Georg Bohn

HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease)

Autosomal recessive severe congenital neutropenia (SCN) constitutes a primary immunodeficiency syndrome associated with increased apoptosis in myeloid cells, yet the underlying genetic defect remains unknown. Using a positional cloning approach and candidate gene evaluation, we identified a recurrent homozygous germline mutation in HAX1 in three pedigrees. After further molecular screening of individuals with SCN, we identified 19 additional affected individuals with homozygous HAX1 mutations, including three belonging to the original pedigree described by Kostmann. HAX1 encodes the mitochondrial protein HAX1, which has been assigned functions in signal transduction and cytoskeletal control. Here, we show that HAX1 is critical for maintaining the inner mitochondrial membrane potential and protecting against apoptosis in myeloid cells. Our findings suggest that HAX1 is a major regulator of myeloid homeostasis and underline the significance of genetic control of apoptosis in neutrophil development.

p14-MP1-MEK1 signaling regulates endosomal traffic and cellular proliferation during tissue homeostasis.

The extracellular signal-regulated kinase (ERK) cascade regulates proliferation, differentiation, and survival in multicellular organisms. Scaffold proteins regulate intracellular signaling by providing critical spatial and temporal specificity. The scaffold protein MEK1 (mitogen-activated protein kinase and ERK kinase 1) partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in mice, we now demonstrate that the p14–MP1-MEK1 signaling complex regulates late endosomal traffic and cellular proliferation. This function its essential for early embryogenesis and during tissue homeostasis, as revealed by epidermis-specific deletion of p14. These findings show that endosomal p14–MP1-MEK1 signaling has a specific and essential function in vivo and, therefore, indicate that regulation of late endosomal traffic by extracellular signals is required to maintain tissue homeostasis.

A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3′ untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

Perturbation of fetal liver hematopoietic stem and progenitor cell development by trisomy 21

The 40-fold increase in childhood megakaryocyte-erythroid and B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in perturbing fetal hematopoiesis. Here, we show that compared with primary disomic controls, primary T21 fetal liver (FL) hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors are markedly increased, whereas granulocyte-macrophage progenitors are reduced. Commensurately, HSC and megakaryocyte-erythroid progenitors show higher clonogenicity, with increased megakaryocyte, megakaryocyte-erythroid, and replatable blast colonies. Biased megakaryocyte-erythroid–primed gene expression was detected as early as the HSC compartment. In lymphopoiesis, T21 FL lymphoid-primed multipotential progenitors and early lymphoid progenitor numbers are maintained, but there was a 10-fold reduction in committed PreproB-lymphoid progenitors and the functional B-cell potential of HSC and early lymphoid progenitor is severely impaired, in tandem with reduced early lymphoid gene expression. The same pattern was seen in all T21 FL samples and no samples had GATA1 mutations. Therefore, T21 itself causes multiple distinct defects in FL myelo- and lymphopoiesis.

Severe congenital neutropenia: new genes explain an old disease.

Purpose of reviewThis review summarizes the recent advances in the diagnosis and molecular characterization of isolated and syndromal forms of severe congenital neutropenia. Recent findingsIt has become evident that severe congenital neutropenia comprises several genetically distinct entities. In 1999, mutations were identified in the neutrophil elastase gene ELA2. ELA2 mutations have been found in cyclic, sporadic and autosomal dominant neutropenia. Recently, homozygous mutations in the antiapoptotic gene HAX1 were found in patients with autosomal recessive severe congenital neutropenia. Ongoing linkage studies suggest that more and, as yet unidentified, genes may be involved in the pathophysiology of severe congenital neutropenia. In other patients, congenital neutropenia is not an isolated finding but is associated with other abnormalities, in particular, lymphoid immunodeficiency and pigmentation defects such as Chédiak–Higashi syndrome, Griscelli syndrome type 2, Hermansky–Pudlak syndrome type 2, or deficiency of the endosomal adaptor p14. The molecular identification of these disorders originating from mutations in lysosome (related) proteins has advanced our knowledge of intracellular protein trafficking. SummaryRecent insights into the molecular etiology of severe congenital neutropenia provide the opportunity for a definitive genetic classification system. Based on this knowledge, disease-related risks may be recognized and optimized therapeutic options may become available.

