Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report

The proband (Fig. 1, V: 2) is 1-month-old Saudi boy born normally at term to first 24-year- old parents. Four maternal aunts (Fig. 1, IV: 1, 4, 7, 8.) had died at the age of four, five, three and six weeks respectively, in a remote medical facility with no available records. However, all are said to have presented with microcephaly and abnormal movements similar to the index (see below). The mother was G2P1 (IUFD at 28 weeks gestation) +0. No history of exposures. Antenatal ultrasound (US) scan showed microcephaly. Apgar score was 9 and 10 at one and five minutes, respectively. Birth weight 2675gm (10^th percentile), head circumference 29 cm (−3SD). Examination showed microcephaly, sloping forehead, short neck, and micrognathia (Fig. 2a). Shortly after birth, he developed abnormal movements in the form of bursts of tonic/clonic movements provoked by non-habituating glabellar and root of the nose tapping, sound and light (see Additional files 1, 2 and 3). There was hyperreflexia, hypertonia and arthrogryposis of the lower limbs. He developed frequent apneas necessitating mechanical ventilation. Treatment with clonazepam was initiated and later phenobarbitone was added to control the abnormal movements. Laboratory investigations (Table 2) showed normal metabolic screen including plasma and CSF asparagine, glutamine, aspartate and glutamate.CSF neurotransmitters, 5HIAA, 3-OMD, and HVA were normal. Brain MRI (Fig. 3) revealed cerebral atrophy, simplified gyral pattern and hypoplastic cerebellum and pons. EEG showed multifocal discharges, fast spiking in the left hemisphere favoring cortical dysplasia, and frontal spikes. The findings favor epileptic encephalopathy in a transitional phase with predominant seizure burdens. Whole exome sequence (WES) revealed nonsense mutation in the ASNS gene, (NM_001178076.1: c.970C > T p. (Arg324*). He died at the age of six weeks in status epilepticus.

Is a 4- year-old Saudi boy delivered normally at term to a 23-year-old primigravida lady and her 25-year-old first cousin husband (Fig. 1, IV:1). Antenatal US scan revealed microcephaly but pregnancy was uneventful otherwise. Apgar score was 9 and 10 at one and five minutes, respectively. Birth weight 2790 gm (25^th percentile), length 51 cm (50^th percentile) and head circumference 30 cm (−2.6SD). He was admitted to the Neonatal Intensive Care Unit (NICU) because of microcephaly and abnormal movements. Clinical examination showed microcephaly, staring anxious look, sloping forehead, receding chin and relatively large ears. Neurological examination revealed tonic/clonic rapid movements of both upper and lower limbs, with positive head retraction reflex (HRR). The attacks were provoked by glabellar and tip of the nose tapping, were non-habituating and precipitated by sounds. Additional files 4, 5, and 6 show this in more detail. Hypertonia with exaggerated reflexes and arthrogryposis of both upper and lower limbs were also noted. The rest of the systemic examination revealed no abnormality. He was initially managed by phenobarbitone. Clonazepam and levetiracetam were later added due to the intractable movements. At the age of four years he was found to have profound global developmental delay and spastic quadriplegia with severe contractures of both upper and lower limbs (Fig. 2b). He also had cortical blindness and hyperekplexic activities could still be elicited by glabellar and root of the nose tapping, light and sounds [see Additional files 1, 2, 3, 4, 5 and 6]. His growth parameters were severely retarded: his weight 5.3 Kg (−6.8SD) and head circumference was 35 cm (−10.1SD).

