The role of ZIC3 in vertebrate development.

ZIC3 is a C2H2 zinc finger transcription factor that is involved in early patterning of the vertebrate embryo. Human patients with mutations in the X-linked ZIC3 gene have a complex developmental phenotype that includes laterality defects, congenital heart disease, and lumbosacral and anal anomalies, including neural tube defects. Similar phenotypes are found in the bent tail (BN) mouse, a spontaneous mutation that is associated with a submicroscopic deletion of the ZIC3 locus, as well as in a ZIC3 null allele generated through homologous recombination. These findings suggest that ZIC3 plays important roles during development in establishing a proper left-right axis and in midline neural patterning. This review will summarize our current understanding of the role of ZIC3 in patterning of the vertebrate embryo, based on studies in model organisms such as Xenopus and the mouse. In addition, a comparison of ZIC3 with other vertebrate ZIC family members will be provided.

Inborn errors of sterol biosynthesis.

The known disorders of cholesterol biosynthesis have expanded rapidly since the discovery that Smith-Lemli-Opitz syndrome is caused by a deficiency of 7-dehydrocholesterol. Each of the six now recognized sterol disorders-mevalonic aciduria, Smith-Lemli-Opitz syndrome, desmosterolosis, Conradi-Hünermann syndrome, CHILD syndrome, and Greenberg dysplasia-has added to our knowledge of the relationship between cholesterol metabolism and embryogenesis. One of the most important lessons learned from the study of these disorders is that abnormal cholesterol metabolism impairs the function of the hedgehog class of embryonic signaling proteins, which help execute the vertebrate body plan during the earliest weeks of gestation. The study of the enzymes and genes in these several syndromes has also expanded and better delineated an important class of enzymes and proteins with diverse structural functions and metabolic actions that include sterol biosynthesis, nuclear transcriptional signaling, regulation of meiosis, and even behavioral modulation.

X-Linked dominant disorders of cholesterol biosynthesis in man and mouse.

The X-linked dominant male-lethal mouse mutations tattered and bare patches are homologous to human X-linked dominant chondrodysplasia punctata and CHILD syndrome, rare human skeletal dysplasias. These disorders also affect the skin and can cause cataracts and microphthalmia in surviving, affected heterozygous females. They have recently been shown to result from mutations in genes encoding enzymes involved in sequential steps in the conversion of lanosterol to cholesterol. This review will summarize clinical features of the disorders and describe recent biochemical and molecular investigations that have resulted in the elucidation of the involved genes and their metabolic pathway. Finally, speculations about possible mechanisms of pathogenesis will be provided.

The X-linked mouse mutation Bent tail is associated with a deletion of the Zic3 locus.

Bent tail (BN:) is a spontaneous, semi-dominant mutation on the mouse X chromosome that produces tail deformities and, rarely, open neural tube defects. Analysis of 292 normal male and affected male and female progeny from an intraspecific back-cross involving BN: supports a gene order of cen-DXMit89-18.5 +/- 2.3 cM-DXMit166-1.4 +/- 0.7 cM-BN:-1.0 +/- 0.6 cM-DXMit140 -4.8 +/- 1.3 cM-DXBay6-tel. A high frequency of sex chromosomal non-disjunction, unrelated to the BN: mutation, was also identified in the background strain. Refined genetic and physical mapping of the BN: critical region demonstrate that the mutation is associated with a <170 kb submicroscopic deletion that includes the anonymous microsatellite marker DXMit208 as well as the entire Zic3 locus. Human mutations in ZIC3 are associated with left-right axis malformations (MIM 306955, 208530, 207100). Abnormalities of abdominal and thoracic situs were also detected in viable BN: males and females. The presence of anal and spinal abnormalities in some of the human patients and the deletion of Zic3 in BN: mice support a key role for this gene in neural tube development and closure.

Comparative genome sequence analysis of the Bpa/Str region in mouse and Man.

