Pathogenic RAB34 variants impair primary cilium assembly and cause a novel oral-facial-digital syndrome.

Oral-facial-digital syndromes (OFDS) are a group of clinically and genetically heterogeneous disorders characterized by defects in the development of the face and oral cavity along with digit anomalies. Pathogenic variants in over 20 genes encoding ciliary proteins have been found to cause OFDS through deleterious structural or functional impacts on primary cilia. We identified by exome sequencing bi-allelic missense variants in a novel disease-causing ciliary gene RAB34 in four individuals from three unrelated families. Affected individuals presented a novel form of OFDS (OFDS-RAB34) accompanied by cardiac, cerebral, skeletal and anorectal defects. RAB34 encodes a member of the Rab GTPase superfamily and was recently identified as a key mediator of ciliary membrane formation. Unlike many genes required for cilium assembly, RAB34 acts selectively in cell types that use the intracellular ciliogenesis pathway, in which nascent cilia begin to form in the cytoplasm. We find that the protein products of these pathogenic variants, which are clustered near the RAB34 C-terminus, exhibit a strong loss of function. Although some variants retain the ability to be recruited to the mother centriole, cells expressing mutant RAB34 exhibit a significant defect in cilium assembly. While many Rab proteins have been previously linked to ciliogenesis, our studies establish RAB34 as the first small GTPase involved in OFDS and reveal the distinct clinical manifestations caused by impairment of intracellular ciliogenesis.

Loading of cell cultures with cholesterol-dextran particles as a new functional test for Niemann-Pick type C disease.

Deuterium-labeled cholesterol-dextran particles (d4-CholDex), prepared by co-precipitation, were internalized by cultured human skin fibroblasts and HEK293 cells. Subcellular particles from d4-CholDex-treated HEK293 cells were fractionated on iodixanol gradients. More than 60% of d4-cholesterol (d4-UC) in the gradient co-fractionated with lysosomal markers and NPC1. This and formation of d4-cholesteryl esters (d4-CE) in the cells suggests that d4-CholDex is lysosomally processed. In accordance with these findings, we observed an increase in lysosomal cholesterol content by fluorescence microscopy in CholDex-loaded cells. Fibroblast cultures including 13 NPC1-deficient, four heterozygous and six control lines were treated with d4-CholDex at final d4-UC concentration of 0.05 mg/ml (127.98 µmol/L) for 3 h and chased for 48 h in medium without d4-CholDex. Concentrations of d4-UC and d4-CE in harvested cells were measured by tandem mass spectrometry (MS/MS). d4-UC/d4-CE ratios were elevated in NP-C lines compared to controls (n = 6, mean = 4.36, range = 1.89-8.91), with the highest ratios in severe NP-C1 phenotypes and the lowest in adolescent/adult type patients. There were overlaps between NP-C1 forms: early infantile (n = 1, mean = 48.6), late infantile (n = 4, mean = 36.3, range = 20.6-54.0), juvenile (n = 5, mean = 24.7, range = 13.4-38.3), adolescent/adult (n = 3, mean = 14.5, range = 11.7-19.8). The ratios in NP-C1 heterozygotes were mildly elevated (n = 4, mean = 16.4, range = 14.9-17.4) and comparable to patients with adolescent/adult NP-C1. The test can be useful in evaluation of suspected NP-C patients with inconclusive results of biomarker or molecular tests. Its advantages include standardized preparation of particles with longer shelf life at 4 °C, quantitative results, and no requirement for radioactive chemicals.

Pigmentary retinopathy can indicate the presence of pathogenic LAMP2 variants even in somatic mosaic carriers with no additional signs of Danon disease.

