Nitrogen enhances the effect of pre-drought priming against post-anthesis drought stress by regulating starch and protein formation in wheat.

Drought stress is one of the most serious environmental stress factor constraining crop production across the globe. Among cereals, wheat grains are very sensitive to drought as a small degree of stress can affect the enzymatic system. This study aimed to investigate whether nitrogen and pre-anthesis drought priming could enhance the action of major regulatory enzymes involved in starch accumulation and protein synthesis in bread wheat (Triticum aestivum L.). For this purpose, cultivars YM-158 (medium gluten) and YM-22 (low gluten) were grown in rain-controlled conditions under two nitrogen levels, that is, N180 (N1) and N300 (N2). Drought priming was applied at the jointing stage and drought stress was applied 7 days after anthesis. Drought stress reduced starch content but enhanced protein content in grains. N2 and primed plants kept higher contents of nonstructural carbohydrates, fructans, and sucrose; with higher activity of sucrose-phosphate synthase in flag leaves. Furthermore, N2 and priming treatments showed higher sink ability to develop grains by showing higher sucrose-to-starch conversion activities of adenosine diphosphate-glucose pyrophosphorylase, uridine diphosphate glucose pyrophosphorylase, sucrose-synthase, soluble-starch synthase, starch branching enzyme, and granule-bound starch synthase as compared to N1 and non-primed treatments. The application of N2 and primed treatment showed a greater ability to maintain grain filling in both cultivars as compared to N1 and non-primed crops. Our study suggested that high nitrogen has the potential to enhance the effect of pre-drought priming to change source-sink relationships and grain yield of wheat under drought stress during the filling process.

Physiological and proteomic analyses revealed the response mechanisms of two different drought-resistant maize varieties.

BACKGROUND: Drought stress severely limits maize seedling growth and crop yield. Previous studies have elucidated the mechanisms by which maize acquires drought resistance and contends with water deficiency. However, the link between the physiological and molecular variations among maize cultivars are unknown. Here, physiological and proteomic analyses were conducted to compare the stress responses of two maize cultivars with contrasting drought stress tolerance. RESULTS: The physiological analysis showed that the drought-tolerant SD609 maize variety maintains relatively high photochemical efficiency by enhancing its protective cyclic electron flow (CEF) mechanism and antioxidative enzymes activities. Proteomics analysis revealed that 198 and 102 proteins were differentially expressed in SD609 and the drought-sensitive SD902 cultivar, respectively. GO and KEGG enrichments indicated that SD609 upregulated proteins associated with photosynthesis, antioxidants/detoxifying enzymes, molecular chaperones and metabolic enzymes. Upregulation of the proteins related to PSII repair and photoprotection improved photochemical capacity in SD609 subjected to moderate drought stress. In SD902, however, only the molecular chaperones and sucrose synthesis pathways were induced and they failed to protect the impaired photosystem. Further analysis demonstrated that proteins related to the electron transport chain (ETC) and redox homeostasis as well as heat shock proteins (HSPs) may be important in protecting plants from drought stress. CONCLUSIONS: Our experiments explored the mechanism of drought tolerance and clarified the interconnections between the physiological and proteomic factors contributing to it. In summary, our findings aid in further understanding of the drought tolerance mechanisms in maize.

Evaluation of immunoprotective effects of recombinant protein and DNA vaccine based on Eimeria tenella surface antigen 16 and 22 in vivo.

