key: cord-0253434-0hvod270
authors: Hebert, A.; Simons, A.; Schuurs-Hoeijmakers, J. H.; Koenen, H.; Zonneveld-Huijssoon, E.; Henriet, S. S.; Schatorje, E. J.; Hoppenreijs, E. P.; Leenders, E. K.; Janssen, E. J.; Santen, G. W.; de Munnik, S. A.; van Reijmersdal, S. V.; van Rijssen, E.; Kersten, S.; Netea, M. G.; Smeets, R. L.; van de Veerdonk, F. L.; Hoischen, A.; van der Made, C. I.
title: Trio-based whole exome sequencing in patients with suspected sporadic inborn errors of immunity: a retrospective cohort study
date: 2022-04-18
journal: nan
DOI: 10.1101/2022.03.30.22272929
sha: cdde2232daad22b7d92ec15faa38b9776f1e4c15
doc_id: 253434
cord_uid: 0hvod270

Background De novo variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) analysis in patients with suspected inborn errors of immunity (IEI). Methods This study explored the potential added value of systematic assessment of DNVs in a retrospective cohort of 123 patients with a suspected sporadic IEI who underwent patient-parent trio-based WES. Results A likely molecular diagnosis for (part) of the immunological phenotype was achieved in 12 patients with the diagnostic in silico IEI WES gene panel. Exome-wide evaluation of rare, non-synonymous DNVs affecting coding or splice site regions led to the identification of 14 candidate DNVs in genes with an annotated immune function. DNVs were identified in IEI genes (NLRP3 and RELA) and potentially novel candidate genes, including PSMB10, DDX1, KMT2C and FBXW11. The FBXW11 canonical splice site DNV, in a patient with autoinflammatory disease, was shown to lead to defective RNA splicing, increased NF-{kappa}B p65 signalling, and elevated IL-1{beta} production in primary immune cells. Conclusions This retrospective cohort study advocates the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI. Furthermore, we have provided functional evidence supporting a causal role for FBXW11 loss-of-function mutations in autoinflammatory disease. Funding This research was supported by grants from the European Union, ZonMW and the Radboud Institute for Molecular Life Sciences.

Cohort characteristics 2 This retrospective cohort study systematically re-analysed patient-parent trio whole exome sequencing (WES) 3 data of 123 patients with suspected inborn errors of immunity (IEI) with the aim to identify (likely) pathogenic 4 de novo single nucleotide variants (SNVs) or small insertion-deletions (indels) (Figure 1 ). Included IEI patients 5 had a median age of 9 years (IQR 2-17) and two-thirds of the cases were below 18 years of age (Table 1A) .

The sex distribution among patients was roughly equal. Classification of IEI phenotypes according to the 7 International Union of Immunological Societies (IUIS) indicated that most cases presented with 8 autoinflammatory syndromes, followed by immune dysregulation and combined, predominantly syndromal 9 immunodeficiencies (14). Eight patients remained unclassified due to limited clinical data.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Whole exome sequencing data from these patient-parent trios was subjected to standardised variant filtering 3 to retain candidate de novo variants. Subsequently, non-synonymous DNVs were systematically evaluated at 4 variant and gene level for their potential involvement in the patient's immunological phenotype. 

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Genetic variants reported after routine diagnostic whole exome sequencing analysis 1 Following routine diagnostic WES, potential disease-causing SNVs and/or copy number variants (CNVs) were 2 reported in 36 index patients (Table 1B) . Twenty-four patients were carriers of recessive disease alleles, 3 previously characterised risk factors, variants of uncertain significance (VUS) or (likely) pathogenic variants 4 affecting established disease genes other than those associated with IEI (Table 1B) . Of note, three of these 5 patients carried de novo CNVs (patient 21, 69 and 115).

In 12 patients, (likely) pathogenic SNVs were identified in known IEI genes that (partially) explain the 7 patient's immunological phenotype (Table 1B , details shown in Table 2A ). While the majority of variants was 8 inherited, one patient with Muckle-Wells syndrome (patient 59) carried a de novo missense variant in NLRP3 9 (NM_001079821.2:c.1049C>T p.(Thr350Met)). This variant has previously been described in patients with pathogenic (35, 36).

Overall, routine diagnostic WES analysis provided a likely molecular diagnosis for (part) of the 13 phenotype in 19 patients (15.4%) ( Table 1B, Table 2A ).

