The gene for achondroplasia maps to the telomeric region of chromosome 4p.

Achondroplasia is the most common type of genetic dwarfism. It is characterized by disproportionate short stature and other skeletal anomalies resulting from a defect in the maturation of the chondrocytes in the growth plate of the cartilage. We have now mapped the achondroplasia gene near the telomere of the short arm of chromosome 4 (4p16.3), by family linkage studies using 14 pedigrees. A positive lod score of z = 3.35 with no recombinants was obtained with an intragenic marker for IDUA. This localization will facilitate the positional cloning of the disease gene.

Cytochrome P450s: coupling development and environment.

There is growing evidence that some members of the cytochrome P450 superfamily could be involved in the regulation of basic developmental processes such as pattern formation, morphogenesis, cell differentiation and growth. This development calls attention to the myriad small molecules metabolized by cytochrome P450s, some of which might function as the morphogens proposed by the Local Source-Dispersed Sink hypothesis. This new information also suggests a mechanism for the developmental toxicity of drugs and environmental pollutants: such compounds could interfere with normal development by altering the spatial and temporal expression patterns of cytochrome P450s required for normal development.

Effect of two mutations of human CYP1B1, G61E and R469W, on stability and endogenous steroid substrate metabolism.

CYP1B1 is linked to normal eye development by the disease phenotype, primary congenital glaucoma (PCG). CYP1B1 mRNA was expressed in a number of human fetal tissue cDNA libraries, supporting the suggestion of its involvement in tissue development. Highest expression levels were found in thymus and kidney, followed by spleen. A considerably lower level was observed in lung, cardiac and skeletal muscle. No expression was detected in liver or brain. CYP1B1 is able to metabolize steroid hormones. Testosterone was a poor substrate and activity with progesterone was 6-fold higher, but estradiol was the preferred substrate, exhibiting a greater metabolite profile with CYP1B1 than with CYP1A2. Major metabolites were A-ring hydroxylations (75-80%). Others were 15alpha-, 6alpha-, 16alpha- and 6beta-hydroxy metabolites. Two CYP1B1 mutations found in families with the PCG phenotype in which incomplete penetrance is seen were expressed in Escherichia coli. G61E, a hinge region mutation, and R469W, a heme region mutation, were shown to code for holoenzymes. G61E had greatly diminished stability, while the R469W holoenzyme, if anything, was stabilized. Both mutants showed compromised catalytic activity. The extents of isomeric site activity diminution were not proportional, resulting in alterations in the metabolite profiles. The results suggest that if a metabolite of CYP1B1 or elimination of a metabolite by CYP1B1 is necessary for normal embryonic or fetal tissue development, the appearance of these two mutations could result in developmental abnormalities. The altered activities of the mutants and ability of CYP1B1 to respond to external challenge may be the basis for the observed incomplete penetrance.

A common FGFR3 gene mutation is present in achondroplasia but not in hypochondroplasia.

Achondroplasia is the most common type of genetic dwarfism. It is characterized by disproportionate short stature and other skeletal anomalies resulting from a defect in the maturation of the chondrocytes in the growth plate of the cartilage. Recent studies mapped the achondroplasia gene on chromosome region 4p16.3 and identified a common mutation in the gene encoding the fibroblast growth factor receptor 3 (FGFR3). In an analysis of 19 achondroplasia families from a variety of ethnic backgrounds we confirmed the presence of the G380R mutation in 21 of 23 achondroplasia chromosomes studied. In contrast, the G380R mutation was not found in any of the 8 hypochondroplasia chromosomes studied. Furthermore, linkage studies in a 3-generation family with hypochondroplasia show discordant segregation with markers in the 4p16.3 region suggesting that at least some cases of hypochondroplasia are caused by mutations in a gene other than FGFR3.

Enhanced expression of CYP1B1 in Escherichia coli.

