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2017 Short-term Fellowship Report

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by Tiziana Lorenzini

Short-term fellowship report

I sincerely thank ESID for supporting me in this educational experience. I spent my period of stay at the Center for Chronic Immunodeficiency of Freiburg, Germany, under the supervision of Prof. Dr. Bodo Grimbacher. I'd also like to thank Prof. Dr. Alessandro Plebani who promoted my internship. I had the opportunity to enhance my knowledge in the field of PID, in both clinical and laboratory research. I focused my study on NF-kappaB signaling pathway and I tried to characterize the phenotype of patients with heterozygous NFKB1 mutations.


The NF-κB (nuclear factor of kappa light polypeptide gene enhancer in B cells) signaling pathway is involved in several biological processes, including cell survival and proliferation, innate  and adaptive immune response. NF-κB transcription factors are homo- or heterodimers of five subunits: NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB and c-Rel. NFKB1 gene encodes for the precursor p105, which is processed to p50, while NFKB2 gene encodes for the precursor p100, which is processed to p52. NF-κB1 belongs to the canonical pathway, which controls a broader immune and inflammatory response than the main component of the non-canonical pathway, NF-κB2, involved in B-cell survival and activation, BAFF- and CD40-mediated, respectively.

The five members of NF-κB transcription factor family share a Rel homology domain (RHD) which mediates dimerization, proteins interaction and DNA-binding. However, p50 NF-κB1 and p52-NF-κB2 lack the transactivation domain so their dimerization with RelA, RelB and c-Rel is required to induce gene expression. The dimer p50:p65 activates the transcription of genes involved in cell survival and inflammation, while p50 homodimers function as transcriptional repressors or as anti-inflammatory response inducers.

In resting cells, NF-κB dimers are sequestered within the cytoplasm in an inactive state by inhibitory proteins (IκB). In addition, p105 and p100 function as inhibitory proteins, retaining the active subunits in the cytoplasm. Upon stimulation, inhibitory proteins are phosphorylated and degraded by the proteasome, releasing NF-κB heterodimers which can translocate to the nucleus and activate gene transcription. Processing of p105 and p100 also releases p52 and p50 respectively, allowing them to translocate to the nucleus in association with other Rel subunits.

The NFKB1 gene encodes the full length transcript p105, which spans 3452 bp and contain 25 exons, generating a 971 amino acid protein (105 kDa), and the p50 protein, which spans amino acids 1-430 of p105. p50 transcript contains the Rel homology domain (RHD), composed by the N-terminal domain (NTD), dimerization domain (DimD) and the nuclear localization sequence (NLS). Adjacent to the RHD is the glycine-rich region (GRR) which stabilizes p50 preventing its proteolytic degradation. p105 transcript is composed by the p50 transcript, the ankyrin repeat domain (ARD), by which inactive dimers are sequestered in the cytoplasm, and the death domain, which contains two serine residues phosphorylated by IKKs (inhibitory κB kinases) after stimulation, allowing ubiquitination and proteolytic degradation of p105 by the proteasome and release of p50.

In 2015, Fliegauf et al. identified, by whole-exome sequencing (WES), three heterozygous NFKB1 mutations in 20 patients with Common Variable Immunodeficiency. Two of them (c.730+4A>G and c.835+2T>G) were splice-site mutations causing in-frame skipping of an exon (8 and 9 respectively) and internal deletion of the RHD; the last (c.465dupA) was a frameshift mutation in exon 7 resulting in a severely truncated protein that retain only the N-terminal part of the RHD. All of them resulted in a functional NF-κB1 p50 haploinsufficiency, as they led to a rapid degradation of the mutant p105 which could not be processed to the mutant p50, undetectable in affected individuals.

Subsequently, other NFKB1 mutations have been identified by Boztug et al., Schipp et al., and Maffucci et al.

Lougaris et al. described a frameshift mutation (c.1517delC) downstream the nuclear localization predicted to cause a premature translation termination with a lack of expression of the p105 precursor from the mutant allele but a p50 aberrant expression.

Recently, Kaustio et al. reported three novel NFKB1 mutations: two of them (the missense c.A667G and the stop-gain c.C936T) localized in the RHD, the third (the missense c.C1659G) was the first identified in the ankyrin repeat domain (ARD). The missense mutations were not associated with a depletion of p50 and p105, while the stop-gain mutation caused a significant reduction also in endogenous p50 and p105. The missense mutation c. C1659G increased NF-κB reporter activity after stimulation while the other mutations failed. The nuclear localization of the ARD-mutant protein was normal, while the other mutant proteins showed an impaired nuclear localization. The ARD-mutant protein displayed a decreased phosphorylation after stimulation.

The aim of my project was to characterize the large spectrum of NFKB1 mutations, in order to broaden the phenotype of the disease and to define how the different NFKB1 variants affect the NF-κB signaling pathway. We have identified NFKB1 mutations by next generation sequencing in a cohort of patients with primary antibody deficiency, immune dysregulation and autoinflammation and I planned to characterize these novel variants with functional studies.

Data collection and analysis

We enrolled a cohort of patients with NFKB1 mutations, identified by next generation sequencing, in a world-wide collaboration. Clinical and laboratory data were collected by the attending physicians using a questionnaire including the medical history, the physical examination and the immunological assessment. Genetic analysis, demographic characteristics, mortality rate, age and symptom at onset, age at diagnosis and penetrance rate were studied in detail. Respiratory, gastrointestinal and skin involvement, autoimmunity and immune dysregulation, infections, malignancies and other complications were analyzed. The immunological characterization defined a disease primary affecting antibody response, but with additional immune defects. I analyzed the treatment options, in order to aid the management of patients with NFKB1 mutations. These results will be published in a large cohort study involving several collaborators.

Functional tests

In the second part of my study, I focused on the functional characterization of the NFKB1 mutations which had not been characterized previously. For my experiments, I transfected cells with mutant constructs and I used Western blotting, fluorescence microscopy and gene reporter assay. I studied the frameshift NFKB1 mutations located downstream of the nuclear localization sequence (NLS) and the missense mutations located within the RHD and the ARD.

I evaluated the expression, nuclear translocation and transcriptional activity of mutant form of NFKB1 in unstimulated cells and after NFκB signaling pathway induction.


NFKB1 deficient patients show a heterogeneous phenotype, ranging from primary antibody deficiency to immune dysregulation and autoinflammation. The disease penetrance and expressivity are incomplete. NFKB1 function is tightly regulated and the mechanisms by which heterozygous NFKB1 mutations affect the protein function are wider than the premature protein degradation leading to a functional haploinsufficiency. In this study, I summarized the clinical phenotype of NFKB1 mutations, characterizing a large cohort of affected individuals, and I tried  to understand the pathomechanisms that lead to the disease.