Case Study – CES

24-05-2019

Clinical Exome Sequencing

Introductions:

Diagnostic odyssey caused due a negative clinical exome

result in a patient with high suspicion of a genetic condition
can cause frustration for families and physicians alike. In
addition, families experience anxiety due to the time taken
for next generation sequencing and high costs associated
with the test. Utilizing advanced analytical tools and well
curated databases can improve identification of variants. We
demonstrate that in cases with high suspicion of a genetic
condition identification of variants in a suspected gene can Name – Proband X
provide greater insight to physicians and improve accuracy Age – 01 months
of diagnosis at comparatively lesser time.
Gender – Male

Proband profile: Recommended Test – LifeCell

CES
1-month-old male presented with clinical indications of
Previous genetic
lethargy, poor appetite, status epilepticus, recurrent
investigations, if any – None
encephalopathy, peripheral vascular insufficiency, and
compromised peripheral circulation in all limbs with anuria. CES Reports – Hemizygous
He was found to have decreased urine output levels and VoUS variant reported in OTC
elevated levels of serum ammonia. MRI finding was gene
suggestive of symmetrical bulky bilateral thalami and basal
ganglia hyperintensities with patchy hyperintensity in bilateral cerebral cortical gyri. Proband
was suspected to have a metabolic or urea cycle disorder.

Initial results of sequencing:

Clinical exome sequencing was initially performed by an outsourced lab M. The libraries were
sequenced to mean >80-100X coverage on Illumina sequencing platform. The results were
reported to be negative.
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24-05-2019

Secondary analysis at LifeCell:

A secondary analysis was performed in-house we identified a hemizygous intronic variation in

intron 5 of the OTC gene (chrX: 38260946; G>A). The intronic OTC variant is not present in
both the 1000 genomes and gnomAD databases. In-silico analysis of this nucleotide change
which is at 265 bp downstream from the 3' end of exon 5 revealed that it creates a novel splice
acceptor site (splice site score=90.5) which can lead to the premature truncation of protein.

Genetic Counseling Recommendations:

 Validation of the variant by Sanger sequencing was recommended.

 Testing for the variant in the parents and other affected, unaffected members can help
further identify clinical significance

How is our analysis different?

Commonly, mutations in the exonic region that affect the protein function are associated with
clinical manifestations. However, evidence suggests that mutations that affect splicing can be
Case Study – CES
24-05-2019

present in exonic, intronic or splice junctions. Such mutations can also be found in deep intronic
regions, many of which have been identified to be pathogenic. For this reason, at LifeCell we
utilise advanced NGS data analysis software Golden Helix that allows identification of splice
site predictions for exonic and intronic variants not near splice sites. This can help uncover
variants in intronic regions. In addition, VarSeq which is an integrated software provides a
filtering and annotation engine to sift through large variant data sets. The analysis pipeline
automates workflow and analysis of variants for gene panels, exomes, and whole genomes.
Further, it provides access to a wide selection of public databases which Golden Helix curates
and updates on a quarterly basis. Updated data will ensure timely and accurate classification of
variants as new data is published in literature.

References:

Engel K, Nuoffer JM, Mühlhausen C, Klaus V, Largiadèr CR, Tsiakas K, Santer R, Wermuth B, Häberle J. Analysis of mRNA
transcripts improves the success rate of molecular genetic testing in OTC deficiency. Mol Genet Metab. 2008 Jul;94(3):292-
7. doi: 10.1016/j.ymgme.2008.03.009. Epub 2008 Apr 25.

F. Pagani, F.E. Baralle. Genomic variants in exons and introns: identifying the splicing spoilers Nat. Rev. Genet., 5 (2004),
pp. 389-396