Validation of a novel patient reported tool to assess the impact of treatment in erythropoietic protoporphyria: the EPP-QoL.

BACKGROUND: A novel treatment has been developed for erythropoietic protoporphyria (EPP) (a rare condition that leaves patients highly sensitive to light). To fully understand the burden of EPP and the benefit of treatment, a novel patient reported outcome (PRO) measure was developed called the EPP-QoL. This report describes work to support the validation of this measure. METHODS: Secondary analysis of trial data was undertaken. These analyses explored the underlying factor structure of the measure. This supported the deletion of some items. Further work then explored the reliability of these factors, their construct validity and estimates of meaningful change. RESULTS: The factor analyses indicated that the items could be summarised in terms of two factors. One of these was labelled EPP Symptoms and the other EPP Wellbeing, based on the items included in the domain. EPP Symptoms had evidence to support its reliability and validity. EPP Wellbeing had poor psychometric properties. CONCLUSIONS: Based on the analysis it was recommended to drop the EPP Wellbeing domain (and associated items). EPP Symptoms, despite limitations in the development of items, showed evidence of validity. This work is consistent with the recommendations of a task force that provided recommendations regarding the development, modification and use of PROs in rare diseases.

Erythropoietic protoporphyria: time to prodrome, the warning signal to exit sun exposure without pain-a patient-reported outcome efficacy measure.

PURPOSE: Patients with erythropoietic protoporphyria (EPP), a severe painful photodermatosis, experience prodromal sensations when exposed to sunlight, which are the "warning signals" to exit the sun, as prolonged exposure causes an excruciatingly painful phototoxic attack. The unique prodromal cutaneous sensations are reversible and differ from the severe burning pain attack lasting 2-7 days. Previously, afamelanotide treatment was studied using time to pain or time outside as primary outcome measures. Since patients have an ingrained fear of sunlight, these measures did not capture the full treatment effect. We retrospectively characterized and evaluated time to prodrome (TTP) as a safer, patient-reported outcome (PRO) measure in afamelanotide-treated patients. METHODS: Structured interviews recorded TTP before and during afamelanotide treatment in retrospective US and Dutch cohort studies. RESULTS: Thirty-one US and 58 Dutch EPP patients participated. Before afamelanotide treatment, 54.8% US and 39.7% Dutch patients reported TTP onset <10 minutes in direct sunlight. In both studies, patients' TTP's were significantly longer during afamelanotide treatment (p < 0.0001). All US patients' TTP increased; no TTP was <10 minutes. Among Dutch patients 81% improved; only 10.3% reported TTPs < 10 minutes. CONCLUSION: EPP patients reported substantial improvements in TTP during afamelanotide treatment. TTP could provide a safer, PRO-based efficacy endpoint for assessing future EPP treatments.

Osteoporosis in patients with erythropoietic protoporphyria.

BACKGROUND: Erythropoietic protoporphyria (EPP) is a rare metabolic disease with painful photosensitivity due to protoporphyrin IX accumulation. OBJECTIVES: To evaluate bone mineral density (BMD) and known osteoporosis risk factors in patients with EPP. METHODS: Patients with EPP attending the Erasmus MC outpatient clinic who had undergone BMD measurements were included. Plasma 25 hydroxy (OH) vitamin D, alkaline phosphatase, parathyroid hormone and total protoporphyrin IX levels were measured; information on lifestyle, sunlight exposure and a bone-relevant physical exercise index [Bone Physical Activity Questionnaire (BPAQ) score] was obtained via questionnaires. BMD scores and the prevalence of osteopenia and osteoporosis in the EPP population were compared with a reference population. RESULTS: Forty-four patients with EPP (23 female, 21 male; mean age 37·6 years) were included. The mean SDs of the T-scores were -1·12 for the lumbar spine and -0·82 for the femoral neck (both P < 0·001). Osteopenia was present in 36%; osteoporosis in 23%. Based on the reference population the expected prevalence was 15% and 1%, respectively. Prevalence of vitamin D deficiency was 50% (defined as a 25-OH vitamin D level < 50 nmol L-1 ). Mean self-reported BPAQ score was 19·4 units (reference interval 19-24). Multiple linear regression analysis showed a significant influence of vitamin D deficiency and bone-relevant physical exercise score on BMD in patients with EPP. CONCLUSIONS: The prevalence of osteoporosis and osteopenia is greatly increased in patients with EPP. Alkaline phosphatase (related to vitamin D deficiency) and amount of weight-bearing exercise are significantly correlated with low BMD in this population.

