Genome sequencing for the diagnosis of intellectual disability as a paradigm for rare diseases in the French healthcare setting: the prospective DEFIDIAG study

Abstract

Background: Intellectual disability (ID) is the leading cause of patient referral to medical genetic departments in French academic hospitals. Whole genome sequencing (WGS) as a first diagnostic approach is expected to achieve a higher diagnostic yield than the French national reference strategies (RefStrategy) (fragile X expansion testing, chromosomal microarray analysis, and 44 ID genes panel), given its broad and more homogeneous coverage, its ability to identify copy number, structural and intergenic/deep intronic events.

Methods: DEFIDIAG is a national, prospective pilot investigation, carried out in the framework of the French initiative for genomic medicine (Plan France Médecine Génomique 2025), aimed at comparing the diagnostic yield of WGS trio analysis (WGS-trio) (index case, father, mother) with the RefStrategy in real-life conditions of clinical and laboratory workflows. Both strategies were applied in a blinded fashion in 1239 ID probands (50% were already-tested, 50% were never-tested) with no definitive genetic diagnosis. Among them, a subgroup of 187 patients were randomized to undergo WGS-solo (proband only) in addition to WGS-trio and RefStrategy.

Results: Four hundred forty two likely pathogenic/pathogenic single-nucleotide variants were identified (for 231 genes) as well as 171 variants of uncertain significance warranting clinical or functional reassessment for a potential reclassification (VUS +) (for 142 genes), 79 likely pathogenic/pathogenic copy number variants and 10 likely pathogenic/pathogenic structural variants. The diagnostic yield for likely pathogenic/pathogenic variants increased from 17.3% with the RefStrategy to 41.9% with WGS-trio in the never-tested patient cohort. An increase of 13.9% was observed in all categories by adding the VUS + , thus raising the yield to 56% for WGS-trio. Overall, WGS-solo enabled the identification of likely pathogenic/pathogenic variants in 29.9% of cases (increasing to 41.1% when including VUS +) compared to 21.9% with the RefStrategy. In addition, following recent reports of de novo variants in the non-coding spliceosomal RNU4-2 gene as a common cause of ID, this gene was subsequently analyzed, leading to the identification of pathogenic de novo variants in 7 patients.

Conclusions: As a first line test for ID diagnosis, WGS (including for solo situations) proved to be more effective than the reference strategy, in the context of real-life hospital settings in France.

Trial registration: Prospectively registered with ClinicalTrials.gov under the identifier NCT04154891 (07/11/2019).

Keywords: Centers of expertise; Diagnostic yields; Intellectual disability; Multidisciplinary meetings; Real-life hospital setting; Short-read sequencing; Solo; Trio; Whole genome sequencing (WGS).

Fig. 1

Overview of the DEFIDIAG study. A schematic overview of the DEFIDIAG study is presented. The starting point is patients with ID and their parents (the trio), referred to the medical genetics department of their local university hospital. Two distinct patient groups were defined, both of which underwent WGS in trio (WGS-trio) in addition to the reference strategy (Fragile X analysis, CMA, and the 44GPS genes panel). Patient group 1 included patients who had never undergone any genetic analysis (n = 583), for whom WGS-trio was performed as the first-line investigation (WGS first). Among them, a randomized subgroup of 187 patients also underwent WGS in solo (WGS-solo). Patient group 2 included patients who had already undergone previous genetic investigations, but without identification of the cause of their ID (n = 606), for whom WGS-trio was performed as the second- or third-line investigation (WGS after). Each patient included underwent both WGS and the reference strategy, with blinded interpretation. 44GPS: 44-ID genes panel; CMA: chromosomal microarray analysis; WGS: whole genome sequencing

Fig. 2

Diagnostic yields of the RefStrategy, WGS-trio, and WGS-solo in the 1239 ID patients. A McNemar tests between diagnostic yields of WGS-trio compared to the RefStrategy, the 44 ID genes panel (44GPS), fragile X expansion analysis and chromosomal micro array analysis (CMA) in already explored patients (AlreadyTested, dark blue bars) and never explored patients (NeverTested, light blue bars) (p < 10⁻³). B Diagnostic yields of 44GPS, WGS-trio, and WGS-solo according to likely pathogenic/pathogenic variants (light blue bars) and VUS + (yellow bars), in NeverTested. C McNemar tests between diagnostic yields of WGS-solo compared to WGS-trio and to RefStrategy in the subgroup of 187 randomized NeverTested patients