Laboratory tests including hematologic indices, renal function, liver function, and electrolytes were all normal. Metabolic screen, plasma amino acids, lactate, and ammonia were unremarkable. Chromosome study revealed normal male karyotype. MRI brain (Fig. 4) showed microcephaly, thin and smooth cortex with simplified gyral pattern [4], delayed myelination, dilatation of the ventricles, global brain atrophy and hypoplastic cerebellum and pons. EEG showed very low amplitude bilaterally without epileptiform discharges. Only sporadic sharp transient spikes, most likely myogenic in origin, were noted. Genetic testing, utilizing whole exome sequencing (WES), showed a novel homozygous ASNS gene mutation: NM_001178076.1, missense c.944A > G, p. (Tyr315Cys). Both parents are heterozygous carriers. This change was absent in 615 in-house Saudi exomes, and is predicted pathogenic by PolyPhen (0.992), SIFT (1.0) and CADD (28.7) [5]. The family was offered genetic counselling.

We report two novel mutations in the ASNS gene in two Saudi patients from first cousin marriages who presented with congenital microcephaly, hyperekplexia, cerebral atrophy, simplified gyral pattern, and hypoplastic cerebellum and pons (Figs. 3 and 4). The phenotype is consistent with the recently described ASNSD (OMIM# 615574). ASNS encodes an asparagine synthetase enzyme involved in the synthesis of asparagine from glutamine and aspartate [6]. The neurological impairment resulting from ASNS mutation can be explained by asparagine depletion in the brain or by accumulation of aspartate /glutamate leading to enhanced excitation and neuronal damage [1]. To date five pathogenic mutations in the ASNS gene have been identified (Table 1).

All cases of asparagine synthetase deficiency were diagnosed by molecular genetics as there is no reliable biochemical test for diagnosing the disorder. Alfadhel et al. [2] reported low levels of asparagine in the CSF of two siblings with ASNSD confirmed by molecular genetics, while Ruzzo et al. [1] reported low asparagine levels in the CSF of only two of the five patients with the disorder. Our two patients had normal plasma and CSF asparagine, glutamine, aspartate, and glutamate and CSF neurotransmitters (Table 2). Therefore, this condition cannot be ruled out by normal plasma and CSF asparagine, aspartate and glutamate levels (Table 3) in a patient presenting with congenital microcephaly, and unexplained encephalopathy in the form of intractable seizures or hyperekplexia [1].

ASNSD is a very rare disorder with a prevalence of <1/1000000 worldwide (ORPHA 391376); and hitherto, only 12 cases have been reported. We suspect this condition is underdiagnosed due to lack of recognition and the risk of confusing the hyperekplexia phenotype for nonspecific neonatal epilepsy, and also due to lack of a specific laboratory test and the difficulty of performing molecular genetic testing in suspected cases except in research centers or referral hospitals. Given that there is no reliable biochemical test, sequencing of ASNS is the mainstay of diagnosis. While our two patients were diagnosed using WES, this gene can easily be targeted by Sanger sequencing or added to a gene panel approach as described before [7]. The clinical and radiographic presentation of our patients, namely congenital microcephaly, hyperekplexia, brain malformation, and (in long term survivors) severe psychomotor retardation and cortical blindness is identical to that observed in the patients reported by Ruzzo et al. [1] with ASNS gene mutation. Interestingly, we previously [8] reported six patients with a similar presentation of hyperekplexia, microcephaly and brain malformations who later proved to have cathepsin deficiency (CTSD) known to be associated with congenital ceroid lipofuscinosis neuronal 10 (CLN 10) [9, 10]. Our observation raises the intriguing possibility of a link between asparagine synthetase deficiency and cathepsin deficiency, although this will require future research. Both disorders should be considered in microcephalic neonates who present with seizures or hyperekplexia at or before birth, and molecular genetic testing needs to be performed for pertinent and timely genetic counseling. This will pave the way for adopting effective preventive and therapeutic approaches like preimplantation genetic diagnosis (PGD), or early termination of affected pregnancies, which helped many families in this region with high prevalence of autosomal recessive disorders [11]. Therapeutic approach by supplementation with asparagine in ASNSD seems attractive. However, the prenatal onset of the microcephaly and early postnatal presentation make such treatment unlikely to be curative unless started prenatally [1].