Molecular and computational analyses of genes involved in mannose 6-phosphate independent trafficking.

The newly-synthesized lysosomal enzymes travel to the trans-Golgi network (TGN) and are then driven to the acidic organelle. While the best-known pathway for TGN-to-endosome transport is the delivery of soluble hydrolases by the M6P receptors (MPRs), additional pathways do exist, as showed by the identification of two alternative receptors: LIMP-2, implicated in the delivery of ß-glucocerebrosidase; and sortilin, involved in the transport of the sphingolipid activator proteins prosaposin and GM2AP, acid sphingomyelinase and cathepsins D and H. Disruption of the intracellular transport and delivery pathways to the lysosomes may result in lysosomal dysfunction, predictably leading to a range of clinical manifestations of lysosomal storage diseases. However, for a great percentage of patients presenting such manifestations, no condition is successfully diagnosed. To analyse if, in this group, phenotypes could be determined by impairments in the known M6P-independent receptors, we screened the genes that encode for LIMP-2 and sortilin. No pathogenic mutations were identified. Other approaches will be needed to clarify whether sortilin dysfunction may cause disease.

Lysosomal multienzymatic complex-related diseases: a genetic study among Portuguese patients.

The functional activity of lysosomal enzymes sialidase, ß-galactosidase and N-acetylaminogalacto-6-sulfate-sulfatase in the cell depends on their association in a multienzyme complex with cathepsin A. Mutations in any of the components of this complex result in functional deficiency thereby causing severe lysosomal storage disorders. Here, we report the molecular defects underlying sialidosis (mutations in sialidase; gene NEU1), galactosialidosis (mutations in cathepsin A; gene PPGB) and GM1 gangliosidosis (mutations in ß-galactosidase; gene GLB1) in Portuguese patients. We performed molecular studies of the PPGB, NEU1 and GLB1 genes in biochemically diagnosed Portuguese patients. Gene expression was determined and the effect of each mutation predicted at protein levels. In the NEU1 gene, we found three novel missense mutations (p.P200L, p.D234N and p.Q282H) and one nonsense mutation (p.R341X). In the PPGB gene, we identified two missense mutations, one novel (p.G86V) and one already described (p.V104M), as well as two new deletions (c.230delC and c.991-992delT) that give rise to non-functional proteins. We also present the first molecular evidence of a causal missense mutation localized to the cathepsin A active site. Finally, in the GLB1 gene, we found six different mutations, all of them previously described (p.R59H, p.R201H, p.H281Y, p.W527X, c.1572-1577InsG and c.845-846delC). Seven novel mutations are reported here, contributing to our knowledge of the mutational spectrum of these diseases and to a better understanding of the genetics of the lysosomal multienzymatic complex. The results of this study will allow carrier detection in affected families and prenatal molecular diagnosis, leading to the improvement of genetic counseling.

Origin and spread of a common deletion causing mucolipidosis type II: insights from patterns of haplotypic diversity.

Mucolipidosis II (ML II alpha/beta), or I-cell disease, is a rare genetic disease in which activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes, GNPTAB and GNPTG. A spectrum of mutations in GNPTAB has been recently reported to cause ML II alpha/beta. Most of these mutations were found to be private or rare. However, the mutation c.3503_3504delTC has been detected among Israeli and Palestinian Arab-Muslim, Turkish, Canadian, Italian, Portuguese, Irish traveller and US patients. We analysed 44 patients who were either homozygous or compound heterozygous for this deletion (22 Italians, 8 Arab-Muslims, 1 Turk, 3 Argentineans, 3 Brazilians, 2 Irish travellers and 5 Portuguese) and 16 carriers (15 Canadians and 1 Italian) for three intragenic polymorphisms: c.-41_-39delGGC, c.18G>A and c.1932A>G as well as two microsatellite markers flanking the GNPTAB gene (D12S1607 and D12S1727). We identified a common haplotype in all chromosomes bearing the c.3503_3504delTC mutation. In summary, we showed that patients carrying the c.3503_3504delTC deletion presented with a common haplotype, which implies a common origin of this mutation. Additionally, the level of diversity observed at the most distant locus indicates that the mutation is relatively ancient (around 2063 years old), and the geographical distribution further suggests that it probably arose in a peri-Mediterranean region.

