From July 2019 through May 2023, 247 probands with suspected rare genetic disease and their family members were enrolled in the iHope program at one of three sites in Lima, Peru: Hospital Edgardo Rebagliati Martins (HNERM) (n = 108), the Instituto Nacional de Ciencias Neurológicas (INCN) (n = 88), and the Instituto Nacional de Salud Niño-San Borja (INSNSB) (n = 51) (Table 1 and Supplementary Data 1).

The median age at enrollment was 8.7 years (IQR, 3.1–15.8 years), and 130 (52.6%) were male. The cohort consisted mostly of children (199/247, 80.2%). Trio or higher-order family structures, including the affected child and both unaffected parents, were most common (180/247, 72.9%), but variable family structures were submitted depending on the expected inheritance mode and family members available for testing. Patients traveled an average of 195 km for genetics evaluation (range 0–1011 km), 36.8% (91/247) of whom resided outside of the capital city of Lima (Fig. 1). The median time from symptom onset to cGS testing was 5 years (IQR, 2.4–11.8 years). By site, INCN had the longest median time from symptom onset to cGS testing at 9 years (IQR, 3.8–18.1 years), followed by HNERM (5.4 years, IQR, 2.5–9.5 years) and INSNSB (2.2 years, IQR, 1.1–4.8 years).

The distribution of patients participating in the iHope program is shown across Peru. Participating individuals are shown as purple circles, with the size of the circle denoting the number of patients from a given ZIP code. More than a third of patients (37%) were drawn from the capital city, Lima, which is shown in the inset in the lower left. Geographic barriers potentially impacting patient accessibility to clinical sites in Lima include both the Andes mountains (shown as the high-elevation band in dark brown) and the Amazon rainforest (shown in dark green). The cartographic and geographic data used to generate this figure were drawn from GPL3-licensed sources, as detailed in the “Statistical analysis” section.

All patients were evaluated by a clinical geneticist and had signs and symptoms suggestive of a rare, genetic disorder, consistent with current professional guidelines, and limited access to molecular testing (see “Methods”)20. Additional patient selection criteria considered by the ordering clinicians included a broad differential diagnosis (222/247; 89.9%), no other available next-generation sequencing test (198/247; 80.2%), non-diagnostic prior testing (180/247; 72.9%), a diagnostic odyssey lasting more than two years (173/247; 70.0%), a phenotypic presentation assessed by the clinician as severe (169/247; 68.4%), clinical suspicion of a disorder with an available treatment (91/274; 36.8%), parents of reproductive age (85/247; 34.4%), acute illness or ICU admission (29/247; 11.7%), and/or a first-degree relative with a similar clinical presentation (41/247; 16.6%) (Fig. 2a). Some differences in patient selection criteria were observed across sites (Supplementary Table 1).

a The total proportion of patients, and the proportion from each site, that were associated with each of iHope program selection criteria. Patients could be associated with more than one selection criteria. Broad differential diagnosis was the most common rationale for program inclusion. b The proportion of iHope patients with genetic testing prior to cGS stratified by site and type of genetic test. WES: whole exome sequencing; HNERM Hospital Nacional Edgardo Rebagliati Martins, INCN Instituto Nacional de Ciencias Neurológicas, INSNSB Instituto Nacional de Salud Niño-San Borja.

Although the majority of patients had at least one genetic test prior to the iHope-related consult and cGS (175/247 [70.9%]), the most frequently ordered test was a karyotype (132/247 [53.4%]). Only 34.4% (85/247) had access to other genetic tests (Fig. 2b). The number of genetic investigations performed per patient ranged from 0 to 4, with most patients having pursued one (129/247 [52.2%]) prior test. Significant differences in the genetic tests ordered by site were observed. For example, 85.2% (92/108) of patients from HNERM pursued karyotype, compared to 3.4% (3/88) at INCN and 72.6% (37/51) at INSNSB (Fisher’s exact test (FET), p < 0.001). Microarray testing was more frequently pursued at INSNSB (11/51 [21.6%]) compared to HNERM (6/108 [5.6%]) and INCN (3/88 [3.4%]) (FET, p = 0.001), with the same trend noted for panel testing (INSNSB 15/51 [29.4%] vs HNERM 10/108 [9.3%] vs INCN 11/88 [12.5%], FET p = 0.003). Single gene testing was more frequently pursued at INCN (23/88 [26.1%]) compared to HNERM (3/108 [2.8%]) and INSNSB (1/51 [2.0%], FET p < 0.001) (Fig. 2b).

