De Novo Truncating Mutations in AHDC1 in Individuals with Syndromic Expressive Language Delay,Hypotonia, and Sleep Apnea

Clinical whole-exome sequencing (WES) for identification of mutations leading to Mendelian disease has been offered to the medical community since 2011. Clinically undiagnosed neurological disorders are the most frequent basis for test referral, and currently, approximately 25% of such cases are diagnosed at the molecular level. To date, there are approximately 4,000 “known” disease-associated loci, and many are associated with striking dysmorphic features, making genotype-phenotype correlations relatively straightforward. A significant fraction of cases, however, lack characteristic dysmorphism or clinical pathognomonic traits and are dependent upon molecular tests for definitive diagnoses. Further, many molecular diagnoses are guided by recent gene-disease association discoveries. Hence, there is a critical interplay between clinical testing and research leading to gene-disease association discovery. Here, we describe four probands, all of whom presented with hypotonia, intellectual disability, global developmental delay, and mildly dysmorphic facial features. Three of the four also had sleep apnea. Each was a simplex case without a remarkable family history. Using WES, we identified AHDC1 de novo truncating mutations that most likely cause this genetic syndrome.     

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12_{E31Q/D32N/W59F}) where the residues Glu³¹, Asp³², and Trp⁵⁹ were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12_{E31Q/D32N/W59F} mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12_{E31Q/D32N/W59F} activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu³¹, Asp³², and Trp⁵⁹ in FKBP12 and Gln³¹, Asn³², and Phe⁵⁹ in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.     

A Vaccine against CCR5 Protects a Subset of Macaques upon Intravaginal Challenge with Simian Immunodeficiency Virus SIVmac251

As an alternative to targeting human immunodeficiency virus (HIV), we have developed vaccines targeting CCR5, a self-protein critically involved in HIV replication and pathogenesis. By displaying peptides derived from CCR5 at high density on the surface of virus-like particles, we can efficiently induce high-titer IgG antibodies against this self-molecule. Here, we investigated whether prophylactic immunization of rhesus macaques with a particle-based vaccine targeting two regions of macaque CCR5 could prevent or suppress vaginal infection with highly virulent SIVmac251. Twelve macaques were vaccinated with a bacteriophage Qß-based vaccine targeting macaque CCR5 (Qß.CCR5). Six control animals were immunized with the Qß platform alone. All animals immunized with Qß.CCR5 developed high-titer anti-CCR5 antibody responses. Macaques were vaginally challenged with a high dose of SIVmac251. The mean peak viral RNA levels in the vaccinated groups were 30-fold lower than in the control group (10^6.8 versus 10^8.3 copies/ml plasma). Three of the 12 vaccinated macaques dramatically suppressed simian immunodeficiency virus (SIV) replication: peak viral loads were low (10³ to 10⁴ RNA copies/ml), and SIV RNA became undetectable from 6 weeks onward. No viral RNA or DNA could be detected in colon and lymph node biopsy specimens collected 13 months after challenge. In vivo depletion of CD8⁺ cells failed to induce a viral rebound. However, once anti-CCR5 antibody responses had waned, the 3 animals became infected after intravaginal and/or intravenous rechallenge. In conclusion, vaccination against CCR5 was associated with dramatic suppression of virus replication in a subset (25%) of macaques. These data support further research of vaccination against CCR5 to combat HIV infection.     

Analysis of Strains Lacking Known Osmolyte Accumulation Mechanisms Reveals Contributions of Osmolytes and Transporters to Protection against Abiotic Stress

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K⁺ glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high- and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.