Clinical spectrum
The clinical findings of the 15 individuals are summarized in Table 1. Comparison of the clinical presentation of this cohort with previously reported cases is summarized in Table 2. Pedigrees are shown in Fig. 1a and detailed clinical reports are provided in Supplementary File 2 and Supplementary Figure 1.
Our cohort of new cases comprised 15 individuals (ten females and five males) from 12 unrelated families. Consanguinity was reported in seven of them. All documented birth measurements were within the normal range. Postnatal adaptation and development in the first weeks of life were unremarkable in 14 of the 15 children. Only one child (individual 5) developed postnatal complications in the form of respiratory distress and pulmonary hypertension. In the following months, recurrent upper respiratory tract infections, anemia and hepatomegaly were noted, and she died prematurely of viral pneumonia at two years of age. All other individuals were aged between 30 months and 19 years at the last follow-up. Thirteen of the 15 individuals had no respiratory or pulmonary symptoms. At the last clinical examination, four individuals (ages five, six, eight and 19) had microcephaly (SD below −2). Short stature and decreased body weight were observed in seven and nine individuals, respectively, with five individuals having both. All 15 individuals showed global developmental delay and later intellectual disability (ID), which in most cases was classified as moderate or severe. Nine children were non-verbal at the last assessment, and five could speak only a few rudimentary words. In four cases, speech regression occurred after 1–3 years of age, and the siblings (individual 1 and 2) had motor regression starting from the age of six. Behavioral abnormalities (hyperactivity, impulsive, aggressive, anxious and autistic behavior) were reported in seven individuals. All 15 individuals had variable neuromuscular involvement: 12 had truncal hypotonia, five individuals were never able to walk and four developed hyperkinetic movement disorder, spasticity or hyperreflexia. Two depended on a wheelchair for long distances and five had an unsteady, wide gait. Seizures occurred in 11 individuals, with age of onset ranging from nine months to 13 years. In individual 5, epileptiform discharges were documented but no clinical seizure was observed. In six individuals, multiple antiepileptic drugs were tried due to intractable seizures and two children were fitted with a vagus nerve stimulation (VNS) device. Brain abnormalities (dilated ventricles, corpus callosum hypoplasia, mild cortical atrophy and delayed myelination) were diagnosed in five individuals, while six had unremarkable brain MRI scans. Other less common findings included strabismus (n = 3), diarrhea and/or malabsorption (n = 3), recurrent infections (n = 2), anemia (n = 2), congenital heart defect (n = 2) and unilateral renal reflux (n = 1).
Molecular findings
In addition to the recurrent missense variant p.(Asp148Tyr), we identified nine novel variants, including one start-loss variant, three missense variants, two single amino acid in-frame deletions, two nonsense and one C-terminal frameshift variant. The localization of the variants, their level of conservation and the pathogenicity prediction using different in silico tools are summarized in Fig. 1b–d and Supplementary Table 2. The results of the segregation analysis are shown in Supplementary Fig. 2. While RT-qPCR showed no clear difference in NHLRC2 expression levels between patient and control LCLs (Fig. 2a), Western blot showed a clear reduction in NHLRC2 protein levels in all patient samples tested compared to control samples (Fig. 2c, d). Consistent with the RT-qPCR results, Sanger sequencing of cDNA from patients’ LCLs confirmed all variants at the mRNA level (Fig. 2b and Supplementary Fig. 3).
In one individual with early pulmonary distress and severe multisystem involvement (individual 5), the recurrent missense variant p.(Asp148Tyr) was detected in trans with an N-terminal nonsense variant p.(Gln50*). In seven individuals with overall less severe neurological manifestations and without pulmonary symptoms, ES detected the known pathogenic variant p.(Asp148Tyr) in homozygous state. The homozygous NHLRC2 start-loss variant c.1A>G was detected in four individuals from two unrelated families (A and F) in association with a severe and progressive neurological phenotype without pulmonary disease. The variant is absent in controls according to the gnomAD database and the closest in-frame alternative translation start codon is located at c.433, p.145. By analyzing the individual vcf files we confirmed that they share the same disease haplotype (Supplementary Fig. 4). NHLRC2 protein levels, extracted from LCL-derived cells of individuals 1 and 2, were strongly reduced compared to control samples (Fig. 2c, d).
In individual 4, presenting with neuroregression and epileptic encephalopathy, a nonsense variant p.(Leu584*) was detected in combination with a rare missense variant p.(Asp692Tyr) affecting a moderately conserved residue located in the ß-strand domain. The nonsense variant is predicted to undergo nonsense-mediated decay (NMD) and Western blotting showed a strong reduction in NHLRC2 protein levels compared to control samples (Fig. 2c, d).
