Of 103 families deemed eligible for the iHope Program after chart review, sixty probands (58.3%) were successfully contacted and attended a “Genome Day”, a clinic visit dedicated to cWGS testing consent and phenotyping. Of those individuals who did not participate, the majority was due to inability to re-contact the family to offer cWGS (n = 38; 79%). The remaining five families were successfully contacted and initially expressed interest in cWGS but did not follow-up with a visit to clinic for a Genome Day (Fig. 1). Six Genome Days were held between August 2016 and March 2018 in which 60 probands and their families (one proband-only, 14 duos, 42 trios, and three quads) provided informed consent and blood samples for cWGS (Table 1). No families who attended a Genome Day and participated in the informed consent process declined to participate in cWGS testing. The mean rounded proband age was 7.6 years at the time of blood draw (ranging from four months to 21 years). Most (n = 41; 68.3%) probands had no prior cytogenetic or molecular genetic testing. Of the probands that underwent some genetic testing prior to cWGS, karyotype analysis was the most commonly completed test (n = 19). Three probands who underwent karyotype analysis also pursued other genetic testing, including single gene analysis (n = 1), methylation studies to assess for Prader-Willi syndrome (n = 1), and chromosomal microarray (n = 1). The most common indications for testing included congenital anomalies, developmental delay, seizures/epilepsy, growth restriction, and intellectual disability, with clinician categorization showing 76.7% (46/60) of proband phenotypes were consistent with a suspected pattern of malformation and 23.3% (14/60) with a primary neurologic presentation.

Fig. 1 Case selection criteria. Chart review of previously evaluated individuals was performed by the clinician team. Probands who were diagnosed with a recognizable pattern of malformation (e.g., isolated Down syndrome), received counseling, and discharged from clinic were excluded from referral to the iHope Program. Probands with acquired disease (e.g.,: suspected environmental exposures) or isolated features (e.g.,: individuals with cleft lip with or without cleft palate) were typically also excluded. Probands with prior non-diagnostic molecular or cytogenic testing were included if all other criteria were met. Resulting families who were eligible for the iHope Program were contacted, offered cWGS, and scheduled to attend a Genome Day. Upon completion of a Genome Day visit, whole-blood samples were transported to the clinical laboratory for cWGS. Dx: diagnosis; cWGS: clinical whole genome sequencing Full size image

Table 1 Cohort demographic data Full size table

A genomic finding congruent with the reported phenotype was identified in 41 of 60 probands, resulting in an overall diagnostic yield of 68.3% (Table 2). In 36 probands, pathogenic or likely pathogenic variants were reported in genes or regions of the genome with significant phenotypic overlap, which were considered positive molecular diagnoses. Three probands were considered to have likely positive molecular diagnoses, where variants of unknown significance (VUS) were reported in well-characterized genes, which were confirmed to contribute to the proband’s diagnosis by subsequent clinical feedback (P6, P20, P36). Two cases were considered partial molecular diagnoses, as the identified pathogenic variant was hypothesized to explain the proband’s phenotype only in part (P17 and P31). Of note, molecular diagnoses were achieved for 80% (12/15) of all duo and proband only cases. A diagnostic result from cWGS was obtained for 76.1% of all probands in the patterns of malformation phenotype group compared with 42.9% of all probands in the primary neurologic phenotype group (p = 0.0455). All individuals in this cohort received analysis for ACMG secondary findings, and pathogenic secondary findings were reported in three cases (P6, P12, and P39). These include one maternally inherited pathogenic BRCA2 variant identified in a trio case, one de novo pathogenic deletion encompassing the entire STK11 gene in a trio case, and one pathogenic deletion involving exons 1–10 of the PMS2 gene in the proband but not in the proband’s mother identified in a duo case.

Table 2 Molecular diagnoses reported by variant type Full size table

A range of variant types were observed in the 41 cases in which primary molecular diagnoses were achieved. These included SNVs (n = 18; 43.9%), CNVs ranging from 26 kb–18 Mb (n = 10; 24.4%), multiple terminal CNVs suggestive of derivative chromosomes (n = 5; 12.2%), aneuploidies (n = 2; 4.9%), absence of heterozygosity (AOH) consistent with uniparental isodisomy (UPD) (n = 1; 2.4%), indel (n = 2; 4.9%), a compound heterozygous variant pair involving multiple variant types (n = 1; 2.4%), a dual diagnosis of SNV and aneuploidy (n = 1; 2.4%), and one case with at least four molecular diagnoses including a SNV and a compound heterozygous pair involving multiple variant types (n = 1; 2.4%), as summarized in Fig. 2.