Conditions for the optimal expression of the human CYP1B1 hemoprotein in Escherichia coli have been investigated. CYP1B1 cDNA was prepared from a retinal cDNA template and used to generate cDNA fragments with modified 5'-sequences reported to allow enhanced expression in E. coli DH5alpha. Plasmids were constructed, using the pCWori+ expression vector and were used to examine necessity for thiamine, delta-aminolevulinic acid (ALA), and IPTG. The optimal shaking speed in an orbital incubator was 150 rpm at 30 degrees C. Higher speeds resulted in increased cell death and lower speeds resulted in lower expression of cytochrome P450. IPTG was necessary for this expression system, which makes use of the lac repressor, but levels above 0.5 mM were without additional benefit. We were able to show thiamine to be unnecessary in this expression system, although included by others expressing CYP1B1. ALA has been reported to enhance expression of several different forms of cytochrome P450. We examined the dependence of CYP1B1 expression on ALA. The expression proved to be highly dependent upon this heme precursor, with levels of CYP1B1 increasing approximately 20-fold, to 920 nmol/l in the presence of up to 2.5 mM ALA. The question of whether heme synthesis and apoprotein synthesis were coupled was then investigated. It could be shown that although heme synthesis was not limiting (CYP101 holoenzyme expression in the absence of ALA was four times higher than the ALA-supported CYP1B1 holoenzyme expression), it was necessary for optimal expression of CYP1B1. CYP1B1 protein synthesis appears to be coupled to heme precursor availability, as seen by SDS-PAGE, because in the absence of heme precursor apocytochrome P450 1B1 does not accumulate.

Minor and trace sterols in marine invertebrates 55. The isolation, structure elucidation and synthesis of ergosta-5, 24(28), 25-trien-3 beta-ol.

The isolation, structure determination and synthesis of ergosta-5, 24(28), 25-trien-3 beta-ol, as well as the synthesis of its 28-14C analog--a possible biosynthetic precursor of several marine sterols--is described.

Cell membrane localization of sterols with conventional and unusual side chains in two marine demosponges.

Subcellular fractionation by differential centrifugation was performed on two previously unstudied marine sponges that predominantly contain either conventional (Reniera sp.) or unconventional (Pseudaxinyssa sp.) sterols. Direct evidence for the presence of unconventional sterols with C24 alkylated side chains in the cellular membranes of Pseudaxinyssa sp. is provided, but the presence of unconventional sterols in sponge membranes is shown not to be a universal feature of the Porifera. Possible structural and functional roles of unconventional lipid molecules in sponge cell membranes are discussed.

Sterols and their biosynthesis in some freshwater bivalves.

A number of free sterols and sterol esters of three freshwater mussels was separated and identified. A slow rate of biosynthesisde novo of sterols was demonstrated inAnodonta cygnea. Injected cholesterol was found to undergo esterification, oxidation, Δ(22)-dehydrogenation and C-24 alkylation. Methyl-[(14)C]methionine was proved to be incorporated in C-24 alkylsterols. Abnormally large amounts of cholesterol injected inA. cygnea were metabolized toward restoration of the normal composition of sterols. This was achieved by intensified metabolism of cholesterol, mainly by conjugation, oxidation and Δ(22)-dehydrogenation.

Fatty acids as biological markers for bacterial symbionts in sponges.

Analyses of fatty acids with carbon numbers between C12 and C22 are reported for five Great Barrier Reef sponges. These analyses indicate that phototrophic cyanobacterial symbionts (blue-green algae) present in three of the sponges are chemically distinct, whereas the other two sponges do not contain cyanobacterial symbionts. All the sponges contain other, nonphototrophic bacteria. The fatty acid analyses indicate that the non-phototrophic bacterial populations present in the different sponges are distinct in both their chemical compositions and their abundances. Nonphototrophic bacteria are estimated to account for between 60 and 350 micrograms/g (extractable fatty acids:tissue wet weight), whereas cyanobacteria account for between 10 and 910 micrograms/g. One sponge (Pseudaxinyssa sp.) contains a relatively large amount of the isoprenoid acid, 4, 8, 12-trimethyltridecanoic acid; this acid is presumed to be derived from phytol, a degradation product of chlorophyll. This sponge also contains relatively large amounts of the nonmethylene interrupted fatty acid, octadeca-5,9-dienoic acid. Analyses of interior and cyanobacteria-rich surface tissues of this sponge indicate that these two acids are probably not associated with the symbiotic cyanobacteria.

Roles of cytochrome p450 in development.

Cytochrome P450 (CYP) forms are ubiquitous in nature, appearing in almost all phyla, with many forms appearing in any organism. About 50 different forms have been identified in man, and some of these are found in the embryo, some showing temporal dependence. Many of the forms of cytochrome P450 present in one species have homologues in other species. For example, CYP1A2 is present in many species, including man, rabbits, rodents, fish and fowl. The amino acid sequence identity of these homologues is often in excess of 70%. CYP26, too, has more than 61% identity in amino acid sequence between fish, fowl and mammals. In view of the high degree of conservation of sequence as well as of enzymatic activities, it is only reasonable to assume that such strong conservation of sequence also reflects a conservation of function. Since the 'xenobiotic metabolizing' enzymes predate the production of the many xenobiotics they are known to metabolize, perhaps it is reasonable to consider endobiotics as natural substrates for their metabolism. Of the identified forms of cytochrome P450 that are present in embryonic tissue, we consider the possibility that they serve the organism in support of morphogenesis of the embryonic tissue. These forms may either function to generate morphogenic molecules or to keep regions free of them, thereby creating temporal and spatial regions of morphogen action and supporting region-specific changes in cells. One known morphogen, retinoic acid, has the enzymes retinal dehydrogenase (RALDH) and CYP26 maintaining its actions, the former responsible for its generation and the latter for its elimination. Another form of cytochrome P450, CYP1B1 appears also to be involved in differentiation of tissue, with its absence resulting in primary congenital glaucoma. However, the nature of the morphogen it may maintain still remains to be elucidated.

