Infantile hypotonia with psychomotor retardation and characteristic facies-1 (IHPRF1, MIM#615419) is a rare autosomal recessive disorder with an onset at birth or early in infancy, with a typical severe course. Individuals affected by IHPRF1 are generally characterized by moderate-severe hypotonia, global developmental delay, and dysmorphic features (Al-Sayed et al., 2013; Bramswig et al., 2018; Karimi, et al., 2020) IHPRF1 is caused by homozygous truncating variants in NALCN gene (MIM#611549) on chromosome 13q33 resulting in a loss-of-function (LoF) effect (Bramswig et al., 2018; Bend et al., 2016) NALCN encodes for sodium leak channel, non-selective (NALCN) protein, a voltage-independent, nonselective cation channel that belongs to a family of voltage-gated sodium and calcium channels regulating the resting membrane potential and excitability of neurons with passive, intracellular movement of sodium ions (National Library of Medicine: NCBI, 2024). Novel evidence suggests that NALCN plays a role also in other pathways such as pain sensation (Zhang and Wei, 2023). NALCN LoF variants lead to reduced sodium leak causing hyperpolarization of the resting membrane potential explaining the hypoexcitability phenotype that characterizes IHPRF1 disease (Bramswig et al., 2018; Bend et al., 2016). Interestingly, heterozygous pathogenic variants in NALCN gene are associated, with another disorder predominantly characterized by congenital contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD, MIM#616266) that owns a significant phenotypic overlap degree with IHPRF1, but also shows distal arthrogryposis (Chong et al., 2015).

Other important IHPRF1 features are seizures, brain anomalies (cortical atrophy and/or thin corpus callosum, e.g.,), muscle wasting, strabismus and/or nystagmus, scoliosis, feeding difficulties, constipation, sleep disturbance (secondary to altered circadian rhythm) and breathing abnormalities. Dysmorphic features frequently reported in IHPRF1 are prominent forehead with triangular face, microcephaly, micrognathia, smooth philtrum, thin upper lip, low-set and large ears, slender nose, and strabismus (Al-Sayed et al., 2013; Liu et al., 2013; Bramswig et al., 2018). Periodic breathing and sleep apnea, that usually increase in severity during sleep, are frequently reported both in patients with IHPRF1 and CLIFAHDD (Lozic et al., 2016; Gal et al., 2016; Bourque et al., 2018; Campbell et al., 2018; Maselli et al., 2022; Winczewska-Wiktor et al., 2022) and are generally alleviated by oxygen supplementation or in some cases with support of Non-Invasive Ventilation (NIV) (Lozic et al., 2016).

To date 48 patients have been reported with IHPRF1 (Al-Sayed et al., 2013; Köroğlu et al., 2013; Farwell et al., 2015; Gal et al., 2016; Angius et al., 2018; Bourque et al., 2018; Bramswig et al., 2018; Campbell et al., 2018; Carneiro et al., 2018; Takenouchi et al., 2018; Karimi, et al., 2020; Ope et al., 2020; Maselli et al., 2022; Khan et al., 2022; Abul-Husn et al., 2023; Tehrani Fateh et al., 2023; Susgun et al., 2024) with 38 different NALCN variants from 26 unrelated and 22 related patients (33 different families) in homozygosity or compound heterozygosity. Here, we report on five new IHPRF1 patients enrolled in the framework of an international multicentric collaboration study, four with homozygous variants and one with compound heterozygous deleterious variants in NALCN gene, in order to: I. further delineate the IHPRF1 genotype-phenotype correlation and its clinical management, II: broaden the spectrum of biallelic variants in NALCN gene, and III: assess the mechanistic consequences of these variants on the protein structure and function by resorting to specialized in silico pathogenicity predictors and ad hoc structural analyses.

Pathogenetic variants of the NALCN gene, in homozygosity or compound heterozygosity with a putative LoF role, are associated with IHPRF1, that is inherited in an autosomal recessive manner, in contrast to heterozygous pathogenic variants in NALCN with a GoF effects, that are associated with CLIFAHDD. According to Monteil et al. (2024), variants reported in CLIFAHDD are mostly missense and are concentrated within the pore-forming S5 and S6 helices, while IHPRF1 variants are broadly distributed and the majority are truncating variants (nonsense, frameshift or deletions) (Monteil et al., 2024). LoF variants in IHPRF1 seem to cause hyperpolarization of the resting membrane potential explaining the hypoexcitability phenotype that characterizes IHPRF1 disease with profound hypotonia (Bend et al., 2016; Bramswig et al., 2018). On the other hand, most of the literature seems to indicate that the putative mechanism underlying CLIFAHDD is the gain of function of NALCN explaining the presence of arthrogryposis caused by hyperexcitability of the motor unit, secondary to the depolarization of the resting membrane potential, resulting in a hypercontracted phenotype (Bramswig et al., 2018; Bouasse et al., 2019). However, according to Bend et al. (2016), some variants reported in CLIFAHDD, seem to have a loss of function mechanism acting as dominant negative in functional assays on C. elegans (CLIFAHDD-dominant Antimorphic) (Bramswig et al., 2018).