The progress of human and mouse genome sequencing programs presages the possibility of systematic cross-species comparison of the two genomes as a powerful tool for gene and regulatory element identification. As the opportunities to perform comparative sequence analysis emerge, it is important to develop parameters for such analyses and to examine the outcomes of cross-species comparison. Our analysis used gene prediction and a database search of 430 kb of genomic sequence covering the Bpa/Str region of the mouse X chromosome, and 745 kb of genomic sequence from the homologous human X chromosome region. We identified 11 genes in mouse and 13 genes and two pseudogenes in human. In addition, we compared the mouse and human sequences using pairwise alignment and searches for evolutionary conserved regions (ECRs) exceeding a defined threshold of sequence identity. This approach aided the identification of at least four further putative conserved genes in the region. Comparative sequencing revealed that this region is a mosaic in evolutionary terms, with considerably more rearrangement between the two species than realized previously from comparative mapping studies. Surprisingly, this region showed an extremely high LINE and low SINE content, low G+C content, and yet a relatively high gene density, in contrast to the low gene density usually associated with such regions.

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.

The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.

Medical complications in long-term survivors with X-linked myotubular myopathy.

OBJECTIVES: X-linked myotubular myopathy (MTM1) is a rare developmental disorder of skeletal muscle characterized by the presence of central nuclei in biopsy specimens from affected male subjects. Until recently, the disorder was usually fatal within the first year of life. This study was undertaken to determine the outcome in long-term survivors (>1 year of age) with MTM1. METHODS: Clinical data were obtained on 55 male subjects from 49 independent North American families for which a mutation was identified in the X-linked myotubularin gene by direct genomic sequencing. Medical records were reviewed and families were interviewed to ascertain features at birth, length of survival, developmental milestones, and medical complications. RESULTS: Seventy-four percent (26 of 35) of the affected male subjects over the age of 1 year are living (range, 1 to 27 years); 80% remain completely or partially ventilator-dependent. In the absence of significant hypoxia, cognitive development is normal, and the muscle disorder appears nonprogressive. Several patients have had other medical problems not previously reported to be associated with MTM1. These include pyloric stenosis (4 male subjects from 3 families), spherocytosis (2 patients), gallstones (4 patients), kidney stones or nephrocalcinosis (2 patients), a vitamin K responsive bleeding diathesis (2 patients), and height >/=90% for age (40% of the patients). Six patients have had biochemical evidence of liver dysfunction, and 2 patients died after significant liver hemorrhage. CONCLUSIONS: These data suggest that the prognosis for X-linked MTM may not be as poor as previously reported. However, at least some long-term survivors appear at risk for medical complications involving other organ systems, and patients should be carefully monitored for these potentially life-threatening complications. The pleiotropic symptoms demonstrated in these patients strongly suggest that the function of the MTM1 protein is not limited to developing muscle cells.

Physical mapping of the mouse genome.

This review is intended to provide an overview of techniques and a source of reagents for physical mapping of the mouse genome. It focuses on those applications, methods, or resources unique to the mouse and on the generation of comparative physical maps. The reference list is not comprehensive; rather, recent reviews on each topic and selected representative examples are given.

Characterization of mutations in the myotubularin gene in twenty six patients with X-linked myotubular myopathy.

A candidate gene, myotubularin, involved in the pathogenesis of X-linked myotubular myopathy (MTM1) was isolated recently. Mutations originally were identified in 12% of patients examined for 40% of the coding sequence, raising the possibility that additional genes could be responsible for a proportion of X-linked cases. We report here the identification of mutations in 26 of 41 independent male patients with muscle biopsy-proven MTM, by direct genomic sequencing of 92% of the known coding sequence of the myotubularin gene. Eighteen patients had point mutations, including one A/G transition found in four patients which alters a splice acceptor site in exon 12 and leads to a three amino acid insertion. Six patients had small deletions involving <6 bp, while two larger deletions encompassed two or six exons, respectively. No differences were noted among the types of mutations between familial and sporadic cases. However, all of the five patients with a mild phenotype had missense mutations. While 50% of the mutations were found in exons 4 and 12, and three distinct mutations were found in more than one patient, no single mutation accounted for more than 10% of the cases. The low frequency of large deletions and the varied mutations identified suggest that direct mutation screening for molecular diagnosis may require gene sequencing.

Genomic structure of a novel LIM domain gene (ZNF185) in Xq28 and comparisons with the orthologous murine transcript.

