Abstract - Text

Abstract-Text

In routine diagnostics, short-read sequencing is the dominating technique for exome analysis. State-of-the-art short-read sequencing provides high-quality data at an impressive throughput. However, the limited read length is a drawback leaving cases unsolved. Here, we present two routine diagnostic cases that could not be conclusively solved and therefore long-read Nanopore sequencing was applied to get further insights.

The first individual is a child with intellectual disability, autism, facial dysmorphism and seizures. By exome sequencing, we identified a single basepair deletion leading to a frameshift in SETD1A (c.1143del, p.(Tyr382Thrfs*115)). SETD1A associated neurodevelopmental disorder (MIM #619056) has a high phenotypic overlap with the child"s symptoms. We could exclude the variant in the mother, but segregation analysis was restricted as the father already died. Consequently, we were interested in phasing the variant as finding it on the maternal allele would mean it occurred de novo. For phasing, we used a SNP about 5 kb downstream of the variant. A long-range PCR spanning the variant and the SNP and subsequent Nanopore sequencing was performed. As the variant was found on the paternal allele, it is still unclear if the variant occurred de novo or was inherited from the father. Now, analysis of further paternal relatives is ongoing to check for the variant.

The second individual was a newborn with a leukodystrophy, muscular hypotonia, poor head control, failure to thrive and motor developmental delay. We identified two missense variants in the gene NFU1. Pathogenic variants in NFU1 are associated with a mitochondrial dysfunction syndrome, which is a severe autosomal recessive disorder of systemic energy metabolism resulting in respiratory failure, lack of neurologic development and early death. By segregating the parents, the first variant (c.565G>A, p.(Gly189Arg)) was determined to be maternally inherited while the second variant (c.545G>T, p.(Arg182Leu)) occurred de novo. Hence, the variants could not be phased. Furthermore, an RNA sample was not available as the patient deceased meanwhile. Hence, by long-range PCR, a product of about 6.5 kb size containing both variants was obtained and subsequently sequenced on a Nanopore device. As the variants were not observed at the same reads, they were pinpointed as compound-heterozygous and confirmed as disease-causing. As a result, the recurrence risk was assessed as very low (<1%) which was relevant to the parents as they had a lasting wish for a child.

The presented families are unique, but the field of human genetics often handle rare disorders and special constellations. Thus, long-read sequencing is a valuable addition to the toolbox of routine genetic diagnostic methods. Long-read sequencing has specific advantages not only when phasing variants as presented here but also for the analysis of repeat expansions and structural variants.