Coccidiosis triggered by Eimeria tenella is accompanied by haemorrhagic caecum and high morbidity. Vaccines are preferable choices to replace chemical drugs against coccidiosis. Surface antigens of apicomplexan parasites can adhere to host cells during the infection process. Therefore, truncated fragments coding E. tenella surface antigen 16 (EtSAG16) and 22 (EtSAG22) were cloned into pET-28a prokaryotic vector to express recombinant protein 16 (rEtSAG16) and 22 (rEtSAG22), respectively. Likewise, pEGFP-N1-EtSAG16 and pEGFP-N1-EtSAG22 plasmids were constructed using pEGFP-N1 eukaryotic vector. Further, pEGFP-N1-EtSAG4-16-22 multiple gene plasmid carrying EtSAG4, 16 and 22 were designed as cocktail vaccines to study integral immunoprotective effects. Western blot and RT-PCR (reverse transcription) assay were performed to verify expressions of EtSAG16 and 22 genes. Immunoprotective effects of recombinant protein or DNA vaccine were evaluated using different doses (50 or 100 µg) in vivo. All chickens in the vaccination group showed higher cytokine concentration (IFN-γ and IL-17), raised IgY antibody level, increased weight gain, lower caecum lesion score and reduced oocyst shedding compared with infection control groups (p < 0.05). The highest anticoccidial index (ACI) value 173.11 was from the pEGFP-N1-EtSAG4-16-22 plasmid (50 µg) group. In conclusion, EtSAG16 and 22 might be alternative candidate genes for generating vaccines against E. tenella infection.

Ferrocene-Appended Iridium(III) Complexes: Configuration Regulation, Anticancer Application, and Mechanism Research.

A series of ferrocene-appended half-sandwiched iridium(III) phenylpyridine complexes have been designed and synthesized. These complexes show better anticancer activity than cisplatin widely used in clinic under the same conditions. Meanwhile, complexes could effectively inhibit cell migration and colony formation. Complexes could interact with protein and transport through serum protein, effectively catalyzing the oxidation of nicotinamide-adenine dinucleotid and inducing the accumulation of reactive oxygen species (ROS, 1O2), which confirmed the anticancer mechanism of oxidation. Furthermore, laser scanning confocal detection indicates that these complexes can enter cells followed by a non-energy-dependent cellular uptake mechanism, effectively accumulating in the lysosome (Pearson's colocalization coefficient: ∼0.90), leading to lysosome damage, and reducing the mitochondrial membrane potential (MMP). Taken together, ferrocene-appended iridium(III) complexes possess the prospect of becoming a new multifunctional therapeutic platform, including lysosome-targeted imaging and anticancer drugs.

Identification of novel ATP7A mutations and prenatal diagnosis in Chinese patients with Menkes disease.

Menkes disease (MD) is a fatal X-linked multisystem disease caused by mutations in ATP7A. In this study, clinical and genetic analysis was performed in 24 male MD patients. Development delay, seizures, kinky coarse hair, and dystonia were found in 24, 22, 24, and 24 patients, respectively. Serum ceruloplasmin/copper tested in 19 patients was low. Abnormal classic features of MD presented in the MRI/MRA of 19 patients. Seventeen mutations of ATP7A were identified in 22 patients. Twelve were novel mutations including three small deletion/insertion, one missense mutation, two nonsense mutations, three splicing-site mutations, and three gross deletions. Twenty-two patients were genetically diagnosed; neither point mutation nor deletion/duplication was found in two of them. c.2179G > A found in five patients might be a hot-spot mutation. Prenatal molecular diagnosis was performed for five unrelated fetuses (1 female and 4 male), which found four fetuses to be wild type and one male carried the same mutation as the proband. This study of the largest sample of Chinese MD patients examined to date discovered the unique phenotype and genotype spectrum in Chinese patients with 12 novel mutations of ATP7A, and that c.2179G > A might be a hot-spot mutation in MD patients. Five successful prenatal diagnosis contributed important information for MD families.

[Clinical and ATP7A gene analysis of three infants with Menkes disease and prenatal diagnosis for a fetus at risk].

