Genome sequencing in families with congenital limb malformations

Abstract

The extensive clinical and genetic heterogeneity of congenital limb malformation calls for comprehensive genome-wide analysis of genetic variation. Genome sequencing (GS) has the potential to identify all genetic variants. Here we aim to determine the diagnostic potential of GS as a comprehensive one-test-for-all strategy in a cohort of undiagnosed patients with congenital limb malformations. We collected 69 cases (64 trios, 1 duo, 5 singletons) with congenital limb malformations with no molecular diagnosis after standard clinical genetic testing and performed genome sequencing. We also developed a framework to identify potential noncoding pathogenic variants. We identified likely pathogenic/disease-associated variants in 12 cases (17.4%) including four in known disease genes, and one repeat expansion in HOXD13. In three unrelated cases with ectrodactyly, we identified likely pathogenic variants in UBA2, establishing it as a novel disease gene. In addition, we found two complex structural variants (3%). We also identified likely causative variants in three novel high confidence candidate genes. We were not able to identify any noncoding variants. GS is a powerful strategy to identify all types of genomic variants associated with congenital limb malformation, including repeat expansions and complex structural variants missed by standard diagnostic approaches. In this cohort, no causative noncoding SNVs could be identified.

The performance of genome sequencing as a first-tier test for neurodevelopmental disorders

“…Using ES, on average 1 in 3 patients receives a genetic diagnosis [ 1 , 2 ]. For patients in whom the genetic etiology remains elusive, it has been shown that GS can help to provide a diagnosis, as it allows for comprehensive variant calling of all types of variation without the technical biases and limitations observed in ES [ 4 – 6 , 8 – 13 ]. From our prospective parallel study including 150 patient-parent trios, we conclude that GS does not result in a significantly higher diagnostic yield (30.0%) than an ES-based SOC pathway (28.7%).…”

“…Another explanation for the seemingly lower than initially expected GS diagnostic yield may be the limited scope of variant interpretation, i.e., only focusing on variants affecting coding sequence using an “exome-from-genome” approach. Whereas examples of non-coding disease-causing variants most certainly exist [ 8 , 34 – 36 ], the contribution of such non-coding variants for NDD have so far been estimated to be rare [ 12 , 37 , 38 ]. In addition, interpretation and establishing pathogenicity of variants in non-coding space is not straightforward [ 39 ], and often involves labor-intensive and time-consuming (functional) follow-up experiments to determine putative clinical relevance [ 34 , 38 , 40 ].…”

“…This accurate and comprehensive analysis of all different variant types has great potential for further increasing the diagnostic yield. In rare disease patients in whom standard of care (SOC) testing failed to provide a molecular diagnosis, GS has been shown multiple times to reveal pathogenic variants leading to genetic diagnoses that were not identified at time of routine diagnostic work-up [ 8 – 14 ]. However, many of these studies were conducted in highly selected patient cohorts, making it difficult to draw firm conclusions on the potential of increased diagnostic yields when GS is employed in a prospective routine care setting [ 8 – 10 ].…”

“…In rare disease patients in whom standard of care (SOC) testing failed to provide a molecular diagnosis, GS has been shown multiple times to reveal pathogenic variants leading to genetic diagnoses that were not identified at time of routine diagnostic work-up [ 8 – 14 ]. However, many of these studies were conducted in highly selected patient cohorts, making it difficult to draw firm conclusions on the potential of increased diagnostic yields when GS is employed in a prospective routine care setting [ 8 – 10 ]. While other studies have concluded that GS is technically suitable as first genetic test based on randomized control trials with standard of care [ 11 – 16 ], those studies have not allowed for a systematic comparison at the individual patient level for (type of) diagnoses gained or missed.…”

Developmental genomics of limb malformations: Allelic series in association with gene dosage effects contribute to the clinical variability

“…During the past decade, elucidation of the molecular etiology of CLM has been facilitated by advances in high-throughput family-based genomics, rare variant allele studies, computational tools, and population variation studies, driving the molecular diagnostic “solved rate” of a random CLM-related cohort to be reported with a range of 10%–35% in different populations. 7 , 8 , 9 , 10 , 11 To date, more than 150 “disease trait genes or loci” have been attributed to CLM in affected individuals and families. A diverse mutational landscape comprised of single-nucleotide variant (SNV) alleles, small insertion or deletion (indel) events, simple sequence repeat expansions, and locus copy number variant (CNV) alleles involving coding or non-coding regulatory regions has been elucidated.…”