Aceruloplasminemia presents as Type 1 diabetes in non-obese adults: a detailed case series.

AIM: To detect features that might lead to the early diagnosis and treatment of aceruloplasminemia, as initiation of treatment before the onset of neurological symptoms is likely to prevent neurological deterioration. METHODS: The PubMed and OMIM databases were searched for published cases of aceruloplasminemia. Diagnostic criteria for aceruloplasminemia were undetectable or very low serum ceruloplasmin, hyperferritinemia and low transferrin saturation. Clinical, biochemical and radiological data on the presentation and follow-up of the cases were extracted and completed through e-mail contact with all authors. RESULTS: We present an overview of 55 aceruloplasminemia cases, including three previously unreported cases. Diabetes mellitus was the first symptom related to aceruloplasminemia in 68.5% of the patients, manifesting at a median age of 38.5 years, and often accompanied by microcytic or normocytic anaemia. The combination preceded neurological symptoms in almost 90% of the neurologically symptomatic patients and was found 12.5 years before the onset of neurological symptoms. CONCLUSIONS: There is a diagnostic window during which diabetes and anaemia are present although there is an absence of neurological symptoms. Screening for aceruloplasminemia in adult non-obese individuals presenting with antibody-negative, insulin-dependent diabetes mellitus and unexplained anaemia is recommended. The combination of ferritin and transferrin saturation provides a sensitive initial measure for aceruloplasminemia.

Vitamin D deficiency in patients with erythropoietic protoporphyria.

BACKGROUND: The main symptom of patients with erythropoietic protoporphyria (EPP) is painful photosensitivity, starting within minutes of sun exposure and leading to sun-avoidance. As 80-100% of vitamin D is synthesized under the influence of sunlight, we investigated whether the avoidance of sunlight exposure in the Dutch EPP patient population causes vitamin D deficiency. Furthermore, we studied the relation between vitamin D levels, total erythrocyte protoporphyrin and quality of life. METHODS: In a cross-sectional study of 48 Dutch EPP patients (mean age 41.4 years; range 16-77; 23 male, 25 female), we assessed serum 25-hydroxyvitamin D (25(OH)D) levels between June and November 2007, as well as total erythrocyte protoporphyrin (TEP) levels and Dermatology Life Quality Index (DLQI) scores. RESULTS: Mean serum 25(OH)D was 66 nmol/L (range 18-140, quartiles 36, 87). Twenty-two patients (46%; 15 male, 7 female) were vitamin D deficient. There was a significant difference (p = 0.029) in mean serum 25(OH)D between female (mean 75 nmol/L, range 18-140) and male patients (mean 55 nmol/L, range 18-115). The level of serum 25(OH)D showed a negative correlation with total erythrocyte protoporphyrin (TEP) (Pearson rank correlation (r(p)) = -0.337; p = 0.034). Serum 25(OH)D was inversely associated with scores of the Dermatology Life Quality Index (DLQI) (Spearman's rho correlation (r(s)) = -0.486; p = 0.001). CONCLUSIONS: The prevalence of vitamin D deficiency is high in the Dutch EPP population, especially in male patients, and correlates with the severity of EPP. Screening for and treatment of vitamin D deficiency should therefore be implemented in the care of these patients.

Evidence for an ancestral founder of the common R116W mutation in the hydroxymethylbilane synthase gene in acute intermittent porphyria in The Netherlands.

