Biallelic and monoallelic variants in PLXNA1 are implicated in a novel neurodevelopmental disorder with variable cerebral and eye anomalies

Purpose To investigate the effect of PLXNA1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and to functionally characterize the zebrafish homologs plxna1a and plxna1b during development. Methods We assembled ten patients from seven families with biallelic or de novo PLXNA1 variants. We describe genotype–phenotype correlations, investigated the variants by structural modeling, and used Morpholino knockdown experiments in zebrafish to characterize the embryonic role of plxna1a and plxna1b. Results Shared phenotypic features among patients include global developmental delay (9/10), brain anomalies (6/10), and eye anomalies (7/10). Notably, seizures were predominantly reported in patients with monoallelic variants. Structural modeling of missense variants in PLXNA1 suggests distortion in the native protein. Our zebrafish studies enforce an embryonic role of plxna1a and plxna1b in the development of the central nervous system and the eye. Conclusion We propose that different biallelic and monoallelic variants in PLXNA1 result in a novel neurodevelopmental syndrome mainly comprising developmental delay, brain, and eye anomalies. We hypothesize that biallelic variants in the extracellular Plexin-A1 domains lead to impaired dimerization or lack of receptor molecules, whereas monoallelic variants in the intracellular Plexin-A1 domains might impair downstream signaling through a dominant-negative effect.

Family A: A: II-2 is a 6 year old female of Caucasian descent who exhibited mild developmental delay and had experienced a first febrile seizure at age 15 months and later suffered from nonfebrile seizures and one episode of status epilepticus. Overall, she experienced 15 seizures from age 15 months to 4y7m, all manifested as clonic seizures, and has since been seizure free under phenobarbitone (PB) monotherapy. Although she has been seizure free for almost 18 months, it is uncertain if she benefited from PB medication because some of her last seizures had occurred when she was already being treated with this medication. Her mother (I-2) had a history of a single febrile seizure in early infancy and the proband's older brother (II-1) had also experienced two febrile seizures at age 3 y. Cognitive testing at age 6 years revealed a full scale IQ of 84, with difficulties in executive functions and praxis functions. Psychodiagnostics testing revealed a normal profile. Neurological examination was normal. There was no clinical sign of any hearing impairment.
Family C: C: II-2 is a 4-year-old female of Caucasian descent referred to the Genetics clinic. She showed GDD for her age, is non-verbal and has autistic behaviors. She has severe unilateral sensorineural hearing loss. Physical examination noted ptosis, cupped ears, small teeth, and microcephaly. Brain MRI showed abnormal morphology of the corpus callosum, suggesting dysgenesis or mild presentation of "mega" corpus callosum Clinical history of the parents noted that the father reported having speech delay that required speech therapy in school and both parents graduated from high school. The proband has two siblings that show similar clinical features. Proband's elder sister (C: II-1) is currently 7 years of age and shows GDD and urinary reflux, younger sister (C: II-3) is currently 21 months of age and also shows GDD and nystagmus.
Family D: D: II-1 is a 12 year old male of Turkish descent that was referred to the Genetics clinic with features of facial dysmorphism and hyperactivity ( Figure 1B). He was born at term via normal delivery with a birth weight of 3.2 kg (28 th percentile) after an uncomplicated pregnancy. He showed global developmental delays (GDD) from early childhood including motor delay. Clinical examination at 12 years of age showed normal anthropometric measurements: weight of 51 kg (81 st percentile), height of 143 cm (20 th percentile) and head circumference of 54.5 cm (69 th percentile). Physical examination noted downslanting palpebral fissures, ptosis, sparse lateral eyebrows, flattened nasal bridge, enlarged earlobes, short neck, vertical scar (present since birth and not related to any trauma), and hypopigmented lesions on the torso that measured less than 1 cm ( Figure 1C). Diagnostic laboratory work up included metabolic screening, karyotyping, and array CGH which were all reported as normal. Brain MRI revealed dysmorphic ventricular system and prominent Virchow-Robins (perivascular) spaces at the level of the semiovale in both cerebral hemispheres ( Figure 1D). Evaluation by a child psychiatrist at 12 years of age showed ADHD, mild autistic features, and an intelligence quotient (IQ) of 77. Ophthalmological evaluation revealed optic disc hypoplasia. D: II-2 is a 6 year old female ( Figure 1E), sister to 1: II-1. She was evaluated for developmental delay/intellectual disability (DD/ID). She was born full term after an uncomplicated pregnancy with a birth weight of 2.8 kg (12 th percentile). She did not cry immediately after birth and was in the NICU postnatal for 15 days due to difficulty swallowing and mild respiratory distress. She showed GDD during the first months of life. Growth parameters at 6 years of age showed weight of 16 kg (3 rd percentile), height of 113 cm (37 th percentile), and head circumference of 51.5 cm (69 th percentile). Physical examination showed dysmorphic features including strabismus, sparse lateral eyebrows, flattened nasal bridge, large earlobes, hypopigmented lesions on the torso measuring less than 1 cm ( Figure 1F), and foot drop. Laboratory metabolic testing revealed hypothyroidism. Brain MRI showed absent corpus callosum and colpocephalic dilation ( Figure   1G,H). Electromyography (EMG) and nerve conduction studies (NCS) were performed due to the observation of foot drop and showed bilateral mild axonal neuropathy involving the peroneal nerves. Array CGH showed a de novo 506 kb deletion in the 15q11.2 region, which was absent in the affected brother. Her IQ was evaluated to be borderline normal with a score of 85.
