Successful application of genome sequencing in a diagnostic setting: 1007 index cases from a clinically heterogeneous cohort

Abstract

Despite clear technical superiority of genome sequencing (GS) over other diagnostic methods such as exome sequencing (ES), few studies are available regarding the advantages of its clinical application. We analyzed 1007 consecutive index cases for whom GS was performed in a diagnostic setting over a 2-year period. We reported pathogenic and likely pathogenic (P/LP) variants that explain the patients' phenotype in 212 of the 1007 cases (21.1%). In 245 additional cases (24.3%), a variant of unknown significance (VUS) related to the phenotype was reported. We especially investigated patients which had had ES with no genetic diagnosis (n = 358). For this group, GS diagnostic yield was 14.5% (52 patients with P/LP out of 358). GS should be especially indicated for ES-negative cases since up to 29.6% of them could benefit from GS testing (14.5% with P/LP, n = 52 and 15.1% with VUS, n = 54). Genetic diagnoses in most of the ES-negative/GS-positive cases were determined by technical superiority of GS, i.e., access to noncoding regions and more uniform coverage. Importantly, we reported 79 noncoding variants, of which, 41 variants were classified as P/LP. Interpretation of noncoding variants remains challenging, and in many cases, complementary methods based on direct enzyme assessment, biomarker testing and RNA analysis are needed for variant classification and diagnosis. We present the largest cohort of patients with GS performed in a clinical setting to date. The results of this study should direct the decision for GS as standard second-line, or even first-line stand-alone test.

Fig. 1

a The index case presented with intrauterine growth retardation, microcephaly, dysmorphism, with clinical suspicion of a lysosomal disease. GS detected a heterozygous intronic deletion (NM_024312.4(GNPTAB):c.2915+4_2915+9del, inherited from the father) and a maternally inherited heterozygous insertion within exon 13 of the GNPTAB gene (mucolipidosis type II). Corresponding IGV images are shown with the small intronic deletion (left) and large exonic insertion (right). The exonic insertion was confirmed by PCR and gel electrophoresis, index and mother presented a larger band corresponding to the allele with the insertion. b The index patient presented with neurodevelopmental delay, microcephaly, abnormal skin pigmentation, reticular rash and photophobia. GS identified a heterozygous missense variant (NM_000057.2(BLM):c.3164G>C, p.(Cys1055Ser), Sanger traces are shown) and a heterozygous deletion encompassing exons 11 and 12 of the BLM gene (indicated by red arrows, IGV). Note comparation in IGV of ES and GS data (only index, upper lane) in the corresponding region of the BLM gene. c The index patient presented with neurodevelopmental delay, short stature, facial dysmorphism (hypertelorism, low set ears) and cardiovascular malformation (coarctation of the aorta and persistent ductus arteriosus). A structural variant was detected by GS: a balanced translocation between chromosomes 1p (left panel) and 3p (right panel) with break points definitions at chr1:3,300,737 and chr3:51,573,020. The break points are likely affecting the PRDM16 and RAD54L2 genes which have been implicated in cardiomyopathy and neurodevelopmental delay. Upper panel: Additional confirmation was performed, by agarose gel electrophoresis (1.5%) of PCR amplified products of control fragment and translocation testing fragment. Control fragment shows amplification corresponding to size 403 bp for two controls samples (CTRL_1, CTRL_2) and patient. Translocation testing fragment shows amplification corresponding to size 803 bp only for patient sample. d Index case presented with failure to thrive, fatigue, metabolic acidosis, polyuria, polydipsia. GS did not detect any abnormality in the nuclear DNA. A large heteroplasmic deletion was detected in the mitochondrial DNA (chrM:8637–16072, indicated by red arrows) confirming the diagnosis of mitochondrial deletion syndrome.

Fig. 2. GS diagnostic yield in 1007 index cases.

a Overall diagnostic yield in 1007 index cases. b GS diagnostic yield in 649 ‘naive’ cases (no previous ES). c Variant classification and diagnostic yield are influenced by GS test design (trio/other vs. solo). Singleton GS testing resulted in increased number of VUS reported due to difficulties phasing the alleles and recognition of de novo status, which indirectly affects diagnostic yield.