PURPOSE: Danon disease (DD) is a rare X-linked disorder caused by pathogenic variants in LAMP2. DD primarily manifests as a severe cardiomyopathy. An early diagnosis is crucial for patient survival. The aim of the study was to determine the usefulness of ocular examination for identification of DD. METHODS: Detailed ocular examination in 10 patients with DD (3 males, 7 females) and a 45-year-old asymptomatic female somatic mosaic carrier of a LAMP2 disease-causing variant. RESULTS: All patients with manifest cardiomyopathy had pigmentary retinopathy with altered autofluorescence and diffuse visual field loss. Best corrected visual acuity (BCVA) was decreased (<0.63) in 8 (40%) out of 20 eyes. The severity of retinal pathology increased with age, resulting in marked cone-rod involvement overtime. Spectral-domain optical coherence tomography in younger patients revealed focal loss of photoreceptors, disruption and deposition at the retinal pigment epithelium/Bruch's membrane layer (corresponding to areas of marked increased autofluorescence), and hyperreflective foci in the outer nuclear layer. Cystoid macular oedema was seen in one eye. In the asymptomatic female with somatic mosaicism, the BCVA was 1.0 bilaterally. An abnormal autofluorescence pattern in the left eye was present; while full-field electroretinography was normal. CONCLUSIONS: Detailed ocular examination may represent a sensitive and quick screening tool for the identification of carriers of LAMP2 pathogenic variants, even in somatic mosaicism. Hence, further investigation should be undertaken in all patients with pigmentary retinal dystrophy as it may be a sign of a life-threatening disease.

Danon disease is an underdiagnosed cause of advanced heart failure in young female patients: a LAMP2 flow cytometric study.

AIMS: Danon disease (DD) is a rare X-linked disorder caused by mutations in the lysosomal-associated membrane protein type 2 gene (LAMP2). DD is difficult to distinguish from other causes of dilated or hypertrophic cardiomyopathy (HCM) in female patients. As DD female patients regularly progress into advanced heart failure (AHF) aged 20-40 years, their early identification is critical to improve patient survival and facilitate genetic counselling. In this study, we evaluated the prevalence of DD among female patients with non-ischemic cardiomyopathy, who reached AHF and were younger than 40 years. METHODS AND RESULTS: The study cohort comprised 60 female patients: 47 (78%) heart transplant recipients, 2 (3%) patients treated with ventricular assist device, and 11 (18%) patients undergoing pre-transplant assessment. Aetiology of the cardiomyopathy was known in 15 patients (including two DD patients). LAMP2 expression in peripheral white blood cells (WBC) was tested by flow cytometry (FC) in the remaining 45 female patients. Whole exome sequencing was used as an alternative independent testing method to FC. Five additional female DD patients (two with different novel LAMP2 mutations) were identified by FC. The total prevalence of DD in this cohort was 12%. HCM phenotype (57% vs. 9%, * P = 0.022) and delta waves identified by electrocardiography (43% vs. 0%, ** P = 0.002) were significantly more frequent in DD female patients. CONCLUSIONS: Danon disease is an underdiagnosed cause of AHF in young female patients. LAMP2 expression testing in peripheral WBCs by FC can be used as an effective screening/diagnostic tool to identify DD in this patient population.

Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations.

BACKGROUND: Niemann-Pick type C (NP-C) is a rare neurovisceral genetic disorder caused by mutations in the NPC1 or the NPC2 gene. NPC1 is a multipass-transmembrane protein essential for egress of cholesterol from late endosomes/lysosomes. To evaluate impacts of NPC1 mutations, we examined fibroblast cultures from 26 NP-C1 patients with clinical phenotypes ranging from infantile to adult neurologic onset forms. The cells were tested with multiple assays including NPC1 mRNA expression levels and allele expression ratios, assessment of NPC1 promoter haplotypes, NPC1 protein levels, cellular cholesterol staining, localization of the mutant NPC1 proteins to lysosomes, and cholesterol/cholesteryl ester ratios. These results were correlated with phenotypes of the individual patients. RESULTS: Overall we identified 5 variant promoter haplotypes. Three of them showed reporter activity decreased down to 70% of the control sequence. None of the haplotypes were consistently associated with more severe clinical presentation of NP-C. Levels of transcripts carrying null NPC1 alleles were profoundly lower than levels of the missense variants. Low levels of the mutant NPC1 protein were identified in most samples. The protein localised to lysosomes in cultures expressing medium to normal NPC1 levels. Fibroblasts from patients with severe infantile phenotypes had higher cholesterol levels and higher cholesterol/cholesteryl ester ratios. On the contrary, cell lines from patients with juvenile and adolescent/adult phenotypes showed values comparable to controls. CONCLUSION: No single assay fully correlated with the disease severity. However, low residual levels of NPC1 protein and high cholesterol/cholesteryl ester ratios associated with severe disease. The results suggest not only low NPC1 expression due to non-sense mediated decay or low mutant protein stability, but also dysfunction of the stable mutant NPC1 as contributors to the intracellular lipid transport defect.