Ophthalmological evaluation revealed optic disc hypoplasia, similar to her brother.
Family E: E: II-1 is a 4 year old male of Pakistani descent referred to the clinic with elevated creatine kinase (CK) and autistic features. His early motor and speech development had been normal however his speech had regressed significantly at the age of 2 years and 7 months (went from using 20-30 words consistently to only 1-2 words). He subsequently also developed autistic behavioral features and typical complex motor stereotypies. He was normally grown, not weak and had an age-appropriate gait. He had lower limb hyperreflexia, a hypopigmented macule with an irregular border in the midline of his lower back, a hyperpigmented "streak" along his left arm, and additional hyperpigmented macules on his right temple, and lower back. Brain MRI revealed several high signal foci in the subcortical white matter bilaterally, without associated enhancement or restricted diffusion, with an appearance suggestive of demyelination caused by previous viral infection. He also had normal spinal MRI, cardiac, eye and renal findings/imaging results. He also presented with persistently elevated serum creatine kinase (CK) (2,500 -3,000 U/L; normal range 200 U/L) with consistently elevated CK also detected in his mother (CK levels 264 U/L) and father (CK levels 782 U/L). Given the history of consanguinity in this family, and high level of homozygosity by decent identified by SNP array (10% AOH spread over 17 autosomal regions: total 288 Mb), it is plausible that individual E: II-1 may have more than one recessive disorder, and that the homozygous PLXNA1 missense variant p.(Arg188Trp) accounts for his neurodevelopmental phenotype only. DMD MLPA and DM1 test results were normal and prior PathWest gene panel testing (which includes over 300 known neuromuscular genes) had not revealed a diagnosis. Of additional note is the fact that both parents, who are consanguineous, and healthy/not weak also have multiple non-specific pigmentary skin changes. Individual E: II-2 was a spontaneous miscarriage.
Family F: F: II-1 is a female child of unrelated parents. Her mother had previous history of three 1st trimester miscarriages and was diagnosed with epilepsy at the age of 21 years (generalized tonic-clonic seizures) and also had ulcerative colitis. The mother was on Lamotrigine and Mesalazine during the pregnancy. The pregnancy was a natural conception. At the 20 week scan, the baby was noted to have enlarged cardiac ventricles and there were some concerns about right-sided heart failure with tricuspid regurgitation. For breech presentation she was delivered by elective caesarean section. Preterm delivery at 34 weeks and 6 days of gestation was due to fetal cardiac enlargement. There was also a history of intrauterine growth retardation and she weighed 4lbs at birth. She was intubated and ventilated for two weeks requiring CPAP for a further two weeks. She was a hypotonic baby in the neonatal period. There was significant feeding difficulty requiring NG feeding. There was gastroesophageal reflux and constipation. Episodes of twitching and blank spells started in the neonatal period which were thought to potentially represent seizures. EEG at the time showed cerebral dysmaturity suggestive of cortical dysfunction, frequent sharp wave discharges over the left central region, less frequently in the similar distribution on the right. She was clinically diagnosed with absence seizures. Brain MRI was normal. She was subsequently found to have moderate conductive hearing loss due to a glue ear and was given a softband hearing aid to improve language development. Her generalized hypotonia persisted and there was also joint hypermobility. Cardiac investigations following delivery revealed a dilated cardiomyopathy which was improving on treatment, myxomatous mitral valve with mitral regurgitation were detected. Feeding difficulties persisted. She required PEG feeding but by the age of 7 years was managing to have most of her food orally other than requiring 600ml of high calorie milk to boost her nutritional intake. She sat without support at 9 months. She walked at the age of approximately 2 years 4 months. At 11 months of age her head circumference was 44.5cm (2nd to 9th centile) weight 6.74kg (0.4th to 2nd centile) and height 67cm (0.4th to 2nd centile). At the age of 8-1/2 years, her height was 119.6cm (3rd) and her weight 25.7kg (33rd).
