Syndromic and non-syndromic etiologies causing neonatal hypocalcemic

IntroductionNewborn infants are found to be at a higher risk of experiencing brain disorders, encephalopathies, and seizures. The neonatal hypocalcemic seizures (HS) are a group of disorders with diverse and complex etiology. The diagnosis of neonatal HS is currently based only on patients’ clinical presentation and laboratory examination (1–3). Early diagnosis and rapid treatment are essential in patients with neonatal seizures (4). Identification of etiology and immediate initialization of effective therapy could help clinicians to manage the severity of additional comorbidities.Calcium is the one of most important ion in neonatal nerve conduction. Hypocalcemia, either symptomatic or asymptomatic, is a common metabolic disorder in newborns. Symptomatic hypocalcemia manifests in the form of common clinical symptoms, such as jitteriness, muscle jerking, and seizures. After birth, the serum level of calcium in newborns is affected by several factors, such as parathyroid hormone secretion, dietary intake, renal reabsorption function, skeletal calcium storage, and vitamin D level. In a healthy 2-day term infant, calcium levels decrease to a physiological nadir of 7.5–8.5 mg/dL (5). Hypocalcemia with PTH elevation can be caused by increased phosphate load, vitamin D deficiency, vitamin D metabolism defect, renal dysfunction, hypomagnesemia, genetic mutations resulting in end-organ resistance to PTH, or critical illness (6–9). Hypocalcemic seizures are can be classified as early onset (manifestation before 5 days of life) or late onset (manifestation after 5 days of life). Early-onset seizures can commonly occur in both preterm and term newborns. Late-onset seizures have a more complex and varied etiology. Syndromic neonatal HS include DiGeorge syndrome and other genetic disorders (often not identified). Non-syndromic neonatal HS include parathyroid gland disorders and acquired causes such as vitamin D deficiency. However, the etiology remains unknown in some cases. A previous retrospective review summarized clinical and laboratory characteristics as well as outcomes of neonates presenting with transient late-onset hypocalcemia and reported that the majority of patients had low vitamin D levels (6). All patients responded to magnesium and calcium supplementation and the administration of calcitriol and low phosphate formula (6). The outcome of vitamin D deficiency is relatively benign; however, in syndromic HS, it is unclear and noteworthy.The use of whole exome sequencing (WES) to diagnose neonatal HS has been rarely reported. The application of WES to other unknown causes of seizures can help further elucidate the etiologies, initialize early treatment, predict outcomes, prevent a potential diagnostic odyssey, and greatly improve our understanding of the pathophysiology at the molecular level (10, 11). However, WES is rarely utilized in the diagnosis of neonatal HS and the genotype-phenotype correlation remains complicated and not fully understood.In this study, we discuss a case-series of hypocalcemic seizures with various etiologies in newborns. Using the advanced technique of WES, rare etiologies found in neonatal hypocalcemic seizures can be better understood and the outcomes can be predicted. To evaluate the etiologies need more comprehensive care for those neonatal HS.Materials and methodsPatientsFrom 2017 to 2020, 16 patients with neonatal HS, who met the hypocalcemia criteria (serum calcium level < 8 mg/dL [2.0 mmol/L] and ionized calcium level < 4.0 mg/dL [1.0 mmol/L]) were included in the study (6, 12, 13). Intact parathyroid hormone (iPTH) and vitamin D levels were measured for each patient if possible. All patients with HS were administered intravenous calcium, with or without anti-seizure drugs, and then switched to oral medication. Refractory hypocalcemia was defined as a duration of hypocalcemia of ≥ 7 days despite calcium supplementation. Additionally, genetic examination was performed in patients with comorbidities, including congenital heart diseases and other organ or endocrine disorders. Genetic testing comprised chromosome and whole exome sequencing (WES); WES