Introduction:
Introduction: Warsaw breakage syndrome (WABS) is an autosomal recessive disorder caused by biallelic pathogenic variants in DDX11, which encodes a DNA helicase essential for DNA replication and repair. WABS is characterized phenotypically by the triad of congenital microcephaly, pre-and postnatal growth restriction, and congenital sensorineural hearing loss.  This report highlights the challenges in confirming a molecular diagnosis for two daughters of consanguineous parents. 

 

Methods:
Methods: Following negative SNP microarray, whole exome sequencing, and mitochondrial DNA analysis, we performed long-read genome and transcriptome sequencing and DNA methylation analysis on the Oxford Nanopore platform.

 

Results:
Results: This testing identified a homozygous variant of uncertain significance (VUS): NC_000012.12:g.31073186_31074821del, NM_030653.4:c.-910_-5+730del. This deletes the promoter and the first exon of DDX11. RNA studies showed loss of DDX11 expression compared to unrelated controls.  In the gnomAD database, this variant has a frequency of 0.04% and is homozygous in two individuals. Functional studies with 40 ng/mL MMC and 100 ng/mL DEB did not show evidence of  DNA breaks or radial formations.  We discuss additional functional studies assessing sensitivity to G-quadraplex stabilizing compounds as well as the complex genomic structure of the DDX11 locus that likely impedes analysis by and accurate sequence mapping for short read sequencing. The reported variant frequency in gnomAD and the reported homozygous individuals likely reflect mapping errors.     

 

Conclusion:
Conclusions: For individuals failing molecular diagnosis with standard of care short read sequencing, we suggest multi-omic studies with long-read sequencing and transcriptome analysis as an appropriate next step. We envision a future in which integrated long-read genome sequencing, transcriptome sequencing, and epigenome analysis are first tier testing.