Clinical diagnosis by whole-genome sequencing of a prenatal sample

Abstract

Conventional cytogenetic testing offers low-resolution detection of balanced karyotypic abnormalities but cannot provide the precise, gene-level knowledge required to predict outcomes. The use of high-resolution whole-genome deep sequencing is currently impractical for the purpose of routine clinical care. We show here that whole-genome "jumping libraries" can offer an immediately applicable, nucleotide-level complement to conventional genetic diagnostics within a time frame that allows for clinical action. We performed large-insert sequencing of DNA extracted from amniotic-fluid cells with a balanced de novo translocation. The amniotic-fluid sample was from a patient in the third trimester of pregnancy who underwent amniocentesis because of severe polyhydramnios after multiple fetal anomalies had been detected on ultrasonography. Using a 13-day sequence and analysis pipeline, we discovered direct disruption of CHD7, a causal locus in the CHARGE syndrome (coloboma of the eye, heart anomaly, atresia of the choanae, retardation, and genital and ear anomalies). Clinical findings at birth were consistent with the CHARGE syndrome, a diagnosis that could not have been reliably inferred from the cytogenetic breakpoint. This case study illustrates the potential power of customized whole-genome jumping libraries when used to augment prenatal karyotyping.

Figure 1. Clinical Findings Detected with Prenatal Imaging

A transaxial ultrasonogram with a four-chamber view of the heart, obtained at 27.3 weeks of gestation (Panel A), shows a small right ventricle (arrow), as compared with the left ventricle (star), which was first detected at 18.8 weeks; tricuspid atresia was also detected on earlier imaging. A transaxial ultrasonogram obtained at 35.3 weeks of gestation (Panel B) shows polyhydramnios (dashed line), first detected at 30.4 weeks; also noteworthy is the absence of a fluid-filled stomach in the upper abdomen (arrow). An ultrasonogram of the fetal profile (Panel C) and a three-dimensional ultrasonogram of the fetal face (Panel D), both obtained at 34.4 weeks of gestation, show microstomia and protrusion of the upper lip (Panel D, arrow), and a three-dimensional ultrasonogram obtained at 33.3 weeks of gestation (Panel E) shows abnormally clenched hands and flexed arms. A transaxial ultrasonogram of the perineum in a phenotypic male fetus, obtained at 35.3 weeks of gestation, shows only one testicle in the scrotum (Panel F, arrow).

Figure 2. Sequencing and Analysis Timeline

The delineation of a de novo balanced translocation initially reported as 46,XY,t(6;8)(q13;q13)dn is revised, after DNA sequencing, to 46,XY,t(6;8)(q13;q12.2)dn. In step 1A, 2-kb jumping libraries are prepared from genomic DNA, and in step 1B, the final distribution of fragment sizes is shown. In step 2, massively parallel paired-end 25-cycle sequencing of DNA fragments is performed on an Illumina HiSeq 2000. In step 3, computational analyses are performed, including distributed parallel alignment of sequenced reads and clustering of anomalous read pairs (step 3A) and identification of candidate translocation clusters (step 3B). The inset in step 3B shows an example of a theoretical distribution of reads spanning a translocation breakpoint on a derivative chromosome. In step 4, the translocation breakpoint is confirmed by means of a polymerase-chain-reaction assay, and Sanger sequencing informs the precise breakpoint in the initial karyotype. Here, the breakpoint on der(8) is delineated on the Sanger sequencing reads. Chromatogram peaks are shown at the top, and a nucleotide sequence from a fragment crossing the breakpoint is shown below, with chromosome 8 highlighted in pink, chromosome 6 highlighted in brown, and the breakpoint sequence, with microhomology between the chromosomes, highlighted in yellow.

Figure 3. Sequence-Based Delineation of a Balanced De Novo Translocation

Sequencing revealed a balanced translocation disrupting CHD7 at 8q12.2 and disrupting LMBRD1 at 6q13. CHD7 and LMBRD1 are transcribed on opposite strands in the translocation and are incompatible with the formation of a fusion transcript. Normal chromosomes 6 and 8 are shown, as are the derivative chromosomes, after translocation. The breakpoint region is expanded in the middle, showing the cytogenetic band, the genomic coordinates of each chromosome, the precise breakpoint (dashed lines) on each derivative, and the nucleotide sequence of the junction point, including microhomology (yellow) at the breakpoint.