Correction: The Endocytic Adaptor Eps15 Controls Marginal Zone B Cell Numbers.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis

The Human Phenotype Ontology in 2024: phenotypes around the world.

The Human Phenotype Ontology (HPO) is a widely used resource that comprehensively organizes and defines the phenotypic features of human disease, enabling computational inference and supporting genomic and phenotypic analyses through semantic similarity and machine learning algorithms. The HPO has widespread applications in clinical diagnostics and translational research, including genomic diagnostics, gene-disease discovery, and cohort analytics. In recent years, groups around the world have developed translations of the HPO from English to other languages, and the HPO browser has been internationalized, allowing users to view HPO term labels and in many cases synonyms and definitions in ten languages in addition to English. Since our last report, a total of 2239 new HPO terms and 49235 new HPO annotations were developed, many in collaboration with external groups in the fields of psychiatry, arthrogryposis, immunology and cardiology. The Medical Action Ontology (MAxO) is a new effort to model treatments and other measures taken for clinical management. Finally, the HPO consortium is contributing to efforts to integrate the HPO and the GA4GH Phenopacket Schema into electronic health records (EHRs) with the goal of more standardized and computable integration of rare disease data in EHRs

ORPHAcodes use for the coding of rare diseases: comparison of the accuracy and cross country comparability

Abstract Background Estimates of rare disease (RD) population impact in terms of number of affected patients and accurate disease definition is hampered by their under-representation in current coding systems. This study tested the use of a specific RD codification system (ORPHAcodes) in five European countries/regions (Czech Republic, Malta, Romania, Spain, Veneto region-Italy) across different data sources over the period January 2019-September 2021. Results Overall, 3133 ORPHAcodes were used to describe RD diagnoses, mainly corresponding to the disease/subtype of disease aggregation level of the Orphanet classification (82.2%). More than half of the ORPHAcodes (53.6%) described diseases having a very low prevalence (< 1 case per million), and most commonly captured rare developmental defects during embryogenesis (31.3%) and rare neurological diseases (17.6%). ORPHAcodes described disease entities more precisely than corresponding ICD-10 codes in 83.4% of cases. Conclusions ORPHAcodes were found to be a versatile resource for the coding of RD, able to assure easiness of use and inter-country comparability across population and hospital databases. Future research on the impact of ORPHAcoding as to the impact of numbers of RD patients with improved coding in health information systems is needed to inform on the real magnitude of this public health issue

The endocytic adaptor Eps15 controls marginal zone B cell numbers.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis

Eps15 has multiple roles in the immune system.

<p><b>A.</b> Scheme depicting the outline of the competitive bone marrow transplantation experiment. Bone marrow cells from Eps15-WT or -KO mice were mixed with WT bone marrow cells at a 1∶1 ratio and injected into sub-lethally irradiated recipient mice. The origin of the cells in the recipient mice was traced taking advantage of isogenic variation of the CD45 locus: CD45.2 is present in Eps15-WT and Eps15-KO mice, while CD45.1 is present in the wildtype (WT) mice used as competitor donor and as recipient mice. <b>B.–F.</b> Pie charts depicting the relative contribution of CD45.2 (Eps15-WT or Eps15-KO) versus WT CD45.1 positive competitor cells in the bone marrow (<b>B</b>), spleen (<b>C</b>), peritoneum (<b>D</b>), thymus (<b>E</b>) and lymph nodes (<b>F</b>) of recipient mice, as determined by FACS analysis. Significant differences (*p<0.05, **p<0.01) in the competition between Eps15-WT (n = 4) and Eps15-KO (n = 5) CD45.2⁺ cells with CD45.1⁺ WT cells, are indicated by red lines in the pie charts.</p

Eps15-KO mice are viable and show an immunological defect. A.

<p>Scheme depicts the region within the Eps15 wildtype (WT) locus that was altered to generate the knockout (KO) locus. The first coding exon of Eps15 and 2 kb of the 5′ promoter region were replaced by gene targeting with a neomycin (NEO) cassette. The location of the primers (a, b, c) used for genotyping is indicated (not to scale). <b>B.</b> Three primer PCR on tail biopsies gave the expected bands for the WT and KO allele: 515 and 642 bp, respectively. <b>C.</b> Western blot of total spleen lysates from Eps15-WT (WT) or Eps15-KO (KO) mice decorated with an anti-Eps15 antibody, or anti-vinculin antibody as loading control, shows lack of Eps15 protein in Eps15-KO mice. <b>D.</b> Bar graph depicting the percent of mice that have been weaned (black bars) for the indicated genotypes (wildtype (WT), heterozygous (Het), knockout (KO)) from the breeding of Eps15 heterozygous mice. The expected frequency for each genotype is depicted by the grey bars. A total of 231 pups were analyzed. <b>E.</b> Western blot analysis of multiple tissues from Eps15-WT (WT) and Eps15-KO (KO) mice reveals ubiquitous expression of Eps15 and Eps15L1. Total tissue lysates (10 µg protein) were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed for Eps15, Eps15L1 or vinculin, as loading control, as indicated. For brain lysates obtained from Eps15-WT (WT) or Eps15-KO (KO) mice, 10 and 20 µg protein were loaded to allow a better comparison of Eps15 and Eps15L1 protein levels. <b>F.–G.</b> Bar graphs displaying cell surface receptor levels (<b>F</b>) and internalization rate constants Ke (<b>G</b>) for the transferrin receptor (TfR) and the EGF receptor (EGFR) in Eps15-WT (white bars) and Eps15-KO (black bars) primary mouse fibroblasts. <b>H.</b> Bar graph depicting the number of white blood cells (WBC), lymphocytes (Lym), monocytes (Mo), granulocytes (Gr) and eosinophils (Eo) in the peripheral blood of 3-month-old Eps15-WT (WT, white bars, n = 10) and Eps15-KO (KO, black bars, n = 10) mice as determined by hematological analysis. <b>I.</b> Bar graph depicting the number of B cells (B), T cells (T), granulocytes (Gr), monocytes (Mo) and natural killer cells (NK) in the peripheral blood of 3 month old Eps15-WT (WT, white bars, n = 10) and Eps15-KO (KO, black bars, n = 10) mice as determined by FACS analysis. Statistical significance was assessed using Student’s t-test. Significant differences are indicated as * = p<0.05, *** = p<0.005.</p

