Biallelic Cys141Tyr variant of SEL1L is associated with neurodevelopmental disorders, agammaglobulinemia, and premature death

Suppressor of lin-12-like–HMG-CoA reductase degradation 1 (SEL1L-HRD1) ER-associated degradation (ERAD) plays a critical role in many physiological processes in mice, including immunity, water homeostasis, and energy metabolism; however, its relevance and importance in humans remain unclear, as no disease variant has been identified. Here, we report a biallelic SEL1L variant (p. Cys141Tyr) in 5 patients from a consanguineous Slovakian family. These patients presented with not only ERAD-associated neurodevelopmental disorders with onset in infancy (ENDI) syndromes, but infantile-onset agammaglobulinemia with no mature B cells, resulting in frequent infections and early death. This variant disrupted the formation of a disulfide bond in the luminal fibronectin II domain of SEL1L, largely abolishing the function of the SEL1L-HRD1 ERAD complex in part via proteasomal-mediated self destruction by HRD1. This study reports a disease entity termed ENDI-agammaglobulinemia (ENDI-A) syndrome and establishes an inverse correlation between SEL1L-HRD1 ERAD functionality and disease severity in humans.


Genetic analysis
The informed consent was obtained from the parents of the patient and this study was approved by Johannes Kepler University Ethics Committee (Approval No: 1253/2021).The venous blood samples were collected for genomic DNA extraction from individual (III-3, III-4, IV-4 and IV-6).
The Human Genome CGH Microarray (4x44K Agilent Technologies, USA) designed with enhanced coverage on known genes (24 Kb) were performed in Patients 3 and 5, and parents.Subsequently, SurePrint G3 Human Genome CGH+SNP 2x400K Microarray was performed in Patients 3 and 5, to increase the resolution with the median spacing of 4.5 Kb in Refseq genes.
Appropriate sex-matched samples included in SureTag Complete DNA Labeling Kit (Agilent Technologies, USA) were used as references.DNA labeling, hybridization and washing were performed according to the manufacturer's protocols (Agilent).Microarray slides were scanned using SureScan Microarray Scanner.Data were analyzed with default algorithm settings using Cytogenomics Software (Agilent Technologies, USA).Pathogenicity of all identified CNVs variants was verified in the UCSC Genome Browser, Database of Genomic Variants (DGV), International Standards for Cytogenomic Arrays (ISCA) database and DECIPHER.
Trimmed reads were then mapped against the human genome (GRCh37 / hg19; match score 1, mismatch cost 2, insertion and deletion cost 3, length fraction 0.5 and similarity fraction 0.8, non-specific match handling was set to map randomly) and locally realigned (multi-pass realignment 3).Variants were called using the low frequency variant detection tool (required significance 1%, minimum coverage 10, minimum count 2, minimum frequency 1%, base quality filter with neighborhood radius 5, minimum central quality 20, and minimum neighborhood quality 15, direction and position filters with direction frequency 5%, relative read direction filter significance 1%, and read position filter significance 1%).Called variants were then processed for storage in a database (MariaDB 10.1, MariaDB Corporation, USA).Variants were annotated using the Ensemble variant effect predictor (VEP) tool (1) and also separately the combined annotation dependent depletion (CADD) tool (2).VEP annotated variants were then filtered against their allele frequency (AF): variants having AF>1% in any (sub-) population were filtered out and only variants with AF£1% or unknown AF were further processed.Remaining variants were then filtered against their CADD PHRED score (³10) and affected genes were annotated by HGNC multi-symbol checker, UniProtKB, and OMIM.Genes were also queried for pathway involvement by reactome (3), for gene-gene interactions by string-db (4), and gene expression by BioGPS (5), and Bgee (doi 10.1007/978-3-540-69828-9_12).

Immunohistochemistry
Duodenum samples of patient 3 and samples with inconspicuous cases or other intestinal disorders (Supplemental Table 3) were used for immunohistochemistry staining.

Immunofluorescence
Human primary skin fibroblasts were seeded to a maximum of 50% confluency on culture slides.
After overnight fixation in neutral buffered 4% formaldehyde solution at 4°C cells were washed 3

Immunoprecipitation (IP)
HEK293T cells transfected with the indicated plasmids were snap-frozen in liquid nitrogen and whole cell lysate was prepared in IP lysis buffer [150 mM NaCl, 0.2% Nonidet P-40 (NP40), 0.1% Triton X-100, 25 mM Tris-HCl pH 7.5] at 4°C, supplemented with protease inhibitors, protein phosphatase inhibitors, and 10 mM N-ethylmaleimide.A total of ~5 mg protein lysates were incubated with 10 μl anti-FLAG agarose (Sigma, #A2220) overnight at 4°C with gentle rocking.The incubated agaroses were washed with IP lysis buffer for three times and eluted in SDS sample buffer at 95 o C for 5 min followed by SDS-PAGE and Immunoblot.