Functional analysis of Ectodysplasin-A mutations causing selective tooth agenesis.

Mutations of the Ectodysplasin-A (EDA) gene are generally associated with the syndrome hypohidrotic ectodermal dysplasia (MIM 305100), but they can also manifest as selective, non-syndromic tooth agenesis (MIM300606). We have performed an in vitro functional analysis of six selective tooth agenesis-causing EDA mutations (one novel and five known) that are located in the C-terminal tumor necrosis factor homology domain of the protein. Our study reveals that expression, receptor binding or signaling capability of the mutant EDA1 proteins is only impaired in contrast to syndrome-causing mutations, which we have previously shown to abolish EDA1 expression, receptor binding or signaling. Our results support a model in which the development of the human dentition, especially of anterior teeth, requires the highest level of EDA-receptor signaling, whereas other ectodermal appendages, including posterior teeth, have less stringent requirements and form normally in response to EDA mutations with reduced activity

Genomic rearrangements of the PRPF31 gene account for

PURPOSE. To determine whether genomic rearrangements in the PRPF31 (RP11) gene are a frequent cause of autosomal dominant retinitis pigmentosa (adRP) in a cohort of patients with adRP. METHODS. In a cohort of 200 families with adRP, disease-causing mutations have previously been identified in 107 families. To determine the cause of disease in the remaining families, linkage testing was performed with markers for 13 known adRP loci. In a large American family, evidence was found of linkage to the PRPF31 gene, although DNA sequencing revealed no mutations. SNP testing throughout the genomic region was used to determine whether any part of the gene was deleted. Aberrant segregation of a SNP near exon 1 was observed, leading to the testing of additional SNPs in the region. After identifying an insertion-deletion mutation, the remaining 92 families were screened for genomic rearrangements in PRPF31 with multiplex ligation-dependent probe amplification (MLPA). RESULTS. Five unique rearrangements were identified in the 93 families tested. In the large family used for linkage exclusion testing, an insertion-deletion was found that disrupts exon 1. The other four mutations identified in the cohort were deletions, ranging from 5 kb to greater than 45 kb. Two of the large deletions encompass all PRPF31 as well as several adjacent genes. The two smaller deletions involve either 5 or 10 completely deleted exons. CONCLUSIONS. In an earlier long-term study of 200 families with adRP, disease-causing mutations were identified in 53% of the families. Mutation-testing by sequencing missed large-scale genomic rearrangements such as insertions or deletions. MLPA was used to identify genomic rearrangements in PRPF31 in five families, suggesting a frequency of approximately 2.5%. Mutations in PRPF31 now account for 8% of this adRP cohort

Genomic rearrangements of the PRPF31 gene account for 2.5% of autosomal dominant retinitis pigmentosa

PURPOSE: To determine whether genomic rearrangements in the PRPF31 (RP11) gene are a frequent cause of autosomal dominant retinitis pigmentosa (adRP) in a cohort of patients with adRP. METHODS: In a cohort of 200 families with adRP, disease-causing mutations have previously been identified in 107 families. To determine the cause of disease in the remaining families, linkage testing was performed with markers for 13 known adRP loci. In a large American family, evidence was found of linkage to the PRPF31 gene, although DNA sequencing revealed no mutations. SNP testing throughout the genomic region was used to determine whether any part of the gene was deleted. Aberrant segregation of a SNP near exon 1 was observed, leading to the testing of additional SNPs in the region. After identifying an insertion–deletion mutation, the remaining 92 families were screened for genomic rearrangements in PRPF31 with multiplex ligation-dependent probe amplification (MLPA). RESULTS: Five unique rearrangements were identified in the 93 families tested. In the large family used for linkage exclusion testing, an insertion–deletion was found that disrupts exon 1. The other four mutations identified in the cohort were deletions, ranging from 5 kb to greater than 45 kb. Two of the large deletions encompass all PRPF31 as well as several adjacent genes. The two smaller deletions involve either 5 or 10 completely deleted exons. CONCLUSIONS: In an earlier long-term study of 200 families with adRP, disease-causing mutations were identified in 53% of the families. Mutation-testing by sequencing missed large-scale genomic rearrangements such as insertions or deletions. MLPA was used to identify genomic rearrangements in PRPF31 in five families, suggesting a frequency of approximately 2.5%. Mutations in PRPF31 now account for 8% of this adRP cohort

Identification of IOMA-class neutralizing antibodies targeting the CD4-binding site on the HIV-1 envelope glycoprotein

A major goal of current HIV-1 vaccine design efforts is to induce broadly neutralizing antibodies (bNAbs). The VH1-2-derived bNAb IOMA directed to the CD4-binding site of the HIV-1 envelope glycoprotein is of interest because, unlike the better-known VH1-2-derived VRC01-class bNAbs, it does not require a rare short light chain complementarity-determining region 3 (CDRL3). Here, we describe three IOMA-class NAbs, ACS101-103, with up to 37% breadth, that share many characteristics with IOMA, including an average-length CDRL3. Cryo-electron microscopy revealed that ACS101 shares interactions with those observed with other VH1-2 and VH1-46-class bNAbs, but exhibits a unique binding mode to residues in loop D. Analysis of longitudinal sequences from the patient suggests that a transmitter/founder-virus lacking the N276 glycan might have initiated the development of these NAbs. Together these data strengthen the rationale for germline-targeting vaccination strategies to induce IOMA-class bNAbs and provide a wealth of sequence and structural information to support such strategies

Complementary antibody lineages achieve neutralization breadth in an HIV-1 infected elite neutralizer.