Menkes disease is a rare X-linked recessive disorder characterized by multi-systemic disorder of copper deficiency caused by ATP7A gene mutation. In this study, the clinical and laboratory features of three patients with Menkes disease were analyzed. Prenatal diagnosis had been performed for a fetus of a family. Three patients were admitted at the age of 8-9 months due to severe epilepsies and marked delayed psychomotor development. Significantly light complexion, pudgy cheeks and sparse fuzzy wooly hair were observed. On their cranial MR imaging, cortical atrophy, leukoencephalopathy, basal ganglia damage and tormesity of the intracranial vessels were found. Their plasma ceruloplasmin decreased to 70.2, 73.5 and 81 mg/L, significantly lower than normal range (210-530 mg/L). c.3914A>G (p. D1305G) was detected on ATP7A gene of case 1 and 2. A novel mutation, c.3265G>T (p.G1089X) was found in case 3. Both of them were firstly found in Chinese patients of Menkes disease. The mother of case 1 was tested at 20 weeks of pregnancy. Karyotype and ATP7A gene studies of the amniocytes were performed for the prenatal diagnosis of her fetus. Normal male karyotypes without c.3914A>G mutation on ATP7A gene was showed. Postnatal genetic analysis and normal development confirmed the prenatal diagnosis.

Bipolaronic Motifs Induced Spatially Separated Catalytic Sites for Tunable Syngas Photosynthesis From CO2.

Photocatalytic reduction of CO2 into syngas is a promising way to tackle the energy and environmental challenges; however, it remains a challenge to achieve reaction decoupling of CO2 reduction and water splitting. Therefore, efficient production of syngas with a suitable CO/H2 ratio for Fischer-Tropsch synthesis can hardly be achieved. Herein, bipolaronic motifs including Co(II)-pyridine N motifs and Co(II)-imine N motifs are rationally designed into a crystalline imine-linked 1,10-phenanthroline-5,6-dione-based covalent organic framework (bp-Co-COF) with a triazine core. These featured structures with spatially separated active sites exhibit efficient photocatalytic performance toward CO2-to-syngas conversion with a suitable CO/H2 ratio (1:1-1:3). The bipolaronic motifs enable a highly separated electron-hole state, whereby the Co(II)-pyridine N motifs tend to be the active sites for CO2 activation and accelerate the hydrogenation to form *COOH intermediates; whilst, the Co(II)-imine N motifs increase surface hydrophilicity for H2 evolution. The photocatalytic reductions of CO2 and H2O thus decouple and proceed via a concerted way on the bipolaronic motifs of bp-Co-COF. The optimal bp-Co-COF photocatalyst achieves a high syngas evolution rate of 15.8 mmol g-1 h-1 with CO/H2 ratio of 1:2, outperforming previously reported COF-based photocatalysts.

Comparative Physio-Biochemical and Transcriptome Analyses Reveal Contrasting Responses to Magnesium Imbalances in Leaves of Mulberry (Morus alba L.) Plants.

Magnesium (Mg) deficiency is a major factor limiting the growth and development of plants. Mulberry (Morus alba L.) is an important fruit tree crop that requires Mg for optimal growth and yield, especially in acid soils. However, the molecular mechanism of Mg stress tolerance in mulberry plants remains unknown. In this study, we used next-generation sequencing technology and biochemical analysis to profile the transcriptome and physiological changes of mulberry leaves under different Mg treatments (deficiency: 0 mM, low: 1 mM, moderate low: 2 mM, sufficiency: 3 mM, toxicity: 6 mM, higher toxicity: 9 mM) as T1, T2, T3, CK, T4, T5 treatments, respectively, for 20 days. The results showed that Mg imbalance altered the antioxidant enzymatic activities, such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), and non-enzymatic, including soluble protein, soluble sugar, malondialdehyde (MDA), and proline (PRO), contents of the plant. The Mg imbalances disrupted the ultrastructures of the vital components of chloroplast and mitochondria relative to the control. The transcriptome data reveal that 11,030 genes were differentially expressed (DEGs). Genes related to the photosynthetic processes (CAB40, CAB7, CAB6A, CAB-151, CAP10A) and chlorophyll degradation (PAO, CHLASE1, SGR) were altered. Antioxidant genes such as PER42, PER21, and PER47 were downregulated, but DFR was upregulated. The carbohydrate metabolism pathway was significantly altered, while those involved in energy metabolism processes were perturbed under high Mg treatment compared with control. We also identified several candidate genes associated with magnesium homeostasis via RT-qPCR validation analysis, which provided valuable information for further functional characterization studies such as promoter activity assay or gene overexpression experiments using transient expression systems.

