Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification

The genetic analysis of congenital skull malformations provides insight into normal mechanisms of calvarial osteogenesis. Enlarged parietal foramina (PFM) are oval defects of the parietal bones caused by deficient ossification around the parietal notch, which is normally obliterated during the fifth fetal month. PFM are usually asymptomatic, but may be associated with headache, scalp defects and structural or vascular malformations of the brain. Inheritance is frequently autosomal dominant, but no causative mutations have been identified in non-syndromic cases. We describe here heterozygous mutations of the homeobox gene MSX2 (located on 5q34-q35) in three unrelated families with PFM. One is a deletion of approximately 206 kb including the entire gene and the others are intragenic mutations of the DNA-binding homeodomain (RK159-160del and R172H) that predict disruption of critical intramolecular and DNA contacts. Mouse Msx2 protein with either of the homeodomain mutations exhibited more than 85% reduction in binding to an optimal Msx2 DNA-binding site. Our findings contrast with the only described MSX2 homeodomain mutation (P148H), associated with craniosynostosis, that binds with enhanced affinity to the same target. This demonstrates that MSX2 dosage is critical for human skull development and suggests that PFM and craniosynostosis result, respectively, from loss and gain of activity in an MSX2-mediated pathway of calvarial osteogenic differentiation.     

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.     

Structure and function of eukaryotic peptide transporters

The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in the kidney. Recently, molecular biology has had a very substantial impact on the study of peptide transport, and here we review the molecular and functional information available within the framework of physiology. To this end we consider not only the mammalian peptide transporters and their tissue distribution and regulation but also those from other species (including Caenorhabditis elegans) which make up the proton-dependent oligopeptide transport superfamily. In addition, understanding the binding requirements for transported substrates may allow future design and targeted tissue delivery of peptide and peptidomimetic drugs. Finally, we aim to highlight some of the less well understood areas of peptide transport, in the hope that it will stimulate further research into this challenging yet exciting topic.     

Mutations of PVRL1, encoding a cell-cell adhesion molecule/herpesvirus receptor, in cleft lip/palate-ectodermal dysplasia

Cleft lip, with or without cleft palate (CL/P), is one of the most common birth defects, occurring in 0.4 to 2.0 per 1,000 infants born alive. Approximately 70% of CL/P cases are non-syndromic (MIM 119530), but CL/P also occurs in many single-gene syndromes, each affecting a protein critical for orofacial development. Here we describe positional cloning of the gene responsible for an autosomal recessive CL/P-ectodermal dysplasia (ED) syndrome (CLPED1; previously ED4; ref. 2), which we identify as PVRL1, encoding nectin-1, an immunoglobulin (Ig)-related transmembrane cell-cell adhesion molecule that is part of the NAP cell adhesion system. Nectin-1 is also the principal cell surface receptor for alpha-herpesviruses (HveC; ref. 7), and the high frequency of CLPED1 on Margarita Island in the Caribbean Sea might result from resistance of heterozygotes to infection by these viruses.     

Intraprotein radical transfer during photoactivation of DNA photolyase

Amino-acid radicals play key roles in many enzymatic reactions. Catalysis often involves transfer of a radical character within the protein, as in class I ribonucleotide reductase where radical transfer occurs over 35 A, from a tyrosyl radical to a cysteine. It is currently debated whether this kind of long-range transfer occurs by electron transfer, followed by proton release to create a neutral radical, or by H-atom transfer, that is, simultaneous transfer of electrons and protons. The latter mechanism avoids the energetic cost of charge formation in the low dielectric protein, but it is less robust to structural changes than is electron transfer. Available experimental data do not clearly discriminate between these proposals. We have studied the mechanism of photoactivation (light-induced reduction of the flavin adenine dinucleotide cofactor) of Escherichia coli DNA photolyase using time-resolved absorption spectroscopy. Here we show that the excited flavin adenine dinucleotide radical abstracts an electron from a nearby tryptophan in 30 ps. After subsequent electron transfer along a chain of three tryptophans, the most remote tryptophan (as a cation radical) releases a proton to the solvent in about 300 ns, showing that electron transfer occurs before proton dissociation. A similar process may take place in photolyase-like blue-light receptors.