She had evolving picture of significant learning difficulties and now attends special educational needs school at the age of 9 years. She can hold a conversation. She has reasonable comprehension. She knows some numbers and she can copy words. Her particular strength is her memory. She is noted to have a high forehead, mild hypertelorism, extensive joint hypermobility, hypeperfused palms. Her x-rays showed fusion of her capitate and hamate in the right hand, asymmetrical carpal bone development between the two wrists, slender ribs, narrow iliac wings, hypoplastic pubic rami. She has recurrent dislocation of her elbows.
Family G: G: II-1 was 4.5 years of age at the time of assessment. She has a dizygotic healthy twin sister. They were born prematurely at 33 weeks of gestation. The affected individual presented with GDD, epilepsy, bilateral sensorineural hearing loss aided with bilateral implantation of cochlear implants, impaired vision, unilateral facial palsy, dysmorphic right auricle, dextrocardia, and esophageal atresia with tracheoesophageal fistula, tracheomalacia, and hypocortisolism ( Figure 2B). Ophthalmological evaluation revealed optic disc hypoplasia. The EEG was initially normal. Onset of generalized tonic-clonic seizures was observed at three years of age. The Sleep-EEG showed no sharp waves, but generalized intermittent dysrhythmia under treatment with Levetiracetam which was initiated at three years of age. Brain MRI showed  She was able to walk at 5 years of age and showed significant speech delay. Currently, she is not able to read or write and her language is limited to a few words or short and simple sentences.
She also developed atonic seizures from birth to 12 years of age. Physical examination showed macrocephaly, enophthalmia, and mild face hypotrophy. Other neurological features included spastic paraparesis, and pyramidal signs. Brain MRI showed periventricular leukoencephalopathy, basal ganglia calcifications, and subtentorial atrophy.  Family I: I: II-2 is a 2 year-old female child to consanguineous Pakistani parents. The parents and her sister have been reported to be healthy (Pedigree in Figure S1). She showed GDD and non-verbal communication. At eight months of age, she presented with generalized tonic-clonic seizures. Initially she was treated with valproate but seizures continued to occur about four times per month with a duration of approximately five minutes. Levetiracetam was later added and she became seizure-free nine months. The medication was subsequently withdrawn which resulted in early seizures recurrence. She is restless in her behavior. Overall, her trunk musculature is slightly hypotonic ( Figure S1B). Brain MRI showed under-opercularization of Sylvian fissures, thinning of the body of CC, concern for cortical dysplasia over precentral gyrus, and white matter signal abnormality consistent with delayed myelination (Figure S1D,E). ES was performed using methods as previously described. 8 ES revealed a homozygous c.231C>G, p.(Asn77Lys) variant in PLXNA1 in individual I: II-2 ( Figure S1C). This missense variant was reported in gnomAD with a MAF of 0.00199 %, but was not reported homozygously. Prediction tools classified the variant to be deleterious (Polyphen-2: probably damaging; SIFT: deleterious; CADD: 23.8). When performing the segregation we found the patient's father to be heterozygous but the healthy mother and the healthy sister were homozygous for the c.231C>G, p.(Asn77Lys) variant. Thus, c.231C>G, p.(Asn77Lys) cannot explain the phenotype in this family. However, we did not find any other candidate gene from ES and we cannot rule out that it might contribute to a phenotype.
Family J: This consanguineous family has been recently described in a large scale study of more than 2,000 families from Saudi Arabia. 9 In that study, PLXNA1 has been suggested as a recessive candidate gene for the neurodevelopmental phenotype of the affected child in family J; however, no functional or additional human genetic data supported this finding. Here, we extend the phenotypic description of the affected child of family J. J: II-3 is a boy born full term by normal spontaneous vertex delivery with a birthweight of 3 kg with history of NICU admission for 2 days.
The mother noticed that he is delayed in catching his motor milestones at the age of 9 months for which she sought medical advice and was diagnosed as hypotonia. He sat at the age of 1 year and 7 months, crawled at the age of 2 years, started to walk with support at the age of 2 ½ years.
He can now speak two-word sentences. He can hold objects and he can transfer from 1 hand to another. He scribbled, used spoon and fork. He has history of 1 admission to hospital for respiratory infection. There is no history of seizure. He is a product of 2nd degree cousin with other 3 healthy children and no family history of similar condition. He is conscious, alert, and interactive. His weight is 8.6 kg below 3rd percentile, height is 86.5 cm on 5th percentile. His head circumference is 48 cm on 25th percentile. There is no apparent cranial nerve palsy. He has decreased muscle bulk and he has generalized hypotonia. Deep tendon reflexes are almost absent. There is no tongue fasciculation. He is weak more distally. Chest, heart and abdominal examinations are unremarkable. Brain MRI showed mild prominence of extra CSF spaces appropriate to his age.
