Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells

The mechanism of disease-associated trinucleotide repeat instability involves cis-acting factors (cis-elements) in the vicinity of the repeat, but the nature of these elements is unknown. One cis-element may be the location of the replication origin relative to the repeat. We have used an SV40 DNA replication system to investigate the effect of the location of replication initiation on (CTG)(n)*(CAG)(n) stability in primate cells. Depending on the distance between the SV40 replication origin and the repeat tract, templates with 79 repeats yield predominantly expansions or predominantly deletions or remain intact. All templates with 17 repeats are stable. Thus, cis-elements that affect the sites of Okazaki fragment initiation relative to the repeat are crucial determinants of instability. This model system recapitulates the bias for expansions observed in many of the diseases associated with trinucleotide repeats. Our results might explain the variable amounts of CTG/CAG instability that are observed in different chromosomal contexts.     

Mutations in the gene encoding the lamin B receptor produce an altered nuclear morphology in granulocytes (Pelger-Huët anomaly)

Pelger-Hu?t anomaly (PHA; OMIM *169400) is an autosomal dominant disorder characterized by abnormal nuclear shape and chromatin organization in blood granulocytes. Affected individuals show hypolobulated neutrophil nuclei with coarse chromatin. Presumed homozygous individuals have ovoid neutrophil nuclei, as well as varying degrees of developmental delay, epilepsy and skeletal abnormalities. Homozygous offspring in an extinct rabbit lineage showed severe chondrodystrophy, developmental anomalies and increased pre- and postnatal mortality. Here we show, by carrying out a genome-wide linkage scan, that PHA is linked to chromosome 1q41-43. We identified four splice-site, two frameshift and two nonsense mutations in LBR, encoding the lamin B receptor. The lamin B receptor (LBR), a member of the sterol reductase family, is evolutionarily conserved and integral to the inner nuclear membrane; it targets heterochromatin and lamins to the nuclear membrane. Lymphoblastoid cells from heterozygous individuals affected with PHA show reduced expression of the lamin B receptor, and cells homozygous with respect to PHA contain only trace amounts of it. We found that expression of the lamin B receptor affects neutrophil nuclear shape and chromatin distribution in a dose-dependent manner. Our findings have implications for understanding nuclear envelope-heterochromatin interactions, the pathogenesis of Pelger-like conditions in leukemia, infection and toxic drug reactions, and the evolution of neutrophil nuclear shape.     

Urocortin-deficient mice show hearing impairment and increased anxiety-like behavior

Urocortin is a member of the corticotropin-releasing hormone peptide family and is found in many discrete brain regions. The distinct expression pattern of urocortin suggests that it influences such behaviors as feeding, anxiety and auditory processing. To better define the physiological roles of urocortin, we have generated mice carrying a null mutation of the urocortin gene. Urocortin-deficient mice have normal basal feeding behavior and stress responses, but show heightened anxiety-like behaviors in the elevated plus maze and open-field tests. In addition, hearing is impaired in the mutant mice at the level of the inner ear, suggesting that urocortin is involved in the normal development of cochlear sensory-cell function. These results provide the first example of a function for any peptidergic system in hearing.     

Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia

Familial hypomagnesemia with secondary hypocalcemia (OMIM 602014) is an autosomal recessive disease that results in electrolyte abnormalities shortly after birth. Affected individuals show severe hypomagnesemia and hypocalcemia, which lead to seizures and tetany. The disorder has been thought to be caused by a defect in the intestinal absorption of magnesium, rather than by abnormal renal loss of magnesium. Restoring the concentrations of serum magnesium to normal values by high-dose magnesium supplementation can overcome the apparent defect in magnesium absorption and in serum concentrations of calcium. Life-long magnesium supplementation is required to overcome the defect in magnesium handling by these individuals. We previously mapped the gene locus to chromosome 9q in three large inbred kindreds from Israel. Here we report that mutation of TRPM6 causes hypomagnesemia with secondary hypocalcemia and show that individuals carrying mutations in this gene have abnormal renal magnesium excretion.     

Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations

Mutations in BRCA1 and BRCA2 confer a high risk of breast and ovarian cancer, but account for only a small fraction of breast cancer susceptibility. To find additional genes conferring susceptibility to breast cancer, we analyzed CHEK2 (also known as CHK2), which encodes a cell-cycle checkpoint kinase that is implicated in DNA repair processes involving BRCA1 and p53 (refs 3,4,5). We show that CHEK2(*)1100delC, a truncating variant that abrogates the kinase activity, has a frequency of 1.1% in healthy individuals. However, this variant is present in 5.1% of individuals with breast cancer from 718 families that do not carry mutations in BRCA1 or BRCA2 (P = 0.00000003), including 13.5% of individuals from families with male breast cancer (P = 0.00015). We estimate that the CHEK2(*)1100delC variant results in an approximately twofold increase of breast cancer risk in women and a tenfold increase of risk in men. By contrast, the variant confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutations. This suggests that the biological mechanisms underlying the elevated risk of breast cancer in CHEK2 mutation carriers are already subverted in carriers of BRCA1 or BRCA2 mutations, which is consistent with participation of the encoded proteins in the same pathway.     

Biological and biomedical implications of the co-evolution of pathogens and their hosts

Co-evolution between host and pathogen is, in principle, a powerful determinant of the biology and genetics of infection and disease. Yet co-evolution has proven difficult to demonstrate rigorously in practice, and co-evolutionary thinking is only just beginning to inform medical or veterinary research in any meaningful way, even though it can have a major influence on how genetic variation in biomedically important traits is interpreted. Improving our understanding of the biomedical significance of co-evolution will require changing the way in which we look for it, complementing the phenomenological approach traditionally favored by evolutionary biologists with the exploitation of the extensive data becoming available on the molecular biology and molecular genetics of host-pathogen interactions.     

Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits

The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A-D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by D-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by D-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B 'NMDARs' constitute a type of excitatory glycine receptor.     

Three-dimensional X-ray structural microscopy with submicrometre resolution

Advanced materials and processing techniques are based largely on the generation and control of non-homogeneous microstructures, such as precipitates and grain boundaries. X-ray tomography can provide three-dimensional density and chemical distributions of such structures with submicrometre resolution; structural methods exist that give submicrometre resolution in two dimensions; and techniques are available for obtaining grain-centroid positions and grain-average strains in three dimensions. But non-destructive point-to-point three-dimensional structural probes have not hitherto been available for investigations at the critical mesoscopic length scales (tenths to hundreds of micrometres). As a result, investigations of three-dimensional mesoscale phenomena--such as grain growth, deformation, crumpling and strain-gradient effects--rely increasingly on computation and modelling without direct experimental input. Here we describe a three-dimensional X-ray microscopy technique that uses polychromatic synchrotron X-ray microbeams to probe local crystal structure, orientation and strain tensors with submicrometre spatial resolution. We demonstrate the utility of this approach with micrometre-resolution three-dimensional measurements of grain orientations and sizes in polycrystalline aluminium, and with micrometre depth-resolved measurements of elastic strain tensors in cylindrically bent silicon. This technique is applicable to single-crystal, polycrystalline, composite and functionally graded materials.