LiDAR-aid Inertial Poser: Large-scale Human Motion Capture by Sparse Inertial and LiDAR Sensors.

We propose a multi-sensor fusion method for capturing challenging 3D human motions with accurate consecutive local poses and global trajectories in large-scale scenarios, only using single LiDAR and 4 IMUs, which are set up conveniently and worn lightly. Specifically, to fully utilize the global geometry information captured by LiDAR and local dynamic motions captured by IMUs, we design a two-stage pose estimator in a coarse-to-fine manner, where point clouds provide the coarse body shape and IMU measurements optimize the local actions. Furthermore, considering the translation deviation caused by the view-dependent partial point cloud, we propose a pose-guided translation corrector. It predicts the offset between captured points and the real root locations, which makes the consecutive movements and trajectories more precise and natural. Moreover, we collect a LiDAR-IMU multi-modal mocap dataset, LIPD, with diverse human actions in long-range scenarios. Extensive quantitative and qualitative experiments on LIPD and other open datasets all demonstrate the capability of our approach for compelling motion capture in large-scale scenarios, which outperforms other methods by an obvious margin. We will release our code and captured dataset to stimulate future research.

The Effects of 1-Deoxynojirimycin from Mulberry on Oxidative Stress and Inflammation in Laying Hens and the Direct Effects on Intestine Epithelium Cells In Vitro.

The intestine is highly vulnerable to various factors and has been proposed as a promising determinant for poultry health. Phytogenic or plant-derived feed additives can be used to help improve intestinal health. In this study, we aimed to investigate the effects of DNJ on the antioxidative parameters, including malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and inflammatory cytokines (IL-6, IL-1ß, and TNF-α), in plasma and intestinal tissues using layers supplemented with or without the DNJ extract of mulberry leaves (DNJ-E) via the ELISA method. A total of 192 healthy Hy-Line Brown layers, aged 47 weeks old, were used to conduct a 56-day study. All hens were randomly separated into four groups as follows: a basal diet containing 0 mg/kg DNJ-E(CON), 50 mg/kg, 100 mg/kg, and 150 mg/kg DNJ-E. Furthermore, the potential mechanism by which DNJ influences intestinal function was also investigated in in vitro cultured intestinal epithelium cells (IEC) with quantification methods including the use of a cell counting kit-8 (CCK8), ELISA, qRT-PCR, and ROS detection. The results showed that CAT in plasma significantly increased following 50 mg/kg DNJ-E supplementation. Moreover, 50 mg/kg DNJ-E supplementation was associated with increases in T-SOD in the jejunum and ileum. However, there was no significant difference in inflammatory cytokines between groups in in vivo experiments. Subsequent in vitro IEC studies revealed that cell viability increased significantly following 5 µM and 10 µM DNJ treatments while decreasing significantly following 20 µM DNJ treatment. Antioxidative parameters improved at 5 µM and 10 µM DNJ concentrations. However, there were no ameliorative effects on antioxidant parameters observed under 20 µM DNJ treatment. The expression levels of Nrf2 mRNA increased significantly under DNJ treatment. DNJ treatment was associated with significant changes in the expression of genes of inflammatory cytokines. In conclusion, our study revealed that DNJ could improve oxidative stress and inflammation responses in the chicken intestine. These findings provide a theoretical reference for the development of functional feed additives that regulate intestinal health and lay the foundation for systematically revealing the mechanism of DNJ.

Melatonin Enhances Drought Tolerance by Regulating Leaf Stomatal Behavior, Carbon and Nitrogen Metabolism, and Related Gene Expression in Maize Plants.