14 Table 1B. Genetic findings after routine diagnostic panel analysis. Genetic variants reported after routine 15 diagnostic whole exome sequencing analysis of the 123 patients included in this cohort of inborn errors of 16 immunity. Table 1Btable supplement 2. (patients 98 and 118). However, these patients did not share phenotypic features and the function of the 1 proteins encoded by these genes could not be linked to the respective patient phenotype.

2 Subsequently, all non-synonymous candidate DNVs were systematically evaluated based on 3 information on variant and gene level, leading to the selection of 14 candidate DNVs potentially causing IEI 4 (Table 2A and B), including the above-mentioned variant in the known IEI gene NLRP3. The 13 novel IEI 5 candidate DNVs were found in patients with different IEI phenotypes, although three subtypes reoccurred: 6 predominantly antibody deficiency (hypogammaglobulinemia), autoinflammatory disorder and bone marrow 7 failure. Candidate DNVs that were considered most promising based on variant and gene level metrics are 8 presented in more detail in the following paragraphs.

In a patient with an autoinflammatory phenotype characterised by mucocutaneous ulceration of mouth and genital area (patient 119, Table 2B ), a DNV was located in the canonical splice acceptor site preceding 11 exon 10 of RELA (NM_021975.3:c.959-1G>A). The guanine to adenine change was predicted to compromise the splice acceptor site by transferring it to the first guanine of exon 10, leading to an out-of-frame exon. The 13 resulting frameshift was therefore assumed to cause a reduction in functional RelA protein by nonsense-14 mediated decay. RelA is also known as p65 and is critically involved in nuclear factor kappa-light-chain- 

(NM_021975:c.559+1G>A), likely leading to a premature stop codon and haploinsufficiency (46) . Both, the 20 phenotype and proposed mutational mechanism in our patient, match this description. Although RELA has 21 already been reported as an IEI gene in a previous IUIS classification (48), it was not yet listed in the IEI in silico gene panel of our Department of Human Genetics (25), because evidence was considered insufficient 23 at the time. Based on these arguments, this DNV has only now been classified as pathogenic, which could 24 carry implications for therapy with anti-tumour necrosis factor alpha (TNFα) inhibitors (47).

In addition, a private de novo missense variant in PSMB10 was observed in a patient with clinically 26 diagnosed Omenn syndrome with severe combined immunodeficiency (SCID), ectodermal dysplasia, 27 alopecia, hypodontia and anonychia (patient 1, Table 2B ). The clinical phenotype of this patient has been 28 previously reported (28). The DNV was predicted to be pathogenic based on the majority of variant and gene 29 level metrics. In additional data that was available from a previously performed single nucleotide polymorphism 30 (SNP) micro-array, it was shown that the genomic location of PSMB10 was spanned by a partial somatic 31 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 18, 2022. ; 13 Furthermore, in a patient with common variable immunodeficiency (CVID) due to a B cell maturation viral infections, psoriasis and alopecia areata, another candidate DNV was identified in DDX1 (patient 49, 

Another frameshift DNV was carried by a patient with a syndromal combined immunodeficiency 21 characterised by recurrent ear infections, developmental delay, low-average intelligence level and facial dysmorphism (patient 78, Table 2B ). The variant was predicted to lead to a loss-of-function (LoF) of the KMT2C 23 protein, which acts as a histone methyltransferase. In humans, de novo LoF mutations in KMT2C are 24 associated with Kleefstra syndrome 2, a neurodevelopmental disorder (60). Two of the six individuals 25 described in that study were reported to have recurrent respiratory infections (60). The occurrence of 26 immunological symptoms in patients with mutations in chromatin-regulating genes is increasingly being 27 recognized in the field of intellectual disability (ID) (66, 67). Therefore, more in-depth characterisation of 28 patients with KMT2C mutations and predominant ID phenotypes might indicate (mild) immunological 29 phenotypes that overlap with the phenotype of our patient, in support of pathogenicity of the observed DNV.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022.

Lastly, a DNV was identified in FBXW11, carried by a patient with an autoinflammatory disorder 1 characterised by recurrent periodic fever and severe headaches (patient 53, Table 2B ). FBXW11 encodes a 2 component of SCF (SKP1-CUL1-F-box) E3 ubiquitin ligase complex, TrCP2, that is involved in the regulation 3 of NF-κB signalling through the ubiquitination of several of its components (61, 68). An important function of 4 both the TrCP1 and TrCP2 isoforms is the regulation of IκBα degradation, leading to subsequent activation of 5 NF-κB and release of pro-inflammatory cytokines (69, 70). The identified DNV affected the canonical splice 6 acceptor site preceding exon 12 (NM_012300.2:c.1468-2A>G). This DNV was predicted to lead to skipping of 7 exon 12 based on splicing prediction by the Alamut Visual Software and to be deleterious by all utilised in silico 8 prediction tools. The predicted RNA splicing defect leading to an in-frame, shortened RNA transcript was 9 confirmed in Epstein-Barr virus (EBV) transformed B cells from the patient (Figure 2 ).

