Cleavage of IgG1 in gingival crevicular fluid is associated with the presence of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVES: Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of Porphyromonas gingivalis and other periodontopathogens. MATERIAL AND METHODS: GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis and five periodontally healthy individuals. The bacterial loads of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia were analysed by real-time polymerase chain reaction, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA. RESULTS: Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. Porphyromonas gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (P < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of Tannerella forsythia and Prevotella intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of Porphyromonas gingivalis (r = 0.425, P < 0.01). The presence of Kgp (range 0.07-10.98 ng/mL) was associated with proteolytic fragments of IgG1 (P < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp. CONCLUSION: In patients with periodontitis, cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by Porphyromonas gingivalis.

Region and Field Level Distributions of Aster Yellows Phytoplasma in Small Grain Crops.

Aster yellows (AY), a disease of small grain crops caused by aster yellows phytoplasma (AYp), produces disease symptoms similar to barley yellow dwarf (BYD). From 2003 to 2005, small grain production fields in Minnesota and North Dakota were surveyed to determine the incidences of AY and BYD. In-field spatial patterns of AY-infected plants also were investigated. Plants collected along a five-point transect line were tested for AYp using nested polymerase chain reaction (PCR) and quantitative real-time PCR assays, and extracted plant sap was tested for serotypes PAV and RPV of Barley yellow dwarf virus (BYDV) using enzyme-linked immunosorbent assays. During 2003, 2004, and 2005, AYp was detected in plants from 49, 15, and 7% of tested fields, respectively, whereas BYDV was found in plants from 2, 0, and 5% of fields, respectively. Average amplicon count number indicated an in-field spatial trend for greater incidence of AYp and increased populations of AYp in plants located near field edges, with comparably low copy numbers at transect point locations toward the direction of field center. AY is likely a common but largely undetected disease on small grain crops in the Upper Midwest.

Medicare coverage for patients with diabetes. A national plan with individual consequences.

The prevalence of diabetes in the U.S. Medicare population is growing at an alarming rate. From 1980 to 2004, the number of people aged 65 or older with diagnosed diabetes increased from 2.3 million to 5.8 million. According to the Centers for Medicare and Medicaid (CMS), 32% of Medicare spending is attributed to the diabetes population. Since its inception, Medicare has expanded medical coverage of monitoring devices, screening tests and visits, educational efforts, and preventive medical services for its diabetic enrollees. However, oral antidiabetic agents and insulin were excluded from reimbursement. In 2003, Congress passed the Medicare Modernization Act that includes a drug benefit to be administered either through Medicare Advantage drug plans or privately sponsored prescription drug plans for implementation in January 2006. In this article we highlight key patient and drug plan characteristics and resources that providers may focus upon to assist their patients choose a coverage plan. Using a case example, we illustrate the variable financial impact the adoption of Medicare part D may have on beneficiaries with diabetes due to their economic status. We further discuss the potential consequences the legislation will have on diabetic patients enrolled in Medicare, their providers, prescribing strategies, and the diabetes market.

Latency of cathepsin B secreted by human colon carcinoma cells is not linked to secretion of cystatin C and is relieved by neutrophil elastase.

The lysosomal cysteine proteinase cathepsin B is shown to be secreted by ten human colon carcinoma cell lines and to accumulate in culture media as a latent enzyme. The cell lines also secrete a physiological inhibitor of cathepsin B, cystatin C. A significant correlation was found between secretion of the latent enzyme and the inhibitor (r = 0.755, P < 0.01). The aim of the present study was to modulate the respective secretion of the two antagonists to test whether or not latency of cathepsin B was due to the concomitant secretion of the inhibitor. SW480 colon carcinoma cells were treated with the acidotropic agent ammonium chloride, phorbol 12-myristate 13-acetate, and the inflammatory cytokines TGF-beta, TNF-alpha, and IL-1 beta. Ammonium chloride significantly increased latent cathepsin B levels without affecting the constitutive secretion of cystatin C. Phorbol 12-myristate 13-acetate induced a 4- to 5-fold increase in secreted latent cathepsin B, but did not alter significantly the accumulation of cystatin C in media. The cytokines, TGF-beta, TNF-alpha, and IL-1 beta, had no major effect on the expression of these two antagonists. Latent cathepsin B released from human carcinoma cells could be efficiently activated by neutrophil elastase at neutral pH. It is concluded that latent cathepsin B is a true proenzyme rather than an enzyme-inhibitor complex. In addition, our data from neutrophil elastase activation experiments indicate that a proteolytic system for activation of the tumor cell-secreted latent enzyme may exist in vivo.

