The sodium leak channel nonselective protein (NALCN) is a regulator of the pacemaker neurons that are responsible for rhythmic behavior (including respiration), maintaining the resting membrane potential, and are required for action potential production. NALCN-null mice show early death associated with disrupted respiratory rhythms, characterized by frequent and profound apneas. We report 3 children (2 siblings) with compound heterozygous mutations in NALCN associated with developmental impairment, hypotonia, and central sleep-disordered breathing causing apneas. Supplemental oxygen normalized the respiratory rhythm. NALCN mutations have been previously reported to cause severe hypotonia, speech impairment, and cognitive delay as well as infantile neuroaxonal dystrophy and facial dysmorphism. Nonsynonymous changes in the 2 affected extracellular loops may be responsible for the deleterious effect on the stability of the respiratory rhythm. Although oxygen is known to be a stabilizer of respiratory rhythm in central apnea in children, its role in NALCN dysfunction requires further investigation.
The sodium leak channel nonselective protein (NALCN) is a transmembrane, pore-forming protein that facilitates the intracellular movement of sodium. The NALCN gene is widely expressed in the central nervous system1 and is a critical component in the maintenance of resting membrane potential in excitable cells. An excellent recent review of NALCN function is provided by Cochet-Bissuel et al.1
Case Patient 1 (Family I.1)
The elder sister of our siblings was born by emergency cesarean delivery after a failed induction of labor (postterm) for oligohydramnios. The parents are nonconsanguineous and white. Her birth weight was 3230 g (z: −0.07) and occipitofrontal circumference was 34 cm (z: 0.1). Hypotonia with poor sucking reflex necessitated nasogastric feeding. Bottle feeding was established with specialized teats. Weight gain was slow and required calorie supplementation. At the age of 6 years, her BMI was 12.6. She showed a disturbance of her sleep-wake cycle. On occasion, she went without sleep for 48 hours. She has been diagnosed with attention-deficit/hyperactivity disorder. A diagnosis of global developmental delay was reached by 1 year and 3 months, she now attends a school for children with additional needs and has severe intellectual impairment. She has strabismus and hypermetropia requiring corrective lenses. Mild craniofacial dysmorphism is apparent with myopathic features (Fig 1A). Before this, plagiocephaly gave rise to pseudohemihypertrophy of the face, which has resolved. At age 7 years, she had an adenotonsillectomy after reported obstructed breathing at night. This had no effect on the underlying respiratory rhythms described below.
Case Patient 2 (Family I.2)
The male, younger sibling (Distributed European Community Individual Patient Healthcare Electronic Record [DECIPHER] number 257768) was born by elective cesarean at 39 weeks. His birth weight was 3232 g (z: −0.3) and occipitofrontal circumference was 36 cm (z: 1.21). Similar issues to his sibling were apparent from birth, including hypotonia and feeding difficulties, but the early use of specialized teats allowed an earlier discharge from the hospital. At age 8 years, his BMI was 13.7. Although he was sitting at 6 months old, further milestones were delayed. Speech and social skills are profoundly affected, and he has been diagnosed with autism spectrum disorder and learning disability. An MRI of the brain performed at age 3 years was normal. He attends a school for children with additional needs. He has strabismus and hypermetropia.
Case Patient 3 (Family II.1; DECIPHER Number 263524)
Case patient 3 was born at 41 weeks’ gestation weighing 2850 g (z −0.88). She was noted to be hypotonic and required support feeding in the neonatal period. She had marked plagiocephaly and facial asymmetry, which is still evident when she smiles. She has a tall forehead with bitemporal narrowing and deep-set eyes. A brain MRI scan at age 3 years was reported normal. She underwent adenotonsillectomy at age 2 years and tympanostomy-tube insertion at age 5 years for glue ear.
She was delayed in her developmental milestones and sat at 1 year old. By 19 months old, she was walking with 1 hand being held but did not speak. An assessment at age 3 years highlighted difficulties with intelligibility and her expressive language, and this persisted until age 10 years. She started mainstream school with educational support but transferred to a school with additional support for learning at age 10 years. Child and Adolescent Mental Health Services offer support for behaviors and anxiety. At age 10 years, her BMI was 26.7 but this has plateaued on a weight-management program. She had normal echocardiogram, ophthalmology, and audiology assessment results. She has joint hypermobility.
A phenotypic summary of these and other published case reports is provided in Supplemental Table 1.