The other candidate DNVs will not be described in detail here, as there is insufficient evidence to 11 suggest pathogenicity or a genotype-phenotype relationship. Future discovery of cases with DNVs in the 12 presented genes and overlapping clinical phenotypes could encourage further in-depth research into the 13 possible mutational mechanisms.

14 Functional validation of FBXW11 de novo variant

In addition to systematic DNV analysis, we have selected the candidate DNV in FBXW11 for functional 16 validation as part of this study to provide further evidence for a causal genotype-phenotype relationship (patient 17 53, Table 2B ). As such, the putative effects on NF-κB signalling and the downstream production of pro-18 inflammatory cytokines were investigated in peripheral blood mononuclear cells (PBMC) extracted from the 19 patient and a healthy control. In unstimulated PBMC of the patient showed higher levels of phosphorylated 20 NF-kB p65 compared to the control. Ex vivo stimulation of these PBMC with phorbol 12-myristate 13-acetate 21 (PMA) and ionomycin for 30 minutes led to higher NF-κB activation, reflected by p65 phosphorylation CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101/2022.03.30.22272929 doi: medRxiv preprint

We investigated the potential benefit of exome-wide trio-based sequencing over routine single whole exome 2 sequencing (WES) analysis in a retrospective cohort of 123 patients with sporadic, suspected inborn errors of 3 immunity (IEI). Systematic analysis of de novo single nucleotide variants (SNVs) and small insertion-deletions 4 (indels) led to the identification of 14 candidate de novo variants (DNVs), of which two were in known IEI genes 5 and classified as pathogenic (NLRP3, RELA). Of the 12 variants in potentially novel candidate genes for IEI, 6 four were considered to be most likely pathogenic (PSMB10, DDX1, KMT2C, FBXW11) based on gene and 7 variant level metrics. Additionally, we have provided functional evidence that the FBXW11 splice site DNV led 8 to skipping of exon 12 resulting in the transcription of an altered protein product and subsequent downstream 9 activation of NF-κB signalling with higher IL-1β production capacity.

We have performed a systematic, exome-wide DNV analysis in selected patients with sporadic, 11 suspected IEI. On average, these patients carried 0.89 non-synonymous DNVs in coding regions, a rate 12 comparable to other, larger studies, indicating that an enrichment or depletion of DNVs in IEI patients is unlikely 13 (8). Based on gene and variant level information, 14 DNVs (11.4%) were considered potential disease-causing the 12 (likely) pathogenic inherited variants in known IEI genes and the three reported de novo copy number 16 variants (CNVs) with unknown pathogenicity. Six of the candidate DNVs (4.9%) were considered likely or 17 possibly pathogenic variants, while the consequence of the other nine DNVs (6.5%) was uncertain. Two DNVs 18 were in IEI genes (NLRP3, RELA) listed in the most recent IUIS classification and were classified as pathogenic 19 (35, 36, 48 

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022.

suggesting a strong (cellular) effect of this variant. In addition, a de novo frameshift variant in the highly 1 intolerant DDX1 (pLI 0.994) was identified in a patient with hypogammaglobulinemia, hematopoietic cell 2 lineage abnormalities and recurrent infections. Although a causal genotype-phenotype relationship remains 3 unclear, it has been reported that DDX1 plays a role in NF-κB signalling, type I interferon responses, and the 4 regulation of hematopoietic stem and progenitor cell homeostasis (59, 65). Furthermore, the de novo frameshift 5 variant in KMT2C was detected in a patient with combined immunodeficiency and a neurodevelopmental 6 phenotype, displaying partial phenotypic overlap with Kleefstra syndrome type 2 that has already been 7 associated with de novo mutations in KMT2C (60).

