A novel RAB39B mutation and concurrent de novo NF1 mutation in a boy with neurofibromatosis type 1, intellectual disability, and autism: a case report

Background Mutations in RAB39B at Xq28 causes a rare form of X-linked intellectual disability (ID) and Parkinson’s disease. Neurofibromatosis type 1 (NF1) is caused by heterozygous mutations in NF1 occurring de novo in about 50% of cases, usually due to paternal gonadal mutations. This case report describes clinical and genetic findings in a boy with the occurrence of two distinct causative mutations in NF1 and RAB39B explaining the observed phenotype. Case presentation Here we report a 7-year-old boy with multiple café-au-lait macules (CALMs) and freckling, severe macrocephaly, peculiar facial gestalt, severe ID with absent speech, epilepsy, autistic traits, self-harming, and aggressiveness. Proband is an only child born to a father aged 47. Parents did not present signs of NF1, while a maternal uncle showed severe ID, epilepsy, and tremors.By RNA analysis of NF1, we identified a de novo splicing variant (NM_000267.3:c.6579+2T>C) in proband, which explained NF1 clinical features but not the severe ID, behavioral problems, and aggressiveness. Family history suggested an X-linked condition and massively parallel sequencing of X-exome identified a novel RAB39B mutation (NM_171998.2:c.436_447del) in proband, his mother, and affected maternal uncle, subsequently validated by Sanger sequencing in these and other family members. Conclusions The case presented here highlights how concurrent genetic defects should be considered in NF1 patients when NF1 mutations cannot reasonably explain all the observed clinical features.

in humans [4]. Rab39b knockout mice showed reduced cortical neurogenesis, macrocephaly, and autistic behaviors, similarly to patients with mutations in RAB39B [5]. The Ras-related protein Rab-39B is a small neuronspecific GTPase that contributes to synapse formation and maintenance by regulating organization and dynamics of intracellular membranes and vesicular membrane traffic [6]. With its effector, the protein interacting with C-kinase 1, Rab-39B regulates availability of AMPA receptor, important for synaptic plasticity [7]. Furthermore, the complex formed by C9orf72, WDR41, and SMCR8 was found to act as a GDP/GTP exchange factor for Rab-8A and Rab-39B, suggesting that Rab-39B might be involved in autophagy regulation [8]. However, precisely how RAB39B loss-of-function or increased dosage [9] can perturb neuronal development leading to cognitive impairment needs further clarification.
Neurofibromatosis type 1 (NF1; MIM 162200) is caused by dominantly inherited mutations in NF1 (MIM 613113), a complex gene encoding for neurofibromin, a GTPase-activating protein that negatively regulates Ras/ MAPK signaling pathway [10]. In about 50% of cases, mutations occur de novo, with NF1 exhibiting one of the highest single locus mutation rates known in humans (1 × 10 − 4 per gamete per generation) [11]. De novo mutations are mainly linked to paternal age and to the increasing number of cell divisions in the male germ line [12].
Here, we describe a boy with clinical diagnosis of NF1 complicated by severe macrocephaly, dysmorphic features, severe ID with absent speech, epilepsy, and ASD. Based on his family history, these neurological features could not be only assigned to NF1 and an extensive genetic characterization was carried out. To our knowledge, this is the first case in which two distinct causative mutations in NF1 and RAB39B explained the observed phenotype.