Normal motor activity in aged Eps15-KO mice.

<p>Motoractivity was measured in aged (>20-month old) mice according to the modified SHIRPA protocol.</p

Eps15-KO mice show increased marginal zone B cell numbers.

<p><b>A.</b> Dot plots of bone marrow cells from 4-month-old Eps15-WT and Eps15-KO mice stained for B220 and IgM to identify pre−/pro-B cells (B220⁺IgM⁻), immature (B220⁺IgM⁺) and mature (B220^highIgM⁺) B cells. Pre−/pro-B cells were further analyzed for expression of CD43 to identify pro- (CD43⁺) and pre- (CD43⁻) B cells. <b>B.–C.</b> Dot plots depicting the total number of mature (B220⁺⁺), immature (Imm.) and pre−/pro- and B cells (<b>B</b>) and pre- and pro-B cells (<b>C</b>) in the bone marrow of Eps15-WT (WT, white symbols, n = 5) and Eps15-KO (KO, black symbols, n = 6) mice. <b>D.</b> Top panel: dot plot of thymocytes from 2-month Eps15-WT and Eps15-KO mice stained for CD19 and TCRβ to identify B and T cells, respectively. Middle panel: dot plots for thymocytes from 4-month-old Eps15-WT and Eps15-KO mice staind for CD4 and CD8 to identify thymocyte subpopulations. Bottom panel: the CD4/CD8 double negative population (DN) was further analyzed for immature thymocytes using CD44 and CD25. <b>E.–G.</b> Dot plots depicting the total number of the following cell populations in the thymi of Eps15-WT (WT, white symbols) and Eps15-KO (KO, black symbols) mice: (<b>E</b>) TCRβ⁺ and CD19⁺ cells (n = 3), (<b>F</b>) DN (double negative, CD4⁻CD8⁻), DP (double positive, CD4⁺CD8⁺), CD4⁺ and CD8⁺ (n = 4) and (<b>G</b>) of DN1 (CD44⁺CD25⁻), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁻) cells (n = 4). <b>H</b>. Dot plot of peritoneal B cells from 2–4 month old Eps15-WT and Eps15-KO mice stained for CD19 and B220 gated for CD19⁺B220^low B1 and CD19⁺B220^high B2 B cells. B1 B cells were further stained and gated for CD5 to identify CD5⁺ B1a and CD5⁻ B1b B cells. <b>I.–J.</b> Dot plots depicting the percentage of B1 and B2 (I) and B1a and B1b (J) B cells in the peritoneum of Eps15-WT (WT, white symbols) and Eps15-KO (KO, black symbols) mice (n = 8). <b>K.</b> Dot plot of splenocytes from Eps15-WT and Eps15-KO mice stained for CD19 and gated for CD21 and CD23 to identify Fo B (CD19⁺/CD21⁺/CD23⁺) and MZ B (CD19⁺/CD21⁺/CD23⁻) cells. <b>L.</b> Dot plots depicting the total number of CD19+, Fo B and MZ B cells in the spleens of Eps15-WT (WT, white symbols) and Eps15-KO (KO, black symbols) mice (n = 5). Values are depicted as mean±standard error mean. Statistical significance was assessed using Student’s t-test and significant differences are indicated as * = p<0.05.</p

FACS analysis of the B cell populations present in the lymph nodes three months after competitive bone marrow transplantation.

<p>Distribution of CD45.1+ and CD45.2+ cells in the lymph node of recipient mice 3 months after bone marrow transplantation. CD45.1+ WT and CD45.2+ WT or CD45.2+ KO donor cells were mixed at a 1∶1 ratio prior to injection into recipient CD45.1+ mice. The percentage of total cells gated and the percentage of CD45.1+ or CD45.2+ cells for any given gate are shown. Significance was assessed using Student’s t-test and p-values are indicated as *p<0.05, **p<0.01, ***p<0.001.</p