Spinal muscular atrophy caused by a novel Alu-mediated deletion of exons 2a-5 in SMN1 undetectable with routine genetic testing.

BACKGROUND: Spinal muscular atrophy (SMA) is an inherited neuromuscular disease affecting 1 in 8,000 newborns. The majority of patients carry bi-allelic variants in the survival of motor neuron 1 gene (SMN1). SMN1 is located in a duplicated region on chromosome 5q13 that contains Alu elements and is predisposed to genomic rearrangements. Due to the genomic complexity of the SMN region and genetic heterogeneity, approximately 50% of SMA patients remain without genetic diagnosis that is a prerequisite for genetic treatments. In this work we describe the diagnostic odyssey of one SMA patient in whom routine diagnostics identified only a maternal heterozygous SMN1Δ(7-8) deletion. METHODS: We characterized SMN transcripts, assessed SMN protein content in peripheral blood mononuclear cells (PBMC), estimated SMN genes dosage, and mapped genomic rearrangement in the SMN region. RESULTS: We identified an Alu-mediated deletion encompassing exons 2a-5 of SMN1 on the paternal allele and a complete deletion of SMN1 on the maternal allele as the cause of SMA in this patient. CONCLUSION: Alu-mediated rearrangements in SMN1 can escape routine diagnostic testing. Parallel analysis of SMN gene dosage, SMN transcripts, and total SMN protein levels in PBMC can identify genomic rearrangements and should be considered in genetically undefined SMA cases.

Alu-mediated Xq24 deletion encompassing CUL4B, LAMP2, ATP1B4, TMEM255A, and ZBTB33 genes causes Danon disease in a female patient.

Cullin 4B (CUL4B), lysosomal-associated membrane protein Type 2 (LAMP2), ATP1B4, TMEM255A, and ZBTB33 are neighboring genes on Xq24. Mutations in CUL4B result in Cabezas syndrome (CS). Male CS patients present with dysmorphic, neuropsychiatric, genitourinary, and endocrine abnormalities. Heterozygous CS females are clinically asymptomatic. LAMP2 mutations cause Danon disease (DD). Cardiomyopathy is a dominant feature of DD present in both males and heterozygous females. No monogenic phenotypes have been associated with mutations in ATP1B4, TMEM255A, and ZBTB33 genes. To facilitate diagnostics and counseling in CS and DD families, we present a female DD patient with a de novo Alu-mediated Xq24 rearrangement causing a deletion encompassing CUL4B, LAMP2, and also the other three neighboring genes. Typical to females heterozygous for CUL4B mutations, the patient was CS asymptomatic, however, presented with extremely skewed X-chromosome inactivation (XCI) ratios in peripheral white blood cells. As a result of the likely selection against CUL4B deficient clones, only minimal populations (~3%) of LAMP2 deficient leukocytes were identified by flow cytometry. On the contrary, myocardial LAMP2 protein expression suggested random XCI. We demonstrate that contiguous CUL4B and LAMP2 loss-of-function copy number variations occur and speculate that male patients carrying similar defects could present with features of both CS and DD.

LAMP2 exon-copy number variations in Danon disease heterozygote female probands: Infrequent or underdetected?

Danon disease (DD) is an X-linked disorder caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene (Xq24). DD is characterized by cognitive deficit, myopathy, and cardiomyopathy in male patients. The phenotype is variable and mitigated in females. The timely identification of de-novo LAMP2 mutated family members, many of whom are heterozygous females, remains critical for their treatment and family counseling. DD laboratory testing builds on minimally invasive quantification of the LAMP2 protein in white blood cells and characterization of the specific mutation. This integrative approach is particularly helpful when assessing suspect female heterozygotes. LAMP2 exon-copy number variations (eCNVs) were so far reported only in X-hemizygous male DD probands. In heterozygous female DD probands, the wild-type allele may hamper the identification of an eCNV even if it results in the complete abolition of LAMP2 transcription and/or translation. To document the likely underappreciated rate of occurrence and point out numerous potential pitfalls of detection of the LAMP2 eCNVs, we present the first two DD heterozygote female probands who harbor novel multi-exon LAMP2 deletions. Critical for counseling and recurrence prediction, we also highlight the need to search for somatic-germinal mosaicism in DD families.