In this study, we reported five male patients from four different families of whom four carried homozygous variants (patient 2, 3, 4, and 5) and one (patient 1) inherited a compound heterozygosity in the NALCN gene. Only the nonsense variant c.2563C > T (p.Arg855Ter) retrieved in patient 1 was already previously reported by Karimi et al. (2020), the others (1 splicing, 1 missense, 1 frameshift, and 1 nonsense) are retrieved for the first time. To date, 36 different variants have been reported in the literature in homozygosis or compound heterozygosis associated to IHPRF1, and among them, 12 nonsense, 9 missense, 9 frameshift, 5 splicing, and 1 synonymous variant (Figure 3; Supplementary Table S2). Frequencies of the main clinical features are comparable with those previously reported in literature (Table 1; Figure 1). Considering the clinical phenotype of our patients is it possible to observe that the whole cohort experienced feeding difficulties (needing in some cases NG or G tube) and FTT. This finding is consistent with the literature on IHPRF1 (Al-Sayed et al., 2013; Bramswig et al., 2018; Campbell et al., 2018). Interestingly, patient 1, after 6 months of linear growth, presented with severe FTT, in the presence of adequate caloric intake. Finding at brain MRI of a large arachnoid cyst in the suprasellar region needing surgical corrections led to the clinical diagnosis of DS. DS is a rare cause of FTT in infants associated with central nervous system tumors in the hypothalamic region. Clinical presentation includes severe emaciation, lipodystrophy, normal linear growth, and histories of poor weight gain starting at about 6 months of age despite having normal caloric intake (Burr et al., 1976; Trapani et al., 2022). Other features described but more inconstant are hyperalertness, hyperkinesia, hydrocephalus, nystagmus, visual field defects, and vomiting. The DS is characterized by the absence of specific laboratory findings related to hypothalamic dysfunction and it is often a diagnosis of exclusion after a long differential diagnostic work-up (Burr et al., 1976; Fleischman et al., 2005). MRI is considered the gold standard for the diagnosis of a cerebral mass causing DS (Hoffmann et al., 2014; Trapani et al., 2022). Treatment includes surgical resection, radiation therapy, and chemotherapy, and it is often followed by weight gain (Kim, 2012; Tosur et al., 2017). Significantly, during the diagnostic workup for patient 1 an elevation of urinary oligosaccharides with increased levels of glucose tetrasaccharide (Glc4) was reported. Glc4 (Glca1-6Glca1-4Glca1-4Glc), is a glycogen-derived dextrin that correlates with the extent of glycogen accumulation in skeletal muscle (Young et al., 2009). Glc4 is described in the literature as the most sensitive biochemical biomarker of Glycogen storage diseases (GSDs) type II (acid α-glucosidase deficiency, Pompe disease, PD, OMIM #232300) a metabolic disorder affecting mainly the liver and muscle. It was described in patients performing a whole-body muscle magnetic resonance imaging a correlation between muscle strength, the amount of fatty infiltration in muscle, and urinary Glc4 (Khan et al., 2020). The degree of elevation appears to correlate with the severity of the clinical phenotype and the stage of Pompe disease (Piraud et al., 2020; Heiner-Fokkema et al., 2020), but it can be elevated in other conditions associated with increased glycogen storage, including certain leukemias and sarcomas, Duchenne muscular dystrophy and acute pancreatitis (Young et al., 2009). Glc4 levels were markedly increased also in three disorders belonging to the autophagy machinery: Vici syndrome, Yunis-Varon syndrome, and Danon disease, all sharing a cardiomuscular involvement with increased glycogen storage and variable vacuoles accumulation detectable on light microscopy at the level of skeletal and cardiac muscle (Semeraro et al., 2021; Mastrogiorgio et al., 2021). Since muscular wasting and severe hypotonia are frequently reported in IHPRF1, we hypothesized that Glc4 could be a marker of the neuromuscular damage in this condition, so we measured Glc4 also in patient 2 but the examination tested negative. Interestingly, the urinary glucosaccharides exam was repeated in patient 1 after neurosurgery and amelioration of the growth pattern, with Glc4 levels normalization. This finding led us to hypothesize that Glc4 elevations may be considered as a DS biomarker which could be associated likewise retrieved in patient 1, with severe muscular wasting and FTT. Of note leptin levels in our patient were suppressed in several determinations, with a slight increase after surgery and weight gain. Leptin is a pleiotropic hormone primarily secreted in adipose tissue by adipocytes and is implicated in a wide range of biological functions that control different processes such as the regulation of body weight and energy expenditure, stimulates thermogenesis, and reduces appetite, reproductive function, immune response, and bone metabolism (Greco et al., 2021). In recent years, leptin has been widely studied in obesity and anorexia nervosa, and its plasma levels are related to BMI (Miller, 2011). In literature, DS is associated in large case series with low levels of leptin and concomitant increase of ghrelin (Brauner et al., 2006; Trapani et al., 2022). In some cases, reductions in leptin levels have been reported after surgical lesions’ excision probably due to a direct secretion by the tumor mass (Velasco et al., 2014). In humans, decreasing leptin concentrations in response to food deprivation are responsible for the starvation-induced suppression of the hypothalamic-pituitary-gonadal axes (Sainsbury et al., 2002). Low levels of leptin have been observed also in patients with FTT (Shaoul et al., 2003). According to Monteil et al. (2024) leptin seems to act as a positive regulator of NALCN. Recent evidence demonstrated how leptin is a potent respiratory stimulant, by activating NALCN to depolarize neurons type 1 of the solitary tract (NTS) that express the long form of leptin receptor (LepRn), providing a glutamatergic pathway that relays excitation to premotor breathing control areas in the ventral respiratory group (rVRG) which harbors bulbospinal inspiratory premotor neurons (Do et al., 2020). Do et al. (2020) also showed that the leptin-induced depolarization in type-1 cells was abolished in NALCN-cKO mice (selective deletion of NALCN in LepRb neurons), with an increase of breathing irregularities, concluding that NTS LepRb neurons contribute to the stability of the respiratory pattern and reduce the occurrence of both central and obstructive apneas. Moreover, previous studies demonstrated that injection of leptin into NTS of rats stimulates respiratory output (Inyushkina et al., 2010). This observation led us to speculate on the hypothesis that low levels of leptin in the context of IHPRF1 or CLIFAHDD can worsen the clinical picture of these patients. In this perspective, good management of FTT and weight loss could ameliorate the phenotype of the patients with IHPRF1, in particular for periodic breathing. Patient 1 presented a progressive increase of leptin values after surgery and weight gain (from 0.06 ng/mL to 1.18 ng/mL, even if still under reference value) and this was accompanied by improvement of clinical condition and periodic breathing. The patient had already started oxygen supplementation so it is difficult to assess if the increase in leptin levels may have improved this aspect. We suggest that further studies on leptin levels in a larger cohort of patients with IHPRF1 and CLIFAHDD with FTT are needed to better assess the role of this hormone in the variability of clinical features.