Fig. 2 Proportion of variant types observed in cases where molecular diagnoses were achieved. Number of probands with small variants (including SNVs and indels), copy number/chromosomal variants (including CNVs, derivative chromosomes, aneuploidies, and UPD), and multiple variant types (SNVs and another variant type in a single case) are noted. SNV: single nucleotide variant, indel: insertions and deletions, UPD: uniparental disomy, CNV: copy number variant Full size image

No molecular diagnosis was achieved in 19 cases. In four negative cases, birth injury or teratogenic exposures including monozygotic twin-related injury, valproic acid embryopathy, fetal alcohol syndrome, and Zika virus-related embryopathy, were noted by the clinician team as among the differential diagnoses. In one negative case, the proband is suspected to have a genetic disorder involving mosaicism given her hyperpigmented and hypopigmented patchy skin lesions on the legs and skeletal asymmetry including hypoplasia of the left humerus and shoulder girdle. In some negative cases (n = 4; 6.7%), variants of interest were reported for clinical consideration (Table 3). These probands are not considered to have received molecular diagnoses, but clinicians have characterized these variants of clinical interest as either possibly contributory (n = 3) or secondary (n = 1) molecular findings in relation to the proband’s phenotype.

Table 3 Reported variants of clinical interest reported by variant type Full size table

Surveys assessing the clinical utility of cWGS results were completed by the clinical team for all 60 probands. Of the 41 probands who received a molecular diagnosis, cWGS results produced new clinical diagnoses in 80.5% of cases (n = 33) and confirmed clinical diagnoses in 19.5% of cases (n = 8). A change in the proband’s clinical course due to cWGS findings was reported in 48.8% of cases (n = 20). These included referrals to specialists to assesses for co-morbidities (n = 8; e.g., neurology, ophthalmology, audiology) or for imaging or functional testing (n = 6; e.g., renal ultrasound, brainstem auditory evoked response test, echocardiogram, electrocardiogram, electroencephalogram). Muscle biopsy was avoided in three probands who received molecular diagnoses, reducing potential morbidity and clinical resource burden. Some findings (n = 3) led to additional clinical investigations related to the molecular diagnosis, including abnormal eye movements further investigated for possible seizures and examination of muscle tone in association with progressive spastic paraplegia. One family received information regarding the creation of an augmentative communication system for a child with Angelman syndrome, a condition in which there is typically poor prognosis for the development of expressive verbal language. One patient was transitioned to palliative care after molecular diagnosis of neuronal ceroid lipofuscinosis. Finally, current or future screening for malignancies and/or tumors/polyps was discussed for the proband with a deletion encompassing exons 1–10 of the PMS2 gene (P6), for the proband with an entire gene deletion of STK11 (P39), and for the proband and mother with a pathogenic BRCA2 variant (P12) identified by cWGS as secondary findings.

Among cases where no molecular diagnoses were achieved, cWGS was reported to be helpful to the proband’s clinical care in at least six probands and their families. In two cases where the proband’s brain imaging suggested possible leukodystrophy, cWGS was reported to be helpful to clinicians in communicating a significantly reduced likelihood for suspected specific diagnoses. In three negative cases, clinicians were motivated to expedite additional clinical work-up to further investigate the proband’s phenotype, including ophthalmology exams and a muscle biopsy. For one proband (P43) with no molecular diagnosis, a variant of clinical interest was submitted to GeneMatcher12 and yielded contact from three institutions and enrollment in a clinical study of patients with suspected causative variants in the USP7 gene.

Post-test genetic counseling was modified for 49 families (100% of probands with molecular diagnoses; 81.7% of the cohort) following cWGS results. Among cases with molecular diagnoses, the most frequently cited reason for the change in post-test counseling was the ability to share information about recurrence risk/options for preconception testing or prenatal diagnosis (n = 37) and prognostic indications (n = 21). Among negative cases, the most frequently cited reasons for change in post-test counseling was ability to counsel regarding decreased likelihood of genetic conditions (n = 3).