This study aimed to evaluate the diagnostic yield and the impact of targeted gene panel sequencing in a selected cohort attending a LMIC tertiary paediatric neurology outpatient service. The overall study diagnostic yield (DY) was 53% (66/124 patients). It was highest for neuromuscular disorders 64% (16/25) followed by cerebral palsy spectrum disorders 54% (9/16) and epilepsies 44% (28/63).
To date, few studies have investigated the diagnostic yield of molecular testing in paediatric neurological disorders. The overall rate of molecular diagnosis in previous studies range from 20 to 60%. However, the results of previous studies are not generalizable, due to heterogeneous study designs, sample size, inclusion criteria, and specific diagnostic techniques. To the best of our knowledge, this is the first study that specifically investigated the diagnostic yield of commercially available gene panels in a large cohort attending a tertiary paediatric neurology outpatient service in a LMIC. The overall diagnostic yield in this study is similar to previously reported paediatric cohorts in HICs (See Supplementary Table 3)
Clinical interpretation of genetic test results is increasingly complicated by variants of uncertain significance (VUS) that have an unknown impact on health. High volumes of returned VUS (47/124 patients) in the study similarly complicated interpretation of results. This is true across disease category and across panels, as our population is understudied and underrepresented in global databases (5). Reclassification can clarify a variant's clinical significance and it is increasingly facilitated by the availability of updated information about human genetic diversity, especially among underrepresented populations. This furthermore highlights the importance of studies, which include LMICs and diverse populations.
The most common genes identified in the study were SCN1A (n = 8), CDKL5 (n = 4), DMD (n = 4), KCNQ2 (n = 3), RYR1 (n = 3). These genes have been well described in other international paediatric studies. SCN1A, CDKLA5 and KCNQ2 were also among the top five genes with the highest yield using NGS in studies focusing on paediatric epilepsy in a study by Mei et al. (6). A recent study from the South Africa using a panel of 71 DEE-associated genes identified Pathogenic/Likely pathogenic candidate single nucleotide variant or short indels in 12% of cases (28/234 patients) (7). Similar to our study, SCN1A proved the most prevalent gene in this category (n = 13). Of interest was the failure to identify any KCNQ2 cases despite the gene’s inclusion in the testing panel. The three KCNQ2 cases identified in our study highlight the importance of including this gene in any DEE or Epilepsy panel, as it is relevant and potentially allows clinicians to offer precision therapy (sodium blockers).
Spinal muscular atrophy is a commonly encountered neuromuscular disease in our region. Studies have shown that the birth incidence of SMA in black South Africans is higher than one in 3574 (8). Local testing for SMN1 does not include a determination of SMN2 copy numbers. The latter may be employed to correlate with the disease phenotype, predict disease evolution, and stratify patients that are eligible for gene therapy. The Invitae SMA panel used in this study offered the additional advantage of identifying the SMN2 copy numbers, facilitating easier entry into clinical trials for these patients.
With regards to the three patients where additional panels were requested: Patient 43 clinically had features suggestive of Rett syndrome but a pathogenic variant was only found once a re-requisition was made to the extensive Epilepsy panel. Patient 51 was diagnosed on the main panel (Epilepsy panel) and the additional panel was not helpful. Patient 86’s results still yielded VUS even though the panel was expanded to more than 600 genes.
Interesting cases
Unexplained cause of muscle weakness and hypotonia
A female infant presented with hypotonia, muscle weakness, recurrent chest infections and progressive scoliosis presumed to be related to a congenital myopathy. Investigations including creatine kinase (CK) were normal. Muscle biopsy was deferred due to high risk of anaesthetic complications. Genetic testing via the comprehensive neuromuscular NGS panel identified one pathogenic variant in TTN (OMIM #188840), which is associated with autosomal-dominant dilated cardiomyopathy (OMIM #604145) as well as a group of disorders affecting skeletal muscles, including autosomal-dominant tibial muscular dystrophy and autosomal-recessive limb-girdle muscular dystrophy type 2J (LGMD2J: OMIM #608807), autosomal-recessive centronuclear myopathy (CNM; OMIM # 611705) and autosomal-dominant hereditary myopathy with early respiratory failure (HMERF; OMIM # 603689). This patient is a carrier for a variant in a domain of TTN known to be associated with autosomal recessive TTN-related conditions. The second variant in TTN was not identified on the panel and the clinical significance of his variant remains unknown A definitive diagnosis has thus not been made. Further testing in the form of genome sequencing or transcriptomics may uncover the underlying cause of her myopathy.
