SLC12A2 mutations cause NKCC1 deficiency with encephalopathy and impaired secretory epithelia

Objective To describe the phenotype in 2 sisters with a rare constellation of neurologic symptoms and secretory impairments and to identify the etiology by the use of whole-genome sequencing (WGS). Methods After an extensive workup failed to reveal the cause of disease, in a girl with a previously not reported phenotype, WGS of the proband, her diseased older sister, an older healthy brother, and their parents was performed, and potentially pathogenic variants were analyzed. Results The proband and her older sister both presented with neonatal Staphylococcus aureus parotitis, apneas, disappearance of the Moro reflex, and hypotonia. The proband survived. Her brain MRI showed white matter and basal ganglia abnormalities, and CSF damage biomarkers were increased. At age 8 years, she exhibits a constellation of symptoms including severe neurodevelopmental disorder, hearing impairment, gastrointestinal problems, and a striking lack of tear fluid, saliva, and sweat. Her respiratory mucosa is dry with potentially life-threatening mucus plugging. Through WGS, 2 loss-of-function variants in SLC12A2 were identified that follow an autosomal recessive inheritance pattern. Conclusions Taken together with a single previously reported case and the close resemblance to the phenotypes of corresponding mouse models, our study firmly establishes biallelic variants in SLC12A2 as causing human disease and adds data regarding the neurologic phenotype.

The Na-K-Cl cotransporter sodium-potassium-chloride cotransporter 1 (NKCC1), encoded by the SLC12A2 gene, is widely expressed in neurons and secretory epithelial cells of multiple organs. 1-3 SLC12A2, located on chromosome 5q23.3, has only recently been implicated in human disease. The SLC12 family of solute carriers consists of 9 electroneutral cation-chloride cotransporters (CCCs) involved in epithelial ion transport, gamma-aminobutyric acid (GABA)-mediated neurotransmission, and cell volume regulation. 4 Another SLC12 member, the potassium-chloride cotransporter 2 (KCC2), encoded by SLC12A5, is the main chloride exporter in neurons. 5 As a neuronal chloride importer, NKCC1 is in dynamic balance with KCC2, thus regulating the intraneuronal chloride concentration on which GABA neurotransmission and neuronal excitability depends. 2,6 Four of the CCCs have previously been linked to monogenic disease in humans, 7-10 of which 2 show a neurologic phenotype. 9,10 Autosomal recessive early infantile epileptic encephalopathy results from pathogenic variants in SLC12A5. 9 Biallelic variants in SLC12A6, encoding KCC3, are linked to agenesis of corpus callosum with peripheral polyneuropathy, also called Andermann syndrome. 10 For SLC12A2, there are mouse models exhibiting phenotypes related to NKCC1 deficiency in neurons and epithelia. 4 The first human case of a homozygous deletion in SLC12A2 was recently described. 11 In this study, we report the detailed characterization of 2 sisters with an NKCC1 deficiency syndrome due to novel biallelic variants in SLC12A2.

Subject recruitment
The proband was referred to our hospital at age 9 days. An extensive etiologic workup was performed before her inclusion as a research subject. The proband's 21-month-older sister had been treated and died at another hospital, and fibroblasts were preserved for future diagnostics.
Standard protocol approvals, registrations, and patient consents Written informed consent was obtained from the parents including a consent to disclose photographs of the proband. The study was approved by The Regional Ethical Review Board in Stockholm, Sweden.

Whole-genome sequencing
Whole-genome sequencing (WGS) of the proband, her 2 siblings, and their parents was performed to a sequencing depth of 30× mean coverage using a HiSeq X sequencing instrument and a PCR-free library preparation method. The resulting sequences were analyzed using the Mutation Identification Pipeline (MIP) as previously described. 12 All called variants were scored and ranked using the MIP weighted sum model, which uses multiple parameters, but emphasizes mendelian inheritance patterns, conserved, rare, and protein-damaging variants. The genome was analyzed for potential pathogenic variants in exons or splice regions.

Data availability
The data that support the findings of this study are available from the corresponding author on reasonable request.

