Whole Genome Sequencing: Early Diagnostic Tool in Newborns with Refractory Seizures.

Abstract

A term male infant born at an outlying hospital to nonconsanguineous parents was transferred immediately after birth due to facial dysmorphisms and limb abnormalities (Figs 1A, 1B, 1C).Born to a 24-year-old gravida 1, para 0 white woman.Pregnancy complicated by mild oligohydramnios at 36 weeks’ gestation that resolved with hydration; prenatal ultrasonography findings unremarkable with consistent fetal movement throughout pregnancyCesarean section at 38 weeks’ gestation for breech presentationPrenatal maternal laboratory findings: Group B Streptococcus negative, hepatitis B surface antigen negative, rubella immune, HIV negative, rapid plasma reagin nonreactiveApgar scores: 4 and 9 at 1 and 5 minutes, respectivelyThe infant was cyanotic and hypotonic at birth with no respiratory effort. He required positive pressure ventilation for approximately 2 min and was then transitioned to nasal cannula oxygen. His tone improved by 5 minutes of age. He was transported to a level 3 NICU for evaluation of his dysmorphic features.Heart rate: 160 beats/minRespiratory rate: 40 breaths/minBlood pressure: 57/41 mm HgOxygen saturation: 97% (on high-flow nasal canula 5 L/min, fraction of inspired oxygen 0.3)Temperature: 99.1°F (37.3°C)Birthweight: 2,640 g (10th percentile), length: 48.5 cm (37th percentile), head circumference: 35.8 cm (85th percentile)Head: Cranial molding; microcephaly; retrognathia; micrognathia; long, downward-slanting palpebral fissures; low-set ears; prominent nose; broad nasal bridgeCardiovascular: Regular rate and rhythm; normal S1, S2; no murmursLungs: Clear and equal bilaterally; no respiratory distressChest wall: Pectus carinatum, wide-spaced nipplesAbdomen: Soft, nontender; no masses; 3-vessel cordGenitourinary: Normal male genitalia; testes descended; patent anusSkeletal: Ulnar deviation of bilateral wrists; arachnodactyly; bilateral talipes equinovarus; arthrogryposisSkin: Bruising of bilateral lower extremities; no sacral tufts or dimplesNeurologic: Awake, alert with spontaneous movement of all extremities with increased tone and contractures; intermittent myoclonic movements with tactile stimulationOn the second day after birth, he was observed to have myoclonic events that were associated with vital sign instability and staring that lasted 10 to 15 seconds. Video electroencephalography confirmed epileptiform activity. The seizures were multifocal, brief, and clustering, with multiple episodes in a 12-hour period. He was supported with orogastric feedings and high-flow nasal cannula while an extensive evaluation was initiated.Investigative studies within normal limits: Infectious evaluation: C-reactive protein, blood and urine cultures, cerebrospinal fluid (CSF) meningoencephalitis panelMichigan newborn screenUrine organic acidsChromosomal microarrayCongenital disorders of glycosylationAbnormal laboratory findings with unclear significance: CSF triene/tetraene ratio 0.099 (normal 0.017–0.083)CSF linolenic acid 288 nmol/mL (normal 30–170 nmol/mL)Serum taurine 7 nmol/mL (normal 8–48 nmol/mL)Serum α-amino-n-butyric acid 5 nmol/mL (normal 7–28 nmol/mL)Serum ammonia 40.6 μg/mL (29 μmol/L; normal 75.5–126 μg/mL [54–90 μmol/L])Serum 3-methylhistidine 3 nmol/mL (normal <1 nmol/mL)Radiography (Fig 2) showed prominent cardiothymic silhouette and mild thoracolumbar scoliosisBrain magnetic resonance imaging (MRI) without contrast showed diffuse, mild undersulcation and under-opercularization; mildly prominent extra-axial CSF; thinning of posterior corpus callosum (Figs 3 and 4)The infant continued to have subclinical seizures despite multiple antiepileptic drugs (AEDs), including phenobarbital, levetiracetam, phenytoin, and topiramate. Due to the risk of respiratory depression with additional medications, the care team decided to not increase the AED dosages any further and monitor clinically for seizure activity. Given that the extensive laboratory and radiographic studies were unrevealing, the team decided to perform additional genetic testing. A family history revealed that the parents were nonconsanguineous. A maternal aunt had died at 8 months of age from a presumed seizure disorder present from birth and a maternal uncle had died at several weeks of age of sudden unexpected infant death. Parental consent was obtained for rapid whole genome sequencing (rWGS), which was performed during the infant’s second week of age, and results were obtained within 5 days.Congenital brain malformation (anencephaly, Chiari malformation, holoprosencephaly, Dandy Walker spectrum, agenesis of the corpus callosum)Neuromuscular disorder (spinal muscular atrophy with progressive myoclonic epilepsy)Congenital disorders of glycosylationEssential fatty acid disorderUrea cycle disorderAmino acid disorderChromosomal abnormalitySingle gene or multifactorial genetic disorderSingle gene or multifactorial genetic disorderThe results of the infant’s rWGS showed maternal heterozygous mutation of the c.294dup (p.Leu99ThrfsTer92) variant and paternal heterozygous mutation of the c.638dup (p.Val214GlyfsTer18) variant. These results were consistent with a compound heterozygous BRAT1 mutation that is associated with rigidity and multifocal seizure syndrome, lethal neonatal (RMFSL). A multidisciplinary care conference was held that included the neonatologist, pediatric neurologist, social worker, nurse, and case manager. The team discussed with the parents that this diagnosis is rare, and as a result, it is difficult to provide a definitive prognosis for the infant. Various management