INTRODUCTION: Acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant inborn disorder of heme biosynthesis caused by mutations in the porphobilinogen deaminase (PBGd) gene. The prevalence of AIP in Europe is estimated as 1/10.000-1/20.000. The majority of the known AIP mutations are restricted to only one or just a few AIP families, with the exception of the frequent occurring R116W mutation which is found in 19/80 Dutch AIP families. This mutation has also been reported in 6 other populations (Sweden, Norwegian, i.a.) Recent haplotype analysis of Norwegian and Swedish patients with the R116W mutation show high heterogeneity. The conclusion of that report is that this mutation is abundant due a high mutability of CpG dinucleotides. The Dutch R116W families are well documented with extended pedigrees (up to 1750) which makes it possible to study the haplotypes in these families. AIM: To investigate haplotype heterogeneity in the Dutch R116W families. METHODS: To investigate the haplotype heterogeneity of the Dutch R116W families, intragenic single nucleotide polymorphism's (SNPs) which cover the whole PBGd gene of 8.6 kb were selected. In addition to the intragenic SNPs, microsatellite markers were selected, flanking the genomic region of the PBGD gene covering a distance of 7.48cM in chromosome 11. The 7 SNPs were first analysed in 4 out of 19 R116W families selected for their most complete and informative pedigree. The 7 analysed SNPs revealed a distinctive R116W haplotype and were used to analyse the other 14 families in this study cohort, which mainly consisted of DNA from single patients or families with limited members. RESULTS: The informative SNPs reveal a distinctive haplotype which segregates with the R116W mutation present in the Dutch AIP families (-64T, 1345 G, 2479 G, 3581 G, 6479 T, 7064 C and 8578 A). SNP base nrs a less conserved microsatellite haplotype was observed in these AIP families. CONCLUSION: This common R116W haplotype based on 7 SNPs strongly suggests that the relatively high frequency of the R116W mutation in Dutch AIP patients is due a founder effect (eldest parent in pedigree was born in 1750!!). The high mutability of CpG dinucleotides is not a likely explanation for the abundant presence of the R116W mutation, since it is only reported in a few western countries. The heterogeneity described in the Sweden and Norwegian patients and the homogeneity found in the Dutch R116W carriers is compatible with origin of the mutation in Scandinavia with later introduction into the Netherlands. Due to the high frequency of this R116W mutation within the Dutch AIP families it may be applied to refine estimations of the prevalence of AIP in The Netherlands.

Allele-sharing of cytokine genes in familial inflammatory bowel disease.

BACKGROUND/AIMS: The pathogenesis of inflammatory bowel disease is complex, multifactorial, and involves genetic predisposition. This predisposition is likely to include various chromosomal loci, but simple Mendelian inheritance cannot be excluded in a subset of families with inflammatory bowel disease. METHODOLOGY: We evaluated allele-sharing in 17 sib-pairs with inflammatory bowel disease as an approach to select candidate genes for further studies in individual families. It was determined whether each sib-pair had inherited the same alleles for interleukin-2, interleukin-2 receptor beta, interleukin-4, interleukin-4 receptor, interleukin-10, interleukin-10 receptor, tumor necrosis factor alpha, tumor necrosis factor alpha receptor 1 and 2. RESULTS: The results were very different per individual family. The estimated probability of sharing both alleles identical-by-descent at interleukin-4 receptor, interleukin-10, interleukin-10 receptor, and tumor necrosis factor alpha were 50%, 39%, 40%, and 33% respectively. The LOD score was significant for interleukin-4 receptor (p = 0.04). CONCLUSIONS: In this small group of sib-pairs with inflammatory bowel disease a modestly increased allele-sharing was found for some inflammatory related genes. Different results per family may suggest genetic heterogeneity. This method can be useful as a first step to further evaluation of specific candidate genes which may play a pathogenetic role in individual families.

Cardiac abnormalities in adults with the attenuated form of mucopolysaccharidosis type I.

BACKGROUND: Cardiac involvement in mucopolysaccharidosis type I (MPS I) has been studied primarily in its most severe forms. Cardiac involvement, particularly left ventricular (LV) systolic and diastolic function, in the attenuated form of MPS I is less well known. METHODS: Cardiac function was prospectively investigated in 9 adult patients with the attenuated form of MPS I. All patients underwent 12-lead electrocardiography, 24 h Holter monitoring and two-dimensional echocardiography including tissue Doppler imaging (TDI). Eighteen age- and sex-matched healthy volunteers served as a control group. RESULTS: Aortic, mitral and tricuspid valve thickening was seen in, respectively, 5 (56%), 4 (44%) and 2 (22%) patients. Moderate mitral valve stenosis was seen in 1 patient and moderate aortic stenosis in 2 patients. All patients had mild-to-moderate aortic and mitral valve regurgitation and 6 patients (67%) had mild-to-moderate tricuspid valve regurgitation. Despite normal LV dimensions, ejection fraction and mass index, MPS patients had lower mean systolic mitral annular velocities (6.1 +/- 0.6 vs 9.1 +/- 1.4 cm/s, p < 0.01) compared to normal control subjects. Similarly, mean early diastolic mitral annular velocities were lower in MPS patients (7.8 +/- 0.9 vs 13.3 +/- 3.3 cm/s, p < 0.01). CONCLUSION: MPS I patients with the attenuated phenotype have not only valvular abnormalities but also LV diastolic and systolic abnormalities.