Clinical summary
The proband (II:3) (figure 1) is an 8-year-old girl and the third child of nonconsanguineous parents of Swedish descent. In addition to the deceased older sister (II:2), she has a healthy older brother (II:1).
The older sister was born 21 months before the proband, full term with birth weight 3.0 kg after a normal pregnancy. She was described as hypotonic. At age 9 days, she presented with bacterial parotitis confirmed by ultrasound and growth of Staphylococcus aureus in pus from the parotid gland. She was tired but afebrile, infection parameters were only slightly elevated, and blood cultures negative. The infection was well controlled by antibiotics, but the patient remained hospitalized due to apneas, muscle hypotonia, and feeding problems. It was noted that the Moro reflex that was normal during the first week of life later could not be elicited. During the third week, apneas of unknown cause worsened. A chest x-ray was normal. No viral or infectious agents were detected in CSF, blood, urine, feces, or nasopharynx. Infectious parameters were normal, and CSF showed no pleocytosis and normal albumin. Brain imaging was not performed. At age 22 days, the patient died of asystole during assisted ventilation. Fibroblasts were preserved. Autopsy revealed aspiration of ventricular content into the lungs. There were no malrotation or other internal malformations.
transferred to a neonatal unit due to hypotonia and difficulties breastfeeding. The following course was characterized by bacterial parotitis and S aureus septicemia presenting at day 5, central and obstructive apneas, disappearance of the Moro reflex, and muscle hypotonia. The similarity with the symptoms of the deceased older sister was striking. The patient was admitted to our hospital's pediatric intensive care unit on day 9. She was treated with antibiotics, continuous positive airway pressure, oxygen, caffeine citrate, and frequent inhalations of sodium chloride. The infection was promptly controlled, but apneas persisted until she could be discharged to her local hospital at age 7 weeks. Because of the history of the older deceased sister, an extensive workup for immunodeficiency and metabolic disease was started. Results came back negative as did a genetic test for cystic fibrosis. A neurologic workup was prompted by the muscle hypotonia, central apneas, and the lack of Moro reflex. MRI (figures 2, A-E and 3, A-E) and magnetic resonance spectroscopy (MRS) (supplementary material, figure e-1, A and B, links.lww.com/NXG/A277) of the brain revealed white matter and basal ganglia abnormalities. Lumbar puncture showed elevated albumin and increased damage biomarkers in CSF (supplementary material, table e-1).
The continued course has been without severe infections or other dramatic events. The patient shows dysmorphic facial features (figure 4). Her head circumference and length are around −2.5 SD and weight −3.5 SD. She has severe intellectual disability and does not speak or walk. She has a hearing impairment and a striking lack of tear fluid, saliva, and sweat. Her respiratory mucosa is dry, which necessitates frequent inhalations of hypertonic sodium chloride to prevent life-threatening mucus plugs. She has constipation and an intestinal malrotation. Follow-up MRI investigations of the brain have been performed at 2 months (figure 2, F-J) and 4 years (figure 2, K-O and 3, F-J) and CSF analyses at 4

Molecular genetic analyses
When the proband was 6 years of age, WGS of all family members was performed. The cause of her and her older sister's disease was still unknown, and the parents where planning another pregnancy. Analysis of sequencing data did not reveal any potential pathogenic variants in 870 known metabolic disease genes. When expanding the analysis to the whole genome, no potential disease-causing variants that fit with an autosomal recessive inheritance were detected in any known disease-causing gene. When moving to genes not previously linked to monogenic diseases, 2 highly interesting variants were detected in SLC12A2. At the time, no patients with SLC12A2 mutations had been reported. No other potential diseasecausing variants that fit the segregation among the 5 family members were detected. Both variants were ranked high by the MIP-weighted sum model. The index patient and her deceased older sister were compound heterozygotes for these variants. As the heterozygous c.2006-1G>A variant is positioned in the splice acceptor site, it is predicted to affect normal splicing of exon 13. Splicing of the SLC12A2 transcript (NM_001046.3) was examined by PCR amplification of complementary DNA from exon 12 to exon 14. A PCR product of 172 bp was amplified, which was 102 bp shorter than the expected wildtype allele, and following Sanger sequencing, it was subsequently found to entirely lack exon 13 (figure 5). Sequencing of exon 8 from both the patients' and father's complementary DNA showed a lack of expression from the allele carrying the deletion, (c.1431delT) indicating nonsense mediated decay of this transcript (data not shown).