Mosaic tissue distribution of the tandem duplication of LAMP2 exons 4 and 5 demonstrates the limits of Danon disease cellular and molecular diagnostics.

Alu-mediated tandem duplication of exons 4 and 5 (g.15815_22218dup6404) is a novel mutation that has been detected in the LAMP2 gene (Xq24). This exon copy number variation was found in two brothers with the typical phenotype of Danon disease, including characteristic myocardial changes on magnetic resonance imaging. The 6.4 kb duplication was identified in both boys by a combination of exon dosage qPCR analyses and duplication breakpoint/junction mapping. The rearrangement results in a plethora of abnormal LAMP2 splicing variants and also in use of likely cryptic splice sites in the 3' terminus of LAMP2 gene. Although we found minute amounts of normal LAMP2B and LAMP2A mRNAs, no protein was detectable in peripheral blood leukocytes by flow cytometry in both brothers. Uniquely, the fraction of LAMP2-deficient granulocytes (0.06%) assessed by flow cytometry in the patients' asymptomatic mother substantially differed from the random distribution of X-chromosome inactivation in her leukocytes. This discrepancy was later explained by molecular genetic methods as a consequence of mosaic distribution of the mutation in her somatic tissues. Altogether, we report a novel and mosaically distributed exon copy number rearrangement in the LAMP2 gene and comment on obstacles this genetic setup presents to the overall cellular and molecular diagnostic algorithm of Danon disease. Our observations of the mosaicism in the asymptomatic mother suggest that similarly affected females could be a potentially under-diagnosed Danon disease carrier group and that LAMP2 flow cytometry, because of its supreme sensitivity, can be an efficient method for pedigree screening.

Bioinformatic and biochemical studies point to AAGR-1 as the ortholog of human acid alpha-glucosidase in Caenorhabditis elegans.

Human acid alpha-glucosidase (GAA, EC 3.2.1.20) is a lysosomal enzyme that belongs to the glycoside hydrolase family 31 (GH31) and catalyses the hydrolysis of alpha-1,4- and alpha-1,6-glucosidic linkages at acid pH. Hereditary deficiency of GAA results in lysosomal glycogen storage disease type II (GSDII, Pompe disease). The aim of this study was to assess GH31 proteins in Caenorhabditis elegans (C. elegans) to identify the ortholog of human GAA. Bioinformatic searches for GAA ortholog in C. elegans genome revealed four acid alpha-glucosidase-related (aagr-1-4) genes. Multiple sequence alignment of AAGRs with other GH31 proteins demonstrated their evolutionary conservation. Phylogenetic analyses suggested clustering of AAGR-1 and -2 with acid-active and AAGR-3 and -4 with neutral-active GH31 enzymes. In order to prove the AAGRs' predicted alpha-glucosidase activity, we performed RNA interference of all four aagr genes. The impact on the alpha-glucosidase activity was evaluated at pH 4.0 (acid) and pH 6.5 (neutral), with or without the inhibitor acarbose. AAGR-1 and -2 expressed acidic alpha-glucosidase activity; on the contrary, AAGR-3 not -4 represented the predominant neutral alpha-glucosidase activity in C. elegans. Similar results were obtained in each of aagr-1 and -4 deletion mutants. Moreover, based on our structural models of AAGRs and these biochemical experiments, we hypothesize that the enzymatic sensitivity of AAGR-2 and human maltase-glucoamylase to the inhibitor acarbose is associated with a tyrosine residue in the GH31 active site, whereas acarbose resistance of AAGR-1 and human GAA is associated with the corresponding tryptophane in the active site. Acid-active AAGR-1 may thus represent the ortholog of human GAA in C. elegans.

Two aspartic proteinases secreted by the pathogenic yeast Candida parapsilosis differ in expression pattern and catalytic properties.