NMR structural studies of human cystatin C dimers and monomers.

Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, "trapped" dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance assignments and secondary structure determination were obtained for the monomer using data from triple resonance experiments performed on 13C/15N doubly labeled protein. A marked similarity of the cystatin C secondary structure to that of chicken cystatin was observed. Using uniformly and amino-acid-specific 15N-enriched protein, backbone NH signals were assigned for cystatin C in its dimeric state. Comparison of 1H -15N correlation NMR spectra of the monomer and dimer shows that the three-dimensional structure remains unchanged in the dimer and that only local perturbations occur. These are localized to the amino acid residues comprising the cysteine proteinase binding site. Such a mode of dimerization readily explains the complete loss of the inhibitory activity in the dimer. The NMR results also demonstrate that the dimer is symmetric.

Culture of functionally active human pituitary cells: investigation of gonadotropin regulation.

A method for culturing cells from human pituitaries obtained at autopsy for studies of the secretion of gonadotropins and other hormones is described. The EC50 of GnRH-stimulated LH secretion from human pituitary cells was 1.25 nmol/L, similar to that found with rat pituitary cells (2.45 nmol/L). The GnRH agonist (D-Ala6, N alpha MeLeu7, Pro9-NEt)GnRH was 10-fold more active in stimulating LH release from both human and rat pituitary cells (EC50, 0.21 and 0.35 nmol/L, respectively). The GnRH antagonists (Ac-D-Nal(2)1,D-alpha-Me-4-ClPhe2,D-3-Pal3,D-Arg6,D-Ala10 )GnRH and (D-pGlu1,D-Phe2,D-Trp3,6)GnRH both inhibited 3 nmol/L GnRH-stimulated LH release from human pituitary cells (IC50, 2.40 and 78.6 nmol/L, respectively) with potencies similar to those observed in the rat (IC50, 0.68 and 33.0 nmol/L, respectively). Stimulation of the human pituitary cells with 100 nmol/L GnRH for 44 min resulted in biphasic release of LH and FSH. The initial phase occurred during the first 4 min of stimulation, and the second protracted plateau phase continued for at least the ensuing 40 min. The kinetics of LH and FSH release were identical, with no evidence of differential release at any stage. GnRH-stimulated LH and FSH secretion was Ca2+ dependent. The presence of 3 mmol/L EGTA prevented the gonadotropin response to GnRH, and the Ca2+ ionophore A23187 stimulated release of both LH and FSH. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate also stimulated gonadotropin secretion, indicating that activation of protein kinase-C is able to elicit LH release. GnRH desensitized the cells to subsequent stimulation with GnRH. After initial 2-h incubation with 0.1-100 nmol/L GnRH, LH release during a second 2-h incubation with the same doses was reduced by 50-100%. The reduction in FSH ranged from 63-92%. Depletion of gonadotropin pools may contribute to desensitization, since total LH and FSH cell content decreased 48% and 49%, respectively, after 100 nmol/L GnRH stimulation for 4 h. We conclude that functionally active cells can be cultured from human pituitaries obtained postmortem and that the mechanism of GnRH action and the relative potencies of GnRH analogs closely parallel those in the rat.

Neuroendocrine regulation of thyrotropin release in cultured human pituitary cells.

The regulation of TSH release in man was investigated using cell cultures derived from human pituitaries obtained within 24 h of accidental death. TRH stimulated TSH release in a dose-dependent manner. The ED50 was 2.9 +/- 0.6 (+/- SEM) nmol/L, similar to that reported for rat pituitary cell cultures. The release of TSH was calcium dependent, since the calcium channel antagonist verapamil inhibited TRH-stimulated TSH release, and the calcium ionophore A23187 stimulated TSH release. 12-O-Tetradecanoyl-phorbol-13-acetate stimulated TSH secretion, while dibuytryl cAMP had no effect. Epinephrine and serotonin stimulated TSH release, and dopamine and somatostatin inhibited TRH-stimulated TSH release. These findings have directly demonstrated that the regulation of TSH secretion by hypothalamic neuropeptides and biogenic amines in the human pituitary is similar to that in the rat. The development of a tissue culture system to study thyrotrophs from postmortem human pituitaries provides the means for detailed studies of the regulation of TSH secretion in man.

High-level expression of active human cystatin C in Escherichia coli.

