 billion base pairs (bp). The project was largely based on Sanger sequencing which is capable of sequencing ~1000bp at a time. In recent years, the introduction of Next Generation Sequencing (NGS) has resulted in a precipitous drop in the price of sequencing. The entire human genome can now be sequenced in a matter of days for close to 1000 USD.. Next Generation Sequencing is also called Massive Parallel Sequencing (MPS) because it allows the simultaneous sequencing in parallel of hundreds of millions DNA fragments. This enables the reconstruction of the entire genome – Whole Genome Sequencing (WGS); the determination of the entire coding sequenced of all genes - Whole Exome sequencing (WES); Complete sequencing of selected genes (NGS Panels); and detection of chromosomal aberrations. NGS has been employed in many areas in Medicine including research, personalized-precision medicine, cancer and more. Some of the most significant and commonly used applications are in the field of Reproductive Medicine:
Preconception Population-based Carrier Screening
The objectives of carrier screening is to identify at-risk couples, provide them with genetic counseling, and offer reproductive choices in including prenatal diagnosis or :
Preimplantation Genetic Diagnosis (PGD). Screening should focus on life threatening genetic disease and conditions that are associated with cognitive disability or that have a serious impact on the quality of life. Various tests have gained acceptance in certain countries but with the exception of a handful of pan-ethnic conditions (Fragile X syndrome, Cystic fibrosis, Spinal Muscular Atrophy and hemoglobinopathies), most condition are rare and have ethnic- and race-specific incidences. Moreover, within different populations, there is a unique characteristic set of mutations. A new paradigm is now emerging termed "Expanded Screening" whereby all individuals, regardless of ethnicity, are offered screening for the same set of conditions. This may be achieved using various array based techniques but even the most detailed methodologies carry only a limited set of conditions and mutations and with the assimilation of novel findings require constant modifications. In contrast, NGS has been recently shown to provide a more comprehensive analysis. Hallam et al. have recently validated the clinical utility of NGS for carrier screening. They tested 11,691 IVF patients and detected 447 mutations in carriers, 2 in an affected individual. Of these ~ 25% of the mutations were not included in traditional, limited, mutation panels. If the price of sequencing will continue to drop as anticipated, screening by NGS will also become more cost effective.
Preimplantation Genetic Screening (PGS)
Aneuploidy increases with age and is the leading cause of miscarriage, congenital anomalies and implantation failure. In fact > 50% of human embryos are aneuploid.
It was therefore suggested that preimplantation genetic screening (PGS) would improve clinical outcomes by increasing implantation rates and reducing miscarriage rates, particularly in patients at risk of producing aneuploid embryos, such as those of advanced age, recurrent implantation failure and recurrent miscarriages. Using an ever growing combination of Fluorescence in situ Hybridization (FISH) probes proved to be of little benefit and may even have had detrimental effects on such high risk patients. Several reasons have been put forth as to why PGS with FISH was unsuccessful including the mosaic nature of cleavage stage embryos; the fact that the analysis was of limited to a select number of chromosomes; the limited accuracy of the FISH technique as well as the detrimental effect of biopsy. Recent years have seen the introduction of array based Comparative Genomic Hybridization (aCGH) techniques which enable the analysis of all 24 chromosomes in parallel using thousands of unique BAC probes. The efficacy of this approach has already been demonstrated in randomized controlled trials, albeit in good prognosis patients. Over the last couple of years it has been shown that NGS may achieve similar, if not superior results. Because up to a million DNA fragments are analysed from each embryo, NGS has been shown to have complete concordance with aCGH, providing a higher dynamic range with greater sensitivity (Fiorentino et al. 2014). With the reduction of sequencing costs it is expected that NGS will replace aCGH in the coming years.
Non-invasive prenatal screening (NIPT)
Due to cell turnover, short fragments (~200bp) of Cell-Free DNA (cfDNA) are released into the plasma. In pregnancy ~10% of cfDNA is of fetal origin. CfDNA is now used worldwide as an advanced screening tool for fetal aneuploidy. In a normal euploid pregnancy the amount of fetal DNA from each chromosome is proportional to that of the mother. However in an aneuploid pregnancy there is a slight deviation from the expected level. For example in Down syndrome there is expected to be an increase of fragments form chromosome 21 due to the presence of 3 instead of 2 fetal copies. NGS allows the detection of such slight variations by sequencing and counting of millions of cfDNA fragments from maternal plasma. The high sensitivity and specificity has been demonstrated not only for high risk patients but also recently in average risk patients (Bianchi et al., 2014, Norton et al., 2015). Current testing includes screening for trisomy 13, 18, and 21 as well as sex chromosome anomalies. Moreover, recent developments have shown that NIPT can also detect some submicroscopic copy number variants (CNVs) that are responsible for some genetic syndromes (such as the 22q11.2 deletion responsible for the velocardiofacial syndrome and 15qdeletion responsible for Prader Willi/Angelman syndromes)
Prenatal diagnosis of rare genetic syndromes.
While the genome contains some 3 billion base pairs, only a small fraction - the Exome - constitute the coding sequence of expressed genes. While the Exome consists of only 2% of the genome. It harbors ~ 85% of disease-causing mutations. It is estimated that there are about 7000 Mendelian disorders, and while most are individually rare, collectively they account for ~20% of infant mortality. Next generation sequencing is now commonly employed in order to sequence the exome. This approach called Whole exome Sequencing (WES) is currently employed in the clinical setting in order to detect the genetic cause of many , yet undiagnosed, genetic conditions. The clinical utility of WES has been demonstrated in numerous studies of postnatal cases. For instance, a recent study (Farwell et al., 2014) of 500 families with undiagnosed genetic disease found that 30% had positive or likely positive finding in a characterized gene, and 7.5% had findings in a novel gene. This approach has recently been applied to prenatal diagnosis of fetuses with malformations (Carss et al. 2014). Of 30 non-aneuploid fetuses and neonates with malformations identified by prenatal ultrasound, 3 (10%) had findings that were highly likely to be causative and an additional 5 (17%) had in plausible candidate genes that require additional validation. Currently there are several ongoing studies to examine the utility of WES in prenatal diagnosis and it is expected that its use will expand in the coming years.
Hallam S et al. Validation for Clinical Use of, and Initial Clinical Experience with, a Novel Approach to Population-Based Carrier Screening using High-Throughput, Next-Generation DNA Sequencing J Mol Diagn 2014, 16: 180e189
Fiorentino F et al. Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles. Hum Reprod 2014;29:2802-13.
Bianchi D et al. DNA sequencing versus standard prenatal aneuploidy screening. New Engl J Med 2014;370
Norton ME et al. Cell-free DNA analysis for noninvasive examination of trisomy. New Engl J Med 2015;372
Farwell et al. Enhanced utility of family-centered diagnostic exome sequencing with inheritance model–based analysis: results from 500 unselected families ... Genet Med Nov 2014
Carss KJ et al. Exome sequencing improves genetic diagnosis of structural fetal abnormalities revealed by ultrasound. Hum Mol Genet 2014;23:3269–3277
Director prenatal Genetic Diagnosis Unit
Tel Aviv Sourasky Medical Center
6 Weizmann Street 64239, Israel