Secreted aspartic proteinases (Sap) play a role in the virulence of pathogenic Candida spp. Candida parapsilosis possesses three genes encoding these enzymes: SAPP1, SAPP2, and SAPP3. We analyzed the expression of the SAPP1 and SAPP2 genes and the production of Sapp1p and Sapp2p proteinases in the presence of different nitrogen sources. While the SAPP2 transcript was present under all of the conditions tested, expression of SAPP1 was induced only by the presence of exogenous protein as the sole nitrogen source. The concentration of Sapp1 p in the medium upon induction was at least one order of magnitude higher than the concentration of Sapp2p in all media tested in this study. Enzymological characterization of purified Sapp1 p and Sapp2p demonstrated that Sapp2p has a more restricted substrate specificity and significantly lower catalytic activity than Sapp1p. Homology models of Sapp1p and Sapp2p revealed structural motifs that may be responsible for the differences between these two enzymes. Our results indicate that C. parapsilosis secretes a low level of Sapp2p proteinase with narrow substrate specificity and low proteolytic activity under most conditions, while expression and secretion of a higher amount of catalytically efficient Sapp1p enzymes is triggered in the presence of exogenous protein serving as a nitrogen source.

Photodynamic therapy of nonmelanoma skin cancer with topical hypericum perforatum extract--a pilot study.

Hypericin, the photoactive compound of Hypericum perforatum, is probably the most powerful photosensitizer found in nature. This compound has shown high potency in the photodynamic treatment of tumor cells. However, there is only limited knowledge regarding the photodynamic effect of hypericin on nonmelanoma skin cancer cells. The aim of this prospective study was to investigate the efficacy of photodynamic therapy with topical application of an extract of H. perforatum in actinic keratosis, basal cell carcinoma (BCC) and morbus Bowen (carcinoma in situ). The study was carried out on 34 patients--eight with actinic keratoses (AKs), 21 with BCC and five with Bowen's disease. The extract of H. perforatum was applied on the skin lesions under occlusion and that was followed by irradiation with 75 J cm(-2) of red light 2 h later. The treatment was performed weekly for 6 weeks on average. The percentage of complete clinical response was 50% for AKs, 28% in patients with superficial BCC and 40% in patients with Bowen's disease. There was only a partial remission seen in patients with nodular BCCs. A complete disappearance of tumor cells was found in the histologic preparation of 11% of patients with superficial BCCs and 80% in the patients with Bowen's disease. All patients complained of burning and pain sensations during irradiation. Although the results of this first clinical trial could be regarded as disappointing, there are still possibilities for improvement. Better preparation of the lesions, enhancement of hypericin delivery and other types of light exposure procedures could significantly improve the clinical outcomes of this relatively inexpensive treatment modality.

Changes in GM1 ganglioside content and localization in cholestatic rat liver.

(Glyco)sphingolipids (GSL) are believed to protect the cell against harmful environmental factors by increasing the rigidity of plasma membrane. Marked decrease of membrane fluidity in cholestatic hepatocytes was described but the role of GSL therein has not been investigated so far. In this study, localization in hepatocytes of a representative of GSL, the GM1 ganglioside, was compared between of rats with cholestasis induced by 17alpha-ethinylestradiol (EE) and vehicle propanediol treated or untreated animals. GM1 was monitored by histochemical reaction employing cholera toxin B-subunit. Our findings in normal rat liver tissue showed that GM1 was localized in sinusoidal and canalicular hepatocyte membranes in both peripheral and intermediate zones of the hepatic lobules, and was nearly absent in central zones. On the contrary, in EE-treated animals GM1 was also expressed in central lobular zones. Moreover, detailed densitometry analysis at high magnification showed greater difference of GM1 expression between sinusoidal surface areas and areas of adjacent cytoplasm, caused as well by increased sinusoidal staining in central lobular zone as by decreased staining in cytoplasm in peripheral zone. These differences correlated with serum bile acids as documented by linear regression analyses. Both GM1 content and mRNA corresponding to GM1-synthase remained unchanged in livers; the enhanced expression of GM1 at sinusoidal membrane thus seems to be due to re-distribution of cellular GM1 at limited biosynthesis and could be responsible for protection of hepatocytes against harmful effects of bile acids accumulated during cholestasis.

Estrogen-induced cholestasis results in a dramatic increase of b-series gangliosides in the rat liver.