Case presentation
We report the clinical and genetic characterization of a boy (III.1; Fig. 1), the only child of apparently healthy parents, who was referred to our NF1 Referral Center at age 7 due to the presence of CALMs and freckling.
Proband was born at week 39 of an uneventful pregnancy by C-section because of neonatal macrosomia. Prenatal karyotyping was normal (46, XY). At birth, weight was 3.930 kg (75th centile), length 52 cm (75th centile), and head circumference 35 cm (66th centile). The child presented respiratory distress not requiring intubation. He started walking at 2 years and spoke his first words at 12 months, with reported regression and loss of verbal competence soon after.
Severe ID and ASD was diagnosed at age 3, and pharmacologically treated with risperidone. At this age, he had his first episode of generalized seizures during fever, followed by others while awake and in apyrexia. A sleep electroencephalogram showed rare physiological figures, as sleep spindles and k complex, and epileptiform abnormalities in the right temporal region. Seizures were adequately controlled by levetiracetam. Brain magnetic resonance imaging performed at age 4 showed periventricular white matter hyperintensities, likely due to hypoxia, and revealed T2W hyperintensities in basal ganglia bilaterally, while FLAIR images showed hippocampal and temporomesial hyperintensities bilaterally. Ophthalmological evaluation, electrocardiogram, auditory/visual brainstem potentials were normal. He showed hyperactivity and motor instability, as well as limited social interactions and aggressiveness. At age 6, sleep disorder (insomnia) was observed and chlorpromazine, clonazepam, and melatonin were added to the pharmacological therapy.
At our first consultation, family history revealed that a maternal uncle (II.4; Fig. 1) was also affected by severe ID, absence of speech, and tremors, with global motor impairment. He had been institutionalized for many years and while this study was ongoing died suddenly at the age of 53 from complications of a pulmonary infection, preventing any further clinical examination. Proband was still able to walk, and epilepsy and sleep disorders were well managed pharmacologically. Global motor impairment, severe behavioral disorders, including self-injurious behavior and psychomotor agitation episodes, and food selectivity were noted. At medical examination, he presented with CALMs and freckling, suggestive of NF1 according to NIH diagnostic criteria [10]. He presented restless with absent speech (less than 5 words), macrocephaly (head circumference 56 cm, > +2SD), peculiar facial gestalt, gingival hypertrophy, and generalized hypotonia. Previously performed array-CGH and genetic testing for fragile X syndrome resulted negative.
At last observation, at age 14, his pharmacological therapy included the oral administration of risperidone (0.75 mg BID), levetiracetam (250 mg BID), lorazepam (6 drops BID), clonazepam (12 drops BID) and sodium valproate (250 mg TID), with a moderate control of both seizures and behavioral problems.
Based on clinical signs of NF1 and family history suggestive of an XLID, despite suspicion of neonatal hypoxia, a comprehensive genetic investigation was performed. As the legal representative of a minor, proband's mother gave written informed consent to the study.

Discussion and conclusions
Cognitive impairment commonly affects children with NF1 and includes an IQ in the low average range, learning difficulties and social dysfunction [16,17]. ID was reported for NF1 patients carrying a 17q11.2 microdeletion, [18] while ASD features were observed in up to 30% of children with NF1 and epilepsy in about 4% of NF1 patients [19,20].
We identified a de novo heterozygous mutation in NF1 that affects splicing and was not related to a specific NF1 phenotype or to an impaired cognitive profile [13]. Severe ID with absent speech, associated with epilepsy, ASD, self-injurious behavior, and aggressiveness, could not be explained by the NF1 mutation alone. Although brain imaging showed signs of hypoxia, family history A novel RAB39B mutation (NM_171998.3:c.436_ 447del) was subsequently identified in proband, his carrier mother, and affected maternal uncle, as well as in one maternal aunt (Fig. 1). This in-frame deletion removed four amino acids (p.Gly146_Tyr149del) in the C-terminus region of Rab-39B. To date, only 12 causative variants were reported in RAB39B, mainly associated with variable ID, macrocephaly, and ASD, and in some cases complicated by early-onset Parkinson's disease (Fig. 2) [1-3, 15, 21-23].
The mutation identified in RAB39B explained neurocognitive and neurological features observed in affected male family members, while the unbalanced X inactivation in carrier females was in agreement with their apparently healthy status. The independent occurrence of a de novo NF1 mutation accounted for NF1 signs and could contribute to the severity of neurocognitive features.
In NF1, severe cognitive impairment not linked to a 17q11.2 microdeletion syndrome or not explainable by other causes should not exclude the albeit rare possibility of another concomitant genetic condition. The high mutation rate of NF1 and the relatively not low worldwide prevalence of neurofibromatosis type 1, can explain the occurrence with NF1 of distinct genetic condition in the same patient [27][28][29][30]. This case report clearly underscores the need for more extensive genetic investigation, today possible thanks to massive clinical application of next-generation sequencing-based genetic testing, when the identification of a causative mutation does not fully explain the observed phenotype.