was performed if the results of the chromosome study were negative. We arranged EEG monitoring for all patients with seizures. To clarify whether HS was caused by hypocalcemia or associated complex comorbidities, we defined acute seizures due to hypocalcemia as the cessation of seizures after normalization of serum calcium levels. We reviewed patient charts retrospectively and analyzed clinical presentations and etiologies that led to neonatal HS. Levels of 25-hydroxyvitamin D ≤ 25 ng/mL (62.4 nmol/L) were defined as abnormally lowered (6). Patients with neonatal hypoxic-ischemic encephalopathy (HIE) were as follows: 1. placental abruption, cord prolapse, or fetal distress; 2. Apgar score ≤ 5 in the 10th minute after birth; 3. need for ventilation beyond the tenth minute of life; and 4. acidosis (arterial pH of G (p.A3360G)/c.10323G>C (p.K3441N) mutations inherited from each parent (Figure 1). According to the ACMG guidelines for interpretation of genetic variants (21), the mutation of c.10079C>G (p.A3360G) in the ALMS1 and the variant c.10323G>C (p.K3441N) were predicted to be “likely pathogenic” (PS2, PM2, PM3, PP3, and PP4). According to her phenotype and genetic study, a Alström Syndrome was diagnosed (22). She had 2 major criteria (compound heterozygous ALMS1mutations, nystagmus and dilated cardiomyopathy). One patient with Kleefstra syndrome carried a paired 9 ring chromosome at 3 months of age. His hypocalcemic seizures began 4 days after birth. He had facial dysmorphism, and hearing impairments. The patient’s initial calcium level was 6.0 mg/dL (calcium ion, 0.63 mg/dL). He also had ambiguous genitalia (Figure 2A), scrotal bifida with hypospadias, penoscrotal transposition, congenital heart disease with double-outlet right ventricle, ventricular septal defect, atrial septal defect, and pulmonary stenosis. Initially, phenobarbital and phenytoin were intravenously administered for the HS. His calcium levels returned to normal after 4 days of calcium supplementation; next, oral calcitriol was administered. His MRI revealed hypoplasia of the splenium part of the corpus callosum (Figure 2B). At the age of 2 years and 3 months, his seizures reappeared and were controlled by levetiracetam and pyridoxine. For the five syndromic neonatal HS, the mean seizure onset time in the syndromic neonatal HS group was 5.8 ± 1.3 days, was not different from those seizures in vitamin D deficiency group (5.9 ± 1.9) days.Figure 1 Patient 12 had hypocalcemic seizures which started to appear 8 days after birth in one newborn. The patient’s calcium level was 6.8 mg/dL (ion calcium, 0.71 mg/dL). The patient was diagnosed with Alström Syndrome with WES and was found to carry a compound heterozygous ALMS1 (NM_015120.4) (A) c.10323G>C (p.K3441N) / (B) c.10079C>G (p.A3360G) mutation inherited from one each parent respectively. According to her phenotype, a Alström syndrome was diagnosed.Figure 2 Patient 4 with hypocalcemic seizures that started 6 days after birth, had facial dysmorphism and hearing impairments. The patient’s calcium level was 6.0 mg/dL (ion calcium, 0.63 mg/dL). (A) He had ambiguous genitalia (arrow), scrotal bifida with hypospadias, penoscrotal transposition, congenital heart disease with double outlet right ventricle, ventricular septal defect, atrial septal defect, and pulmonary stenosis. Chromosome study confirmed the ring chromosome 9 and Kleefstra syndrome. (B) His MRI exhibited hypoplasia of splenium part of corpus callosum (arrow).Vitamin D deficiencyAll eight patients with vitamin D deficiency had 25-hydroxyvitamin D levels < 25 ng/mL, with an average of 17.5 ± 2.3 (range: 15 to 22) ng/mL. The mean seizure onset time was 5.9 ± 1.9 (range: 2 to 8) days. Four received only oral calcitriol treatment, and three with oral calcitriol and one antiseizure drug (intravenous phenobarbital). One patient (patient 11) need two antiseizure drugs (intravenous phenobarbital and phenytoin) due to refractory hypocalcemia and seizures. She had dilated cardiomyopathy and persistent pulmonary hypertension of newborn (PPHN) with respiratory failure. Her WES exhibited two ALMS1c.12118-65C>T and c.12118-65C>T (in cis).
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