Genome sequencing increases diagnostic yield in clinically diagnosed Alagille syndrome patients with previously negative test results

Abstract

Purpose: Detection of all major classes of genomic variants in a single test would decrease cost and increase the efficiency of genomic diagnostics. Genome sequencing (GS) has the potential to provide this level of comprehensive detection. We sought to demonstrate the utility of GS in the molecular diagnosis of 18 patients with clinically defined Alagille syndrome (ALGS), who had a negative or inconclusive result by standard-of-care testing.

Methods: We performed GS on 16 pathogenic variant-negative probands and two probands with inconclusive results (of 406 ALGS probands) and analyzed the data for sequence, copy-number, and structural variants in JAG1 and NOTCH2.

Results: GS identified four novel pathogenic alterations including a copy-neutral inversion, a partial deletion, and a promoter variant in JAG1, and a partial NOTCH2 deletion, for an additional diagnostic yield of 0.9%. Furthermore, GS resolved two complex rearrangements, resulting in identification of a pathogenic variant in 97.5% (n = 396/406) of patients after GS.

Conclusion: GS provided an increased diagnostic yield for individuals with clinically defined ALGS who had prior negative or incomplete genetic testing by other methods. Our results show that GS can detect all major classes of variants and has potential to become a single first-tier diagnostic test for Mendelian disorders.

Keywords: Alagille syndrome; JAG1; NOTCH2, diagnostic testing; genome sequencing.

Fig. 1. Flow diagram of the study population.

Genome sequencing (GS) was performed on a cohort of 18 individuals that were identified in our study, Molecular Analysis of Alagille Syndrome (ALGS). Exclusion criteria and results of the study are indicated.

Fig. 2. Schematic of JAG1 inversion identified in proband 12.

The reference genome (upper structure) depicts the 679-kb inverted region, encompassing JAG1 exons 1–3, bounded by dashed lines. The breakpoints extend from intron 3 to a gene desert upstream of the JAG1 promoter. The rearranged structure is shown below. Paired-end reads with abnormal insert size and orientation were used to infer the approximate boundaries of the inversion and soft-clipped reads at the ends of the inversion were used to precisely map the breakpoints at nucleotide-level resolution.

Fig. 3. RNA expression (JAG1) and copy number (NOTCH2) is reduced in patient cell lines harboring novel pathogenic variants.

(a) Droplet digital polymerase chain reaction (ddPCR) performed on complementary DNA (cDNA) made from RNA extracted from lymphoblastoid cell lines of affected individuals showing reduced JAG1 gene expression in the individual with the promoter variant (proband 11) as well as the individual with the inversion (proband 12) and his affected father (proband 12-F), who also has the inversion. Parental samples were not available for proband 11. The negative control is the average of four unaffected individuals with no pathogenic JAG1 variant. An individual with a pathogenic frameshift variant (c.2122_2125del) that is predicted to truncate the JAG1 protein was included as a positive control. Two separate primer/probe sets were used for confirmation, one designed in exon 1 and one designed to cross exons 25–26. Values for all samples were normalized to the internal control, TBP. Error bars for the negative control are plotted as standard deviation. (b) ddPCR showing NOTCH2 copy number in proband 10 and her unaffected parents.

Fig. 4. Genome sequencing (GS) resolves complex structural rearrangements in individuals with clinically defined Alagille syndrome (ALGS).

(a) JAG1 multiplex ligation-dependent probe amplification (MLPA) results for probands 8 and 14. Probe ratio is plotted for each exon. A threshold below 0.75 was used to classify losses and above 1.25 was used to classify gains. Circles represent copy-neutral ratios while squares represent deletions. The SALSA MLPA probemix (P184-C3 JAG1) was purchased from MRC Holland (Amsterdam, Netherlands) and details, including quantification, normalization, and controls can be found through this link: https://www.mrcholland.com/products/18527/Product%20description%20P184-C3-0317%20JAG1-v12.pdf. (b) Schematic of the genomic structure of JAG1 for proband 8, who had noncontiguous deletions of exon 3 and exons 9–26 demonstrated by MLPA, and proband 14, who had noncontiguous deletions of exon 9 and exons 11–12. Dashed lines denote the genomic coordinates (hg38) of the breakpoints and red bars indicate the deleted regions, which are interspersed with nondeleted portions of the gene (shown in gray). (c) Schematic of the genomic rearrangement for probands 8 and 14.