Molecular analysis of the GNPTAB and GNPTG genes in 13 patients with mucolipidosis type II or type III - identification of eight novel mutations.

Mucolipidosis II (ML II) and mucolipidosis III (ML III) are diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage-disease phenotype. GlcNAc-phosphotransferase is a multimeric transmembrane enzyme composed of three subunits (alpha, beta and gamma) encoded by two genes -GNPTAB and GNPTG. Defects in GNPTAB result in ML II and III whereas mutations in GNPTG were only found in ML III patients. We have performed a molecular analysis of the GNPTAB and GNPTG genes in 13 mucolipidosis II and III patients (10 Portuguese, one Finnish, one Spanish of Arab origin and one Indian). Mutations were identified by the study of both cDNA and gDNA. The GNPTAB and GNPTG mRNA expressions were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The study led to the identification of 11 different mutations. Eight of these mutations are novel, six in the GNPTAB gene [c.121delG (V41FfsX42), c.440delC (A147AfsX5), c.2249_50insA (N750KfsX8), c.242G>T (W81L), c.1208T>C (I403T) and c.1999G>T (p.E667X)] and two in the GNPTG gene [c.610-1G>T and c.639delT (F213LfsX7)]. With regard to the mRNA expression studies, the values obtained by qRT-PCR indicate the possible existence of feedback regulation mechanisms between alpha/beta and the gamma subunits.

Molecular analysis of mucopolysaccharidosis type IIIB in Portugal: evidence of a single origin for a common mutation (R234C) in the Iberian Peninsula.

Mucopolysaccharidosis type IIIB (Sanfilippo B disease) is a rare autosomal recessive disorder caused by defective alpha-N-acetylglucosaminidase (NAGLU). We examined the NAGLU gene in 11 MPS IIIB Portuguese patients, having identified five novel (M1K, W147X, G304V, S522P, and R533X) and four previously reported mutations (W168X, R234C, R565W and R643C). R234C attained the high prevalence of 32% of the mutated alleles. Because R234C had already been reported to be common in Spanish patients, a haplotypic analysis was conducted to address the question of its origin in the Iberian Peninsula. Three neutral markers were studied that allowed for the identification of the probable founder haplotype (174-234-G) on which R234C arose. The sharing of the ancestral haplotype by Portuguese and Spanish patients clearly implied a common origin of the mutation in Iberia, through an event that was inferred to have been rather recent. Therefore, the reconstructed history of R234C explains the high incidence of the mutation in Iberian patients with Sanfilippo B disease.

A randomized, double-blind, dose-comparison study of weekly interferon beta-1a in relapsing MS.

BACKGROUND: Interferon beta-1a (IFNbeta-1a; Avonex) is effective for the treatment of relapsing MS; however, the optimal dose of IFNbeta-1a is not known. OBJECTIVE: To determine whether IFNbeta-1a 60 micro g IM once weekly is more effective than IFNbeta-1a 30 micro g IM once weekly in reducing disability progression in relapsing MS. METHODS: In a double-blind, parallel-group, dose-comparison study, 802 patients with relapsing MS from 38 centers in Europe were randomized to IFNbeta-1a 30 micro g (n = 402) or 60 micro g (n = 400) IM once weekly for >/=36 months. The primary endpoint was disability progression, defined as time to a sustained increase of >/=1.0 point on the Expanded Disability Status Scale (EDSS) persisting for 6 months. Additional endpoints included relapses, MRI, safety, immunogenicity, and subgroup analyses of disability progression. RESULTS: Both groups showed equal rates of disability progression (hazard ratio, 0.96; 95% CI, 0.77 to 1.20; p = 0.73). In both groups the proportion of subjects with progression of disability by 36 months estimated from Kaplan-Meier curves was 37%. No dose effects were observed on any of the secondary clinical endpoints. Only one MRI measure at one time point, number of new or enlarging T2 lesions at month 36 compared with month 24, showed a difference favoring the 60- micro g dose. Both doses were well tolerated; however, slightly higher incidences of flulike symptoms and muscle weakness were observed in the 60- micro g group. The incidences of neutralizing antibodies (titers >/= 20) were 2.3% in the 30- micro g group and 5.8% in the 60- micro g group. CONCLUSION: There was no difference between IFNbeta-1a 30 micro g and 60 micro g IM in clinical or MRI measures.

