Clinical utility of whole-genome sequencing for cytogenetically balanced chromosomal abnormalities in genetic diseases

Abstract

Background: Balanced chromosomal abnormalities (BCAs) involve changes in localization or orientation of a chromosomal segment without visible gain or loss of genetic material. It cannot be detected through chromosomal microarray analysis. BCAs occur at a frequency of 1 in 500 newborns and are associated with a higher risk of multiple congenital anomalies and/or neurodevelopmental disorders, especially when there is a de novo change. Whole genome sequencing (WGS) has the advantage of higher resolution of detecting breakpoints at single nucleotide level. The application of this new technology for BCAs can improve the detection of the underlying genetic mechanism of the cause of paediatric patients. Methods: In this pilot study we recruited 10 families with fetuses/ babies affected by de novo BCAs from 2008 to 2018. These cases were retrospectively identified from the Prenatal Diagnostic Laboratory, Tsan Yuk Hospital with archival DNA or frozen cells for DNA extraction. The cases had no known teratogenic exposure and major structural anomalies on prenatal ultrasound. WGS was performed using Illumina pair-ended short read sequencing. Results: Of these 10 families, 8 reciprocal translocation and 2 inversion were identified by conventional G-band karyotype analysis. Pair-ended short read WGS successfully identified the BCA and their breakpoints in all 10 cases with improved resolution to single nucleotide level. In 5 of the 10 cases, extra DNA was available for validating the WGS findings using orthogonal methods including Sanger sequencing (n=4), Nanopore sequencing and PacBio sequencing (n=1). For each case, we assessed bioinformatically for the presence of cryptic deletion/duplication, gene disruption or Topologically associating domain (TAD) disruption. Gene disruption was identified in 2 cases leading to definitive diagnoses for two families with proband affected by: 1) X-linked epilepsy (disruption of PCDH19) and 2) microcephalic osteodysplastic primordial dwarfism type II (MOPDII) (a SNV and gene disruption on PCNT by inversion with phase analysis). Discussion and conclusion: Compared to karyotyping, WGS can precisely detect BCA breakpoints down to nucleotide level and identify disrupted disease-causing genes. This information allows accurate and personalized disease risk prediction for BCAs; otherwise, the family is given a general 6-9% risk for neurodevelopmental problems later in life when a de novo BCA is identified. Moreover, with accurate breakpoint identification in WGS, the geneticist is able to further predict the disease outcome, for instance, extremely high risk of developing X-linked epilepsy in a female baby and early identification of the predominant skeletal disease in MOPDII in the 2 aforementioned cases identified with gene disruption. These specific findings from WGS give further advantage over traditional karyotyping, which allows the practice of personalized medicine.