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.

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.

Long-term expression of human argininosuccinate synthetase in mice following bone marrow transplantation with retrovirus-transduced hematopoietic stem cells.

Amphotropic and ecotropic packaging cell lines were used to obtain high titers (greater than 10(6) colony forming units/ml) of retroviruses encoding human argininosuccinate synthetase, and these viruses were used to transduce murine bone marrow cells using cocultivation in vitro. The bone marrow cells were transplanted into lethally irradiated recipient mice, and argininosuccinate synthetase activity was measured in peripheral blood. Transduction with amphotropic retrovirus resulted in short-term expression for a period of 1-8 weeks, and no animals expressed the human gene after 25 weeks. Over 60% of the animals transplanted with cells transduced with ecotropic retrovirus expressed the human gene 44 weeks post-transplant, although the level of expression varied over a wide range. Analysis of the DNA from transplanted animals demonstrated the presence of the human sequence in expressing animals, and S1 nuclease analysis of RNA confirmed the presence of the human RNA transcripts. Analysis of granulocyte/macrophage (GM) colonies derived from the bone marrow of transplanted, expressing animals revealed a correlation between the level of expression of the transduced gene with the percentage of GM colonies carrying the human gene sequence. These data demonstrate the feasibility of obtaining long-term expression of genes introduced into bone marrow cells using retroviral vectors and the feasibility of obtaining expression of a gene not normally expressed in bone marrow.

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.

Effects of autoprothrombin II-A on epinephrine-induced platelet aggregation of normal- and coumadin-treated dogs.

Platelet-rich plasma from dogs and from coumadin-treated dogs aggregated at the same optimum concentration of epinephrine. Neither protein C nor its active form called autoprothrombin II-A was necessary for aggregation of dog platelets with epinephrine. For platelet aggregation, suboptimal concentrations of epinephrine were potentiated by addition of purified autoprothrombin II-A. The latter, by itself, induced platelet aggregation in high concentration.

Multiple congenital anomaly/mental retardation (MCA/MR) syndrome with Goldenhar complex due to a terminal del(22q).

We present a case of terminal del(22q) with Goldenhar complex including hemifacial microsomia, bilateral epibulbar dermoids, preauricular tags with sensorineural hearing loss, vertebral anomalies, and CNS and renal malformations. The case illustrates causal heterogeneity of the Goldenhar complex and a previously unreported associated chromosome deletion.

Quality control in immunohistochemistry. Report of a workshop sponsored by the Biological Stain Commission.

Although immunohistochemical methods are increasingly applied in diagnostic histopathology, there has been little standardization or quality control of immunoreagents; and published reports have not standardized Material and Methods for meaningful comparisons of results among clinicians. The Biological Stain Commission-sponsored workshop was convened to address the following issues: a manufacturers' testing program for probity of commercial antibodies, development of a manual for performance criteria and quality control assurance procedures, standardization of package inserts, standardization of information provided in the Materials and Methods sections of publications, establishment of a reagent and procedure clearing house, study of the effects of different fixation regimes on tissue antigens, and investigation of the environmental conditions needed for antigen-antibody interaction. The recommendations of the ad hoc committee and their implications for the future are discussed.

Teratogenic effects of first-trimester cyclophosphamide therapy.

Intravenous cyclophosphamide was administered for severe exacerbation of systemic lupus erythematosus to a patient not known to be in the first trimester of pregnancy. The patient received no other medication except prednisone. Her neonate was born with multiple anomalies, including absent thumbs, cleft palate, low-set ears, and multiple eye abnormalities. These anomalies probably reflect teratogenic effects of cyclophosphamide, and indicate that judgment is required before its use in the first trimester. Furthermore, this case illustrates the need for effective contraception and repetitive pregnancy testing when potentially teratogenic agents are administered to presumably nonpregnant women in the reproductive age group.

Sexually transmitted papillomaviral infection in the male: VI. Simultaneous urethral cytology-ViraPap testing of male consorts of women with genital human papillomaviral infection.

A series of 122 males at high risk for development of human papillomavirus (HPV) infection were prospectively studied with simultaneous urethral brushing cytology and ViraPap, to establish their relative sensitivity and specificity in relation to the diagnosis of HPV infection in the urethral canal. The prevalence of disease in this high-risk population was 10 percent. The sensitivity of DNA hybridization was determined to be 67 percent, with a 100 percent specificity, as compared with a 25 percent sensitivity of cytologic analysis. Physical examination alone had a 75 percent sensitivity. Physical examination with ViraPap urethral analysis identified all patients with urethral HPV-associated disease. These results indicate that clinical examination alone misses one quarter of the HPV-involved patients.

D-2-hydroxyglutaric aciduria in neonate with seizures and CNS dysfunction.

D-2-Hydroxyglutaric aciduria was documented in a newborn who presented with seizures, hypotonia, cortical blindness, a movement disorder, and developmental delay. Her clinical presentation differs from that of patients with L-2-hydroxyglutaric aciduria and a single previously reported patient with D-2-hydroxyglutaric aciduria. Cerebrospinal fluid levels of gamma-aminobutyric acid were elevated, while biogenic amine metabolites were normal. The movement disorder in our patient and in those with L-2-hydroxyglutaric aciduria suggests involvement of the basal ganglia in the disease process. Prenatal diagnosis of an affected fetus was accomplished during a subsequent pregnancy.

The taming of immunohistochemistry: the new era of quality control.

The most critical factor for interpreting the results of immunohistochemistry is verification of antibody sensitivity and specificity. While some manufacturers supply material data sheets with this information, many do not. This paper describes a well-defined quality assurance program for testing immune reagents. This program can be used to provide commercial suppliers of antisera with analyses of their products destined for government licensure applications. This paper illustrates the protocol and explains the testing philosophy developed over the last eight years.

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.

Escherichia coli K-12 clones that overproduce dam methylase are hypermutable.

A strain of Escherichia coli K-12 that overproduces dam methylase 50-fold was found to be hypermutable, and mutations which resulted in loss of excess methylase activity restored mutation frequencies to wild-type levels. These results are consistent with involvement of this deoxyribonucleic acid methylase in mismatch correction.

Physical mapping of the loci Gabra3, DXPas8, CamL1, and Rsvp in a region of the mouse X chromosome homologous to human Xq28.

Using pulsed-field gel electrophoresis, a 3 million-bp physical map containing the X-linked loci Gabra3, DXPas8, CamL1, and Rsvp has been constructed for a segment of the mouse X chromosome homologous to human Xq28. Detailed mapping was performed using single and double digestions with rare-cutter restriction enzymes. Gabra3 and DXPas8 have been shown to be physically linked within a maximal distance of 1600 kb, DXPas8 and CamL1 within 750 kb, and CamL1 and Rsvp within 450 kb. In addition, several CpG islands have been detected in the region encompassing CamL1 and Rsvp. These studies confirm a gene order of cen-Gabra3-DXPas8-CamL1-Rsvp-tel determined by genetic mapping in interspecific backcrosses (A.S. Ryder-Cook et al., 1988, EMBO J. 7: 3017-3021; G.E. Herman et al., 1991, Genomics 9: 670-677). Physical distances for the loci studied agree with the calculated genetic distances. Assuming that there is conserved linkage between man and mouse in the region, the physical mapping data presented here may help to clarify the uncertain gene order for some human Xq28 loci.