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BACKGROUND: Children's interstitial lung diseases (chlLD) comprise a broad spectrum of diseases. Besides the genetically defined surfactant dysfunction disorders, most entities pathologically involve the alveolar surfactant region, possibly affecting the surfactant proteins SP-B and SP-C. Therefore, our objective was to determine the value of quantitation of SP-B and SP-C levels in bronchoalveolar lavage fluid (BALF) for the diagnosis of chlLD. METHODS: Levels of SP-B and SP-C in BALF from 302 children with chlLD and in controls were quantified using western blotting. In a subset, single-nucleotide polymorphisms (SNPs) in the SFTPC promoter were genotyped by direct sequencing. RESULTS: While a lack of dimeric SP-B was found only in the sole subject with hereditary SP-B deficiency, low or absent SP-C was observed not only in surfactant dysfunction disorders but also in patients with other diffuse parenchymal lung diseases pathogenetically related to the alveolar surfactant region. Genetic analysis of the SFTPC promoter showed association of a single SNP with SP-C level. CONCLUSION: SP-B levels may be used for screening for SP-B deficiency, while low SP-C levels may point out diseases caused by mutations in TTF1, SFTPC, ABCA3, and likely in other genes involved in surfactant metabolism that remain to be identified. We conclude that measurement of levels of SP-B and SP-C was useful for the differential diagnosis of chlLD, and for the precise molecular diagnosis, sequencing of the genes is necessary.

Patients with interstitial lung disease due to surfactant protein C (SFTPC) mutations are rare and not well characterised.We report on all subjects collected over a 15-year period in the kids-lung register with interstitial lung disease and a provenSFTPCmutation. We analysed clinical courses, interventions and outcomes, as well as histopathological and radiological interrelations.17 patients (seven male) were followed over a median of 3 years (range 0.3–19). All patients were heterozygous carriers of autosomal dominantSFTPCmutations. Three mutations (p.L101P, p.E191 K and p.E191*) have not been described before in the context of surfactant protein C deficiency. Patients with alterations in the BRICHOS domain of the protein (amino acids 94–197) presented earlier. At follow-up, one patient was healthy (2 years), six patients were “sick-better” (2.8 years, range 0.8–19), seven patients were “sick-same” (6.5 years, 1.3–15.8) and three patients were “sick-worse” (0.3 years, 0.3–16.9). Radiological findings changed from ground-glass to increasing signs of fibrosis and cyst formation with increasing age. Empiric treatments had variable effects, also in patients with the same genotype.Prospective studies with randomised interventions are urgently needed and can best be performed in the framework of international registers.

Knowledge about the clinical spectrum of lung disease caused by variations in the ATP binding cassette subfamily A member 3 (ABCA3) gene is limited. Here we describe genotype-phenotype correlations in a European cohort.We retrospectively analysed baseline and outcome characteristics of 40 patients with two disease-causing ABCA3 mutations collected between 2001 and 2015.Of 22 homozygous (15 male) and 18 compound heterozygous patients (3 male), 37 presented with neonatal respiratory distress syndrome as term babies. At follow-up, two major phenotypes are documented: patients with (1) early lethal mutations subdivided into (1a) dying within the first 6 months or (1b) before the age of 5 years, and (2) patients with prolonged survival into childhood, adolescence or adulthood. Patients with null/null mutations predicting complete ABCA3 deficiency died within the 1st weeks to months of life, while those with null/other or other/other mutations had a more variable presentation and outcome. Treatment with exogenous surfactant, systemic steroids, hydroxychloroquine and whole lung lavages had apparent but many times transient effects in individual subjects.Overall long-term (5 years) survival of subjects with two disease-causing ABCA3 mutations was20%. Response to therapies needs to be ascertained in randomised controlled trials.