Expression of the human cysteine proteinase inhibitor, cystatin C (CysC) in the cytoplasm of Escherichia coli was studied using a cDNA fragment encoding the cysteine proteinase inhibitor controlled by the phage lambda pR/cI857 system. The yield of CysC was low, probably due to proteolytic degradation. By fusing the cysC cDNA to a DNA fragment encoding the signal peptide of the E. coli outer membrane protein A, it was possible to produce a substantial amount of CysC in the periplasm. The processing of the signal peptide was shown to be quantitative and to result in CysC with the correct N-terminal amino acid. Yields higher than 1000 micrograms CysC/ml can be obtained by initiating the product formation at a moderate temperature (40 degrees C) late in an optimized fermentation process. A method that gives selective extraction of the periplasmic proteins and at the same time stabilizes CysC has been used.

Structural organization, expression and chromosomal mapping of the mouse cystatin-C-encoding gene (Cst3).

Cystatin C (CstC) is a potent cysteine-proteinase inhibitor. The structure of the mouse CstC-encoding gene (Cst3) was examined by sequencing a 6.1-kb genomic DNA containing the entire gene, as well as 0.9 kb of 5' flanking and 1.7 kb of its 3' flanking region. The sequence revealed that the overall organization of the gene is very similar to those of the genes encoding human CstC and other type-2 Cst, with two introns at positions identical to those in the human gene. The promoter area does not contain typical TATA or CAAT boxes. Two copies of a Sp1-binding motif, GGGCGG, are present in the 5' flanking region within 300 bp upstream from the initiation codon. A hexa-nucleotide, TGTTCT, which is a core sequence of the androgen-responsive element (ARE), is found in the promoter region. This region also contains a 21-nucleotide sequence, 5'-AGACTAGCAGCTGACTGAAGC, which contains two potential binding sites for the transcription factor, AP-1. The mouse Cst3 mRNA was detected in all of thirteen tissues examined by Northern blot analysis. Cst3 was mapped in the mouse to a position on distal chromosome 2.

Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C.

Recombinant cystatin C producing clones were isolated from a human placenta lambda gt11 cDNA library. The cDNA insert of one of the clones, containing 777 base pairs, encodes the complete mature cystatin C (120 amino acids) and a hydrophobic leader sequence of 26 amino acids, indicating an extracellular function of the inhibitor. The deduced protein sequence confirms the protein sequence of cystatin C isolated from human urine, but differs in one position from the sequence of the cystatin C fragment deposited as amyloid in hereditary cerebral hemorrhage with amyloidosis.

Efficient production of native, biologically active human cystatin C by Escherichia coli.

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the lambda PR promoter, an optimized Shine-Dalgarno sequence and the lambda cI 857 repressor. When induced at 42 degrees C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

Two-step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop.

Stopped-flow kinetics showed that the inhibition of the lysosomal cysteine proteinase, cathepsin B, by its endogenous inhibitor, cystatin C, occurs by a two-step mechanism, in which an initial, weak interaction is followed by a conformational change. The initial interaction most likely involves binding of the N-terminal region of the inhibitor to the proteinase. Considerable evidence indicates that the subsequent conformational change is due to the inhibitor displacing the occluding loop of the proteinase that partially obscures the active site. The presence of this loop, which allows the enzyme to function as an exopeptidase, thus complicates the inhibition mechanism, rendering cathepsin B much less susceptible than other cysteine proteinases to inhibition by cystatins.

Cystatin inhibition of cathepsin B requires dislocation of the proteinase occluding loop. Demonstration By release of loop anchoring through mutation of his110.

Cystatins A and C were both shown to inhibit cathepsin B by a two-step mechanism, involving an initial weak interaction followed by a conformational change. Disruption of the major salt bridge anchoring the occluding loop of cathepsin B to the main body of the enzyme by mutation of His110 to Ala converted the binding to an apparent one-step reaction. The second step of cystatin binding to cathepsin B must therefore be due to the inhibitor having to alter the conformation of the enzyme by displacing the occluding loop to allow a tight complex to be formed. Cystatin A was appreciably less effective in displacing the loop than cystatin C, resulting in a considerably lower overall inhibition rate constant.

Interactions of papaya proteinase IV with inhibitors.

Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by 'papain' reported previously. PPIV is slowly bound by human alpha 2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues.

Cathepsin B and cysteine proteinase inhibitors in human lung cancer cell lines.