Neuropsychological effects of interferon beta-1a in relapsing multiple sclerosis. Multiple Sclerosis Collaborative Research Group.

Cognitive dysfunction is common in multiple sclerosis (MS), yet few studies have examined effects of treatment on neuropsychological (NP) performance. To evaluate the effects of interferon beta-1a (IFNbeta-1a, 30 microg administered intramuscularly once weekly [Avonex]) on cognitive function, a Comprehensive NP Battery was administered at baseline and week 104 to relapsing MS patients in the phase III study, 166 of whom completed both assessments. A Brief NP Battery was also administered at 6-month intervals. The primary NP outcome measure was 2-year change on the Comprehensive NP Battery, grouped into domains of information processing and learning/memory (set A), visuospatial abilities and problem solving (set B), and verbal abilities and attention span (set C). NP effects were most pronounced in cognitive domains vulnerable to MS: IFNbeta-1a had a significant beneficial effect on the set A composite, with a favorable trend evident on set B. Secondary outcome analyses revealed significant between-group differences in slopes for Brief NP Battery performance and time to sustained deterioration in a Paced Auditory Serial Addition Test processing rate, favoring the IFNbeta-1a group. These results support and extend previous observations of significant beneficial effects of IFNbeta-1a for relapsing MS.

Interstitial rejection, vascular rejection, and diffuse thrombosis of renal allografts. Predisposing factors, histology, immunohistochemistry, and relation to outcome.

Histological and immunohistochemical analyses were made of biopsy specimens from 50 consecutive patients who experienced putative graft rejection. The mean age of the patients was 44.5 years (range, 17-69 years) and 26 were men. There were 67 evaluable allograft specimens, which were grouped according to the histological diagnosis: group 1, acute tubulointerstitial rejection (n = 42); group 2, acute vascular rejection (n = 18); and group 3, diffuse thrombosis (n = 7). Over a follow-up period of 21-57 months, the mean number of rejection episodes was 1.7, 2.8, and 3.3 in groups 1, 2, and 3, respectively. Allograft loss occurred in 7 out of 30, 10 out of 16, and 4 out of 4 patients in groups 1, 2, and 3, respectively. The following histological parameters differed significantly (P < 0.05) among the groups: interstitial edema, congestion of peritubular capillaries, glomerular thrombosis, and glomerular ischemia (group 3 > group 2 > group 1). Interstitial bleeding was seen more often in group 2 and 3 tissues than in group 1 specimens (P < 0.01). Immunohistochemical analyses showed that vascular rejection was associated with WT14 staining for monocytes and macrophages around the tubuli and with interstitial deposition of complement factor 3. With regard to serology, positive anti-endothelial cell antibody-dependent cellular cytotoxicity was associated with vascular rejection and thrombosis of the graft in all patients tested, and with graft loss in 75%. Pre-existent positive anti-IgG immunofluorescence on peritubular capillaries in pretransplant biopsy specimens incubated with patient serum was found in only 3 of the 50 patients, but was associated with graft loss in 2 of the 3. Cytomegalovirus infection was associated with a higher percentage of graft loss. There were significant intergroup differences in panel reactive antibodies before transplantation (P < 0.001), with higher titers in groups 2 and 3. The findings in relation to interstitial rejection are compatible with cellular rejection, while the data on vascular rejection support a humorally mediated pathogenesis.

Dietary fish oil in renal transplant recipients treated with cyclosporin-A: no beneficial effects shown.