Another promising candidate DNV in a potentially novel IEI gene was identified in a patient with 9 periodic fever and was located in the highly conserved FBXW11 (pLI 0.976). This DNV affected the canonical 10 splice acceptor site preceding exon 12 and was shown to create a splice defect leading to exon skipping with 11 a shortened transcript that retained expression at the RNA level. Exon 12 encodes a component of the WD40

repeat domain, which is involved in substrate recognition (71). De novo missense and nonsense variants in 13 FBXW11 have been previously described in patients with a neurodevelopmental syndrome with abnormalities 14 of the digits, jaw and eyes (72). These variants have been shown to compromise substrate recognition or 15 binding of the Wnt and Hedgehog signalling developmental pathways. In our patient with a distinct encodes β-TrCP2, a component of the SCF (SKP1-CUL1-F-box) E3 ubiquitin ligase complex that mediates the ubiquitination of IκBα and consequently stimulates canonical NF-κB signalling (73) . It was previously shown 19 that the abundance of β-TrCP, which includes the highly homologous isoform β-TrCP1, affects the steady-20 state concentration of NF-κB and its dynamics on stimulation (73) . In peripheral blood mononuclear cells 21 (PBMC) extracted from the patient, we demonstrated that the phosphorylation of the NF-κB subunit p65 was constitutively higher in monocytes and CD8+ T cells as compared to a healthy control, which suggests a 23 functional effect of the FBXW11 variant. This effect is further substantiated by the observation of increased 24 p65 phosphorylation and downstream production of IL-1 after stimulation with pathogens and a TLR3 ligand 25 in the patient. However, a note of caution should be made regarding n=1 studies, as we cannot exclude that 26 the difference is due to normal inter-individual biological variability. These results suggest that NF-κB signalling 27 was aberrantly increased in the patient, a mechanism that has been shown to be involved in the pathogenesis 28 of other monogenic autoinflammatory disorders known as relopathies (74). Although it is likely that the de novo 29 variant in FBXW11 plays a role in the immunological phenotype, further experiments addressing the effect of 30 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10. 1101 /2022 this DNV on IκBα degradation, substrate recognition and TrCP protein abundance should be undertaken to 1 provide conclusive evidence.

To our knowledge, two other cohort studies have systematically performed trio-based sequencing in 3 IEI patients as part of their study design, although patients were not pre-selected based on sporadic 4 phenotypes (9, 75). Stray-Pedersen et al. conducted a large international cohort study to investigate the 5 usefulness of WES in IEI patients from 278 families, which included 39 patient-parent trios (9). The authors 6 reported a molecular diagnosis in 40% of the patients, including 15 (13.6%) de novo mutations, of which 4 7 were identified by trio-based analysis and 11 after segregation analysis. The additional value of trio-based 8 sequencing is indicated by the higher detection rate compared to that of the single cases followed by 

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. (78)) Based on this study and evidence from others, including those from other rare disease fields, we 2 suggest that trio-based sequencing should be part of the routine evaluation of patients with a sporadic IEI 3 phenotype (Box 1). An exome-wide analysis should be conducted to identify potentially novel disease genes 4 in cases with a negative diagnostic WES result in whom a strong clinical suspicion for an underlying monogenic 5 cause remains. Thus far, the relative proportion of DNVs among IEI patients with a genetic diagnosis, 6 estimated to be around 6-14%, seems modest compared to other rare disease fields (i.e., >80% in 7 neurodevelopmental disorders (NDDs)) (79). There are several explanations for this difference that suggest 8 that the true contribution of DNVs is higher than currently appreciated. Most importantly, much more 9 experience has been gained with DNV assessment in the field of NDDs. Despite a steep increase in the total 10 diagnostic rate (6, 80, 81) and the identification of 285 developmental disorder (DD)-associated DNVs, 11 modelling suggests that more than 1000 DD-associated genes still remain to be discovered (8). As more trio-12 based sequencing data will be generated from suspected IEI patients, the field should undertake larger-scale 13 analyses that leverage existing statistical models from the field of NDDs/DDs, including models for gene/exon 

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101/2022.03.30.22272929 doi: medRxiv preprint many genes following AD and X-linked (dominant) inheritance patterns have been described in recent years 1 (48). The genes affected in these disorders possess high intolerance for loss-of-function mutations and 2 essential biological functions. As expected, the DNVs reported in this category to date act through mechanisms 3 of haploinsufficiency (i.e. RELA, pLI 0.999), dominant-negative interference (i.e. IKZF1, pLI 0.999 (82); STAT3, 4 pLI 1.000 (83)) or complete deficiency in hemizygotic males (i.e. WAS, pLI 0.999 (84); IL2RG, pLI 0.992 (85)).