Hepatic ganglioside composition was investigated in normal and cholestatic Wistar rats. Cholestasis was induced by 17alpha-ethinylestradiol (EE; 5 mg/kg body weight s.c. for 18 days). As compared with controls, the EE administration resulted in severe cholestasis, as indicated by biochemical as well as morphological signs. Gangliosides isolated from the liver tissue were separated by TLC, with resorcinol-HCl detection and densitometric evaluation. As compared with controls, the total hepatic lipid sialic acid content in cholestatic rats was increased almost 2-fold (44.3 +/- 15.2 vs 79.1 +/- 9.0 nmol/g wet weight of liver tissue, p < 0.01). This increase was primarily due to the increase of ganglioside GD1a (3.6 +/- 1.0 vs 11.8 +/- 3.0 nmol/g wet weight of liver tissue, p = 0.001), as well as to the enormous up-regulation of b-series gangliosides GD3 (0.08 +/- 0.03 vs 2.0 +/- 1.2 nmol/g wet weight of liver tissue, p = 0.002), GD1b (0.1 +/- 0.06 vs 5.4 +/- 1.6 nmol/g wet weight of liver tissue, p = 0.002) and GT1b (0.06 +/- 0.03 vs 6.4 +/- 2.6 nmol/g wet weight of liver tissue, p = 0.002). As the majority of gangliosides are concentrated in cell membranes, our findings suggest that dramatic increase of b-series gangliosides might contribute to the protection of hepatocytes against the deleterious effects of cholestasis.

Structure-based specificity mapping of secreted aspartic proteases of Candida parapsilosis, Candida albicans, and Candida tropicalis using peptidomimetic inhibitors and homology modeling.

Secreted aspartic proteases (Saps) of pathogenic Candida spp. represent a specific target for antifungal drug development. We synthesized a series of peptidomimetic inhibitors with different isosteric groups and modifications at individual positions and tested them with purified Saps from C. albicans (Sap2p), C. tropicalis (Sapt1p), and C. parapsilosis (Sapp1p). The kinetic parameters indicated that all three proteases prefer binding of inhibitors containing bulky hydrophobic residues between positions P3 and P3'. The most divergent specificity was found for Sapp1p. The sequence alignment of Sap2p, Sapt1p, and Sapp1p, and homology modeling of Sapp1p with the crystal structure of Sapt1p and the complex of Sap2p with a peptidomimetic inhibitor showed that the overall folds of Sap2p, Sapt1p, and Sapp1p are similar. However, the N- and C-terminal loops formed by disulfide bonds between residues 47-53 and 258-292 are significantly shorter in Sapp1p, and a unique insertion following Tyr 129 in Sapp1p results in the formation of a loop that can interact with inhibitor residues. These Sapp1p structural differences might lead to its altered susceptibility to inhibition.

Identification of bilirubin reduction products formed by Clostridium perfringens isolated from human neonatal fecal flora.

Urobilinoids belong to the heterogenous group of degradation products of bilirubin formed in the gastrointestinal tract by intestinal microflora. Among them urobilinogen and stercobilinogen with their respective oxidation products, urobilin and stercobilin, are the most important compounds. The aim of present study was to analyze the products of bacterial reduction of bilirubin in more detail. The strain of Clostridium perfringens isolated from neonatal stools, capable of reducing bilirubin, was used in the study. Bacteria were incubated under anaerobic conditions with various native as well as synthetic bile pigments, including radiolabeled unconjugated bilirubin (UCB). Their reduction products were extracted from media and separated following thin layer chromatography. Pigments isolated were analyzed by spectrophotometry, spectrofluorometry and mass spectrometry. In a special set of experiments, bilirubin diglucuronide was incubated with either bacterial lysate or partially purified bilirubin reductase and beta-glucuronidase to reveal whether bilirubin glucuronides may be directly reduced onto conjugated urobilinoids. A broad substrate activity was detected in the investigated strain of C. perfringens and a series of bilirubin reduction products was identified. These products were separated in the form of their respective chromogens and further oxidized. Based on their physical-chemical properties, as well as mass spectra, end-catabolic bilirubin products were identified to belong to urobilinogen species. The reduction process, catalyzed enzymatically by the studied bacterial strain, does not proceed to stercobilinogen. Bilirubin diglucuronide is not reduced onto urobilinoid conjugates, glucuronide hydrolysis must precede double bond reduction and thus UCB is reduced much faster.