Physiological significance and expression of P450s in the developing eye.

Expression of 10 CYP orthologs (Families 1-3) in developing mouse conceptus is constitutive. These forms have specific temporal and spatial expression. Studies on CYP1B1 indicate its requirement for normal eye development, both in human and mouse. The distribution of the enzyme in the mouse eye is in three regions, which may reflect three different, perhaps equally important, functions in this organ. Its presence in the inner ciliary and lens epithelia appears to be necessary for normal development of the trabecular meshwork and its function in regulating intraocular pressure. Its expression in the retinal ganglion and inner nuclear layers may reflect a role in maintenance of the visual cycle. Its expression in the corneal epithelium may indicate a function in metabolism of environmental xenobiotics.

Milroy disease and the VEGFR-3 mutation phenotype.

Primary congenital lymphoedema (Milroy disease) is a rare autosomal dominant condition for which a major causative gene defect has recently been determined. Mutations in the vascular endothelial growth factor receptor 3 (VEGFR-3) gene have now been described in 13 families world-wide. This is a review of the condition based on the clinical findings in 71 subjects from 10 families. All 71 individuals have a mutation in VEGFR-3. Ninety per cent of the 71 individuals carrying a VEGFR-3 mutation showed signs of oedema, which was confined in all cases to the lower limbs. In all but two cases onset of swelling was from birth. Other symptoms and signs included cellulitis (20%), large calibre leg veins (23%), papillomatosis (10%), and upslanting toenails (10%). In males, hydrocoele was the next most common finding after oedema (37%). Thorough clinical examination of these patients indicates that there are few clinical signs in addition to lower limb oedema. Rigorous phenotyping of patients produces a high yield of VEGFR-3 mutations.

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.

Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24.

INTRODUCTION: Lymphoedema-distichiasis syndrome (LD) (OMIM 153400) is a rare, primary lymphoedema of pubertal onset, associated with distichiasis. Causative mutations have now been described in FOXC2, a forkhead transcription factor gene. Numerous clinical associations have been reported with this condition, including congenital heart disease, ptosis, varicose veins, cleft palate, and spinal extradural cysts. SUBJECTS: We report clinical findings in 74 affected subjects from 18 families and six isolated cases. All of them were shown to have mutations in FOXC2 with the exception of one family who had two affected subjects with lymphoedema and distichiasis and linkage consistent with the 16q24 locus. RESULTS: The presence of lymphoedema was highly penetrant. Males had an earlier onset of lymphoedema and a significantly increased risk of complications. Lymphatic imaging confirmed the earlier suggestion that LD is associated with a normal or increased number of lymphatic vessels rather than the hypoplasia or aplasia seen in other forms of primary lymphoedema. Distichiasis was 94.2% penetrant, but not always symptomatic. Associated findings included ptosis (31%), congenital heart disease (6.8%), and cleft palate (4%). Other than distichiasis, the most commonly occurring anomaly was varicose veins of early onset (49%). This has not been previously reported and suggests a possible developmental role for FOXC2 in both venous and lymphatic systems. This is the first gene that has been implicated in the aetiology of varicose veins. CONCLUSION: Unlike previous publications, the thorough clinical characterisation of our patients permits more accurate prediction of various phenotypic abnormalities likely to manifest in subjects with FOXC2 mutations.

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.

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.

Human microphthalmia associated with mutations in the retinal homeobox gene CHX10.

Isolated human microphthalmia/anophthalmia, a cause of congenital blindness, is a clinically and genetically heterogeneous developmental disorder characterized by a small eye and other ocular abnormalities. Three microphthalmia/anophthalmia loci have been identified, and two others have been inferred by the co-segregation of translocations with the phenotype. We previously found that mice with ocular retardation (the or-J allele), a microphthalmia phenotype, have a null mutation in the retinal homeobox gene Chx10 (refs 7,8). We report here the mapping of a human microphthalmia locus on chromosome 14q24.3, the cloning of CHX10 at this locus and the identification of recessive CHX10 mutations in two families with non-syndromic microphthalmia (MIM 251600), cataracts and severe abnormalities of the iris. In affected individuals, a highly conserved arginine residue in the DNA-recognition helix of the homeodomain is replaced by glutamine or proline (R200Q and R200P, respectively). Identification of the CHX10 consensus DNA-binding sequence (TAATTAGC) allowed us to demonstrate that both mutations severely disrupt CHX10 function. Human CHX10 is expressed in progenitor cells of the developing neuroretina and in the inner nuclear layer of the mature retina. The strong conservation in vertebrates of the CHX10 sequence, pattern of expression and loss-of-function phenotypes demonstrates the evolutionary importance of the genetic network through which this gene regulates eye development.

Papillary thyroid carcinoma associated with papillary renal neoplasia: genetic linkage analysis of a distinct heritable tumor syndrome.

