Mansoor Sarfarazi

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.

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.

Localization of the fourth locus (GLC1E) for adult-onset primary open-angle glaucoma to the 10p15-p14 region.

One of the major causes of blindness is primary open-angle glaucoma, which affects millions of elderly people worldwide. Genetic studies have so far mapped three loci for the adult-onset form of this condition to the 2cen-q13, 3q21-q24, and 8q23 regions. Herein, we report the localization of a fourth locus, to the 10p15-p14 region, in one large British family with a classical form of normal-tension open-angle glaucoma. Of the 42 meioses genotyped in this pedigree, 39 subjects (16 affected) inherited a haplotype compatible with their prior clinical designation, whereas the remaining 3 were classified as unknown. Although a maximum LOD score of 10.00 at a recombination fraction of straight theta=.00 was obtained with D10S1216, 21 other markers provided significant values, varying between 3.77 and 9.70. When only the affected meioses of this kindred were analyzed, LOD scores remained statistically significant, ranging from 3.16 (D10S527) to 3.57 (D10S506). Two critical recombinational events in the affected subjects positioned this new locus to a region of approximately 21 cM, flanked by D10S1729 and D10S1664. However, an additional recombination in a 59-year-old unaffected female suggests that this locus resides between D10S585 (or D10S1172) and D10S1664, within a genetic distance of 5-11 cM. However, the latter minimum region must be taken cautiously, because the incomplete penetrance has previously been documented for this group of eye conditions. A partial list of genes that positionally are considered as candidates includes NET1, PRKCT, ITIH2, IL2RA, IL15RA, IT1H2, hGATA3, the mRNA for open reading frame KIAA0019, and the gene for D123 protein.

Genetic linkage of the Marfan syndrome, ectopia lentis, and congenital contractural arachnodactyly to the fibrillin genes on chromosomes 15 and 5

BACKGROUND The large glycoprotein fibrillin is a structural component of elastin-containing microfibrils found in many tissues. The Marfan syndrome has been linked to the fibrillin gene on chromosome 15, but congenital contractural arachnodactyly, which shares some of the physical features of the syndrome, has been linked to the fibrillin gene on chromosome 5. METHODS Using specific markers for the fibrillin genes, we performed genetic linkage analysis in 28 families with the Marfan syndrome and 8 families with four phenotypically related disorders--congenital contractural arachnodactyly (3 families), ectopia lentis (2), mitral-valve prolapse syndrome (2), and annuloaortic ectasia (1). RESULTS Genetic linkage was established between the Marfan syndrome and only the fibrillin gene on chromosome 15, with a maximum lod score of 25.6 (odds for linkage, 10(25.6):1). Ectopia lentis was also linked to the fibrillin gene on chromosome 15, whereas congenital contractural arachnodactyly was linked to the fibrillin gene on chromosome 5. There was no linkage of mitral-valve prolapse to the fibrillin gene on chromosome 5; studies of chromosome 15 were not informative. Annuloaortic ectasia was not linked to either fibrillin gene. CONCLUSIONS The Marfan syndrome appears to be caused by mutations in a single fibrillin gene on chromosome 15. Diagnosis of the Marfan syndrome by genetic linkage and analysis is now feasible in many families.

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.

Predictors of axial and peripheral bone mineral density in healthy children and adolescents, with special attention to the role of puberty

The role of various physical and lifestyle factors in determining axial bone mineral density (BMD) at the lumbar segment of the spine, as measured by dual-photon absorptiometry, and peripheral BMD at the distal third of the radius, as measured by single-photon absorptiometry, was assessed in 299 healthy white children of both sexes, aged 6 to 18 years. The BMD measurements were correlated with age, height, weight, body mass index, and pubertal status. Peripheral and axial BMD were highly correlated with age, height, weight, and pubertal stage, and more weakly with body mass index. Approximately 76% of the observed changes in peripheral BMD were accounted for by age, height, weight, and pubertal stage, with weight being the single strongest predictor. Up to 80% of the variation in axial BMD was explained by weight and pubertal stage, with pubertal stage being the strongest single predictor. After adjustment for weight, the effect of puberty on axial BMD in both sexes was greatest between middle and late puberty. These data indicate that a large amount of the observed changes on BMD is accounted for by standard measures of growth and development, which are largely genetically determined. Peripheral BMD rose steadily with age. Axial BMD increased steadily before puberty, followed by accelerated increases during puberty, beginning at 10 years of age in girls and 13 years of age in boys. A significant positive effect of dietary calcium intake on peripheral BMD and of physical activity on axial BMD indicated a potentially important impact of physical activity and calcium intake on peak bone mass.

A third locus (GLC1D) for adult-onset primary open-angle glaucoma maps to the 8q23 region

PURPOSE Two genes for adult-onset primary open-angle glaucoma have been mapped to chromosomes 2cen-q13 and 3q21-q24. We studied a family with adult-onset primary open-angle glaucoma in which the disease did not map to these two chromosomal regions. METHODS We ascertained a four-generation family with adult-onset primary open-angle glaucoma in which the disease status of individuals was objectively assigned using defined criteria. Complete ophthalmologic examinations, visual field testing, optic nerve head photographs, and venous blood samples were obtained. Family members were genotyped using polymerase chain reaction amplification of microsatellite polymorphic markers. Linkage analysis was performed and lod scores were calculated. Haplotype transmission data were analyzed. RESULTS A total of 20 subjects in three successive generations agreed to participate in the study. This included samples from eight affected subjects, one glaucoma suspect, one normal individual, and two spouses in generations II and III, and an additional eight individuals in generation IV. The phenotype in this family appears to be variable, with onset of visual field loss in middle age, followed by modest elevation of intraocular pressure and progression of the disease in older individuals. Linkage was established with a group of DNA markers located in the 8q23 region. A lod score value of 3.61 was obtained using marker D8S1471. Three other markers from the same region gave lod score values of over 3.0. Haplotype transmission data identified two recombination events that placed the gene in a 6.3-cM region flanked by D8S1830 and D8S592. The disease-bearing haplotype was inherited by eight affected subjects and three glaucoma suspects. CONCLUSION We present evidence for a third adult-onset primary open-angle glaucoma locus (GLC1D) on chromosome 8q23. The genetic heterogeneity of adult-onset glaucoma is evident from the multiplicity of chromosomal loci associated with this disease.

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.

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.