A complex immunodeficiency is based on U1 snRNP‐mediated poly(A) site suppression

Biallelic mutations in the untranslated regions (UTRs) of mRNAs are rare causes for monogenetic diseases whose mechanisms remain poorly understood. We investigated a 3′UTR mutation resulting in a complex immunodeficiency syndrome caused by decreased mRNA levels of p14/robld3 by a previously unknown mechanism. Here, we show that the mutation creates a functional 5′ splice site (SS) and that its recognition by the spliceosomal component U1 snRNP causes p14 mRNA suppression in the absence of splicing. Histone processing signals are able to rescue p14 expression. Therefore, the mutation interferes only with canonical poly(A)‐site 3′ end processing. Our data suggest that U1 snRNP inhibits cleavage or poly(A) site recognition. This is the first description of a 3′UTR mutation that creates a functional 5′SS causative of a monogenetic disease. Moreover, our data endorse the recently described role of U1 snRNP in suppression of intronic poly(A) sites, which is here deleterious for p14 mRNA biogenesis.

Lethal Graft-Versus-Host Disease in Congenital Neutropenia Caused by p14 Deficiency After Allogeneic Bone Marrow Transplantation From an HLA-Identical Sibling

The molecular heterogeneity of severe congenital neutropenia (SCN) is increasingly recognized and may influence the risk‐benefit assessment of therapeutic strategies. We report on a patient with p14 deficiency who succumbed to severe grade IV graft‐versus‐host disease (GvHD) after a human leukocyte antigen‐identical bone marrow transplantion (BMT) from a sibling donor. Before BMT, in vitro generated p14‐deficient dendritic cells showed a markedly elevated tumor necrosis factor (TNF‐) α production upon toll‐like receptor stimulation. We hypothesize that p14 deficiency predisposes to GvHD through increased TNF‐α production. Adequate genetic testing is needed to prospectively assess potential risk factors for GvHD in defined SCN subgroups. Pediatr Blood Cancer 2008;51:436–438.

Nuclear proteasomes carry a constitutive posttranslational modification which derails SDS-PAGE (but not CTAB-PAGE).

We report that subunits of human nuclear proteasomes carry a previously unrecognised, constitutive posttranslational modification. Subunits with this modification are not visualised by SDS-PAGE, which is used in almost all denaturing protein gel electrophoresis. In contrast, CTAB-PAGE readily visualises such modified subunits. Thus, under most experimental conditions, with identical samples, SDS-PAGE yielded gel electrophoresis patterns for subunits of nuclear proteasomes which were misleading and strikingly different from those obtained with CTAB-PAGE. Initial analysis indicates a novel modification of a high negative charge with some similarity to polyADP-ribose, possibly explaining compatibility with (positively-charged) CTAB-PAGE but not (negatively-charged) SDS-PAGE and providing a mechanism for how nuclear proteasomes may interact with chromatin, DNA and other nuclear components.

High resolution IgH repertoire analysis reveals fetal liver as the likely origin of life-long, innate B lymphopoiesis in humans

The ontogeny of the natural, public IgM repertoire remains incompletely explored. Here, high-resolution immunogenetic analysis of B cells from (unrelated) fetal, child, and adult samples, shows that although fetal liver (FL) and bone marrow (FBM) IgM repertoires are equally diversified, FL is the main source of IgM natural immunity during the 2nd trimester. Strikingly, 0.25% of all prenatal clonotypes, comprising 18.7% of the expressed repertoire, are shared with the postnatal samples, consistent with persisting fetal IgM + B cells being a source of natural IgM repertoire in adult life. Further, the origins of specific stereotypic IgM + B cell receptors associated with chronic lymphocytic leukemia, can be traced back to fetal B cell lymphopoiesis, suggesting that persisting fetal B cells can be subject to malignant transformation late in life. Overall, these novel data provide unique insights into the ontogeny of physiological and malignant B lymphopoiesis that spans the human lifetime.