This study aimed to determine whether dietary supplementation with fish oil has a beneficial effect on graft function and the incidence of rejection in renal allograft recipients treated with cyclosporin A (CsA). Renal function, blood pressure, the incidence of acute rejection episodes, graft survival, and renal histology and immunochemistry were investigated. In a randomized, placebo-controlled, double-blind trial, groups of 25 recipients of primary cadaveric renal allografts who had been treated with CsA took fish oil (30% C20:5 omega-3 and 20% C22:6 omega-3) or coconut oil (63% C8:0 and 36% C10:0) at 6 g/day for 3 months. There were no differences between the two patient groups with regard to HLA matching, panel-reactive antibody titers, or the demographic characteristics of donors or recipients. The GFR and effective RPF were determined at 1, 3, and 12 months after transplantation by simultaneous measurement of (125I-)iothalamate and (131I-)hippuran clearances. At 1 yr after transplantation, patients treated with fish oil showed better renal function than did the control patients, but this difference was not statistically significant. Blood pressure and antihypertensive drug use were similar in both groups. The number of rejection episodes was also similar, and renal histopathological and immunohistochemical studies showed no significant differences between the fish-oil group and the control patients. It is concluded that fish oil, at a dose of 6 g/day, has no beneficial effect after renal transplantation within the time scale of the study.

Evaluation by histology, immunohistology and PCR of protocollized renal biopsies 1 week post-transplant in relation to subsequent rejection episodes.

Renal biopsies were performed 1 week following renal transplantation at a time without clinical evidence of rejection in 43 patients (13 females, mean age 48 years range 18-60 and 30 males, mean age 43 years range 17-59 years). Thirty-six biopsies were available for histological or immunohistochemical analysis. Immunohistochemical analyses were performed with monoclonal antibodies against leukocytes (CD45), monocytes (WT14), complement factor 3 (C3), T-cells (Leu4), T-cell receptor alpha beta and gamma delta, tumour necrosis factor alpha (TNF alpha), IL-2 receptor (IL2-R, TAC), intercellular adhesion molecule-1 (ICAM1) and HLA-DR. The slides were scored semiquantitatively with the observers having no knowledge of clinical or patient data. TNF alpha and IL-2R were also measured by quantative PCR. None of the studied parameters correlated to delayed graft function or graft loss. Histological analysis showed that both focal interstitial infiltrate (18/35) and tubular basement membrane disruption (11/35) were followed by a higher incidence of subsequent rejection (P = 0.03 and 0.02 respectively). Also positivity for WT14 around tubuli (P = 0.02) was associated with subsequent occurrence of rejection. The intensity of staining of ICAM-1 on PTC as well as TAC on proximal tubular cells was associated with the number of subsequent rejection episodes. The association between the IL-2 receptor and subsequent rejection was also found applying PCR to the tissue specimens. We conclude that the presence of focal interstitial infiltrates and tubulitis in 1-week biopsies from well-functioning grafts carries an increased risk of subsequent rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunosuppression in Behçet's disease Clinical management and long-term visual outcome.

The authors review the visual prognosis of 44 patients with Behçet's disease referred to the Ophthalmology and Rheumatology Departments (Hospital S. João-one of the two major referral centers in Northern Portugal), due to ocular complaints or for routine examination, in the last ten years. All fulfilled the clinical criteria for diagnosis of Behçet's disease. Twenty-six were male and 18 female, with mean age 37 years (range 23-66). The mean evolution time since the first clinical manifestations was ten years (range 1-21). Aphthous stomatitis (100%) and genital ulcers (77.3%) were the initial manifestations preceding eye involvement. HLA-B(51)(5) was present in 27 of 36 typed patients (75%). Ocular manifestations were present in 33 patients (75%)-22 patients with panuveitis, eight with hypopyon, four with chronic anterior uveitis and three with episcleritis. Mean age of onset of ocular complications was 32 years (range 20-54). Retinal vaso-occlusive vasculitis was diagnosed in 26 patients (22 with panuveitis and four with posterior uveitis). To prevent ocular relapses, all needed immunosuppression with corticosteroids (drops, depo or systemic), combined with cyclosporine A (CsA) (5 mg/kg/day) in 13 patients and with chlorambucil or cyclophosphamide in six patients, when sight threatening uveitis had previously been refractory to treatment with systemic steroids. The 13 patients under CsA were observed for a period ranging from eight months to five years. Lowdose CsA was found to abrogate the intraocular inflammation, was well tolerated and had no major adverse effects. Regardless of the type of treatment, 21% (14/66) of the eyes lost useful vision five to ten years after initial diagnosis.