Molecular genetics of primary congenital glaucoma.

Molecular genetic studies conducted during the last several years have thrown some light on the basic molecular defects in primary congenital glaucoma (PCG) and the rationale behind the clinical and genetic presentation of this paediatric eye condition. The existence of a hereditary form of PCG segregating as an autosomal recessive trait with high penetrance is now confirmed. The primary molecular defect underlying the majority of PCG cases has been identified as mutations in the cytochrome P4501B1 (CYP1B1) gene. This gene is expressed in tissues of the anterior chamber angle of the eye. Molecular modelling experiments suggest that mutations observed in PCG patients interfere with the integrity of the CYP1B1 molecule as well as its ability to adopt a normal conformation and bind haem. On the basis of these observations, we hypothesised that CYP1B1 participates in the normal development and function of the eye by metabolising essential molecules that are perhaps used in a signalling pathway. Revealing the identity of this molecule is our major objective since it can lead to as yet unknown biochemical cascades controlling the terminal stages of anterior chamber angle development.

Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21.

Primary congenital glaucoma (Buphthalmos) is an autosomal recessive eye disorder, postulated to result from developmental defects in the anterior eye segment. Previously, we reported two chromosomal locations for this condition on 2p21 (GLC3A) and 1p36 (GLC3B) respectively. In this study, heritable mutations of human cytochrome P4501B1 gene (CYP1B1) in affected individuals of five well-characterized families linked to the GLC3A locus are described. CYP1B1 gene has previously been mapped within the GLC3A candidate region and its expression in the trabecular meshwork cells has been demonstrated in this study. Three different homozygous mutations were identified and characterized: a 13 bp deletion in exon III; an insertion of a single cytosine base in exon II; and a larger deletion affecting the 5' end of exon III and the adjacent intronic region. All of these are frameshift mutations that are predicted to remove domains essential for the function of the CYP1B1 protein. Therefore, it is expected that all these mutations result in functional null alleles. The mutations detected in the affected members of these families were not present in 470 chromosomes from randomly selected normal individuals, thus strongly suggesting that CYP1B1 is the gene for the GLC3A locus on 2p21. The results are discussed in the context of the earlier hypothesis that 'drug-metabolizing' enzymes might modulate the processes of growth and differentiation by controlling the steady-state-levels of oxygenated growth-effector molecules.

Isoprenoid biosynthesis in a marine sponge of the Amphimedon genus: incorporation studies with [1-14C] acetate, [4-14C] cholesterol and [2-14C] mevalonate.

1. We present quantitative evidence from incorporation of [1-14C] acetate that the enzymes to synthesise isoprenoids are present in the marine sponge Amphimedon sp. and that efficient carotenoid synthesis takes place. 2. The de novo synthesis of b,b-carotene and (3R,3'R)-zeaxanthin may occur in a chlorophyll a-producing microalgal symbiont with subsequent aromatisation to (3R)-isoagelaxanthin by the sponge itself. 3. Amphimedon sp. contains nuclear-modified sterols derived by modification of conventional dietary sterols.

Comparative expression profiling of 40 mouse cytochrome P450 genes in embryonic and adult tissues.

This study is the first systematic investigation of the gestational age-dependent and adult tissue-specific expression patterns of each known mouse CYP family (40 genes) using normalized cDNA panels and uniform reverse transcriptase polymerase chain reaction-based assays. Twenty-seven of the P450s were constitutively expressed during development. The number gradually increased through the phases of gastrulation E7 (n=14), neural patterning and somitogenesis E11 (n=17), organogenesis E15 (n=20), and fetal period E17 (n=21). Cyp2s1, Cyp8a1, Cyp20, Cyp21a1, Cyp26a1, Cyp46, and Cyp51 were detected in each of the four stages studied. Members of family CYP1 demonstrated complex, nonoverlapping embryonic patterns of expression, indicating that Cyp1a1 and Cyp1a2 may not compensate for Cyp1b1 deficiency associated with abnormal eye development. Multiple Cyp forms were found to be constitutively expressed in each of the adult tissues studied: liver (n=31), kidney (n=30), testis (n=26), lung (n=24), and heart (n=13). The tissue-specific P450-expression profiles reported in this study provide a reference for more focused analysis of the tissue-specific and developmental functions of the cytochrome P450 monooxygenases.