Background Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder predominantly affecting the skin, the eyes and the cardiovascular system. The disease is caused by mutations in the ABCC6 gene and characterized by ectopic calcification and extracellular matrix (ECM) alterations. Matrix metalloproteinases (MMPs) play a pivotal role in the process of ECM remodeling and are likely implied in PXE pathology. The aim of the present study was to investigate the association of single nucleotide polymorphisms (SNPs) in the promoter of the MMP2 gene, and PXE. Methods We evaluated the allelic distribution of five SNPs in the MMP2 promoter in DNA samples from 168 German patients affected by PXE and in 168 healthy, age- and sex-matched control subjects using restriction fragment length polymorphism analysis. Results The alleles c.-1575G, c.-1306C, and c.-790T were more abundant in the PXE patients' group. Furthermore, the haplotype GCTCG was significantly associated with PXE (OR 1.56, 95% CI 1.14–2.12, Pcorrected = 0.026). Conclusions Our results may indicate an involvement of MMP2 in the pathology of PXE. The promoter polymorphisms associated with PXE may lead to increased MMP2 expression and thereby contribute to the elevated proteolytic activity observed in PXE in vitro and in vivo.

Pseudoxanthoma elasticum (PXE) is a heritable disorder affecting the skin, eyes and cardiovascular system. It is caused by mutations in the ABCC6 gene and its clinical picture is highly variable. PXE often leads to severe visual impairment due to the development of choroidal neovascularisation (CNV). CNV in PXE-associated retinopathy is believed to be mediated by the action of vascular endothelial growth factor (VEGF). The objective of the present study was to evaluate a possible impact of variations in the VEGFA gene on ocular manifestations of PXE. For this purpose, we evaluated the distribution of 10 single nucleotide polymorphisms (SNPs) in the promoter and coding region of the VEGFA gene in DNA samples from 163 German patients affected by PXE and in 163 healthy control subjects. Haplotype analysis of SNPs c.-1540A>C, c.-460C>T, c.-152G>A, c.405C>G, c.674C>T, c.1032C>T, c.4618C>T and c.5092C>A revealed that the haplotype CTGGCCCC was associated with PXE (OR 2.05, 95% CI 1.33-3.15, P(corrected) = 0.01). Furthermore, five SNPs showed significant association with severe retinopathy. The most significant single SNP association was c.-460C>T (OR 3.83, 95% CI 2.01-7.31, P(corrected) = 0.0003). Logistic regression analysis identified the c.-460T and the c.674C alleles as independent risk factors for development of severe retinopathy. Our findings suggest an involvement of VEGF in the pathogenesis of ocular PXE manifestations. VEGF gene polymorphisms might prove useful as prognostic markers for the development of PXE-associated retinopathy and permit earlier therapeutic intervention in order to prevent loss of central vision, one of the most devastating consequences of this disease.

Pseudoxanthoma elasticum (PXE, OMIM 177850 and 264800) is a rare heritable disorder predominantly affecting the skin, the eyes and the vascular system. The disease is caused by mutations in the ABCC6 gene and is characterized by calcification and extracellular matrix remodeling, including alterations of the vessel walls. Here, we investigated the cell adhesion molecules ICAM-1 in PXE patients.Soluble ICAM-1 was determined in 58 non-consanguineous PXE patients by quantitative sandwich enzyme immunoassay. The allelic frequencies of the ICAM-1 variant p.K469E were analyzed in patients and age- and sex-matched controls.Soluble ICAM-1 levels were significantly elevated in male and female PXE patients (p0.02 and p0.001, respectively). In addition, the ICAM-1 concentration correlated with the ABCC6 gene status of the PXE patients. The ICAM variant p.K469E genotypes were not different in PXE patients and age- and sex-matched controls.Our data show for the first time increased ICAM-1 concentrations in PXE patients, potentially due to the chronic oxidative stress and elevated protease activity followed by extracellular matrix remodeling which have been previously observed in PXE patients.