Fig. 3

Venn diagrams comparing the strategies in the NeverTested population. A Venn diagrams showing the positive diagnoses in the NeverTested group (N = 583): WGS trio (in blue) vs RefStrategy (in red). B Venn diagrams showing the positive diagnoses in the NeverTested randomized subgroup (N = 187): WGS solo (in green) vs RefStrategy (in red). C Venn diagrams showing the positive diagnoses in the NeverTested group (N = 583): WGS trio (in blue) vs WGS solo (in green). NeverTested: Never explored patients; RefStr: reference strategy; WGS: genome sequencing

Fig. 4

Top 15 ID genes involved in the DEFIDIAG cohort population (likely pathogenic/pathogenic SNVs). Blue bars represent truncating variants, green bars represent missense variants, red bars represent splicing variants, and yellow bars represent in frame deletions or duplications. All variants were de novo, except one BCL11A (2p16.1), one TRIO (5p15.2), one CNKSR2 (Xp22.12), and one MECP2 (Xq28) variant, which were all maternally inherited

Fig. 5

Representation of gene enrichment in DEFIDIAG compared with gene categories in the SysNDD database. Bar diagrams show enrichment of ID-DEFIDIAG-ID genes in each indicated functional category against the SysNDD ID-genes as background. The total number of DEFIDIAG genes per category is displayed in the respective bar. The asterisks indicate statistically significantly enriched categories (Fisher test, Benjamini-Hochberg; ∗ adjusted p < 0.05, ∗ ∗ adjusted p < 0.01, ∗ ∗ ∗ adjusted p < 0.001.). Adapted from SysID database [69]

Fig. 6

Illustrative cases showing patients’ diagnostic odyssey. Patient 1 is a 29-year-old male with severe ID, seizures with continuous spike–waves during slow sleep EEG pattern, facial dysmorphic features (a at age 10 years, b and f at age 26 years), and mildly hypoplastic nails (e). The patient underwent testing for Fragile X syndrome, CMA, and a 556 ID genes panel, all results were negatives. After 22 years of diagnostic wandering, the DEFIDIAG study identified a de novo likely pathogenic heterozygous missense variant in POU3F3 (Chr2(GRCh37):g.105473206 T > C; NM_006236.2(POU3F3):c.1238 T > C; p.(Ile413Thr)). This result highlights the value of WGS compared to ID gene panels, which, although regularly updated, include a limited number of genes. Patient 2 is a 17-year-old male affected by severe ID, autism spectrum disorder, early epilepsy followed by neurological regression, facial dysmorphism (c, d) and short distal phalanges (g). Brain MRI showed thickening of the corpus callosum (arrow) and widening of the vermian sulcus (h). Both CMA and a 207 genes panel targeting epilepsy and cortical malformations yielded negative results. After 17 years of diagnostic wandering, the DEFIDIAG study made it possible to identify a de novo pathogenic chromosome 5 paracentric inversion involving MEF2C (Seq[GRCh37] inv(5)(q14.3q14.3), NC_000005.9:g.88090783_88605087inv) {ISCN 2020}. This clinical case clearly illustrates the superiority of WGS in identifying structural variants. Patient 3 is a 5-year-old female patient presenting with ID and overall developmental delay predominantly affecting language, behavioral and social interaction difficulties, recurrent infections, chronic constipation, and visual impairment. She presents with distinctive craniofacial features, including a prominent forehead and a high anterior hairline (i, j), as well as broad and short hands with tapered fingers and enlarged halluces (k, l). She was enrolled in the DEFIDIAG study, and WGS trio identified a heterozygous de novo pathogenic intragenic inversion in the ADNP gene (Chr20(GRCh37):g. 49515761_49525309inv, NM_001282531.3:c.−89−3923_201 + 2793 inv). This structural variant encompasses exons 3 to 5, involving the two first coding exons with the initiation Met1. RNAseq experiment showed a splice skipping of the inversed exons and in silico analysis suggested that several initiating ATGs would lead to the failure of any in-frame rescuing translation, because of out-of frame ATGs, resulting in haploinsufficiency. Since this inversion is undetectable by exome sequencing, this case emphasizes the added value of whole genome sequencing [110]. Patient 4 is a 9-year-old female born with intrauterine growth restriction and a velar cleft. She achieved independent walking at 23 months, and first spoken words emerged at 20 months. She has since developed mild ID, associated with microcephaly (− 3 SD) and significant anxiety. Feeding remains problematic due to pronounced food selectivity, notably with a consistent refusal to consume fruit and vegetables. She presents with facial features (m, n) including epicanthal folds, hypertelorism, tubular nose with broad and prominent nasal bridge, and large dysplastic ears. The examination of the extremities showed mild, nonspecific morphological anomalies, including slightly low-set thumbs (o, p). 22q11 FISH analysis and CMA were negative and WGS-trio identified a heterozygous de novo pathogenic nonsense variant in TLK2 (Chr17(GRCh37):g.60642437C > T, NM_006852.6(TLK2):c.907C > T p.(Arg303*))