STK11 status and intussusception risk in Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is caused by germline STK11 mutations and characterised by gastrointestinal polyposis. Although small bowel intussusception is a recognised complication of PJS, risk varies between patients. OBJECTIVE: To analyse the time to onset of intussusception in a large series of PJS probands. METHODS: STK11 mutation status was evaluated in 225 PJS probands and medical histories of the patients reviewed. RESULTS: 135 (60%) of the probands possessed a germline STK11 mutation; 109 (48%) probands had a history of intussusception at a median age of 15.0 years but with wide variability (range 3.7 to 45.4 years). Median time to onset of intussusception was not significantly different between those with identified mutations and those with no mutation detected, at 14.7 years and 16.4 years, respectively (log-rank test of difference, chi(2) = 0.58, with 1df; p = 0.45). Similarly no differences were observed between patient groups on the basis of the type or site of STK11 mutation. CONCLUSIONS: The risk of intussusception in PJS is not influenced by STK11 mutation status.

A functional interleukin-10 mutation in Dutch patients with Crohn's disease.

BACKGROUND AND AIMS: Interleukin-10 is an anti-inflammatory and immunomodulatory cytokine. Interleukin-10 deficient mice are prone to develop chronic colitis. Administration of recombinant human interleukin-10 has been proposed to have a beneficial effect in a subgroup of patients with Crohn's disease. Recently, we found an interleukin-10 Gly15Arg mutation in a family with Crohn's disease which is associated with reduced interleukin-10 secretion by in vitro stimulated monocytes and lymphocytes. We hypothesised that this interleukin-10 mutation plays a role in maintaining the inflammatory process in Crohn's disease in some families. PATIENTS AND METHODS: We evaluated interleukin-10 Gly15Arg in 379 patients with Crohn's disease, and 75 unrelated healthy controls. Also, first degree family members of interleukin-10 Gly15Arg carriers were evaluated. Additionally, mutation carriers and their relatives were evaluated for CARD15 R702W, G908R, and 1007fs. RESULTS: Two patients with Crohn's disease were heterozygous for the interleukin-10 Gly15Arg mutation. No homozygotes were found. The Gly15Arg mutation was not observed in the controls. In first degree family members of the Crohn's disease-affected interleukin-10 Gly15Arg carriers, the mutation was found in Crohn's disease-affected as well as in their apparently healthy individuals. All family members carried one or two CARD15 mutation(s). CONCLUSION: The interleukin-10 Gly15Arg mutation is rare in patients with Crohn's disease, and is not associated with the disease in the Netherlands.

Mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADalpha, and MO25alpha, in Peutz-Jeghers syndrome.

Mutations in LKB1 lead to Peutz-Jeghers syndrome (PJS). However, only a subset of PJS patients harbours LKB1 mutations. We performed a mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADalpha, and MO25alpha, in 28 LKB1-negative PJS patients. No disease-causing mutations were detected in the studied genes in PJS patients from different European populations.

Overanticoagulation associated with combined use of lactulose and acenocoumarol or phenprocoumon.

Some medical textbooks on drug interactions take note of the potential interaction between laxatives and coumarin anticoagulants, but epidemiological evidence that this interaction is of practical importance is lacking. We conducted a follow-up study in a large population-based cohort to investigate which laxatives are associated with overanticoagulation during therapy with coumarins. Of the 1124 patients in the cohort, 351 developed an International Normalized Ratio > or = 6.0. The only laxative with a moderate but significantly increased relative risk of overanticoagulation was lactulose (relative risk 3.4, 95% confidence interval 2.2, 5.3). In view of the widespread use of lactulose, especially among the elderly, awareness of this potential drug interaction is required.

A Gly15Arg mutation in the interleukin-10 gene reduces secretion of interleukin-10 in Crohn disease.