HLA and idiopathic uveitis.

Idiopathic uveitis is the most common form of chronic uveitis seen in our area. Characteristically it is a heterogeneous entity in its clinical severity, evolution and therapeutic response, and immune mechanisms are thought to be involved. The phenotype frequencies of HLA antigens were studied in 62 patients with idiopathic uveitis and compared with their frequencies in northern Portugal. The authors also included two groups with a well known immunogenetic background-Behçet's syndrome (BS: n=20) and ankylosing spondylitis (AS: n=18) which are, respectively, the most severe and the most frequent of the rheumatic forms in the area. An increased frequency of HLA B5(B51) in Behçet uveitis (55%; RR=8) was confirmed in the authors' population. They also found in idiopathic uveitis an increased risk for HLA B27 (RR=14). HLA B27 positive idiopathic uveitis was predominantly unilateral (80%) and anterior (97%), whereas B27 negative forms showed a higher prevalence of severe panuveitis. In conclusion, the authors confirmed in their population the association of HLA B27 with uveitis, and HLA B51 with Behçet uveitis. In this last entity an intermediate frequency between northern Europe and the Mediterranean area was observed.

Long-term follow-up of central serous retinopathy in 150 patients.

To study the evolution of the retinal pigment epithelial lesions in the central serous retinopathy, the authors studied 150 patients with ages between 20 and 49 years at the first examination, during periods varying between 6 months and 14 years. It was found a greater incidence of the disease in the males (83.3%); bilaterality in 23.3%; only one point of leakage in 62.7%; 3 different types of diffusion (inkblot 71.4%; mushroom 23.8%; with serous pigment epithelial detachment 4.9%). The final visual acuity was < 20/40 in a quarter of the cases and the frequency of the recurrences was 30%. The laser treatment did not influence the recurrences. In 50 patients with a follow-up superior to 3 years, 8 (16%) developed lesions similar to those described as diffuse retinal pigment epitheliopathy with visual field defects and subnormal EOG. Actuarial calculus suggests that 50% of the patients may get the most severe and extensive form of the disease after 12 years of evolution. The results allow to conclude that the diffuse retinal pigment epitheliopathy is only the terminal state of the most severe cases of central serous retinopathy.

Portal venous gas in a patient with diverticulitis.

Gas in the portal vein is a rare finding associated with a grave prognosis. We present a case of portal venous gas in a 34-year-old man with an abscess due to perforated-sigmoid diverticulitis and Escherichia coli sepsis. successfully treated with sigmoid resection and antibiotic therapy.

Uveitis in northern Portugal.

During the past four years 450 patients (247 female - mean age 36 years and 203 male - mean age 34 years) with intraocular inflammation were evaluated using standard diagnostic criteria, to establish a uveitis survey profile. Patients were divided into four anatomic groups: anterior uveitis (270 cases = 60%), posterior uveitis (108 cases = 24%), panuveitis (54 cases = 12%) and intermediate uveitis (18 cases = 4%). A probable aetiological diagnosis was made in 232 cases (51.5%). Rheumatic diseases were diagnosed most commonly (55 cases = 12.2%), mainly ankylosing spondylitis, as is true for other European countries. Ocular toxoplasmosis was a frequent cause of uveitis, as in Brazil and West Africa, thus requiring a prompt diagnosis and treatment. Behçet's syndrome was associated with HLA B5 (65%) as in Japanese and Mediterranean populations. In our area AIDS retinitis is becoming an important differential diagnosis.

Infectious arthritis caused by Propionibacterium acnes: a report of two cases.

Propionibacterium acnes, a micro-organism with low pathogenicity, rarely causes arthritis in a joint without a prosthesis. This report deals with two cases of P acnes infectious arthritis probably caused by direct penetration of the micro-organism into the joint space during arthrocentesis, despite the use of strict disinfection procedures. P acnes isolated from synovial fluid cannot be dismissed as a bothersome contaminant without due consideration of the clinical status of the individual patient.