Patient phenotypes were diverse and complex, with abnormalities of the nervous system (184/247; 74.5%), skeletal system (143/247; 57.9%), and head or neck (134/257; 54.3%) the most frequently identified Human Phenotype Ontology root ancestor terms overall (Fig. 3). Differences in HPO root ancestor terms by site were observed (Supplementary Table 2).

Summary distribution of top-level Human Phenotype Ontology terms nested beneath “Phenotypic abnormality” (HP:0000118) across the iHope cohort and grouped by clinical site. Patient phenotypes were diverse and complex, and with abnormalities of the nervous system, skeletal system, and head or neck, the most frequently identified Human Phenotype Ontology root ancestor terms overall and for each site. Differences in observed HPO root ancestor terms by site are detailed in Supplementary Table 2. HNERM Hospital Nacional Edgardo Rebagliati Martins, INCN Instituto Nacional de Ciencias Neurológicas, INSNSB Instituto Nacional de Salud Niño-San Borja.

Across the cohort, the diagnostic yield was 54.3% (134/247), with uncertain test results reported in an additional 22.3% (55/247), inclusive of variants of uncertain significance (Fig. 4a). Clinician review of uncertain results endorsed that 69.1% (38/55) were clinically suspected to be likely positive based on clinical correlation with the patient phenotype. Diagnostic yield varied across the sites, with observations of 43.1% at INSNSB (22/51), 53.4% at INCN (47/88), and 60.2% at HNERM (65/108) (Fig. 4a).

a Outcomes of cGS stratified by test result category and by clinical site. b Distribution of variant types reported across the iHope cohort and grouped by site. SNV single nucleotide variant; Indel: insertion or deletion, CNV copy number variant, STR short tandem repeat variant, MT SNV single nucleotide variant in the mitochondrial genome, SMA c.840 C allele in the SMN1 gene not detected, UPD uniparental disomy, HNERM Hospital Nacional Edgardo Rebagliati Martins, INCN Instituto Nacional de Ciencias Neurológicas, INSNSB Instituto Nacional de Salud Niño-San Borja.

Variants related to the indication for testing (248) included SNVs (162), small indels (46), CNVs ranging in size from 3 kb to 77 Mb (30), STRs (5), mitochondrial SNVs (3), regions of homozygosity suggestive of uniparental disomy (1) and spinal muscular atrophy detected by biallelic absence of the c.840 C allele (1) (Fig. 4b).

A total of 121 heterozygous variants in or encompassing single genes associated with disorders following an autosomal dominant mode of inheritance were reported, 87 variants in genes associated with autosomal recessive disorders, 28 variants in genes associated with X-linked disorders, and three variants in the mitochondrial genome (Supplementary Table 3). In 3 cases, chromosomal aneuploidy was detected, including one proband with Trisomy 18 and two probands with mosaic Trisomy 14, which were orthogonally confirmed via chromosomal microarray testing at an outside laboratory. In two probands, two de novo copy number variants were detected in each, with split-read evidence suggestive of a derivative chromosome present in the reportedly healthy parent. The most common recurrent primary diagnoses were Neurofibromatosis (4 probands), dopa-responsive dystonia (3 probands), Alagille syndrome (3 probands), and TTN-related disorders (3 probands).

Positive secondary findings reports were issued for 24 individuals, including ten probands (10/247; 4.0.%) and 14 family members (14/456; 3.1%). In five probands, the reported secondary findings were also included on the primary clinical report due to overlap with the indication for testing, including one proband with a pathogenic FBN1 variant consistent with Marfan syndrome, one proband with bi-allelic BRCA2 variants consistent with Fanconi anemia, two probands with variants in LMNA consistent with dilated cardiomyopathy and one proband with a TTN variant consistent with dilated cardiomyopathy. Reported secondary findings also included variants in FLNC, KCNQ1, LDLR, MEN1, MYBPC3, PALB2, TNNI3, and TP53 (Supplementary Table 4).

