CNTNAP1 mutations cause CNS hypomyelination and neuropathy with or without arthrogryposis

Identification of new CNTNAP1 mutations.

In a large Israeli consanguineous family of Palestinian ancestry (family 1 branch A + B), we identified 2 children (figure 1) with multiple congenital anomalies affecting the skeleton and brain as indicated by fetal sonography. Polyhydramnios occurred during both pregnancies, and children were born at 34- and 35-week gestation, respectively, with normal birth weight. Both children were diagnosed with AMC at birth, with clenched hands, contractures of the elbows, knees, and feet. They were noted to have microcephaly, hypotonia, absent swallow, facial diplegia, low-set ears, cleft palate, and micrognathia.

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Figure 1 Pedigree of family 1 and mutations in CNTNAP1

(A) Pedigree of family 1 branches A–C with multiple consanguineous loops and segregation of the CNTNAP1 variant. The identified variant (position indicated by red arrow) segregated with the disease in all affected patients. (B) Schematic representation of known and new mutations in CNTNAP1: previously described mutations are depicted in black, and new disease-causing mutations are indicated in red. The missense variant p.(Arg764Cys) has been found in a compound heterozygous state together with p.(Gln671*). All other variants were homozygous in affected patients. Blue: extracellular domain; red: helical domain (transmembrane); green: cytoplasmic domain (intracellular) (information of domains from uniprot.org).

Because of profound hypoventilation, both infants required ventilatory support. Both had severe developmental disabilities and never learned to sit, walk, or talk. Later, both developed generalized epilepsy with tonic-clonic seizures.

On neurologic examination at age 9 and 12 years, severe intellectual disability of both children was noted. They had small unresponsive pupils, were unable to make eye contact, did not follow visual stimuli, and did not react to sound stimuli. Both had pronounced contractures of hands, elbows, knees, and feet and showed increased muscle tone of the extremities while the tone of axial muscles was decreased. Reflexes were increased with positive Babinski sign. Both were microcephalic and showed dysmorphic features, including micrognathia, cleft palate, and large, low-set ears (figure 2). Both received nutrition by gastrostomy and underwent tracheostomy.

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Figure 2 Phenotypic features of CNTNAP1 patients

(A–C) Patient 1 with arthrogryposis multiplex congenita (AMC). (A) Contractures of the feet, (B) features including microcephaly, micrognathia, low-set ears, and cachexia, and (C) contractures of the hand and fingers. (D, E) Patient 3 without AMC but with microcephaly, low-set ears, and hypotonia. (F–H) Patient 4 without AMC. (F) No signs of contractures of the fingers, (G) hypotonia, and (H) midline notch upper gum.

Metabolic screening including amino acids, very long chain fatty acids, lactate and ammonia in serum, and organic acids in urine was normal as were CSF findings. Genetic testing excluded SMN1 mutations. Brain MRI of patient 1, taken at the age of 5 years, showed severe hypomyelinating leukodystrophy. There were no signs of myelination even in parts of the brain normally myelinated early after birth such as the posterior limb of the inner capsule. Reduced cerebral, cerebellar, and pontine volume was obvious with pronounced lack of white matter. Furthermore, a thin corpus callosum and mega cisterna magna were noted (figure 3). To determine the genetic basis, we performed whole-exome sequencing on both patients. Following the hypothesis that both patients suffer from the same genetic disorder, only overlapping variants from both exomes were considered and filtered using the strict criteria mentioned in the methods. This resulted in only 9 rare homozygous variants including only 1 truncating variant: a homozygous stop-gain mutation in exon 13 of CNTNAP1: NM_003632.2:c.2015G>A:p.(Trp672*). Sanger sequencing of all family members showed full segregation with the disease (figure 1). The parents were heterozygous for the mutation, and the healthy siblings were either heterozygous or homozygous for the wildtype. The mutation was not found in the ESP6500, 1000 Genomes, or the Exome Aggregation Consortium (ExAC) database.

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Figure 3 MRIs of CNTNAP1 patients and controls

(A) T2-weighted image of healthy 8-day-old child. Signs of beginning myelination within the posterior limb of the inner capsule (PLIC; hypointense signal within the white circle). (B) T2-weighted image of healthy 36-month-old child. Myelination is complete, resulting in hypointense signals of white matter, inner capsule, and corpus callosum. (C) T2-weighted image of patient 4 taken at 4 days of age. No signs of myelination in the PLIC (white circle). No signs of obvious cortex malformation. (E) Sagittal T1 image of patient 4 at 4 days of age indicates normal cerebral, cerebellar, and pontine volume. Small mega cisterna magna is depicted by a white arrow. (D, F, G) T2-weighted images of patient 1 at 5 years of age. (D, G) White matter is hyperintense without signs of myelination; even early myelinating structures are hyperintense without myelin deposits (circle). Cerebral atrophy (white arrow) with pronounced lack of white matter (white star). (F) Midsagittal images show thin corpus callosum (white arrow), mega cisterna magna (white arrow), reduced pontine volume, and cerebellar atrophy.

