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CNV Analyses May Uncover Mendelian Conditions in Undiagnosed Pediatric Patients

NEW YORK – Bringing in copy number variant (CNV) insights can help diagnose mysterious Mendelian genetic conditions in children, particularly when exome sequencing- and/or chromosomal microarray-based tests come up empty-handed, according to a study presented at the Association for Molecular Pathology virtual conference on Tuesday.

"As molecular pathologists in the current clinical workflow, we're not solving as many cases as we'd like for a number of reasons: sometimes exome sequencing is ordered without chromosomal microarray, and sometimes the microarray is ordered, but misses the CNV — for example, if the CNV is below the resolution of the microarray," said University of Toronto anatomical pathology resident Elan Hahn, who was supervised by Children's Hospital Los Angeles (CHLA) clinical pathologist Jianling Ji for the study.

"We decided to try to leverage exome sequencing data to identify CNVs and confirm them with other assays, thereby increasing our diagnostic yield," explained Hahn, noting that the study included surveys to track laboratory directors' perceptions of the analytical workflow in order to gauge its broader feasibility.

"Exome sequencing is the current standard for the diagnosis of Mendelian genetic disorders," Han said. But, he added, while a significant number of cases remain unsolved after exome sequencing, "the majority of the cases still remain undiagnosed."

Based on studies reported so far, the diagnostic yield associated with exome sequencing seems to be on the order of 25 percent to 45 percent, fluctuating somewhat depending on the kind of condition involved and the specific sequencing and analysis approaches used.

Although clinical exome sequencing pipelines are not usually validated for finding suspicious copy number variants or runs of homozygosity that impact individual genes, the team reasoned that it might be possible to diagnose additional Mendelian conditions in children by searching for CNVs in available sequence data, since copy number changes have been implicated in some forms of developmental delay, congenital abnormality, or other pediatric genetic conditions.

"We set out to determine the diagnostic yield and feasibility of CNV and heterozygosity analysis of exome sequencing data from undiagnosed clinical cases," Hahn explained.

Starting from whole-exome sequencing or more targeted gene sequencing on genes related to a patient's phenotype, known as focused exome sequencing, he and his colleagues looked at the possibility of diagnosing unexplained pediatric conditions — involving phenotypes ranging from developmental delay, seizures, autism, or cognitive impairment to immunodeficiency or failure to thrive — with the help of copy number- or heterozygosity-focused analyses.

Of 290 pediatric patients who had been referred for exome or targeted sequencing from the fall of 2019 to May of this year at the CHLA's Center for Personalized Medicine, Hahn noted, 46 of the whole-exome sequenced cases and 155 of the cases assessed by focused exome sequencing remained undiagnosed after initial searches for pathogenic and likely pathogenic variants in the protein-coding sequences considered.

In that set of 201 unresolved cases, the team searched for telling copy number changes using the Biodiscovery NxClinical CNV calling platform, focusing on genes with potential ties to each patient's phenotype based on Human Phenotype Ontology data, Hahn said. Potential microdeletions, duplications, instances of uniparental disomy, or other candidate CNVs were subsequently validated with assays from Thermo Fisher Scientific.

The strategy unearthed suspicious heterozygous or hemizygous deletions in several unresolved pediatric cases, ranging in size from single-exon deletions to those affecting a whole gene. The team also tracked down chromosome loss events, microdeletions and microduplications, heterozygous deletions accompanied by sequence variants, loss of heterozygosity events, and larger CNVs.

Across all 201 cases that had not previously been diagnosed, the researchers identified clinically significant copy number variants — classified as pathogenic or likely pathogenic — in 18 of the pediatric patients, consistent with a diagnostic yield of nearly 9 percent. Another 10 cases were marked by copy number variants currently considered as variants of uncertain significance.

In a child with a clinical history of malignancy, respiratory problems, epilepsy, and other neurological symptoms, for example, the researchers focused in on a gene-level deletion affecting one whole copy of the PHOX2B gene. That deletion also turned up in exome sequence data from the patient's mother and brother during trio exome sequence analyses.

With focused exome sequence data, meanwhile, their copy number-based analysis led to copy number changes consistent with a 7.6-megabase chromosome 1p31 microdeletion syndrome in an infant with macrocephaly and problems eating, while still other CNV, CNV-variant, or heterozygosity changes appeared to explain other pediatric cases.

Participating lab directors reported that it took them under five minutes or between five and 10 minutes to analyze each case, Hahn said, suggesting it should be possible to find candidate CNV or loss-of-heterozygosity events without a large additional investment in time.

"All [survey] respondents reported this as a feasible step to include in a routine clinical workflow," according to Hahn, who said CNV analysis of exome sequences "adds significant value" in the clinical setting.

"We are incorporating the bioinformatic and confirmatory wet lab approaches into our prospective exome sequencing-based studies," he said. "It's only going to get better from here, as additional disease-causing CNVs are discovered."