Papillary thyroid carcinoma usually is sporadic, but may occur in a familial form. The complete clinical and pathological phenotype of familial papillary thyroid carcinoma (fPTC) has not been determined, and the susceptibility gene(s) is unknown. We investigated the clinical and pathological characteristics of an unusually large three-generation fPTC kindred to characterize more fully the clinical phenotype. We performed linkage analysis to determine the chromosomal location of a fPTC susceptibility gene. In addition to the known association of fPTC with nodular thyroid disease, we observed the otherwise rare entity of papillary renal neoplasia (PRN) in two kindred members, one affected with PTC and the other an obligate carrier. The multifocality of PRN in one subject adds weight to the likelihood of a true genetic predisposition to PRN. Both genetic linkage and sequence analysis excluded MET, the protooncogene of isolated familial PRN, as the cause of the fPTC/PRN phenotype. A genome-wide screening and an investigation of specific candidate genes demonstrated that the fPTC/PRN phenotype was linked to 1q21. A maximum three-point log of likelihood ratio score of 3.58 was observed for markers D1S2343 and D1S2345 and for markers D1S2343 and D1S305. Critical recombination events limited the region of linkage to approximately 20 cM. A distinct inherited tumor syndrome has been characterized as the familial association of papillary thyroid cancer, nodular thyroid disease, and papillary renal neoplasia. The predisposing gene in a large kindred with this syndrome has been mapped to 1q21.

Identification of a novel mutation in the paired domain of PAX3 in an Iranian family with waardenburg syndrome type I.

Waardenburg syndrome Type I (WS1) is an autosomal dominant disorder that has previously been associated with mutations in the PAX3 gene on the 2q35 region. In this study, we used an Iranian WS1 family with seven affected individuals in three generations. The phenotypic characteristics of the family include sensorineural deafness, dystopia canthorum, hypopigmented skin patches of the upper limbs, congenital white forelock, confluent white eyebrows, nonpigmented iris, poliosis, and hypopigmentation of the retina. Herein, we report a previously unidentified single-base substitution in exon II (C-->T at position 218) that results in a change of serine to leucine (S73L) in this family. This change was not observed in 100 chromosomes of healthy unrelated individuals. This mutation is within the PAX3 paired domain region, a structure that is highly conserved and implicated in DNA binding. This is the first identification of a PAX3 mutation for this phenotype in the Iranian population. This also provides additional confirmation for the involvement of this gene in the etiology of WS1.

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.

A gene for lymphedema-distichiasis maps to 16q24.3.

Lymphedema-distichiasis (LD) is a dominantly inherited syndrome with onset of lymphedema at or just after puberty. Most affected individuals have distichiasis-fine hairs arising inappropriately from the eyelid meibomian glands-which is evident from birth. A study of three families with LD has shown linkage to chromosome 16q24.3, and subsequent analysis of the region for recombinant genes places the locus between D16S422 and D16S3074, a distance of approximately 16 cM. Possible candidate genes in this interval include the N-proteinase for type 3 collagen, PCOLN3; the metalloprotease PRSM1; and the cell matrix-adhesion regulator, CMAR.

Identification, expression and chromosome localization of a human gene encoding a novel protein with similarity to the pilB family of transcriptional factors (pilin) and to bacterial peptide methionine sulfoxide reductases.

Here we report the isolation, characterization and chromosome localization of a subtracted cDNA (CBS-1) isolated from the human ocular ciliary body which encodes a novel protein. As is deduced from the nucleotide sequence of the cDNA, CBS-1 contains an open reading frame consisting of 182 amino acids, with a molecular weight of 19.5kDa. CBS-1 shares significant nucleotide and amino acid sequence identities (residues 51 to 182) with a hypothetical 15.5kDa protein in the ANSA-GAP intergenic region (yeaA) of Escherichia coli, and the carboxyl terminal region of pilB, a transcription factor involved in the regulation of expression of pili, from Neisseria gonorrhoeae. Interestingly, CBS-1 also shares significant identity with the carboxyl terminus of the peptide-methionine sulfoxide reductase (MsrA), a repair enzyme, from Helicobacter pylori and Streptococcus pneumoniae. However, the amino terminal of CBS-1 (residues 23 to 43), which lacks homology to the amino terminal region of gonococcal pilB or pneumococcal MsrA, exhibits significant identity in a stretch of 20 amino acids, with glycine-rich proteins. By Northern blot, CBS-1, hybridized to a 0.6 to 0.7kb transcript in size, is expressed ubiquitously in many tissues, but most abundantly in the retina and ocular ciliary body, skeletal muscle and heart. An epitope-directed antibody to an amino acid sequence at the carboxyl terminus of CBS-1 recognized a main protein of 19.5kDa in ocular ciliary body extracts, and a 23kDa protein in total extracts from E. coli MC1061 cells, which expresses high levels of MsrA. The CBS-1 gene was mapped to human chromosome 10p12 between markers WI-8535 and WI-4724, and is tightly linked to the two STRP markers of D10S1789 and D10S550. We suggest that the CBS-1 gene encodes a mammalian transcription factor related to the bacterial pilB and certain bacterial MsrA homologues.