It is commonly known that exogenously applied melatonin can alleviate the impact of drought stress, but the mechanism used by melatonin to regulate stomatal behavior and carbon (C) and nitrogen (N) metabolism to increase drought resistance remains elusive. Herein, our aim was to investigate the influence of exogenous melatonin on the regulation of C and N metabolism in maize plants under water deficit. In this study, we analyzed stomatal behavior, the key components of C and N metabolism, and the gene expression and activity of enzymes involved in the C and N metabolism in maize plants. The results showed that the application of melatonin (100 µM) significantly increased maize growth and sustained the opening of stomata, and secondarily increased the photosynthetic capacity in maize. Under drought stress, foliar application of melatonin induced the gene transcription and activities of sucrose phosphate synthetase, ADP-glucose pyrophosphorylase, phosphoenolpyruvate carboxylase, and citrate synthase, resulting in the enhancement of sucrose and starch synthesis and the tricarboxylic acid (TCA) cycle. This enhancement in sugar biosynthesis and the TCA cycle might lead to stronger N assimilation. As anticipated, NO3 - reduction and NH4 + assimilation were also strengthened after melatonin treatment under drought stress. An increase was observed in some key enzymatic activities and transcription involved in nitrogen metabolism, such as that of nitrate reductase, nitrite reductase, glutamate synthase, and glutamine synthetase, in melatonin-treated, drought-stressed maize. Moreover, melatonin attenuated the drought-induced damage by reducing protein degradation and increasing the level of proline. Conclusively, our results indicate that exogenous melatonin enhances drought tolerance in maize via promoting stomatal opening and regulating C and N metabolism and related gene expression.

Physiological and transcriptomic analyses of the effects of exogenous melatonin on drought tolerance in maize (Zea mays L.).

Water deficit inhibits maize (Zea mays L.) seedling growth and yield. Application of exogenous melatonin can improve drought tolerance of corn, but little is known regarding the transcriptional mechanisms of melatonin-mediated drought tolerance in maize. Increased understanding of the effects of melatonin on maize plants under drought stress is vital to alleviate the adverse effects of drought on food production in the future. The aim of this investigation was to use physiological and transcriptome analyses for exploring the possible mechanisms of exogenous melatonin against drought stress in maize. In this study, maize seedlings were subjected to drought stress and some were treated with exogenous melatonin. The physiological results showed that melatonin inhibited H2O2 accumulation and promoted the scavenging of excessive reactive oxygen species to reduce oxidative damage in maize leaves. Transcriptomic analysis identified 957 differentially expressed genes between melatonin and non-melatonin treatment groups. Further detailed analyses suggested that melatonin-regulated genes are mainly related to glutathione metabolism, calcium signaling transduction, and jasmonic acid biosynthesis. Some transcription factor families, such as WRKY, AP2/ERF-ERF, MYB, NAC, and bZIP, were also activated by exogenous melatonin. Moreover, crosstalk between melatonin and other hormones that mediate drought tolerance was observed. In conclusion, the combination of physiological and transcriptome analyses revealed some mechanisms underlying the role of melatonin in alleviating drought; knowledge of these mechanisms may assist in successful maize cultivation under drought stress.

Half-sandwich iridium(III) complexes with α-picolinic acid frameworks and antitumor applications.

Eight half-sandwich iridiumIII (IrIII) complexes of the general formula [(η5-Cpxbiph)Ir(O^N)Cl] (Cpxbiph is tetramethyl(biphenyl)cyclopentadienyl, and the O^N is α-picolinic acid chelating ligand and its derivatives) were synthesized and characterized. Compared with cis-platin widely used in clinic, target IrIII complexes showed at most five times more potent antitumor activity against A549 cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. IrIII complexes could be transported by serum albumin, bind with DNA, catalyze the oxidation of nicotinamide-adenine dinucleotid (NADH) and induce the production of reactive oxygen species, which confirmed the antitumor mechanism of oxidation. IrIII complexes could enter A549 cells followed by an energy-dependent cellular uptake mechanism, meanwhile, target the mitochondria and lysosomes with the Pearson's colocalization coefficient of 0.33 and 0.74, respectively, lead to the lysosomal destruction and the change of mitochondrial membrane potential (ΔΨm), and eventually induce apoptosis.