ADAR-related bilateral striatal necrosis with cyclical transaminitis
A teenage boy presented with a longstanding history of acquired dystonia and recurrent episodes of transaminitis associated with lethargy and vomiting. Clinical examination revealed hyperpigmented macules arranged in reticulated patterns in the face and the dorsal aspects of the extremities of the patient, as well as his mother and brother. Metabolic investigations proved unremarkable, whilst magnetic resonance imaging (MRI) of the brain demonstrated bilateral high signal intensity involving the striatum. The Cerebral Palsy Spectrum Disorders panel revealed that the boy had two variants in RNA-specific adenosine deaminase (ADAR) namely ADAR c.2128_2131dup classified as pathogenic and ADAR c.577C > G, classified as a VUS. On familial testing, the mother and brother had the pathogenic ADAR c.2128_2131dup variant only. The father had the ADAR c.577C > G VUS only, thus confirming that the variants were in trans in the patient. ADAR is associated with autosomal-dominant dyschromatosis symmetrica hereditaria (DSH; OMIM #127400) and autosomal-recessive Aicardi Goutières syndrome (AGS; OMIM #615010). It thus confirmed that the hyperpigmented rash could be attributed to dyschromatosis symmetrica hereditaria (9). ADAR is also an essential molecule for liver homeostasis as it inhibits the pro-inflammatory effects of interferon. Genetic testing therefore allowed molecular insights and may in future assist with more targeted anti-interferon therapies.
Adrenoleukodystrophy
A 5-year old child presented with a left hemiplegia and neurodevelopmental regression. Investigations including MRI brain and very long chain fatty acids suggested a diagnosis of adrenoleukodystrophy. Genetic testing (Invitae leukodystrophy panel) revealed pathogenic variants in 2 genes: ABCD1 (hemizygous) and UGT1A1 (homozygous). ABCD1 (OMIM #300100) on the X chromosome provides instructions for producing the adrenoleukodystrophy protein and UGT1A1 (OMIM #191740) provides instructions for making enzymes called UDP-glucuronosyltransferases. Both have been implicated in genetic white matter disorders.
The phenotype of this patient strongly suggested a diagnosis of X-linked adrenoleukodystrophy: a metabolic disorder caused by impaired peroxisomal beta oxidation that leads to the accumulation of saturated very long chain fatty acids in numerous tissues throughout the body. The majority of males with X-linked adrenoleukodystrophy develop adrenocortical insufficiency in childhood, followed by progressive myelopathy and peripheral neuropathy in adulthood (10)The UGT1A1 gene identified is associated with autosomal-recessive hyperbilirubinemia (11)This finding was deemed of no clinical significance as the boy never presented with jaundice. Importantly, the mother also tested positive for the ABCD1 variant, and was a heterozygous carrier of the UGT1A1 variant. Genetic counselling was of utmost importance here, to explain X-linked inheritance and the 50% chance of recurrence in future male offspring. Patients with X-linked adrenoleukodystrophy benefit from haematopoietic stem cell transplants if they are diagnosed and transplanted before they become symptomatic. While it was already too late for this patient to be considered for a transplant, he had a younger brother, who was asymptomatic and in whom a genetic diagnosis may have facilitated early treatment. The younger brother subsequently tested negative for the pathogenic variant in ABCD1.
A glycogen storage disorder
A male neonate was born at 36 weeks’ gestation via Caesarean-section for pathological cardiotocography (CTG). The mother had polyhydramnios. The baby was noted to be hypotonic with poor respiratory effort even on continuous positive airway pressure and was intubated and ventilated in the neonatal intensive care unit. The baby had minor dysmorphic features including low-set ears and a high-arched palate. No tongue fasciculations were noted. The baby’s power remained 1/5 in the limbs with marked global hypotonia. Cranial ultrasound revealed ventriculomegaly but no other structural abnormalities. Cardiac echocardiography showed a normal structured heart with poor ventricular contractility. Unfortunately, the baby demised after 15 days. NGS results revealed two pathogenic variants: Glycogen branching enzyme (GBE1) (homozygous) and calpain-3 encoding gene (CAPN3) (heterozygous).
GBE1 (OMIM # 607839) is associated with glycogen storage disorder type 4 (GSD IV). Diminished enzyme activity results in a build-up of structurally abnormal glycogen in affected tissues. GSD IV has a highly variable phenotype. Affected systems can include musculoskeletal, cardiac, neurological, and hepatic. The neuromuscular form of GSD IV varies greatly in onset (perinatal, congenital, juvenile, or adult) and severity. This variant explained the patient’s phenotype. This patient was also a carrier for a pathogenic heterozygous variant in CAPN3 (OMIM #114240) related conditions. This result is important for the family and has future reproductive implications. This case therefore demonstrates the importance of NGS for future family planning.
When it is not cerebral palsy
A 4-year old female was referred to the paediatric neurology outpatients service as she and two of her siblings were labelled as having cerebral palsy. The family was visiting the Western Cape Province and presented to Tygerberg Hospital when the patient and one of her siblings developed pneumonia. The treating clinician was concerned that they might have a cerebral palsy mimic as there was no past birth or medical history or radiological findings to correlate with the clinical findings of severe global developmental disability, seizures and upper motor signs. A cerebral palsy spectrum panel was performed and quinoid dihydropteridine reductase (QDPR) (homozygous) pathogenic variant was found. This is a treatable condition using tetrahydrobiopterin (BH4) (12)This result could have a transformative impact on this family and their relatives and newborns can be tested immediately and started on BH4 as soon as possible after birth, thereby optimising their clinical and neurological outcomes. Patient 94 unfortunately passed away shortly after her return to the Eastern Cape.