BACKGROUND: Lipids account for the majority of pulmonary surfactant, which is essential for normal breathing. We asked if interstitial lung diseases (ILD) in children may disrupt alveolar surfactant and give clues for disease categorization. METHODS: Comprehensive lipidomics profiles of broncho-alveolar lavage fluid were generated in 115 children by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Two reference populations were compared to a broad range of children with ILD. RESULTS: Class and species composition in healthy children did not differ from that in children with ILD related to diffuse developmental disorders, chronic tachypnoe of infancy, ILD related to lung vessels and the heart, and ILD related to reactive lymphoid lesions. As groups, ILDs related to the alveolar surfactant region, ILD related to unclear respiratory distress syndrome in the mature neonate, or in part ILD related to growth abnormalities reflecting deficient alveolarisation, had significant alterations of some surfactant specific phospholipids. Additionally, lipids derived from inflammatory processes were identified and differentiated. In children with ABCA3-deficiency from two ILD causing mutations saturated and monounsaturated phosphatidylcholine species with 30 and 32 carbons and almost all phosphatidylglycerol species were severely reduced. In other alveolar disorders lipidomic profiles may be of less diagnostic value, but nevertheless may substantiate lack of significant involvement of mechanisms related to surfactant lipid metabolism. CONCLUSIONS: Lipidomic profiling may identify specific forms of ILD in children with surfactant alterations and characterized the molecular species pattern likely to be transported by ABCA3 in vivo.

Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to tRNA and is critical for protein biosynthesis. We identified biallelic missense mutations in MARS in a specific form of pediatric pulmonary alveolar proteinosis (PAP), a severe lung disorder that is prevalent on the island of Réunion and the molecular basis of which is unresolved. Mutations were found in 26 individuals from Réunion and nearby islands and in two families from other countries. Functional consequences of the mutated alleles were assessed by growth of wild-type and mutant strains and methionine-incorporation assays in yeast. Enzyme activity was attenuated in a liquid medium without methionine but could be restored by methionine supplementation. In summary, identification of a founder mutation in MARS led to the molecular definition of a specific type of PAP and will enable carrier screening in the affected community and possibly open new treatment opportunities.

Background Interstitial lung disease occurring in children is a condition characterized by high frequency of cases due to genetic aberrations of pulmonary surfactant homeostasis, that are also believed to be responsible of a fraction of familial pulmonary fibrosis. To our knowledge, ABCA3 gene was not previously reported as causative agent of fibrosis affecting both children and adults in the same kindred. Methods We investigated a large kindred in which two members, a girl whose interstitial lung disease was first recognized at age of 13, and an adult, showed a diffuse pulmonary fibrosis with marked differences in terms of morphology and imaging. An additional, asymptomatic family member was detected by genetic analysis. Surfactant abnormalities were investigated at biochemical, and genetic level, as well as by cell transfection experiments. Results Bronchoalveolar lavage fluid analysis of the patients revealed absence of surfactant protein C, whereas the gene sequence was normal. By contrast, sequence of the ABCA3 gene showed a novel homozygous G > A transition at nucleotide 2891, localized within exon 21, resulting in a glycine to aspartic acid change at codon 964. Interestingly, the lung specimens from the girl displayed a morphologic usual interstitial pneumonitis-like pattern, whereas the specimens from one of the two adult patients showed rather a non specific interstitial pneumonitis-like pattern. Conclusions We have detected a large kindred with a novel ABCA3 mutation likely causing interstitial lung fibrosis affecting either young and adult family members. We suggest that ABCA3 gene should be considered in genetic testing in the occurrence of familial pulmonary fibrosis.

Pseudoxanthoma elasticum (PXE) is a rare disorder predominantly affecting the skin, the eyes, and the cardiovascular system. The disease is caused by mutations in the ABCC6 gene and characterized by ectopic calcification and extracellular matrix (ECM) alterations. Matrix metalloproteinases (MMPs) play a pivotal role in the process of ECM remodeling. In the present study, we investigated matrix metalloproteinases MMP-2 and MMP-9 in PXE patients compared to healthy controls. We analyzed the serum concentrations of MMP-2 and MMP-9 in a cohort of 69 German PXE patients and in 69 healthy, age-, and sex-matched control subjects using commercially available ELISA assays. We found elevated concentrations of both MMPs in the sera of PXE patients. MMP-2 levels were significantly higher in patients than controls (231 +/- 5.89 vs 202 +/- 5.17 ng/ml, p = 0.0002), as were MMP-9 levels (841 +/- 65.9 vs 350 +/- 30.8 ng/ml, p0.0001). Our findings point to an involvement of matrix metalloproteinases in PXE pathology. ECM remodeling in PXE is reflected by elevated levels of circulating MMP-2 and MMP-9. Those MMPs might, therefore, be applicable as serum markers for the matrix-degradative process in PXE.