BACKGROUND: Genetic susceptibility, probably involving cytokines and their receptors, plays an important role in inflammatory bowel disease (IBD). In this study we examine the potential role of the interleukin-10 (IL-10) gene as a susceptibility gene in IBD. METHODS: We studied 17 sib-pairs with either Crohn disease (CD) or ulcerative colitis. After microsatellite analysis for allele-sharing, the IL-10 gene of sib-pairs who shared alleles was screened for nucleotide alterations in and around exons and the promoter region. The IL-10 promoter polymorphism at position -1082 was also determined. Function was evaluated by measuring IL-10 secretion by peripheral blood mononuclear cells stimulated with lipopolysaccharide or phorbol ester. The activity of recombinant immature wild-type and mutated IL-10 was tested in a proliferation assay with a human monocytic leukaemia cell line (HL60 cells). RESULTS: DNA sequencing revealed a G --> A point mutation in exon 1 at base position 43 in one sib-pair, both affected with CD. It was also found in 2 of their healthy siblings, but not in 75 unrelated healthy controls. This mutation results in a glycine to arginine substitution at amino acid position 15 of the leader sequence (Gly15Arg). The in vitro IL-10 secretion by mononuclear cells of the IL-10 Gly15Arg carriers was about 50% of healthy controls, matched for the -1082 polymorphism in the IL-10 promoter region. Incubation of HL60 cells with recombinant mutated IL-10 showed a markedly reduced cell proliferation compared to wild-type IL-10. CONCLUSION: A Gly15Arg mutation in the leader sequence of IL-10 was found in a multiple CD-affected family. This altered leader sequence decreases IL-10 secretion, thereby reducing the anti-inflammatory effect.

[From gene to disease; 'frame shift'-mutation in the CARD15-gene and Crohn's disease]. / Van gen naar ziekte; 'frame shift'-mutatie in het CARD15-gen en de ziekte van Crohn.

A pericentromeric region on chromosome 16 (IBD1 locus) has been linked with Crohn's disease (CD). Very recently, three genetic variants in the CARD15 gene within the IBD1 locus have been identified which were highly associated with CD. Carriage increases the relative risk of developing CD. One specific mutation (3020insC) leads to a stop codon and truncation of the C-terminal tandem leucine-rich repeats (LRR) of the CARD15 protein. Of all patients with Crohn's disease, 11-19% are heterozygous and 3-7% homozygous for this frameshift mutation. The CARD15 gene is expressed in monocytes. The LRR-domain is thought to be involved in the binding of bacterial lipopolysaccharide and subsequent activation of nuclear factor kappa-B (NF kappa B). NF kappa B plays a central role in the regulation of the expression of other genes involved in the inflammatory response. In vitro, embryonic kidney cells transfected with the CARD15 3020insC mutant showed a reduced activity of NF kappa B after exposure to lipopolysaccharide compared to cells transfected with the wild-type CARD15 gene. How the reduced response to lipopolysaccharide contributes to CD is not yet clear.

Two course illuminating scheme improves aminolevulinic acid photodynamic therapy in cell cultures.

Photodynamic therapy with the pro-drug 5-aminolaevulinic acid (ALA-PDT) is being used for the treatment of Barrett's oesophagus. We postulated that a first early course of ALA-PDT would increase protoporphyrin IX (PPIX) accumulation and thus the efficacy of a second course of ALA-PDT, by manipulating ferrochelatase (FC) and porphobilinogen deaminase (PBG-d) activity. Human EBV-transformed lymphoblastoid cells were used as a model of human tumour cells for the ability to form haem is present in all cells. After a single course of illumination (633 nm, 100 mW/cm2) the FC activity decreased significantly whereas the PBG-d activity did not change. During continued incubation with ALA following the first illumination, cells accumulated up to four times more PPIX than non-illuminated controls [220% +/- 30% versus (vs) 55% +/- 5%; p<0.001]. Two illuminations resulted in more cell death than one illumination (97% +/- 1% vs 80% +/- 2%; p<0.001). Since a second course of ALA-PDT within 3 hr after the first course resulted in a four fold increase in PPIX accumulation and significantly more cell death, we propose that a two course ALA-PDT scheme might improve the efficacy of this treatment for Barrett's oesophagus.

Residual activity of human porphobilinogen deaminase with R167Q or R167W mutations: an explanation for survival of homozygous and compound heterozygous acute intermittent porphyrics.

To find an explanation for survival of homozygous or compound heterozygous variants of acute intermittent porphyria, we studied the three mutant forms of porphobilinogen deaminase (PBG-d) described in the four reported patients with homozygous acute intermittent porphyria. Wild-type human PBG-d and the PBG-d R167W, R167Q and R173Q mutants were expressed in Escherichia coli and the recombinant mutant human enzyme were examined for enzyme activity. Specific antibodies against human PBG-d detected the three human PBG-d mutants. All three had less than 2% of wild-type enzyme activity when examined under customary assay conditions (pH 8.0), but the R167W and R167Q mutants were found to have about 25% of normal activity when assayed at pH 7.0. This residual activity at a more physiological pH provides an explanation for survival when these mutations are inherited in a homozygous or compound heterozygous fashion.