Visual event related potentials modulated by contextually relevant and irrelevant olfactory primes.

Visual evoked potentials were recorded from 16 scalp locations on 10 young subjects during presentation of a series of high-quality photographs on a computer screen. The photographs consisted of equal numbers of pictures of fruit (citrus and non-citrus fruits), flowers (roses and other flowers) and objects (e.g. buildings, vehicles, animals etc.). Every picture was different in order to avoid repetition effects. The pictures were presented under four odour conditions: no odour, rose odour, jasmine odour and citrus odour. In order to keep the subjects alert they were asked to make categorizing decisions for the visual stimuli (e.g. flower or fruit). No decision was required concerning the relationship between the visual stimulus and the odour. As expected, the N400 peak was more negative when the picture stimulus did not match the odour. It is hypothesized that the N400 peak can be used as a measure of relatedness of a sensory stimulus to a previous or on-going prime, irrespective of the mode of the stimuli.

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.

Familial papillary thyroid carcinoma is genetically distinct from familial adenomatous polyposis coli.

Familial papillary thyroid carcinoma (fPTC) is an inherited tumor syndrome characterized by isolated papillary thyroid carcinoma (PTC) in affected subjects. Its etiology is unknown. Large multigeneration families with PTC are very rare, and therefore, modern genetic linkage studies have not been applied extensively to this disorder. Familial adenomatous polyposis coli (FAP) is an inherited tumor syndrome enriched in PTC. FAP is caused by germline mutations of the adenomatous polyposis coli (APC) gene that is located in the 5q21 region. It is not known if fPTC is a phenotypic variant of FAP, or if it is a genetically distinct disorder. We report a large 3-generation fPTC kindred and use linkage analysis to test the hypothesis that fPTC and FAP are genetically distinct. In this kindred there are 25 living informative subjects; 5 have PTC, and 1 is an obligate carrier. Inheritance is autosomal dominant with incomplete penetrance. There is vertical transmission, multifocal disease, an average age of onset of 36 years, and 1 subject has colon cancer. The probability is approximately 1 in 2 billion against the clustering of 5 sporadic PTC cases in this kindred. To test for linkage to the APC gene we used 2 highly polymorphic markers, D5S656 and D5S421, which are located within a maximum distance of 1.7 megabase (Mb) of the APC gene and within an estimated genetic region of less than 1 centimorgan (cM) from each other. After polymerase chain reaction (PCR) amplification 18 family members were genotyped. Construction and inspection of haplotypes showed that the affected subjects do not share the same allelic composition. Using a penetrance ratio of 75%, linkage was excluded at 2 cM and 3 cM on both sides of D5S656 and D5S421, respectively. The combined haplotype of these 2 markers provided an exclusion region of 4 cM. We conclude that fPTC is genetically distinct from FAP.

Mapping of primary congenital lymphedema to the 5q35.3 region.

Primary lymphedema is a chronic tissue swelling, most frequently of the lower limbs, resulting from deficient lymphatic drainage. The variability of the affected phenotype, incomplete penetrance, lack of large families, and possible genetic heterogeneity have hampered the identification of causative genes until now. We carried out a genomewide search, using a four-generation North American family with dominantly inherited primary congenital lymphedema (PCL), otherwise known as "Milroy disease," or "hereditary lymphedema type I" (MIM 153100). Linkage to markers from the 5q35.3 region in this and four additional, British families was established. A minimum of 79 directly scorable haplotypes (37 affected) in five families conspicuously segregated with the most telomeric region of 5q35.3, thus suggesting a major locus for PCL in this vicinity. No recombination was observed with D5S408 (Z = 10.03) and D5S2006 (Z = 8.46) with a combined multipoint score of 16.55. While D5S2073 and WIAF-2213 defined the upper centromeric boundary, no recombinants were obtained for the last telomeric marker of D5S2006. Four unaffected subjects were identified as gene carriers and provided an estimated penetrance ratio of.84 for this condition. A few of the positionally mapped genes in the 5q35 region that may potentially be involved in the etiology of this condition are CANX, FGFR4, HK3, and hnRPH1.

Novel TIGR/MYOC mutations in families with juvenile onset primary open angle glaucoma.

Mutations in the trabecular meshwork induced glucocorticoid response protein (TIGR) or myocilin (MYOC) has recently been shown to cause juvenile onset primary open angle glaucoma (JOAG). In this study, we identified two new mutations (Asp380Ala and Ser502Pro) in two British families and another (Pro370Leu) in a French-Canadian family. These mutations were not present in a total of 106 normal chromosomes. In another Turkish family with JOAG, we also detected a sequence variant that was proven to be an amino acid polymorphism (Arg76Lys). No other sequence changes were found in the entire coding region and splice junctions of the TIGR/MYOC gene in this family. However, it is still possible that mutations either in the TIGR promoter or in another neighbouring gene could cause glaucoma in this JOAG family. Our results confirm the role of the TIGR/MYOC gene in the aetiology of the JOAG phenotype.