Incidental findings in genes not included on the ACMG Secondary Findings gene list but deemed actionable per the laboratory policy of the Illumina Clinical Services Laboratory were reported in a separate category on the primary clinical report in seven probands (7/247, 2.8%). Laboratory policy dictates that variants must be in the molecular state expected to cause disease (e.g., two variants in trans for an autosomal recessive condition, etc.), be classified as pathogenic or likely pathogenic by ACMG variant classification criteria (e.g., for a recessive condition with a compound heterozygous variant pair, each variant must be classified as P/LP), and have a ClinGen Actionability score of six or greater or have available National Comprehensive Cancer Network (NCCN) guidelines to guide the clinician in management recommendations based on the finding21,22,23. Incidental findings included variants in ATM, VWF, PALB2, FH, and G6PD. In two probands, incidental findings were the only reported variants (Supplementary Data 1).

Clinical GS test results impacted clinician diagnostic evaluation in 85.0% (210/247) of cases, including confirmation of a clinical diagnosis or diagnosis in the differential (39.7%, 98/247), establishing a new diagnosis (22.7%, 56/247), identifying variants of potential interest in relation to the patient’s phenotype (14.6%, 36/247), ruling out a suspected diagnosis (12.6%, 31/247) and/or producing an incidental diagnosis (4.9%, 12/247) (Fig. 5a).

a The impact of cGS results on the diagnostic evaluation, b change of management, and c genetic counseling. For each, multiple response options could be endorsed for a single patient. Results are displayed for the cohort overall and by site. HNERM Hospital Nacional Edgardo Rebagliati Martins, INCN Instituto Nacional de Ciencias Neurológicas, INSNSB Instituto Nacional de Salud Niño-San Borja.

Changes in management were reported in 71.3% (176/247) of cases, inclusive of referrals (64.7%, 160/247), therapeutics (26.3%, 65/247), laboratory or physiological testing (25.5%, 63/247), imaging (19%, 47/247), and palliative care (17.4%. 43/247) (Fig. 5b). The need for additional tests or evaluations was eliminated in 33.6% (83/247) of cases.

Clinical GS test results impacted genetic counseling in 72.1% (178/247) of cases, including counseling regarding patient prognosis (70.0%, 173/247), familial recurrence risk estimates (66.8%, 165/247), reproductive screening and testing options (29.6%, 73/247), testing options for other family members (22.3%, 55/247) and clinical screening recommendations for other family member (13.8%, 34/247) (Fig. 5c).

Notable case examples include a 3-month-old female in the intensive care unit on mechanical ventilation from one month of life with liver failure, jaundice, collateral circulation ascites, thrombocytopenia, coagulopathy, respiratory infections, and sepsis with a poor prognosis. At three months of age, cGS revealed a homozygous, pathogenic missense variant, c.443 G > A (p.Arg148Gln) in the GALT gene, consistent with a diagnosis of galactosemia. Her diet was changed to soy formula, resulting in normalized laboratory results (e.g., hemogram and biochemistry), regaining of consciousness, independent breathing, and resolved visceromegaly. As a result, she was discharged from the intensive care unit. Unfortunately, prolonged mechanical ventilation resulted in tracheomalacia, requiring a tracheostomy. Currently, she is two years old, able to stand, babbles, and continues to have a tracheostomy.

In another case, an 8-year-old male presented with walking difficulties and dysarthria since 1 year of age, together with recurrent respiratory infections, ocular telangiectasia, oculomotor apraxia, and head tremor. A compound heterozygous variant pair was identified in ATM, inherited from both parents, consistent with a diagnosis of ataxia–telangiectasia. This genetically confirmed diagnosis allowed the patient to receive IV immunoglobulin to prevent recurrent infections, as well as appropriate counseling for malignancies for the patient and his family members.

In a third case, a 15-year-old female presented with scoliosis, hyperpigmentation along Blaschko’s lines, hemihyperplasia of the right leg, and abnormal intramedullary signal on MRI of the iliac and long bones. She had a clinical diagnosis of Gaucher disease, for which she had been treated via enzyme replacement therapy (ERT) for 5 years. Clinical GS testing corrected the diagnosis to mosaic Trisomy 14, and ERT was discontinued.