Clines, clusters, and the effect of study design on the inference of human population structure.

Previously, we observed that without using prior information about individual sampling locations, a clustering algorithm applied to multilocus genotypes from worldwide human populations produced genetic clusters largely coincident with major geographic regions. It has been argued, however, that the degree of clustering is diminished by use of samples with greater uniformity in geographic distribution, and that the clusters we identified were a consequence of uneven sampling along genetic clines. Expanding our earlier dataset from 377 to 993 markers, we systematically examine the influence of several study design variables--sample size, number of loci, number of clusters, assumptions about correlations in allele frequencies across populations, and the geographic dispersion of the sample--on the "clusteredness" of individuals. With all other variables held constant, geographic dispersion is seen to have comparatively little effect on the degree of clustering. Examination of the relationship between genetic and geographic distance supports a view in which the clusters arise not as an artifact of the sampling scheme, but from small discontinuous jumps in genetic distance for most population pairs on opposite sides of geographic barriers, in comparison with genetic distance for pairs on the same side. Thus, analysis of the 993-locus dataset corroborates our earlier results: if enough markers are used with a sufficiently large worldwide sample, individuals can be partitioned into genetic clusters that match major geographic subdivisions of the globe, with some individuals from intermediate geographic locations having mixed membership in the clusters that correspond to neighboring regions.

STRP screening sets for the human genome at 5 cM density.

BACKGROUND: Short tandem repeat polymorphisms (STRPs) are powerful tools for gene mapping and other applications. A STRP genome scan of 10 cM is usually adequate for mapping single gene disorders. However mapping studies involving genetically complex disorders and especially association (linkage disequilibrium) often require higher STRP density. RESULTS: We report the development of two separate 10 cM human STRP Screening Sets (Sets 12 and 52) which span all chromosomes. When combined, the two Sets contain a total of 782 STRPs, with average STRP spacing of 4.8 cM, average heterozygosity of 0.72, and total sex-average coverage of 3535 cM. The current Sets are comprised almost entirely of STRPs based on tri- and tetranucleotide repeats. We also report correction of primer sequences for many STRPs used in previous Screening Sets. Detailed information for the new Screening Sets is available from our web site: http://research.marshfieldclinic.org/genetics. CONCLUSION: Our new human STRP Screening Sets will improve the quality and cost effectiveness of genotyping for gene mapping and other applications.

A novel locus for adolescent idiopathic scoliosis on chromosome 12p.

Adolescent idiopathic scoliosis (AIS) is a common disorder with strong evidence for genetic predisposition. Quantitative trait loci (QTLs) for AIS susceptibility have been identified on chromosomes. We performed a genome-wide genetic linkage scan in seven multiplex families using 400 marker loci with a mean spacing of 8.6 cM. We used Genehunter Plus to generate linkage statistics, expressed as homogeneity (HLOD) scores, under dominant and recessive genetic models. We found a significant linkage signal on chromosome 12p, whose support interval extends from near 12 pter, spanning approximately 10 million bases or 31 cM. Fine mapping within the region using 20 additional markers reveals maximum HLOD = 3.7 at 5 cM under a dominant inheritance model, and a split peak maximum HLOD = 3.2 at 8 and 18 cM under a recessive inheritance model. The linkage support interval contains 95 known genes. We found evidence suggestive of linkage on chromosomes 1, 6, 7, 8, and 14. This study is the first to find evidence of an AIS susceptibility locus on chromosome 12. Detection of AIS susceptibility QTLs on multiple chromosomes in this and other studies demonstrate that the condition is genetically heterogeneous.

Characterization of low-density lipoprotein cholesterol-lowering efficacy for atorvastatin in a population-based DNA biorepository.