Cell lines derived from human squamous cell (EPCL), large cell (LCLC), and small cell lung cancer (SCLC) lines were investigated for the expression of cathepsin B (Cat B) and cysteine proteinase inhibitors (CPIs). The EPLC and LCLC lines expressed 5- to 50-fold more Cat B activity and contained more mature Cat B of M(r) 27-29 kDa (> 2.5 microg/mg total protein) than the SCLC lines (< 1.0 microg/mg total protein). The LPLC lines also secreted the highest amounts of Cat B precursor of M(r) about 46 kDa. Inhibitory activities against Cat B and papain were associated with high molecular mass (HMM) and low molecular mass (LMM) inhibitory proteins, both in cell extracts and in media. About 75% of the inhibitory activity was associated with HMM inhibitors, the majority of which were kininogens (M(r) > or = 67 kDa). The LMM inhibitors of M(r) 10-15 kDa were cystatin C and stefins A and B, which were quantitated by ELISA: stefins A and B were present in cell extracts and medium in similar concentrations (5-200 ng/10(6) cells), while 80-99% of the cystatin C was released in the medium (10-195 ng/10(6) cells). Phorbol ester (PMA), which induces protein-kinase C mediated signal transduction and enhances cellular differentiation in many non-small cell lung cancer (NSCLC) cell lines, increased intracellular Cat B activity and Cat B protein as well as its secretion in some cell lines but not in others, regardless of their histological type. PMA significantly (P < 0.049) decreased intracellular stefin A concentrations in two EPLC lines and non-significantly in two LCLC lines. PMA decreased secretion of stefin A in all EPLC lines, but not in LCLC lines, while IGF-I significantly increased stefin B secretion in both SCLC lines. These data showed that lung tumor cells produce both cysteine proteinases and cystatins. As the antagonistic molecules are regulated differently in histologically different types of lung tumor cells, it is possible that an imbalance between the proteinases and their specific inhibitors plays a role in progression of certain types of lung tumors in vivo.

A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro.

Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg(8)-Leu(9)-Val(10)-Gly(11) (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg(8)-Leu(9)-Val(10)-Gly(11)-diazomethane (Z-RLVG-CHN(2)) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of (45)Ca and (3)H from mouse calvarial bones prelabeled with [(45)Ca]CaCl(2) and [(3)H]-proline, respectively. Z-RLVG-CHN(2) concentration-dependently inhibited the release of (45)Ca and (3)H in bones stimulated by parathyroid hormone (PTH), with half-maximal inhibition obtained at 1 micromol/L. The inhibitory actions of Z-RLVG-CHN(2) and cystatin C were persistent, whereas action induced initially by calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN(2) and cystatin C on PTH-stimulated (45)Ca release was observed after 6 h, whereas inhibition by calcitonin was seen already after 2 h. In contrast, the inhibitory effects of Z-RLVG-CHN(2) and cystatin C, as well as that of calcitonin, on (3)H release was seen already after 2 h. Z-RLVG-CHN(2), in which the reactive carboxyterminal diazomethane was substituted by nonreactive groups [-OH, -NH(2), or -N(CH(3))(2)], resulted in peptidyl derivatives, which, in contrast to Z-RLVG-CHN(2) and cystatin C, inhibited neither cysteine proteinases nor bone resorption. In contrast to wild-type cystatin C, recombinant human cystatin C with Gly substitutions for residues Arg(8), Leu(9), Val(10), and Trp(106), and with low or nonexistent affinity for cysteine proteinases, did not display any inhibitory effect on bone resorption. These data strongly indicate that Z-RLVG-CHN(2) inhibits bone resorption in vitro by a mechanism that seems primarily to be due to an inhibition of bone matrix degradation via cysteine proteinases. The data also corroborate the hypothesis that cystatin C inhibits bone resorption by virtue of its cysteine proteinase inhibitory capacity.

Antigenicity of cystatin-binding proteins from parasitic protozoan. Detection by a proteinase inhibitor based capture immunoassay (PINC-ELISA).

A novel immunoassay (PINC-ELISA) was designed using proteinase inhibitors of the cystatin superfamily (PINC) in the solid phase, to promote the selective capture of cysteine proteinases. The method was applied in the identification of papain-like antigens from parasitic protozoa. PINC of human origin, namely recombinant cystatin C (r-cystatin C) or low molecular weight kininogen were used in the assays to adsorb proteases contained in cell lysates from various trypanosomatids. The PINC-ELISA was at first optimized with the major cysteine proteinase from Trypanosoma cruzi (known as GP57/51 or cruzipain), an antigen whose serodiagnostic properties were previously established. Cruzipain is selectively adsorbed from crude extracts of T. cruzi onto PINC-coated wells; the finding that antibodies bind to epitopes located away from the sites of interaction with r-cystatin or low molecular weight kininogen has allowed for the screening of antibodies in chagasic sera, the methodology being advantageous in that it dispensed prior purification of the proteinase antigen. The PINC-ELISA was then carried out with lysates originating from Leishmania m. amazonensis (amastigotes) or Leishmania donovani (promastigotes). Complexes between solid-phase r-cystatin C and antigenic ligands in the lysates were again detected. The Leishmania molecules which bound to r-cystatin C, were respectively recognized by serum antibodies from mice chronically infected with L. amazonensis or from patients with visceral leishmaniasis. Direct evidence for the presence of cysteine proteinases in lysates from L. donovani was then obtained, using synthetic fluorogenic substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and localization of retinal cystatin C.