Some heterozygous DNVs can also cause CID through hypermorphic effects at protein level (i.e., RAC2, pLI 6 0.966 (86)). Trio-based sequencing should also be considered in patients with sporadic autoinflammatory 7 syndromes and/or autoimmunity, even when presenting at an adult age that could suggest somatic de novo 8 mutations. In these patients, various pathogenic DNVs in different genes have already been described, 9 originating both from the germline (PLCG2, STAT1) and soma (i.e., NLRP3, UBA1, TLR8) (13, 18, 21-24).

These genes do not necessarily have high constraint for loss-of-function mutations, but they possess 11 nucleotide clusters that are highly conserved and intolerant to variation, encoding protein domains with 12 important regulatory functions.

This explorative study has a number of limitations. First, the sample size precludes a reliable estimation 14 of the prevalence of DNVs among patients with sporadic IEIs. Furthermore, the strict diagnostic rate of both 15 inherited variants and (likely) pathogenic DNVs in our cohort is limited (n=17, 13.8%) compared to other 16 studies. It has been previously reported that the diagnostic yield of WES for IEI patients varies widely from 10-79% (15). The most likely explanation for a relatively low diagnostic yield in our study is the patient selection.

We excluded patients with suspected inherited disease but chose not to apply any other selection criteria in 19 order to study a representative cross-section of suspected IEI patients in our centre in whom WES was 20 performed. As a result, patients were included even if the a priori chance of an IEI was low but should be ruled 21 out in the differential diagnosis (i.e., new-born screening shows low T cell receptor excision circles (TRECs)).

Moreover, compared to other cohorts, the percentage of patients with syndromal CIDs, autoinflammatory 23 syndromes and immune dysregulation was relatively high and could influence the generalisability of our results.

Lastly, the functional effect of most candidate DNVs were not evaluated. As DNVs have a high chance of being 25 deleterious, functional experiments should always be attempted to validate the predicted effect. The candidate 26 DNVs in potentially novel IEI genes were shared on GeneMatcher in order to find similar cases that could 27 motivate further investigation into the underlying mechanisms (1, 87).

In conclusion, we applied trio-based whole exome sequencing in a retrospective cohort of 123 patients 29 with sporadic, suspected IEI, leading to the identification of 14 DNVs with a possible or likely chance of 30 pathogenicity. Amongst the candidate DNVs in potentially novel IEI genes, additional functional evidence was 31 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101/2022.03.30.22272929 doi: medRxiv preprint provided in support of a pathogenic role for the DNV in FBXW11 in a patient with an autoinflammatory 1 phenotype. We advocate the structural implementation of trio-based sequencing in the diagnostic evaluation 2 of patients with sporadic IEI. With decreasing costs of exome sequencing, this approach could improve the 3 diagnostic rate of IEI and advance IEI gene discovery. 4 5 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. 

The authors have no financial or non-financial competing interests. 14 15 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 

Jimenez-Trevino S, Gonzalez-Roca E, Ruiz-Ortiz E, Yague J, Ramos E, Arostegui JI. 

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Rother KI, Hildebrand PW, Brogan P, Kruger E, Aksentijevich I, Goldbach-Mansky R. Additive loss-of-function 23 proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production. J Clin Invest. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 18, 2022. ; 2 Patient characteristics, including sex, five-year age range at sampling and phenotypical IUIS classification were provided, and information on the genetic ancestry, 3 median target coverage, % targets covered with at least 5x or 20x coverage was retrieved from each trio-based whole exome sequencing dataset. Patients included 4 in previous publications are noted. 10 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101/2022.03.30.22272929 doi: medRxiv preprint 

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101/2022.03.30.22272929 doi: medRxiv preprint Table 1Btable supplement 

Panel B shows traces of the three bands from the agarose gel that were cut out and sent for Sanger 6 sequencing. As the splice site variant in the patient was expected to lead to skipping of exon 12, the boundaries 7 between exons 11, 12 and 13 were shown. The second band confirms skipping of exon 12 that results in a 8 shorter transcript of the mutated FBXW11 allele. After CHX treatment, this band shows an increased quantity,

indicating that the mutated allele undergoes nonsense mediated decay, but incomplete. Furthermore, a smaller 10 transcript is formed in the patient, which is shown to contain part of exon 12, but not exon 13. 12 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 

New insights into the generation and role of de novo

Population genetic tools for dissecting innate immunity in humans

Controlling Nuclear NF-kappaB Dynamics by beta-TrCP

Unusual late presentation of X-linked chronic 20 granulomatous disease in an adult female with a somatic mosaic for a novel mutation in CYBB