PDK1 is essential for B cell survival after bone marrow immature B cell stage.

<p>(<b>A</b>) Flow cytometric analyses of apoptosis (as indicated by Annexin V staining) were performed with bone marrow cells of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice. The histograms shown were gated as indicated on the right of each plot. Apoptotic percentage of each B cell lineage in bone marrow of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice (n = 4 mice) presented as mean ± SD. Numbers indicate the percentage of 7AAD and annexin V–double positive cells (top), annexin V–positive cells (right bottom) or 7AAD–annexin V–negative cells (left bottom). (<b>B</b>) Foxo target genes (<i>Aicda</i>, <i>Rag1</i>, <i>Bcl2l11</i> and <i>p27/Kip</i>) and NF-κB target genes (<i>Bcl-2</i> and <i>Bcl-xl</i>) expression levels in IgM-positive bone marrow B cells were analyzed by quantitative RT-PCR analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055378#s2" target="_blank">Materials and Methods</a>. Data are presented as mean ± SD (*<i>p</i><0.05, **<i>p</i><0.01). (<b>C</b>) Flow cytometric analyses of apoptosis markers on B220⁺ B cells were performed with spleen and PLN cells of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice. Data are representative of two independent experiments. (<b>D</b>) Primary B cells were stimulated with LPS and were then infected with Cre expressing retrovirus (MSCV-Cre IRES GFP). The ratio of GFP-positive to GFP-negative B cell one day after infection was set to 1. Each following day the ratios were checked by flow-cytometry. The experiment was done in triplicate and data are presented as mean ± SD. Data are representative of three independent experiments.</p

Peripheral B cell numbers are dramatically reduced in B cell specific <i>Pdk1</i> knockout mice.

<p>(<b>A</b>) Flow cytometric analyses were performed with lymphocytes from spleen and lymph nodes of CD19-Cre⁺<i>PDK1⁺</i>^/+(+/+) and CD19-Cre⁺<i>PDK1</i>^flox/flox (flox/flox) mice. The FACS plots shown are representative of five different experiments. (<b>B</b>) The number of B220⁺ cells in spleen and lymphnode of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice (n = 5 mice) presented as mean ± SD. (<b>C</b>) Spleen and lymph nodes from CD19-Cre⁺<i>PDK1^+/+</i> and CD19-Cre⁺<i>PDK1</i>^flox/flox mice. (<b>D</b>) Structure of Spleen from CD19-Cre⁺<i>PDK1^+/+</i> and CD19-Cre⁺<i>PDK1</i>^flox/flox mice. Paraffin-embedded spleen sections from CD19-Cre⁺<i>PDK1^+/+</i> and CD19-Cre⁺<i>PDK1</i>^flox/flox mice were stained by hematoxylin and eosin (top panel) or anti-B220 (bottom panel).</p

The defect of B cell development in CD19-Cre⁺<i>PDK1</i>^flox/flox mice is caused by B cell intrinsic defects.

<p>Bone marrow cells of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice were transferred into unirradiated Rag1 deficient recipients. Six weeks after transfer, flow cytometric analyses of B cell surface markers were performed with bone marrow cells, spleen cells and lymph node cells from the recipient mice. This is representative of two independent experiments.</p

3-HAA (PDK1 inhibitor) inhibits B cell receptor-meditated NF-κB activation.

<p>(<b>A</b>) HEK293 cell were transfected with plasmids for expression of Myc-PDK1 and HA-PKCβ or HA-PKCθ, the cell lysates were then analyzed for the binding between PDK1 and PKCβ or PKCθ by immunoprecipitation and immunoblotting. In the construct for expression of HA-PKCβ or HA-PKCθ, internal ribosome entry site (IRES) sequence and GFP open reading frame (ORF) is integrated after the PKC ORF. Thus, GFP expression level was used for internal control. (<b>B</b>) The B cells stimulated with anti-IgM were analyzed for the binding between PDK1 and PKCβ by immunoprecipitation and immunoblotting. (<b>C</b>) Luciferase assay with reporter plasmids containing NF-κB binding sites. Expression plasmids of PDK1, PKCβ, or CARMA1-Bcl10-Malt1 (CBM) were cotransfected as indicated. Data are presented as mean ± SD. (<b>D</b>) <i>Bcl-xl</i> gene expression was analyzed through quantitative RT-PCR analysis. Data are presented as mean ± SD. (<b>E</b>) Flow cytometric analyses of apoptosis markers were performed with primary B cells stimulated with or without anti-IgM antibody and with 3-HAA, NF-κB inhibitor peptide (NBD peptide) or DMSO. Numbers indicate the percentage of 7AAD and annexin V–double positive cells (top), annexin V–positive cells (right bottom) or 7AAD–annexin V–negative cells (left bottom). (<b>A</b>), (<b>B</b>), (<b>C</b>), (<b>D</b>) and (<b>E</b>) were representative of two to three experiments.</p

PDK1 is essential for B cell activation.