Broadly neutralizing antibodies (bNAbs) have remarkable breadth and potency against most HIV-1 subtypes and are able to prevent HIV-1 infection in animal models. However, bNAbs are extremely difficult to induce by vaccination. Defining the developmental pathways towards neutralization breadth can assist in the design of strategies to elicit protective bNAb responses by vaccination. Here, HIV-1 envelope glycoproteins (Env)-specific IgG+ B cells were isolated at various time points post infection from an HIV-1 infected elite neutralizer to obtain monoclonal antibodies (mAbs). Multiple antibody lineages were isolated targeting distinct epitopes on Env, including the gp120-gp41 interface, CD4-binding site, silent face and V3 region. The mAbs each neutralized a diverse set of HIV-1 strains from different clades indicating that the patient's remarkable serum breadth and potency might have been the result of a polyclonal mixture rather than a single bNAb lineage. High-resolution cryo-electron microscopy structures of the neutralizing mAbs (NAbs) in complex with an Env trimer generated from the same individual revealed that the NAbs used multiple strategies to neutralize the virus; blocking the receptor binding site, binding to HIV-1 Env N-linked glycans, and disassembly of the trimer. These results show that diverse NAbs can complement each other to achieve a broad and potent neutralizing serum response in HIV-1 infected individuals. Hence, the induction of combinations of moderately broad NAbs might be a viable vaccine strategy to protect against a wide range of circulating HIV-1 viruses

ACS114 targets the silent face and primarily interacts with <i>N</i>-linked glycans on HIV-1 Env.

(a) Magnified view of the ACS114 epitope shown as ribbons. ACS114 HC and LC are color-coded as indicated. Glycans are shown as sticks and contoured by the density of the cryo-EM map at 4σ. Gp120 is color-coded beige. (b) Interaction of ACS114 with the N262, N295, N301 and N332 glycans. Glycans are shown as sticks and contoured by the density of the cryo-EM map at 4σ. Amino acids that interact with the respective glycans are highlighted. (c) Interaction of ACS114 HC with gp120 C4 region. Amino acid interactions between ACS114’s HC and gp120’s C4 are highlighted and based on the density in the cryo-EM map. Predicted hydrogen bonds are shown with a distance d) Comparison of ACS114 to V3-targeting bNAb PGT124 (PDB:6MCO) and 10–1074 (PDB:5T3Z) and silent face-targeting bNAbs VRC-PG05 (PDB:6BF4) and SF12 (PDB:6OKQ). Fabs and trimer are shown as a surface representation. Fabs are colored coded as indicated. The gp41 and gp120 subunits are depicted in grey and light brown, respectively, with the N-linked glycans in light blue. The structures were aligned relative to gp120.</p

BLI binding kinetics.

(TIF)</p

Isolation of four distinct antibody lineages from an HIV-1 infected elite neutralizer using Env-specific B cell sorting.

(a) Longitudinal serum neutralization of viruses from different clades by individual 18877. The geometric mean is given in blue and arrows indicate timepoints where virus (grey) and PBMCs (black) were isolated to obtain env and BCR sequences, respectively. (b) B cells were selected by fluorescence-activated cell sorting (FACS) using fluorescently labeled Env proteins. The gating strategy for the isolation of B cells from PBMCs taken at month 36 post-SC is shown here as an example. (c) Pie-chart displaying the VH gene usage of the isolated BCR sequences (n = 314). Each grey-color coded slice represents a VH gene and the size of the slice is proportional with the amount of BCRs derived from this gene. (d) Phylogenetic analysis of the HC VDJ sequences of four isolated antibody lineages to compare the evolutionary relationship between the various BCRs. The names of the corresponding mAbs are listed on the right. Colors indicate to what lineage the mAbs belong to: IGHV1-2 (blue), IGHV4-34 (green), IGHV4-59 (pink) and IGHV5-51 (red). ACS117 is colored slightly lighter compared to the other members of the IGHV4-34 lineage because ACS117 most likely originated from a different B cell precursor. Scale depicts the nucleotide substitutions per site. (e) Timeline with arrows indicating the timepoints that members from the four different antibody lineages were obtained.</p

HIV-1 Env evolution of the CD4-binding site, silent face, gp120-gp41 interface and V3 epitopes targeted by the various NAbs.

Sequence alignment of longitudinal Envs from donor H18877 with residues surrounding the epitopes of all four antibody lineages are shown. Contact-residues of ACS101, ACS114, ACS122 and ACS130 revealed by high-resolution cryo-electron microscopy structures and neutralization assays are highlighted in different colors as shown below. Amino acids are numbered according to HxB2.</p