The parents noticed an unusual breathing pattern from birth in case patient 1. This was characterized as a pattern of taking 3 breaths normally then “holding her breath” with no respiratory effort for the equivalent of 3 breaths. Initially this was felt to be physiologic apnea, but its persistence led to further investigations. Limited channel cardiopulmonary studies with the recording of airflow by thermistor were performed along with assessments of respiratory effort by inductance plethysmography, pulse, oxygen saturation (SpO2) by pulse oximetry, transcutaneous carbon dioxide (TcCO2) measurement, single lead cardiac monitoring, and video. The airflow measurement was poorly tolerated. Multiple sleep studies demonstrated frequent central apneas ameliorated with oxygen therapy (Fig 1D). A study from 2010 (aged 3 years), revealed a rhythm of 2 breaths followed by a cessation of respiratory effort, giving rise to an SpO2 desaturation of >3% to 80% to 89%. Each event had a mean duration of 10 seconds at a frequency of 39 events per hour. The mean TcCO2 reading was 5.6 kPa. The respiratory rate fell from 26 when awake to 14 when asleep. When supplemented with 1 L per minute of nasal cannula oxygen, there were markedly fewer respiratory pauses and no drop in SpO2. Subsequent studies showed a notable periodicity of the events that is consistent with the effect of different stages of sleep.
Case patient 2 was not noticed to have abnormal breathing rhythm until he was 5 months old. A report from a sleep study performed at age 2 years revealed a rhythm of 1 to 2 breaths then complete cessation of effort for a mean duration of 9 seconds with a frequency of 37 per hour and an associated drop in SpO2. TcCO2 was stable at ∼5.5 kPa. No obstructive events were noted. With supplementation of 1 L per minute of oxygen via nasal cannulae, respiratory pauses were less frequent and not associated with a drop in Spo2. Images are unavailable because of the study quality and data storage procedures in place at his base hospital.
Case patient 3 was referred for assessment at 8 months of age and had a sleep study showing a mixture of central and obstructive episodes. An adenotonsillectomy resolved the obstructive episodes. All subsequent studies have shown the overwhelming feature to be central apnea with >100 events per hour in air and an almost complete abolition of the events with 0.5 L per minute of oxygen. The pattern consists of 3 or 4 breaths followed by a pause with a slight increase in heart rate and a drop in oxygen saturation as shown in Supplemental Fig 2. Sleep state appears to have no effect on the abnormality, and there is no evidence at 10 years of age of any change in sleep breathing abnormality.
All 3 children had negative screen results for common genetic and metabolic causes of hypotonia and congenital central hypoventilation syndrome.
On the basis of the unexplained developmental delay, case patients 2 and 3 were recruited to the Deciphering Developmental Disorders Study (www.ddduk.org). This is a United Kingdom–wide project that has recruited ∼14 000 probands with undiagnosed severe or extreme developmental disorders via regional clinical genetics services in the United Kingdom and Ireland with the aim of using new, genome-wide technologies to identify causative genomic variants. Trio-based, whole-exome sequencing was performed on case patient 2 and his parents as previously described.2 This revealed compound heterozygous mutations in 2 known developmental disorder genes: NALCN and TRAPPC9. The TRAPPC9 mutations were both missense variants; chr8 g.141445217G>A (ENST00000438773 c.853C>T; p.Arg285Trp) was paternally inherited, and chr8 g.141321437G>A (ENST00000438773 c.1532C>T; p.Thr511Met) was maternally inherited. Previous reports of biallelic TRAPPC9 mutations describe mental retardation, microcephaly, corpus callosum hypoplasia, facial asymmetry, and overweight habitus.3,4 He does not have most of these features; however, he is more severely affected than his sibling. She carries only 1 TRAPPC9 variant, and thus, he could display a composite phenotype, which has been reported in 4.9% of the rare diseases investigated with exome sequencing.5 The NALCN mutations are a missense and a nonsense variant; hg19 chr13 g.101756915G>A (ENST00000251127 c.2723C>T; p.Pro908Leu) was paternally inherited, and hg19 chr13 g.102047715C>T (ENST00000376200 c.110G>A; p.Trp37Ter) was maternally inherited. Segregation analysis within the family revealed that the affected sister was also compound heterozygous for the NALCN mutations but not the TRAPPC9 (she carried only the p.The511Met maternally inherited allele).
Case patient 3 was found to have compound heterozygous variants in NALCN and another developmental gene, ALMS1. Both ALMS1 variants were missense; chr2 g.73675690T>C (ENST00000377715 c.2033A>G; p.Tyr678Cys) was paternally inherited, and chr2 g.73747141T>G (ENST00000264448 c.9776A>C; p.Asp3259 Ala) was maternally inherited. ALMS1 mutations cause Alström syndrome, which is characterized by visual impairment, sensorineural hearing loss, insulin insensitivity, cardiomyopathy, and multiple organ failure. The patient has been screened and does not match this phenotype, excluding obesity; thus, it seems that at least 1 ALMS1 variant is benign. Case patient 3 has also inherited a missense and a nonsense NALCN variant; hg19 chr13 g.101742306C>T (ENST00000251127 c.3281G>A; p.Arg1094Gln) was maternally inherited, and hg19 chr13 g.101759854G>A (ENST00000251127 c.2563C>T; p.Arg855Ter) was paternally inherited. For a schematic view of the NALCN mutations in relation to their domains, please see Fig 1E. A more detailed schematic is available in Supplemental Fig 3. It appears significant that both pedigrees have missense variants in the third transmembrane domain of the NALCN, although there are no functional data to confirm that this is the cause of the milder phenotype compared with previous cases.