Genomic structure of HOXD13 gene: a nine polyalanine duplication causes synpolydactyly in two unrelated families.

Synpolydactyly (SPD) is a limb malformation that shows a characteristic manifestation in both hands and feet. This condition is inherited as an autosomal dominant trait with reduced penetrance. We have recently mapped this locus centromeric to the HOXD8 intragenic marker and suggested the HOXD13 gene as a potential candidate for this condition. The genomic structure of HOXD13 established in this study consists of two exons that encodes a polypeptide of 335 amino acids. The downstream exon at the 3' end of this gene contains the homeodomain sequences that are highly conserved. Sixty-three bp upstream of this exon lies a stretch of intronic CA-repeats that proved to be polymorphic in two different populations. The upstream exon encodes 75% of the entire protein and contains a stretch of 15 normal alanines at its 5' end. Sequence comparison at this position in the homozygous affected individuals identified a total of 24 alanine residues that resulted from a duplication of nine polyalanines. In two unrelated SPD families, this duplication was directly transmitted from the affected parents to their affected, but not unaffected, offspring; in one family its size has remained constant for at least 150 years spanning over seven generations. The presence of this duplication confirmed the status of four normal gene carriers, one incomplete penetrance and two affected individuals who were recombinants for HOXD8 or HOXD13-CA repeat markers. This duplication was not present in 150 chromosomes of unrelated healthy subjects of two different populations.

Identification of a single ancestral CYP1B1 mutation in Slovak Gypsies (Roms) affected with primary congenital glaucoma.

Primary congenital glaucoma (PCG) is an autosomal recessive eye disease that occurs at an unusually high frequency in the ethnic isolate of Roms (Gypsies) in Slovakia. Recently, we linked the disease in this population to the GLC3A locus on 2p21. At this locus, mutations in the cytochrome P4501B1 (CYP1B1) gene have been identified as a molecular basis for this condition. Here, we report the results of CYP1B1 mutation screening of 43 PCG patients from 26 Slovak Rom families. A homozygous G-->A transition at nucleotide 1505 in the highly conserved region of exon 3 was detected in all families. This mutation results in the E387K substitution, which affects the conserved K helix region of the cytochrome P450 molecule. Determination of the CYP1B1 polymorphic background showed a common DNA haplotype in all patients, thus indicating that the E387K mutation in Roms has originated from a single ancestral mutational event. The Slovak Roms represent the first population in which PCG is found to result from a single mutation in the CYP1B1 gene, so that a founder effect is the most plausible explanation of its increased incidence. An ARMS-PCR assay has been developed for fast detection of this mutation, thus allowing direct DNA based prenatal diagnosis as well as gene carrier detection in this particular population. Screening of 158 healthy Roms identified 17 (10.8%) mutation carriers, indicating that the frequency of PCG in this population may be even higher than originally estimated.

Sequence analysis and homology modeling suggest that primary congenital glaucoma on 2p21 results from mutations disrupting either the hinge region or the conserved core structures of cytochrome P4501B1.

We recently reported three truncating mutations of the cytochrome P4501B1 gene (CYP1B1) in five families with primary congenital glaucoma (PCG) linked to the GLC3A locus on chromosome 2p21. This could be the first direct evidence supporting the hypothesis that members of the cytochrome P450 superfamily may control the processes of growth and differentiation. We present a comprehensive sequence analysis of the translated regions of the CYP1B1 gene in 22 PCG families and 100 randomly selected normal individuals. Sixteen mutations and six polymorphisms were identified, illustrating an extensive allelic heterogeneity. The positions affected by these changes were evaluated by building a three-dimensional homology model of the conserved C-terminal half of CYP1B1. These mutations may interfere with heme incorporation, by affecting the hinge region and/or the conserved core structures (CCS) that determine the proper folding and heme-binding ability of P450 molecules. In contrast, all polymorphic sites were poorly conserved and located outside the CCS. Northern hybridization analysis showed strong expression of CYP1B1 in the anterior uveal tract, which is involved in secretion of the aqueous humor and in regulation of outflow facility, processes that could contribute to the elevated intraocular pressure characteristic of PCG.