A variant with possible adult-onset disease
A 2-year old female was referred to the paediatric neurology outpatients service via the Audiology Department where she was followed-up for profound bilateral sensory-neural hearing loss requiring cochlear implants. She also presented with acquired microcephaly, global developmental delay and marked dystonia. MRI brain was normal and the Cerebral Palsy Spectrum Disorders panel was requested. One pathogenic variant was identified in ATM Serine/Threonine Kinase (ATM; OMIM #607585)) (heterozygous). The autosomal dominant form of ATM (OMIM #114480) is associated with predisposing to breast cancer and autosomal-recessive inheritance is associated with ataxia-telangiectasia (AT; OMIM #208900). A second disease-causing variant was not identified and clinically, she did not have features of AT. Therefore, the clinical phenotype cannot be explained at this time. However, the patient may be at increased risk for adult-onset breast and ovarian cancers. This is a secondary finding, because of broad testing. We would normally not intentionally test minors for adult-onset diseases, but in this case biallelic pathogenic variants in ATM also result in a childhood-onset disorder (AT; OMIM #208900). The family has received genetic counselling to explain the result.
New-onset refractory status epilepticus (NORSE)
A previously well, developmentally age-appropriate teenager presented with NORSE. MRI brain, metabolic, infectious and autoimmune investigations all proved normal. Electroencephalogram (EEG) video telemetry confirmed left frontal focal-onset seizures with secondary generalisation. Initially, the seizures proved refractory to sodium valproate, levetiracetam, lamotrigine and phenytoin. Fortunately, immunomodulation with corticosteroids and cyclophosphamide brought about marked reduction in the seizure frequency. The Epilepsy panel revealed a heterozygous pathogenic variant in Nitrogen Permease Regulator-like3 (NPRL3; OMIM # 600928). The latter is an important component of the GATOR1 complex and its mutations can promote the activity of the mTOR signalling pathway, thereby causing epilepsy. Sleep related hyper motor epilepsy and frontal lobe epilepsy are the most common clinical presentations, always with a high rate of drug resistance. Identifying the pathogenic variant allowed for a better understanding of the clinical phenotype of NPRL3 related epilepsies. Moreover, the underlying molecular mechanism suggests that mTOR inhibitors such as rapamycin and sodium channel blockers (carbamazepine) may offer potential targeted treatment for drug-resistant cases.
Baby with an excessive startle response
A 3-month old baby with presumed epilepsy was found to have an exaggerated startle response. The Epilepsy panel revealed one pathogenic variant as well as one VUS in Solute Carrier Family 6 Member 5(SLC6A5: OMIM # 604159). Family testing was performed to assist in phasing the variants, and the mother was found to be a carrier of the pathogenic variant in SLC6A5. The father was not available for testing. However, as the results fit the clinical picture of the patient, we are treating her as having Hyperekplexia (OMIM # 614618), and she is currently doing well on a benzodiazepine and behavioural adjustments e.g., avoiding load noise. VUS resolution may be possible with additional information in future.
Utility to the parents
Parents of patients with positive results were given the opportunity to partake in this study by voluntarily answering a six-question questionnaire (Supplementary Table 2). The majority of parents felt that knowing the result brought closure and that if prenatal testing should be available, they would make use of it in future pregnancies. In almost half of the cases, treatment was adjusted to some extent once the pathogenic variant was known. Treatment changes included adjusting anti-seizure medications eg stopping sodium channel blockers like Lamotrigine in patients with SCN1A or adding sodium channel blockers like carbamazepine in patients with KCNQ2 pathogenic variants (4). The questionnaire was given to parents only and not the treating physicians therefore full details of treatment changes were not covered by this study.
Strengths and limitations
The study was unique to a specific population of patients from a single centre. VUS resolution was not performed on all patients, as either one (especially the father) or both biological parents were not available to provide samples. As this was a gene panel testing approach, the analysis was limited to the genes in the panel and the diagnostic assays used. For the genes included in the panel, certain types of variants may be missed (intronic, structural, some deletions/duplications). In addition, cost limitations prohibited more detailed genetic testing (for example, exome or genome for patients with a negative result) or further functional analysis (for example, RNA sequencing or in-vitro ion channel analysis for patients with VUSs in ion-channel genes).
Despite these limitations, our study provides insights into the diagnostic yield of NGS in a resource-constrained, previously non-investigated, LMIC setting with high levels of genetic diversity. Testing allowed clinicians to optimise genetic counselling, patient care and prognostication when pathogenic variants were identified. Caregivers were able to receive closure and make plans for their families.