There have been 3 published case reports of autosomal recessive, inherited NALCN mutation in humans.6,–8 Hypotonia, developmental delay, and constipation are pervasive, but strabismus, poor weight gain, and dysmorphic facies are also common. Dysrhythmic breathing is previously mentioned in 1 family8; we expand on this with formal study and detailed description and add nonconsanguineous families and compound heterozygote genotypes with missense variants in a shared protein domain.
Expression of the NALCN is seen in the retrotrapezoid nucleus that regulates breathing in mice,9 and NALCN knockout mice10 showed pathologic rhythms of breathing characterized by apnea for 5 seconds then deep breathing for 5 seconds at a rate of 5 apneas per minute. This respiratory pattern was predictive of death within 24 hours of birth. Reduced expression of the orthologous gene in snails (Lymnaea stagnalis11) was found to reduce breathing time and frequency in comparison with controls. Nerve-conduction studies showed this to be caused by the abolition of spontaneous firing of the right pedal dorsal 1 neuron that is responsible for respiration.
Chong et al12 described de novo mutations in the NALCN region that cause an autosomal-dominant effect. These individuals were diagnosed with congenital contractures of the limbs and face with hypotonia and developmental delay syndrome. This appears to be a distinct condition, although phenotypic overlap does occur.
Pacemaker neurones are a class of neuron that are proposed to play a critical role in activities such as breathing, sleep-wake cycling, gut motility, and cardiac rhythmogenesis.13 Pacemakers require the resting membrane potential to be maintained within a tight voltage range.14 The maintenance of resting membrane potential is facilitated by the NALCN’s passive, intracellular movement of sodium ions. One such collection of pacemaker cells resides in the pre-Bötzinger complex within the medulla, contributing to the complex network influencing our respiratory rhythm.15,16
Apneas that occur only during sleep pose challenges to the narrative of the case, but there is precedent with conditions such as congenital central hypoventilation syndrome and Brugada syndrome, which also manifest during sleep.17
The role of oxygen in alleviating this problem requires more study, although our sleep studies show similarity with those of periodic breathing of infancy. Premature infants with this condition show marked improvement of breathing regulation with supplemental oxygen therapy.18 The same effect has also been shown on sleep-disordered breathing in Prader-Willi syndrome.19 A knockout animal model observed in hypoxic and hyperoxic environments may offer insight into this relationship.
Dr Campbell conceived the original concept for the article, reviewed the current literature, interviewed the parents in case patients 1 and 2, summarized the clinical history to date, drafted the initial manuscript by incorporating each contribution, and compiled the figure; Dr Urquhart and Prof FitzPatrick conceived the original concept for the article, conducted the investigations, brought the case patients to light, and reviewed and revised the initial manuscript; Dr Gibson identified the sleep-breathing abnormalities in all 3 children, wrote the section concerning the breathing pattern in case patient 3, provided the concurrent images in the figure, and reviewed the article before submission; Dr Joss conducted the investigation, brought case patient 3 to light, wrote the genetics section for that child, provided her picture, and reviewed the manuscript; Dr Somerville provided further clinical information and developmental assessment for case patient 3, wrote the clinical summary for the child, and reviewed the manuscript; Ms Azam performed the genetic segregation analysis in all 3 case patients, contributed to the genetic section, and reviewed the manuscript; and all authors determined the direction of the article via conversations and correspondences and approved the final manuscript as submitted.
FUNDING: No external funding.
We are indebted to the families who have consented to sharing their stories. This article makes use of data generated by the DECIPHER Consortium. A full list of centers that contributed to the generation of the data are available at https://decipher.sanger.ac.uk/ and via e-mail at email@example.com. Funding for the project was provided by the Wellcome Trust. The Deciphering Developmental Disorders Study presents independent research commissioned by the Health Innovation Challenge Fund (grant HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant WT098051). The views expressed in this publication are those of the authors and not necessarily those of the Wellcome Trust or the Department of Health. The study has UK Research Ethics Committee approval (10/H0305/83, granted by the Cambridge South Research Ethics Committee, and GEN/284/12, granted by the Republic of Ireland Research Ethics Committee). The research team acknowledges the support of the National Institute for Health Research through the Comprehensive Clinical Research Network.
POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.
FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.