Genome Sequencing for Diagnosing Rare Diseases

Abstract

Background: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined.

Methods: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center.

Results: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments.

Conclusions: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).

Figure 1.. Examples of Variants or Regions Requiring Genome Sequencing.

Shown are some of the reasons that variants require genome sequencing for detection. These include deep intronic variants (Panel A, arrow), which are unlikely to be reliably detected by typical exome-sequencing methods (>20 bp upstream or downstream from the beginning or end of an exon); variants that are missed with previous exome sequencing because of poor coverage of the region (Panel B, arrow pointing to a region of lower exome coverage in blue as compared with genome coverage in green for the PCSK9 gene, on the gnomAD, version 2, browser); tandem repeat expansions and some structural variants such as copy-neutral inversions; and small (Panel C, approximately 50 to 2000 bp), largely intronic copy-number variants, or complex events involving more than one type of structural variant.

Figure 2.. Diagnostic Yield According to Phenotype and Genetic Ancestry.

Probands were categorized according to the most prominent phenotypic features (Panel A) and genetic ancestry (Panel B). The percentage of families in which diagnoses were made is shown. Genetic ancestry was determined by gnomAD, version 2, random forest model, and a genetic ancestry was assigned to all samples for which the probability of that ancestry was more than 90%. All samples that did not meet the 90% threshold were imputed as “multiple or unassigned.”

Figure 3.. Classification of Families with a Molecular Diagnosis.

Among the 218 families in which a molecular diagnosis or probable molecular diagnosis was obtained by genome sequencing, the types of variants that required genome sequencing for identification (61 families) are shown. A total of 21 (34%) were structural variants, including 11 deletions (2 in noncoding regions), 2 duplications, 4 inversions, 3 complex deletion–duplication events, and 1 mobile element insertion in a noncoding region. In addition, 6 tandem repeat expansions were identified. Of these variants, 19 (31%) were coding variants that had been missed in previous exome sequencing because of poor coverage of the region, which resulted in either no variant at that site or a poor-quality variant that was filtered out by variant quality control and hence overlooked in the analysis process. Most of these variants (13 of 19) were small indels (usually 10 to 15 bp in size), of which 8 were deletions. Fifteen (25%) of the diagnoses requiring genome sequencing involved deep intronic noncoding variants (complemented by RNA sequencing for 8 persons). Two persons had variants that were missed on exome sequencing for more than one reason (a structural variant in a new gene in one case and another case involving a promoted variant plus a new gene contributing to a blended phenotype). Of the 157 families in which the causal variants were detectable by exome sequencing, 94 (59.9%) had had previous exome sequencing that missed the variant, whereas the remaining 63 had not had previous exome sequencing but were diagnosed through the identification of variants expected to be detectable by current exome-sequencing methods. Two diagnostic mitochondrial DNA (mtDNA) variants were identified by genome sequencing and missed with previous exome sequencing, although these variants were also identified by mitochondrial genome sequencing. CNV denotes copy-number variant.

Figure 4.. Reasons for Lack of Diagnosis by Previous Exome Sequencing.

The number of families with diagnoses missed for each reason is shown. The 14 families with deep intronic variants required genome sequencing for identification of diagnoses, and 10 families required additional validation (8 required RNA sequencing and 2 required other functional validation).