<p>(<b>A</b>) Phosphorylation of AKT at T308 and S473 in primary B cells stimulated with AffiniPure F(ab′)2 fragment goat anti-Mouse IgM and with or without 3-HAA were analyzed by immunoblot anlaysis. (<b>B</b>) Phosphorylation of JNK and p38 MAPK and IκBα degradation in primary B cells stimulated with anti-IgM antibody and with or without 3-HAA were analyzed by immunobloting analysis. (<b>C</b>) Flow cytometric analyses of B cell activation markers (CD69 and CD86) were performed with primary B cells stimulated with anti-IgM antibody for 24 hours and with or without 3-HAA. Numbers indicate the percentage of CD69 or CD86 positive cells. DMSO was used for the vehicle control. (<b>A</b>), (<b>B</b>) and (<b>C</b>) are representative of three independent experiments.</p

PDK1 deficiency blocks B cell development at the immature B cell stage.

<p>(<b>A</b>) Flow cytometric analyses of surface markers of B cell lineage development of bone marrow cells from CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice. The FACS plots shown are representative of five different experiments. (<b>B</b>) The number of each B cell lineage cell in bone marrow of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice (n = 5 mice) presented as mean ± SD. (<b>C</b>) PDK1 deletion was confirmed by immunoblot analysis after sorting of the indicated populations. Total cell lysate of 0.4×10⁶ cells from each population was loaded for this analysis.</p

DataSheet1_Systematic analysis of inheritance pattern determination in genes that cause rare neurodevelopmental diseases.docx

Despite recent advancements in our understanding of genetic etiology and its molecular and physiological consequences, it is not yet clear what genetic features determine the inheritance pattern of a disease. To address this issue, we conducted whole exome sequencing analysis to characterize genetic variants in 1,180 Korean patients with neurological symptoms. The diagnostic yield for definitive pathogenic variant findings was 50.8%, after including 33 cases (5.9%) additionally diagnosed by reanalysis. Of diagnosed patients, 33.4% carried inherited variants. At the genetic level, autosomal recessive-inherited genes were characterized by enrichments in metabolic process, muscle organization and metal ion homeostasis pathways. Transcriptome and interactome profiling analyses revealed less brain-centered expression and fewer protein-protein interactions for recessive genes. The majority of autosomal recessive genes were more tolerant of variation, and functional prediction scores of recessively-inherited variants tended to be lower than those of dominantly-inherited variants. Additionally, we were able to predict the rates of carriers for recessive variants. Our results showed that genes responsible for neurodevelopmental disorders harbor different molecular mechanisms and expression patterns according to their inheritance patterns. Also, calculated frequency rates for recessive variants could be utilized to pre-screen rare neurodevelopmental disorder carriers.</p

DataSheet2_Systematic analysis of inheritance pattern determination in genes that cause rare neurodevelopmental diseases.xlsx

Despite recent advancements in our understanding of genetic etiology and its molecular and physiological consequences, it is not yet clear what genetic features determine the inheritance pattern of a disease. To address this issue, we conducted whole exome sequencing analysis to characterize genetic variants in 1,180 Korean patients with neurological symptoms. The diagnostic yield for definitive pathogenic variant findings was 50.8%, after including 33 cases (5.9%) additionally diagnosed by reanalysis. Of diagnosed patients, 33.4% carried inherited variants. At the genetic level, autosomal recessive-inherited genes were characterized by enrichments in metabolic process, muscle organization and metal ion homeostasis pathways. Transcriptome and interactome profiling analyses revealed less brain-centered expression and fewer protein-protein interactions for recessive genes. The majority of autosomal recessive genes were more tolerant of variation, and functional prediction scores of recessively-inherited variants tended to be lower than those of dominantly-inherited variants. Additionally, we were able to predict the rates of carriers for recessive variants. Our results showed that genes responsible for neurodevelopmental disorders harbor different molecular mechanisms and expression patterns according to their inheritance patterns. Also, calculated frequency rates for recessive variants could be utilized to pre-screen rare neurodevelopmental disorder carriers.</p