PURPOSE: Cystatin C is a mammalian cysteine protease inhibitor, synthesized in various amounts by many kinds of cells and appearing in most body fluids. There are reports that it may be synthesized in the mammalian retina and that a cysteine protease inhibitor may influence the degradation of photoreceptor outer segment proteins. In the current study cystatin C was identified, quantitated, and localized in mouse, rat, and human retinas. METHODS: Enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), DNA sequencing, Western blot analysis, and immunohistochemistry have been used on mouse, rat, and human retinas (pigment epithelium included). RESULTS: Cystatin C is present in high concentrations in the normal adult rat retina, as it is throughout its postnatal development. Its concentration increases to a peak at the time when rat pups open their eyes and then remains at a high level. It is mainly localized to the pigment epithelium, but also to some few neurons of varying types in the inner retina. Cystatin C is similarly expressed in normal mouse and human retinas. CONCLUSIONS: Cystatin C was identified and the localization described in the retinas of rat, mouse, and human using several techniques. Cystatin C is known to efficiently inactivate certain cysteine proteases. One of them, cathepsin S, is present in the retinal pigment epithelium and affects the proteolytic processing by cathepsin D of diurnally shed photoreceptor outer segments. Hypothetically, it appears possible that retinal cystatin C, given its localization to the pigment epithelium and its ability to inhibit cathepsin S, could be involved in the regulation of photoreceptor degradation.

Phenotype of an X-linked retinitis pigmentosa family with a novel splice defect in the RPGR gene.

PURPOSE: To assess the clinical phenotype in a Swedish family with X-linked retinitis pigmentosa (XLRP) resulting from a novel splice defect in the RPGR gene. METHODS: RPGR mutation analysis was performed in one family with XLRP, and several individuals from the family were examined clinically. RESULTS: The causative mutation in the family was demonstrated to be a single base-pair change at the splice donor site in intron 7 that resulted in skipping of the complete exon 7 in the mature RPGR transcript. The aberrant mRNA is predicted to produce an RPGR protein with an in-frame deletion of 53 amino acids, corresponding to an RCC1-homology repeat. Clinical studies that included ophthalmological examination and full-field electroretinography showed that this splice mutation resulted in a comparatively less severe form of RP. CONCLUSIONS: Correlation of a causative RPGR genotype with clinical findings in hemizygotes and carrier heterozygotes is an important step toward predictive diagnosis and should assist in the development of gene-based therapies in the future.

Phenotypes in three Swedish families with X-linked retinitis pigmentosa caused by different mutations in the RPGR gene.

PURPOSE: To assess the clinical phenotypes in three Swedish families with X-linked retinitis pigmentosa caused by different mutations in the RPGR gene. METHODS: Three families from different parts of Sweden, including nine patients with retinitis pigmentosa and six female carriers of X-linked retinitis pigmentosa, were examined clinically. Ophthalmologic examination included kinetic perimetry with a Goldmann perimeter using standardized objects I4e and V4e, dark adaptation final thresholds with a Goldmann-Weeker adaptometer, and full-field electroretinograms. RESULTS: The clinical findings in the patients demonstrated a severe form of retinitis pigmentosa with visual handicap early in life. Patients with a microdeletion of exons 8 through 10 of the RPGR gene had a more severe phenotype compared to the patients with single base-pair mutations in the introns 10 and 13 of the RPGR gene, resulting in splicing defects. Furthermore, heterozygous carriers in these families displayed a wide spectrum of clinical features, from minor symptoms to severe visual disability. CONCLUSION: These three families show a variable clinical phenotype resulting from different mutations in the RPGR gene. A microdeletion spanning at least parts of exons 8 through 10 seems to result in a severe phenotype compared to the splice defects. Heterozygous carriers of X-linked retinitis pigmentosa with these specific RPGR genotypes also show a variability of the phenotype; carriers with the microdeletion may be severely visually handicapped.