Breathing abnormalities are frequently reported in both patients with IHPRF1 (Campbell et al., 2018; Maselli et al., 2022) and CLIFAHDD (Gal et al., 2016; Lozic et al., 2016; Bourque et al., 2018; Winczewska-Wiktor et al., 2022). NALCN mutant mice have a severely disrupted respiratory rhythm interrupted by periods of apnea and die within 24 h of birth. Breathing pattern in NALCN null mice is characterized by apnea for 5 s followed by a burst of breathing for 5 s at a rate of 5 episodes of apnea per minute imputable to a complete loss of electrical activity from the fourth cervical root (C4) that innervates the diaphragm with rhythmic electrical signals (Lu et al., 2007). Respiratory rhythms are generated by inspiratory neurons in the pre-Bötzinger complex (preBötC) network of the medulla generating the rhythmic output to cranial and spinal motor neurons. This network is modulated by Substance P that exerts excitatory effects in preBötC neurons by modulating a nonselective cation channel with the properties of NALCN (Lozic et al., 2016; Monteil et al., 2024). Moreover, NALCN is expressed in CO₂/H (+)-sensitive neurons of retrotrapezoid nucleus (RTN) that regulate breathing. In vivo, RTN-specific knockdown of NALCN reduced CO₂-evoked neuronal activation and breathing (Shi et al., 2016). The main treatment for periodic breathing and apnea in the reported cases is based on oxygen supplementation at night (Lozic et al., 2016; Campbell et al., 2018), in some cases requiring NIV (Maselli et al., 2022). Tracheostomy is rarely required (Lozic et al., 2016; Vivero et al., 2017). In our cohort, we reported three patients with breathing abnormalities, patient 1 with periodic breathing and severe central apneas requiring oxygen supplementation, patient 2 with periodic breathing and severe central apneas requiring NIV (discontinued on parent’s decision) and patient 4 with mild central apneas not requiring medical treatment. Interestingly, an increase in urinary homovanillic acid, vanilmandelic acid and NSE values was observed over time in patient 1 during the workup for the presence of brain cysts (before detecting a benign lesion at anatomopathological evaluation) that did not normalize after surgery. Conversely, after the introduction of oxygen supplementation, a decrease in the respective values was observed, with normalization of the homovanillic/vanilmandelic acid ratio (Supplementary Figure S3). Thus, it is possible to hypothesize that the progressive normalization of the urinary homovanillic acid, vanilmandelic acid and their ratio, could be associated to the amelioration of periodic breath, after the low flow oxygen therapy. In literature, several studies suggested an alteration of the sympathetic nervous system with an increase in urinary norepinephrine levels in apneic patients (Dimsdale et al., 1995). To date, most of the studies evaluated this relation in obstructive sleep apnea (OSA). In a recent pediatric systematic review, it was reported that catecholamine levels can be pointed out as a marker for sympathetic nervous system excitability in OSA, and correlate with specific clinical, cardiovascular, neurobehavioral, and metabolic parameters, contributing to increased morbidity (Sica et al., 2021). It is shown that continuous positive airway pressure (CPAP) treatment in patients with OSA reduces catecholamine levels and blood pressure (Green et al., 2021). Sympathetic nervous system activation was observed also in patients with central sleep apnea (McLaren et al., 2019). Recurrent infections, in particular respiratory tract infections, are another important clinical finding among IHPRF1 and CLIFAHDD patients (Bramswig et al., 2018; Karimi, et al., 2020; Tehrani Fateh et al., 2023). In our cohort, we detected two patients with recurrent infections, patient 1 with frequent respiratory infections requiring antibiotic prophylaxis, and patient 3 with multiple episodes of pneumonia. According to Bramswig et al. (2018), recurrent infections in the cohort were not associated with immunological alterations (specific information on test performed was not reported) and were correlated with FTT. Recently, Tehrani Fateh et al. (2023) described a IHPRF1 patient with recurrent urinary tract infections hypothesizing that abnormal contraction of detrusor muscle due to NALCN LoF could lead to incomplete emptying of the bladder with an increased risk of infections. However, patient 1 of the present cohort was found with partial functional defects of T and B lymphocytes that were reported after in vitro stimulation with PHA, OKT3, and CpG and a reduction of NK activity. According to Karimi et al. (2020), male individuals seem to display a more severe phenotype when compared to female individuals, even in the same family. Interestingly, estrogen and progesterone play a role in the regulation of NALCN expression in human myometrial muscle cells suggesting a sex-driven regulation for NALCN. All patients in the present cohort are males and some features such as epilepsy and breathing abnormalities are slightly higher compared to the literature review (which includes both male and female individuals), confirming that the male sex worsens the phenotype in this disease.