Pseudoxanthoma elasticum (PXE) is a hereditary disorder predominantly affecting the eyes, the skin, and the vascular system. The subretinal neovascularization and retinal hemorrhages leading to the loss of central vision in PXE are similar to the process observed in age-related macular degeneration (AMD). The complement factor H (CFH) variant c.1277TC (p.Y402H) is a recently discovered risk factor for AMD. The aim of this study was to analyze whether this CFH variant is a secondary genetic risk factor for PXE. Therefore, the genotypes of CFH c.1277TC (p.Y402H) were determined in 189 German PXE patients and 189 age- and sex-matched controls. The allelic frequencies of the investigated variant did not differ between patients and controls. The frequencies were 33%, 56%, and 11% for wild-type, heterozygous, and homozygous genotypes in the PXE patients and 36%, 51%, and 13% in the control cohort, respectively. Further, no significant associations were identified when allele carriers were analyzed or after adjustment for sex, age, smoking, organ involvement, hypertension, or age at disease onset. No significant genotype-phenotype correlation was detected. In conclusion, our data reliably show that the CFH variant c.1277TC (p.Y402H) is not a genetic risk factor for PXE.

Objectives Recent studies have revealed the involvement of calcification inhibitory proteins in the pathogenesis of pseudoxanthoma elasticum (PXE). Design and methods We analyzed serum concentrations of the calcification inhibitor matrix Gla protein ( MGP ) in a large cohort of patients suffering from PXE ( n = 101), 34 first-degree relatives and 67 healthy controls. Moreover, we determined the distribution of the two MGP promoter polymorphisms c.-7G>A and c.-138T>C in the three cohorts. Results We found significantly lower total MGP concentrations in the sera of PXE patients compared to healthy controls ( p = 0.0002). Furthermore, higher serum MGP concentrations could be correlated with a later PXE onset. Analysis of MGP promoter polymorphism frequencies revealed one MGP haplotype to be a potential protective co-factor in PXE. Conclusions Our findings point to a role of the local calcification inhibitor MGP in PXE manifestation.

Background: Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by progressive calcification and fragmentation of elastic fibers in connective tissues. PXE is caused by mutations in the ABCC6 gene, which encodes the membrane transporter multidrug resistance–associated protein 6. Chronic oxidative stress was recently suggested to play a crucial role in the pathogenesis of the disease. Our aim was to investigate the association of PXE with genetic variation in genes coding for antioxidant enzymes. Methods: We used restriction fragment length polymorphism and allele-specific PCR analyses to evaluate the distribution of single-nucleotide polymorphisms in the genes encoding catalase (CAT), superoxide dismutase 2 (SOD2), and glutathione peroxidase 1 (GPX1) in DNA samples from 117 German PXE patients and 117 healthy age- and sex-matched control individuals. Results: The investigated genetic variants had previously been shown to affect the activities of these antioxidant enzymes. We found a correlation between genotype and age of disease onset for polymorphisms in CAT (c.−262C>T), SOD2 (c.47C>T), and GPX1 (c.593C>T). Furthermore, the age of disease onset was inversely correlated with the number of mutated alleles, indicating a cumulative effect on the time of disease onset [mean (SD) age of 40.9 (13.6) years, 32.4 (16.3) years, and 25.7 (15.9) years for carriers of 0, 1–2, and >2 mutated alleles, respectively; P = 0.03]. Conclusion: Our findings demonstrate that increased oxidative stress due to activity-affecting polymorphisms in genes encoding antioxidant enzymes leads to earlier PXE onset.