Construction of a transcript map in the DXS52 region in Xq28 had previously led to the isolation of a cDNA with a LIM zinc finger domain in the carboxyl terminus. In parallel, the orthologous murine transcript was isolated from the syntenic region. The human and mouse cDNAs have been designated ZNF185 and Zfp185, respectively. By integrating the cDNA sequence with the cosmid-derived genomic sequence the exon-intron structure of the 3' end of the ZNF185 gene was resolved. Comparative sequence analyses of the human genomic sequence with the full-length murine cDNA facilitated prediction of the 5' end of the gene. The selective expression of three transcripts corresponding to the ZNF185 gene and a related gene was shown by Northern and Southern blots. In situ hybridizations revealed a nonubiquitous and stage-specific expression of Zfp185, especially in differentiating connective tissue. Since LIM proteins regulate cellular proliferation and/or differentiation by diverse mechanisms, and some have directly been associated with disease, conceivably ZNF185 may represent a candidate for a disease-causing gene linked to Xq28. Knowledge of the genomic structure will permit detailed mutation analyses.

Comparative mapping on the mouse X chromosome defines a myotubular myopathy equivalent region.

The gene for X-linked myotubular myopathy (MTM1) has been localized to a 300-kb critical region in human Xq28 between IDS and GABRA3. As part of an effort to clone this gene, we developed a YAC contig on the mouse X Chromosome (Chr) which includes loci homologous to those within the human MTM1 critical region. The murine contig consists of 18 YACs and spans 2.5-3.0 Mb. We have aligned the human and murine physical maps by isolating conserved mouse genomic fragments, including CpG islands and trapped exons. We believe that the simultaneous isolation of genes from both mouse and human and continued comparative mapping will prove helpful in the eventual identification of MTM1 and other genes in the region.

A comparative transcription map of the murine bare patches (Bpa) and striated (Str) critical regions and human Xq28.

The X-linked developmental mouse mutations bare patches (Bpa) and striated (Str) may be homologous to human X-linked dominant chondrodysplasia punctata (CDPX2) and incontinentia pigmenti (IP2), respectively, based on their genetic mapping and clinical phenotypes. Bpa and Str have been localized to an overlapping critical region of 600 kb that demonstrates conserved gene order with loci in human Xq28 between DXS1104 and DXS52. As part of efforts to isolate the genes involved in these disorders, we have begun to develop a comparative transcription map spanning this region in both species. Using techniques of cross-species conservation and hybridization, exon trapping, and cDNA selection we have identified four known genes or members of gene families--caltractin, a member of the gamma-aminobutyric acid (GABAA) receptor gene family, a member of the melanoma antigen gene (MAGE) family, and several members of the murine-specific, X-linked lymphocyte regulated gene (Xlr3) family. Trapped exons and, in some cases, longer cDNAs have been isolated for potentially 7-9 additional genes. One cDNA demonstrates highly significant homology with members of the Krüppel family of zinc finger transcription factors. A second novel cDNA demonstrates homology at the 3' end of the predicted amino acid sequence to a LIM domain consensus. Gene order appears conserved among those cDNAs determined to be present in both human and mouse. Three of the murine transcripts appear to be present in multiple copies within the Bpa/Str critical region and could be associated with a predisposition to genomic rearrangements. Reverse transcriptase PCR (RT-PCR) and Northern analysis demonstrate that several of the transcripts are expressed in mid-gestation murine embryos and neonatal skin, making them candidates for the Bpa and Str mutations and their respective homologous human disorders.

The chromosome localization and the HCF repeats of the human host cell factor gene (HCFC1) are conserved in the mouse homologue.

The gene encoding the human host cell factor (HCFC1) has recently been cloned and mapped to Xq28. HCFC1 codes for a family of related polypeptides that apparently arise from posttranslational processing. Six extremely conserved 19-amino-acid (aa)long motifs, unique to HCFC1 and located in the middle of the protein, could play a role in this processing or could be instrumental to the physiological role of the protein. Aternatively, these repeats could have arisen from recent duplications and may not have any specific function. To resolve this issue, we cloned the homologous region from the mouse HCFC1 gene and demonstrated that the 19-aa motifs are extremely conserved in sequence, number, and genomic organization, while the "linker" region between the third and fourth repeat is not. This suggests an important function for these repeats. In addition, by RT-PCR analysis of human RNA and comparison to the human genomic sequence, an alternative transcript including a 44-aa in-frame insertion, deriving from the 3' end of intron 18, was found. The significance of this alternative transcript is unknown, since it was not detectable in the mouse. The mouse HCFC1 gene maps to a region syntenic to Xq28, and, as in human, is in close proximity to the Renin-binding protein gene, in a 100-kb region also including the Licam and Vasopressin receptor type 2 genes.