Two missense variants p.(Gln365Pro) and p.(Phe462Ser), affecting highly conserved residues within the ß-propeller domain, were detected in compound heterozygous state in individual 6, in association with speech regression, mild gait disturbance and medication-responsive epilepsy. While the former variant is listed once in heterozygosity in gnomAD, the latter has an allele frequency of 0.1% in the non-Finnish European population.
In individual 10, presenting with a severe disease including respiratory distress, malabsorption, anemia and recurrent infections, ES detected a single amino acid in-frame deletion affecting the highly conserved residue p.Glu33. Individual 11 was compound-heterozygous for an in-frame deletion p.(Gln333del) and a C-terminal frameshift variant that is unlikely to undergo NMD. She had severe neurological manifestations including epileptic encephalopathy and was previously reported with USP19 as a candidate gene [19].
In vitro studies of missense and in-frame deletion variants
Firstly, to test the impact of the non-truncating variants identified in patients from our study (n = 5), secondly to compare them with previously reported missense variants associated with pulmonary symptoms (n = 4) and thirdly to critically review the strongly reduced NHLRC2 protein levels observed in LCLs, we cloned all nine non-truncating NHLRC2 variants together with a wildtype control into pcDNA3. For reliable detection, we added a C-terminal Flag-tag (Fig. 3a, b, Supplementary Table 1).
Western blot of HEK293 cells transfected with these different NHLRC2 constructs (Fig. 3c, d) showed a reproducible reduction in NHLRC2 protein levels for the recurrent p.(Asp148Tyr) variant. It also showed a strong reduction in mutant NHLRC2 protein levels for the p.(Glu33del) variant identified in the severely affected individual 10 and for the p.(Asp75Val), p.(His143Pro), p.(Pro338Leu) and p.(Glu365Pro) missense variants. The first three missense variants were all identified in trans with the recurrent p.(Asp148Tyr) variant in individuals with a severe multisystem phenotype including respiratory symptoms (Fig. 3e) [6, 7]. The p.(Glu365Pro) variant was detected in trans with the p.(Phe462Ser) variant in individual 6 without pulmonary involvement. It is noteworthy that the p.(Phe462Ser) variant as well as the p.(Gln333del) variant identified in individual 11 in trans with p.(Gln705Leufs*4) and the missense variant p.(Asp692Tyr) detected in trans with p.(Leu584*) in individual 4 showed only slightly reduced protein levels.
To investigate for a possible genotype-phenotype correlation, we compared these findings to variants and their combinations identified in individuals with and without pulmonary disease (Fig. 3e). We observed a correlation of remaining NHLRC2 protein levels with phenotype severity: higher reduction in total NHLRC2 protein level correlated with more severe phenotypes, ranging from milder over more severe neurological symptoms to additional lung disease (Fig. 3f).
In silico modeling of non-truncating variants
To further investigate how the NHLRC variants that still result in stable NHLRC2 protein, as deduced from the in vitro overexpression assays, affect the folding of the protein, we modeled the missense variants p.(Asp148Tyr), p.(Asp692Tyr) and p.(Phe462Ser) and the p.(Gln333del) variant with the AlphaFold tool (Fig. 4). In the recurrent missense variant p.(Asp148Tyr), the replacement of the negatively charged amino acid aspartate by the polar, uncharged tyrosine is predicted to result in the loss of several hydrogen bonds (Fig. 4a, b). Analysis of the internal model accuracy estimates as measured by the pLDDT score at the p.148 position in the wildtype vs. the mutant model revealed a prediction accuracy reduction at the position of the exchange. Notably, this position has previously been predicted with high certainty in the wildtype (Fig. 4c, d) and is also predicted with certainty in other models not affecting the same amino acid position (Supplementary Fig. 5). All four other variants analyzed also lead to the loss of at least one hydrogen bond formed at the wildtype position of the respective variant (Fig. 4e).
Further analysis of the amino acid positions affected by the missense variants with the MaSIFtool suggested possible protein binding sites in the vicinity to amino acids Gln333, Phe462 and Asp692. We therefore modeled the respective variants and their potential effect on these binding sites. While we observed a decrease in the probability of protein-protein interaction at Gln333 and Phe462 for the p.(Gln333del) and p.(Phe462Ser) variants, respectively, we could not detect such an effect for the p.(Asp692Tyr) variant (Fig. 4e–h, Supplementary Figure 5).
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