Finally, all variants cause a markedly significant functional impact, thereby compromising the functionality of the protein complex, as indicated by in silico and structural analyses. Variants introducing premature stop codon may act at different levels, mutant transcripts might undergo nonsense-mediated mRNA decay, or, alternatively, the proteins could be produced but may be severely affected in terms of function and interaction with other elements of the complex. When considering patients harboring nonsense truncating variants in codons 842 (p.Arg842Ter) and 855 (p.Arg855Ter) in the present cohort, namely, patient 1 and patient 2 and the two siblings described by Karimi et al. (2020), it is interesting to observe that all of these four patients seems to display breathing abnormalities that in our two cases are severe, needing in one case oxygen supplementation (patient 1) and NIV (patient 2). We also reported a new missense variant in homozygosity, p.Cys1417Tyr, located into an extracellular soluble loop of the protein. To date, missense variants are less frequently reported as pathogenic, and, only one missense variant c.3860G > T, (p.Trp1287Leu) was reported in homozygosity in three patients from the same family localizing on a site close to the outer region of the channel, with likely milder functional consequences (Al-Sayed et al., 2013). Close to the missense homozygous variant p.Cys1417Tyr described in the present work, a missense variant c.4281C > A, p.Phe1427Leu in compound heterozygosity with a splicing variant (c.4281C > A and c.4103 + 2T > C) in two siblings located in the predicted S6 pore forming segment of domain IV was reported (Bramswig et al., 2018). Of interest, the phenotype of these patients, along with the clinical picture of patient 3 from the present cohort harboring the missense homozygous variant c.4250G>A p.Cys1417Tyr seems to be milder compared with patients harboring truncating variants in NALCN. Further studies and functional in vitro analyses will be necessary to characterize the molecular effects that truncating and missense variants have at the molecular level to enable more accurate genotype-phenotype correlations.

In this study, we focused on WES, in silico pathogenicity predictors, and structural analyses to examine genetic variants retrieved from a new IHPRF1 patients’ cohort. We described five new IHPRF1 patients from four different families, harboring four newly identified and one previously retrieved variant whose deep investigation exhibited a markedly significant functional impact, thereby compromising the functionality of the NALCN complex. The present findings, taken together, widen the pathogenic mechanism and functional spectrum of biallelic variants affecting the NALCN gene, as well as the IHPRF1 syndrome diagnosis and genotype-phenotype correlation.