Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.     

Mutations in SLC6A19, encoding B0AT1, cause Hartnup disorder

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B(0)AT1 (ref. 4). We isolated the human homolog of B(0)AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.     

A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice

Mcm4 (minichromosome maintenance-deficient 4 homolog) encodes a subunit of the MCM2-7 complex (also known as MCM2-MCM7), the replication licensing factor and presumptive replicative helicase. Here, we report that the mouse chromosome instability mutation Chaos3 (chromosome aberrations occurring spontaneously 3), isolated in a forward genetic screen, is a viable allele of Mcm4. Mcm4(Chaos3) encodes a change in an evolutionarily invariant amino acid (F345I), producing an apparently destabilized MCM4. Saccharomyces cerevisiae strains that we engineered to contain a corresponding allele (resulting in an F391I change) showed a classical minichromosome loss phenotype. Whereas homozygosity for a disrupted Mcm4 allele (Mcm4(-)) caused preimplantation lethality, Mcm(Chaos3/-) embryos died late in gestation, indicating that Mcm4(Chaos3) is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4(Chaos3/Chaos3) females succumbed to mammary adenocarcinomas with a mean latency of 12 months. These findings suggest that hypomorphic alleles of the genes encoding the subunits of the MCM2-7 complex may increase breast cancer risk.     

The microRNA-producing enzyme Dicer1 is essential for zebrafish development

MicroRNAs (miRNAs) are produced by the Dicer1 enzyme; the role of Dicer1 in vertebrate development is unknown. Here we report target-selected inactivation of the dicer1 gene in zebrafish. We observed an initial build-up of miRNA levels, produced by maternal Dicer1, in homozygous dicer1 mutants, but miRNA accumulation stopped after a few days. This resulted in developmental arrest around day 10. These results indicate that miRNA-producing Dicer1 is essential for vertebrate development.     

Chromosome stability, in the absence of apoptosis, is critical for suppression of tumorigenesis in Trp53 mutant mice

The p53 protein integrates multiple upstream signals and functions as a tumor suppressor by activating distinct downstream genes. At the cellular level, p53 induces apoptosis, cell cycle arrest and senescence. A rare mutant form of p53 with the amino acid substitution R175P, found in human tumors, is completely defective in initiating apoptosis but still induces cell cycle arrest. To decipher the functional importance of these pathways in spontaneous tumorigenesis, we used homologous recombination to generate mice with mutant p53-R172P (the mouse equivalent of R175P in humans). Mice inheriting two copies of this mutation (Trp53(515C/515C)) escape the early onset of thymic lymphomas that characterize Trp53-null mice. At 7 months of age, 90% of Trp53-null mice had died, but 85% of Trp53(515C/515C) mice were alive and tumor-free, indicating that p53-dependent apoptosis was not required for suppression of early onset of spontaneous tumors. The lymphomas and sarcomas that eventually developed in Trp53(515C/515C) mice retained a diploid chromosome number, in sharp contrast to aneuploidy observed in tumors and cells from Trp53-null mice. The ability of mutant p53-R172P to induce a partial cell cycle arrest and retain chromosome stability are crucial for suppression of early onset tumorigenesis.     

Mutations in a delta 8-delta 7 sterol isomerase in the tattered mouse and X-linked dominant chondrodysplasia punctata. jderry@immunex.com.

Tattered (Td) is an X-linked, semi-dominant mouse mutation associated with prenatal male lethality. Heterozygous females are small and at 4-5 days of age develop patches of hyperkeratotic skin where no hair grows, resulting in a striping of the coat in adults. Craniofacial anomalies and twisted toes have also been observed in some affected females. A potential second allele of Td has also been described. The phenotype of Td is similar to that seen in heterozygous females with human X-linked dominant chondrodysplasia punctata (CDPX2, alternatively known as X-linked dominant Conradi-Hünermann-Happle syndrome) as well as another X-linked, semi-dominant mouse mutation, bare patches (Bpa). The Bpa gene has recently been identified and encodes a protein with homology to 3beta-hydroxysteroid dehydrogenases that functions in one of the later steps of cholesterol biosynthesis. CDPX2 patients display skin defects including linear or whorled atrophic and pigmentary lesions, striated hyperkeratosis, coarse lusterless hair and alopecia, cataracts and skeletal abnormalities including short stature, rhizomelic shortening of the limbs, epiphyseal stippling and craniofacial defects (MIM 302960). We have now identified the defect in Td mice as a single amino acid substitution in the delta8-delta7 sterol isomerase emopamil binding protein (Ebp; encoded by Ebp in mouse) and identified alterations in human EBP in seven unrelated CDPX2 patients.     

Effective targeted gene 'knockdown' in zebrafish

The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted 'knockdown' technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.     

Mutations in IHH, encoding Indian hedgehog, cause brachydactyly type A-1.