ES revealed a homozygous c.2702G>A, p.(Arg901His) variant in PLXNA1 in the individual J: II-3. This missense variant was reported in gnomAD with a MAF of 0.0008 %, but was not reported homozygously. Only one of the prediction tools classified the variant to be deleterious (Polyphen-2: benign; SIFT: benign; CADD: 22.2). However, the parents did not consent for segregation analysis within the family, thereby we cannot ultimately classify this variant as being diseasecausing.
Family K: Furthermore, we identified one additional individual with a biallelic variant in PLXNA1: The boy was a child to consanguineous Pakistani parents that had two additional unaffected children ( Figure S1). Individual K: II-3 showed gross developmental delay and was able to sit at 1.5 years of age. At assessment at 5 years he presented with severe DD, delayed speech, mild nystagmus, brisk reflexes, abnormal movements and he was able to walk with support. He was noted to be aggressive and irritable with features of mild ADHD. Seizures started at one year of age with multiple generalized tonic-clonic seizures per day. Initially seizures in individual K: II-3 were treated with first line drugs including Phenobarbitone and Carbamazepine. Later the regimen was switched to Levetiracetam along with Valproate. His response to treatment was partial. Brain MRI showed abnormal signal intensity in the periventricular white matter bilaterally (hypomyelination), mild dilatation of the ventricles and particularly the left occipital horn also showed slight wavy margins, thinning of corpus callosum, and generous extra-axial CSF ( Figure S1). His response to treatment was partial, seizures persisted and the child recently passed away due to severe pneumonia and status epilepticus.
After informed consent was obtained prior to genetic testing from the family, genomic DNA was extracted from peripheral blood samples according to standard procedures of phenol chloroform extraction. ES was performed as described elsewhere. 10  230000]. Clinical features include progressive mental (95%) and motor (87%) deterioration, coarse facial appearance (79%), growth retardation (78%), recurrent infections (78%), dysostosis multiplex (58%), angiokeratoma corporis diffusum (52%), visceromegaly (44%), and seizures (38%). 11 An abnormal ventricular system was also reported in two additional individuals with biallelic PLXNA1 variants including dysmorphic ventricles (family D: II-1) and colpocephaly (family D: II-2). But the abnormal ventricular system observed in K: II-3 ( Figure S1) could be also caused by Fucosidosis 12 due to his homozygous nonsense variant in FUCA1 and the contribution of biallelic PLXNA1 variant could not be clearly defined. Since, we establish two molecular diagnoses in this individual K: II-3 with a highly overlapping phenotype we cannot draw any conclusion about the contribution of the individual genes. A dual diagnosis, especially in a consanguineous family is not a rare incident. 13 Family L: The boy was a child to Eritrean parents that had the history of two previous miscarriages ( Figure S2A). Because of pathological CTG (cardiotocography), he was born by caesarean section at the 36th week of gestation. Birth parameters are as follows: weight 3.06 kg (21st percentile), birth length: 48 cm (21st percentile), head circumference at birth: 35.5 cm (43rd percentile), APGAR: 3/7/9. Directly after birth, the boy presented with muscular hypotonia. At the age of six months, the muscular hypotonia was no longer present. He had mildly flattened facial profile, ears discretely rotated backwards, and sharp-pointed fingers. Brain MRI was not performed. ES was performed and a NM_032242.3 c.1357C>T p.(Arg453*) PLXNA1 variant was identified and confirmed by Sanger Sequencing (Figure S2E). The parents were tested for the variant using Sanger sequencing and this variant was not found. Paternity test was performed and de novo occurrence could be demonstrated. This variant (g.126710389C>T) resides in exon 2 and is reported in gnomAD with a MAF of 0.003187%, but no homozygous alleles were reported for this variant. The CADD Score is 36. For the truncated p.(Arg453*) Plexin-A1, we removed the C-terminal beyond p.(Arg453) residues and subjected the rest of the sequence to I-Tasser based modeling. The resulting model can be seen in Figure S3.
Since the family has been lost to follow up, we had no information about further development of the child (e.g. history of development delay, seizures, or cerebral anomalies). However, the sponatenously resolved neonatal hypotonia remains unclear. Thus, we cannot classify this variant as being disease-causing.   This variant was confirmed using Sanger sequencing and was found to be absent in the unaffected mother. However, paternal DNA was unavailable for testing. Another variant of interest was found in SETD2 c.5781G>T, p.(Gln1927His) which was similarly absent in the mother. Since we were not able to confirm the de novo occurrence in this family, we cannot classify this variant as being disease-causing.