Further follow-up of the family revealed a 4-week-old child in a distantly related family branch (branch C in figure 1A) who was born at term with a history of polyhydramnios, had similar dysmorphic features with low-set ears, micrognathia, and microcephaly and required controlled ventilation. However, there were no joint contractures or signs of AMC. Genetic testing revealed the identical homozygous CNTNAP1 mutation c.2015G>A:p.(Trp672*).

Screening for CNTNAP1 mutations reveals further non-AMC cases.

An unbiased screening approach using the GEM.app/Genesis database led to the identification of a nonconsanguineous Northern Irish family (family 2) with 2 affected brothers (patients 4 and 5, table), who shared compound heterozygous mutations in CNTNAP1: 1 stop-gain and 1 missense mutation (NM_003632.2:c.2011C>T:p.(Gln671*) and c.2290C>T:p.(Arg764Cys)). The latter changes a highly conserved polar amino acid residue (arginine) to a nonpolar residue (cysteine) (GERP score: 5.84, PhastCons: 1). Different prediction programs (Scale Invariant Feature Transform: 0.03, MutationTaster: 0.999, and PolyPhen: 0.998)^10,–,12 estimate the variant to be pathogenic, and it has never been described in public databases (ExAC, ESP6500, and 1000 Genomes).

Clinical findings were similar as in family 1. Patient 4 was diagnosed with polyhydramnios and reduced fetal movements at 27-week gestation. At birth, he was profoundly hypotonic and required ventilatory support. He was noted to have absent swallow, facial diplegia, complete ptosis, dolichocephaly, epicanthic folds, thickened nares, thickened lips, narrow ridged palate, thickened gums with a notch in the upper midline, micrognathia, and partial bilateral 2–3 toe syndactyly. He was areflexic and preferred to lie in a hyperextended position. Some voluntary limb movements were observed. No arthrogryposis was noted. A full range of passive limb and joint movements was possible. He was able to open his eyes for up to 40 seconds, but this gradually decreased. Tracheostomy was performed at the age of 3 weeks; with this, he was able to breathe unaided. Brainstem evoked responses confirmed bilateral sensorineural hearing loss. At the age of 10 weeks, he developed generalized clonic, brief tonic, and myoclonic seizures. Resting posture was noted to be less hyperextended, elbows and fingers were held flexed, but the range of passive movements remained full. Palliative care was introduced and he died at age 4 months. On brain MRI at the age of 4 days, any signs of beginning myelination were missing (figure 3). There were mega cisterna magna and thin corpus callosum, but clear cerebral hypoplasia or cortical malformations were not detected. Nerve conduction studies and EMG suggested sensory-motor neuropathy and denervation.

Patient 5 was the brother of patient 4. Polyhydramnios and reduced fetal movements were diagnosed at 31-week gestation. At birth, he was profoundly hypotonic with no respiratory effort. He did not swallow and muscle reflexes were absent. No invasive treatment was initiated and he died at 4 hours of age.

Sural nerve biopsy reveals paranodal abnormalities.

Sural nerve biopsy of patient 4 revealed a significant axonal loss which was homogeneous between nerve fascicles. There was a significant decrease of large myelinated fibers and a great number of myelinated fibers with too thin myelin sheaths (figure 4); myelin debris was found in the cytoplasm of some Schwann cells and in a few macrophages in the endoneurial space. Electron microscopic examination of longitudinal sections showed frequent and marked widenings of the nodes of Ranvier typical of a demyelinating neuropathy. Most of the paranodal regions were characterized by loss of attachment sites of the loops, which seemed to be induced by Schwann cell processes penetrating between the axon and the loops (figure 4). Moreover, the typical transverse bands¹³ that cross the paranodal junctional gap were absent.

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Figure 4 Electron microscopy of sural nerves comparing patient 4 and with a healthy control

(A, B) Electron microscopy of sural nerve of patient 4. (A) Transverse section. Severe rarefaction of myelinated fibers; many have a too thin myelin sheath (arrows). (B) Longitudinal section. Abnormal widening of the node of Ranvier. The myelin loops are dissociated by the fingers of Schwann cell cytoplasm (arrows), which penetrate between the loops and the axon. (C, D) Age-matched control images. (C) Transverse section. Normal axonal density. (D) Longitudinal section. Normal node of Ranvier; the loops are closely attached to the axon.