Discussion
We report 2 sisters exhibiting a complex phenotype caused by NKCC1 deficiency due to novel compound heterozygous variants in SLC12A2. Taken together with a recently published case of homozygous deletion in SLC12A2, and the close resemblance to the phenotypes of SLC12A2 knockout mouse models, our study firmly establishes SLC12A2 as a disease gene in humans and adds new data regarding the neurologic phenotype.
Our proband and her sister's neurologic symptoms as neonates were similar. The proband's CSF findings suggested recent and/ or ongoing neuronal damage that in our opinion could not be explained by hypoxia-ischemia or septicemia. She had short apneas and desaturations but was otherwise stable and not clinically septic. The ensuing neurodevelopment of the proband has been severely impaired in both cognitive and motor domains but without regression.
GABA, the main inhibitory neurotransmitter in the mature brain, is excitatory in immature neurons due to high intraneuronal [Cl − ]. This is caused by the high expression of NKCC1 and the immaturity of KCC2. 2,15 During nervous system development, excitatory GABA acts as a trophic factor and affects proliferation, migration, differentiation, synapse maturation, and cell death, 6 thus influencing brain development and CNS network formation. In the CNS of SLC12A2 knockdown mice, neuronal proliferation is decreased, and dendrite development delayed. 16 Based on this, the severe neurodevelopmental impairments in our proband could be of prenatal origin. This is supported by the evolution of the early and follow-up MRI findings (figure 2), together suggestive of hypomyelination. Alternatively, the neonatal white matter findings could, at least partially, be due to a more acute neurometabolic encephalopathy with cytotoxic edema. The latter is supported by the very high damage biomarkers in CSF (supplementary material, table e findings at 4 months and 4 years suggest some degree of ongoing neurodegeneration and disruption of the blood-brain barrier (supplementary material, table e-1).
In brainstem respiratory nuclei of rats, NKCC1 and KCC2 expression shows developmental decrease and increase, respectively, during early postnatal life. 17 The resulting rapid increase of GABA inhibition could destabilize the respiratory network during a critical period. We hypothesize that NKCC1 deficiency contributed to the central apneas in our patients through enhanced neonatal respiratory inhibition or impaired prenatal respiratory network formation. Another striking, and otherwise rare, symptom in our patients is the early permanent loss of the Moro (startle) reflex, also with a potential relation to NKCC1 deficiency in involved brainstem nuclei. 18 NKCC1 is expressed in the basolateral membrane of epithelia in various organs, transporting chloride across the basolateral membrane into the epithelial cells where chloride then can be secreted. 3 The severely reduced saliva in our proband closely resembles the deficient saliva secretion from the parotid gland shown in SLC12A2 knockout mice. 19 The S aureus parotitis of the proband and her sister was assumingly caused by obstruction of the parotid duct due to reduced secretion. Adult SLC12A2 knockout mice do not show the cystic fibrosis-like lung phenotype seen in our proband, presumably due to compensatory mechanisms not found in tracheal epithelia from adult rabbits and humans. 20,21 The impaired hearing and the gastrointestinal problems of our proband are likewise described in SLC12A2 knockout mice. [22][23][24] Recently, a boy with a homozygous 22-kb deletion in SLC12A2 due to paternal parental isodisomy was described as the first convincing case of SLC12A2-related disease. 11 A close resemblance with our proband includes reduced sweat, saliva, and tear fluid, cystic fibrosis-like respiratory problems, intestinal malrotation, a severe neurodevelopmental disorder, hearing impairment, and similar dysmorphic facial features. Whether the boy had any neurologic symptoms in the neonatal period is not reported. Because it is not clearly stated, the neonatal course was presumably not as dramatic as in our patients. The only MRI performed at 9 months is described as showing decreased brain volume involving both gray and white matter, whereas MRI at 4 years in our proband did not show atrophy. Whether CSF analysis was performed is not stated. There are thus to date only 3 patients reported, all of whom are homozygous for completely inactivating mutations in SLC12A2. Further delineation of genotype-phenotype relationships, including potential consequences of partially inactivating mutations, awaits identification of additional cases.
In 2016, a patient carrying a heterozygous de novo truncating variant in SLC12A2 was reported. 25 With first symptoms at 6 months, this female patient developed a multisystem disorder with respiratory, circulatory, gastrointestinal, and endocrine/ metabolic dysfunction. Although the patient's disorder has little resemblance with the phenotype of the 3 known patients with homozygous loss of function mutations or SLC12A2 knockout mice, the possibility remains that the truncating variant was responsible for the phenotype through a different, gain-of-function mechanism. We report 2 sisters, with biallelic loss-of-function variants in SLC12A2 causing an NKCC1 deficiency syndrome with a characteristic constellation of neurologic symptoms and secretory impairment in multiple organs. Identification of additional cases is needed to assess phenotypic variability and genotype-phenotype correlations. The relative roles of prenatal and postnatal disease mechanisms, and whether acute or progressive encephalopathy is part of the syndrome, need to be further explored because this can affect treatment possibilities.