The Marshfield Clinic Personalized Medicine Research Project (PMRP) represents a large population-based biobank located in central Wisconsin. To position the PMRP database for large-scale pharmacogenetic association studies in the context of lipid-lowering therapy, we constructed an electronic phenotyping algorithm to quantify exposure and dose-response for atorvastatin, the most commonly prescribed lipid-lowering agent within this population. The resulting datasets were used to generate five distinct parameters for atorvastatin-induced changes in low-density lipoprotein (LDL) cholesterol level: (i) pretreatment, baseline LDL level [E(0)]; (ii) absolute reduction in LDL; (iii) relative reduction in LDL; (iv) potency [ED(50)]; and (v) maximal efficacy [E(max)]. These parameters will facilitate the efficient application of electronic phenotyping for drug outcomes in the context of large pharmacogenetic association studies.

Mapping autosomal dominant progressive limb-girdle myopathy with bone fragility to chromosome 9p21-p22: a novel locus for a musculoskeletal syndrome.

Progressive myopathy of a limb-girdle distribution and bone fragility is a rare autosomal dominant disorder of unknown etiology. Affected individuals, within this family, present with various combinations of progressive muscle weakness, easy fracturing, and poor healing of long bones. Additional features include premature graying with thin hair, thin skin, hernias, and clotting disorders. Electromyograms show myopathic changes and biopsies reveal non-specific myopathic changes. Skeletal radiographs demonstrate coarse trabeculation, patchy sclerosis, cortical thickening, and narrowing of medullary cavities. We report genetic mapping of this disorder to chromosome 9p21-p22 in a multigenerational family. A genome-wide scan for the disease locus obtained a maximal LOD score of 3.74 for marker GATA87E02 N (D9S1121). Haplotype analysis localized the disease gene within a 15 Mb interval flanked by markers AGAT142P and GATA5E06P. This region also localizes diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH). Identification of the disease gene will be necessary to understand the pathogenesis of this complex disorder.

Human diallelic insertion/deletion polymorphisms.

We report the identification and characterization of 2,000 human diallelic insertion/deletion polymorphisms (indels) distributed throughout the human genome. Candidate indels were identified by comparison of overlapping genomic or cDNA sequences. Average confirmation rate for indels with a > or =2-nt allele-length difference was 58%, but the confirmation rate for indels with a 1-nt length difference was only 14%. The vast majority of the human diallelic indels were monomorphic in chimpanzees and gorillas. The ratio of deletionrcolon;insertion mutations was 4.1. Allele frequencies for the indels were measured in Europeans, Africans, Japanese, and Native Americans. New alleles were generally lower in frequency than old alleles. This tendency was most pronounced for the Africans, who are likely to be closest among the four groups to the original modern human population. Diallelic indels comprise approximately 8% of all human polymorphisms. Their abundance and ease of analysis make them useful for many applications.

Homozygous WNT3 mutation causes tetra-amelia in a large consanguineous family.

Tetra-amelia is a rare human genetic disorder characterized by complete absence of all four limbs and other anomalies. We studied a consanguineous family with four affected fetuses displaying autosomal recessive tetra-amelia and craniofacial and urogenital defects. By homozygosity mapping, the disease locus was assigned to chromosome 17q21, with a maximum multipoint LOD score of 2.9 at markers D17S931, D17S1785, D17SS1827, and D17S1868. Further fine mapping defined a critical interval of approximately 8.9 Mb between D17S1299 and D17S797. We identified a homozygous nonsense mutation (Q83X) in the WNT3 gene in affected fetuses of the family. WNT3, a human homologue of the Drosophila wingless gene, encodes a member of the WNT family known to play key roles in embryonic development. The Q83X mutation truncates WNT3 at its amino terminus, suggesting that loss of function is the most likely cause of the disorder. Our findings contrast with the observation of early lethality in mice homozygous for null alleles of Wnt3. To our knowledge, this is the first report of a mutation in a WNT gene associated with a Mendelian disorder. The identification of a WNT3 mutation in tetra-amelia indicates that WNT3 is required at the earliest stages of human limb formation and for craniofacial and urogenital development.