A new approach for Next Generation Sequencing in prenatal diagnosis applied to a case of Charcot–Marie–Tooth syndrome

In recent years, Next Generation Sequencing (NGS) has become an important tool, not only for gene discovery and research, but also for clinical diagnosis. Genetic diagnosis of rare disorders has developed considerably through the application of massively parallel sequencing methods. To date, a great effort has been made to introduce NGS in noninvasive prenatal testing both for the detection of aneuploidy associated to chromosomes 21, 18 and 13 and, in the latest studies, for single gene disorder analysis. Regarding invasive prenatal diagnosis, NGS has been applied to whole exome or whole genome studies; however, in these cases NGS did not prove useful in routine clinical application, because of many factors, including the necessity of bioinformatics skills during data analysis, the large amount of variants with uncertain clinical significance which were obtained, and the time required for analysis. We have introduced a new target resequencing NGS approach in prenatal diagnosis, called Next Generation Prenatal Diagnosis (NGPD), for the screening of a wide range of genetic disorders in high-risk pregnancies when an ultrasound screening fails to show a definite clinical phenotype, in cases where ultrasound indications must be supported by a molecular genetic diagnosis, or in cases where one or both parents are carriers of a genetic disorder. To the best of our knowledge, this is the first report that describes the use of a multi-gene NGS panel optimized for clinical prenatal diagnosis with a relatively fast turn-around time that can be multiplexed to allow simultaneous analysis of multiple samples. In this report the NGPDmethod was applied for the prenatal genetic diagnosis of Charcot–Marie–Tooth (CMT) disease in a fetus whose mother was affected by early onset CMT. Of all the types of hereditary sensory-motor polyneuropathy, CMT syndrome is the most frequent. From a clinical point of view, the disorder is characterized by distal muscular atrophy associated with malformation of the feet, osteotendinous hyporeflexia and alterations in sensibility. Based on motor conduction velocity, it is possible to identify type 1 (demyelinating form with a particularly reduced conduction speed) and type 2 (axonal form with a normal conduction speed). From a genetic point of view, many genes responsible for CMT have been identified, and transmission can be autosomal dominant, recessive or X linked. At the time of analysis, the 12th week of gestation, the patient was still without a genetic diagnosis. The patient showed postural tremor, sensory impairment and distal muscle atrophy. Weakness and deafness were also present. From the clinical and family history, we could assume a probability of an autosomal dominant-transmitted form of CMT syndrome. At the time of the ultrasound scan no clinical signs emerged. After genetic counseling, the patient underwent a chorionic villous sampling and parents’ blood samples were taken. After exclusion of any chromosomal microdeletions and microduplications associated with CMT disease, through aCGH analysis on CVS and mother’s blood, we used the NGPD method on the same DNA samples. We proceeded directly to trio analysis, followed by confirmation of any mutations identified on the fetus, to reduce time of prenatal diagnosis, which would otherwise have been delayed. Trio analysis was useful not only for identifying variants, but also for studying inheritance patterns. Variants were sorted for autosomal recessive, autosomal dominant, X-linked and de novo mode of inheritance. For the library preparation, we used the Trusight One Sequencing Panel (TSO, Illumina), which enriches for 62 000 exons of 4813 genes. The genes contained in this library were selected by Illumina according to the Human