options were discussed with the family which included, but were not limited to, continuing full support with escalating care if needed and comfort care with home hospice. The parents elected to continue current management in addition to trying a ketogenic diet to attempt to lessen the infant’s seizure burden.At 2 weeks of age, the infant was transferred to a facility with expertise in a ketogenic diet. The infant continued to have myoclonic activity with tactile stimulation, and contractures of his extremities were still present but slightly improved. He continued to have multiple clinically significant seizures and ultimately developed respiratory failure requiring intubation. After 5 days on the ketogenic diet, the infant had not achieved ketosis. The parents elected to proceed with hospice care. He died of cardiorespiratory failure at 1 month of age.We describe a white term male infant born to nonconsanguineous parents with facial dysmorphisms, limb abnormalities, and AED-resistant seizures requiring respiratory and enteral support and who was found to have a compound heterozygous BRAT1 mutation.The infant’s brain MRI on the first day after birth showed diffuse cerebrum underdevelopment. There were no other structural abnormalities indicating a disorder of dorsal induction, ventral induction, or myelination. Serum analysis of essential fatty acids and amino acids was part of the diagnostic evaluation as these components have been implicated in poor neurologic development. As seen in the infant’s laboratory studies, he had elevated levels of essential fatty acids with low levels of ammonia and specific amino acids. The significance of these results was not clear and did not result in a specific diagnosis.BRAT1 mutations have been associated with RMFSL and neurodevelopmental disorder with cerebellar atrophy, with or without seizures. We conducted a literature review and found that since 2012, BRAT1 mutations have been reported in 26 patients, all with a spectrum of neurodevelopmental and phenotypic anomalies but 1 common variable of refractory seizures (Table). Many of these cases have also reported abnormalities on the brain MRI consistent with global underdevelopment of the cerebrum and cerebellum. These findings include mild hypoplasia of the frontal lobes, delayed or decreased myelination, thin corpus callosum, cerebellar hypoplasia, and prominent pericerebral extra-axial space. The cases in which the patient survived long enough to have serial imaging reported a progressive atrophy. The magnitude of the abnormality noted on brain MRIs was not found to be predictive of prognosis. In prior case reports, 12 infants died at or before age 6 six months, 5 before age 2 years, and 1 at age 5 years. (1)(2)(3)(4)(5)(6)(7)(8) Eight patients were reported to have milder symptoms and lived until their childhood years. (3)(9)(10)(11)(12) One study suggested the possibility that homozygous mutations may have a more severe presentation than compound heterozygous. (8) However, our patient presented with severe disease in the neonatal period despite having a compound heterozygous mutation. The extent to which the inheritance pattern affects the severity of the RMFSL may be more complex than initially thought, as patients with both homozygous and compound heterozygous mutations can present with severe symptoms. This raises the question as to whether the true diagnosis is a single gene disorder or multifactorial disorders.In most of the previous case reports, the timing of genetic testing relative to the onset of symptoms was not clear. Many of these infants had progressed to requiring interventions such as gastrostomy tube and/or tracheostomy and underwent multiple studies that yielded results of unknown significance. (4)(5)(6)(8)(12) In infants with AED-refractory seizures in the NICU, the use of rWGS has yet to become an established norm during the evaluation. WGS has become more cost-effective with a shorter test-result time and high analytic specificity and sensitivity of more than 99.5%, making it one of the most comprehensive tests currently available.A spectrum of clinical severity has been found to be associated with RMFSL. Our patient had findings most consistent with a severe form of RMFSL that results in early death at 2 months of age. Several affected individuals, including our patient, have common features of intractable seizures, facial dysmorphisms, and brain underdevelopment. Other congenital brain malformations were less likely in this case as no other structural abnormalities were noted on the brain MRI. Certain neuromuscular disorders were ruled out with a normal newborn screen and no specific laboratory result confirmed a disorder of glycosylation, fatty acid metabolism, or urea cycle or amino acid metabolism. The chromosomal array did not detect any gene duplication or deletion.Although some patients with RMFSL present with later onset of disease, attenuated symptoms, and prolonged survival, others present with early onset, severe symptoms, and early death. This report aims to raise awareness of RMFSL and its variable presentations and to recognize the usefulness of rWGS in cases of intractable seizures from an unknown cause. Early identification of a genetic diagnosis may facilitate discussions between care teams and families and help guide often difficult management decisions. More frequent utilization of rWGS may reveal a higher prevalence of RMFSL than previously reported, which in turn, may create advocacy for research into possible treatments and facilitate the development of a support network for families affected by this rare disease.