Brachydactyly type A-1 (BDA-1; MIM 112500) is characterized by shortening or missing of the middle phalanges (Fig. 1a). It was first identified by Farabee in 1903 (ref. 2), is the first recorded example of a human anomaly with Mendelian autosomal-dominant inheritance and, as such, is cited in most genetic and biological textbooks. Here we show that mutations in IHH, which encodes Indian hedgehog, cause BDA-1. We have identified three heterozygous missense mutations in the region encoding the amino-terminal signaling domain in all affected members of three large, unrelated families. The three mutant amino acids, which are conserved across all vertebrates and invertebrates studied so far, are predicted to be adjacent on the surface of IHH.     

Mutations in ARHGEF6, encoding a guanine nucleotide exchange factor for Rho GTPases, in patients with X-linked mental retardation

X-linked forms of mental retardation (XLMR) include a variety of different disorders and may account for up to 25% of all inherited cases of mental retardation. So far, seven X-chromosomal genes mutated in nonspecific mental retardation (MRX) have been identified: FMR2, GDI1, RPS6KA3, IL1RAPL, TM4SF2, OPHN1 and PAK3 (refs 2-9). The products of the latter two have been implicated in regulation of neural plasticity by controlling the activity of small GTPases of the Rho family. Here we report the identification of a new MRX gene, ARHGEF6 (also known as alphaPIX or Cool-2), encoding a protein with homology to guanine nucleotide exchange factors for Rho GTPases (Rho GEF). Molecular analysis of a reciprocal X/21 translocation in a male with mental retardation showed that this gene in Xq26 was disrupted by the rearrangement. Mutation screening of 119 patients with nonspecific mental retardation revealed a mutation in the first intron of ARHGEF6 (IVS1-11T-->C) in all affected males in a large Dutch family. The mutation resulted in preferential skipping of exon 2, predicting a protein lacking 28 amino acids. ARHGEF6 is the eighth MRX gene identified so far and the third such gene to encode a protein that interacts with Rho GTPases.     

Regulation of left-right patterning in mice by growth/differentiation factor-1

The transforming growth factor-beta (TGF-beta) superfamily encompasses a large group of structurally related polypeptides that are capable of regulating cell growth and differentiation in a wide range of embryonic and adult tissues. Growth/differentiation factor-1 (Gdf-1, encoded by Gdf1) is a TGF-beta family member of unknown function that was originally isolated from an early mouse embryo cDNA library and is expressed specifically in the nervous systemin late-stage embryos and adult mice. Here we show that at early stages of mouse development, Gdfl is expressed initially throughout the embryo proper and then most prominently in the primitive node, ventral neural tube, and intermediate and lateral plate mesoderm. To examine its biological function, we generated a mouse line carrying a targeted mutation in Gdf1. Gdf1-/- mice exhibited a spectrum of defects related to left-right axis formation, including visceral situs inversus, right pulmonary isomerism and a range of cardiac anomalies. In most Gdf1-/- embryos, the expression of Ebaf (formerly lefty-1) in the left side of the floor plate and Leftb (formerly lefty-2), nodal and Pitx2 in the left lateral plate mesoderm was absent, suggesting that Gdf1 acts upstream of these genes either directly or indirectly to activate their expression. Our findings suggest that Gdf1 acts early in the pathway of gene activation that leads to the establishment of left-right asymmetry.     

Deletions and epimutations affecting the human 14q32.2 imprinted region in individuals with paternal and maternal upd(14)-like phenotypes

Human chromosome 14q32.2 carries a cluster of imprinted genes including paternally expressed genes (PEGs) such as DLK1 and RTL1 and maternally expressed genes (MEGs) such as MEG3 (also known as GTL2), RTL1as (RTL1 antisense) and MEG8 (refs. 1,2), together with the intergenic differentially methylated region (IG-DMR) and the MEG3-DMR. Consistent with this, paternal and maternal uniparental disomy for chromosome 14 (upd(14)pat and upd(14)mat) cause distinct phenotypes. We studied eight individuals (cases 1-8) with a upd(14)pat-like phenotype and three individuals (cases 9-11) with a upd(14)mat-like phenotype in the absence of upd(14) and identified various deletions and epimutations affecting the imprinted region. The results, together with recent mouse data, imply that the IG-DMR has an important cis-acting regulatory function on the maternally inherited chromosome and that excessive RTL1 expression and decreased DLK1 and RTL1 expression are relevant to upd(14)pat-like and upd(14)mat-like phenotypes, respectively.     

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa (RP). So far, six loci (USH1A-USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A for USH1B and the gene encoding harmonin for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G-->C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, Delta M1281 and IVS51+5G-->A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.     

CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms

Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation and establishing laterality. Cilia motility defects cause primary ciliary dyskinesia (PCD, MIM244400), a disorder affecting 1:15,000-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive ciliary bending. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD-linked loci. Here we show that the zebrafish cilia paralysis mutant schmalhans (smh(tn222)) encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a-regulated gene product. Screening 146 unrelated PCD families identified individuals in six families with reduced outer dynein arms who carried mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103/Pr46b functions as a tightly bound, axoneme-associated protein. These results identify Ccdc103 as a dynein arm attachment factor that causes primary ciliary dyskinesia when mutated.