Splenic Gaucheroma Leading to Incidental Diagnosis of Gaucher Disease in a 46-Year-Old Man with a Rare GBA Mutation: A Case Report.

BACKGROUND: Gaucher disease is a common lysosomal storage disease caused by the deficiency of the ß-glucosidase enzyme, leading to sphingolipid accumulation in the reticuloendothelial system in Gaucher cells. Clinical findings are quite variable and some patients may remain asymptomatic lifelong. However, even when patients have mild symptoms, there is a significant increase in their quality of life with enzyme replacement therapy. We aimed to reveal the relationship between a rare mutation in the Glucosylceramidase Beta (GBA) gene and clinical signs and symptoms. Another aim of the study was to show the effect of enzyme replacement therapy on the quality of life, even in patients with mild symptoms. CASE PRESENTATION: Here, we report a 46-year-old male diagnosed with Gaucher disease based on splenic Gaucheromas incidentally discovered in a cardiac computerized tomography scan. In GBA gene analysis, the extremely rare R87W mutation was detected in a homozygous state. In retrospect, the patient had nonspecific symptoms such as fatigue and bone pain for a long time, which were substantially ameliorated by enzyme replacement therapy. CONCLUSION: In patients with adult-onset Gaucher disease, the symptoms may be mild, causing significant diagnostic delay. Gaucher disease may be included in the differential diagnosis of abdominal malignancies. Early diagnosis and treatment can improve quality of life and prevent unnecessary procedures.

Clinical-genetic characteristics and treatment outcomes of Turkish children with Gaucher disease type 1 and type 3: A sixteen year single-center experience.

Data from 38 children were retrospectively analyzed to determine the patient characteristics of Turkish children with Gaucher disease (GD) and evaluate the impact of enzyme replacement therapy (ERT) in a pediatric cohort consisting of two different sub-types of the disease, Gaucher disease type 1 (GD1) and type 3 (GD3). Both types were represented equally (GD1/GD3 = 20/18). L444P (35.5%) was the most common mutant allele while L444P/L444P (34.2%) was the most common genotype overall. Compound heterozygosity of N370S and L444P homozygosity were the dominant genotypes in Turkish children with GD1 and GD3, respectively. None of the patients had moderate to severe thrombocytopenia at last follow-up while the percent of patients with anemia decreased from 60% to 5.7% (p < 0.001). Significant improvements in mean liver (from 2.2 to 1.6 MN, p < 0.001) and spleen (from 15.5 to 7.6 MN, p < 0.001) volumes were observed in the first year of ERT. Linear growth was ameliorated as shown by the decrease in the percent of patients having short stature from 34.3% to 13.3% (p < 0.01) at year 5. Erlenmeyer flask deformity, osteopenia and scoliosis were common skeletal findings. Although none of the patients had lung disease at diagnosis, 20% developed radiological findings suggestive of pulmonary involvement. This single center experience is the first comprehensive study from Turkey not only reporting clinical and genetic characteristics of GD patients but also providing information on the outcomes of ERT in two different sub-types of GD. Genotypic background of Turkish children with GD is similar to western populations. Although visceral and hematological therapeutic goals are reached as early as one year of ERT in both sub-types, achieving normal growth takes several more years than suggested in significant number of children with GD.

Impairment of lipophagy by PNPLA1 mutations causes lipid droplet accumulation in primary fibroblasts of Autosomal Recessive Congenital Ichthyosis patients.

BACKGROUND: Autosomal Recessive Congenital Ichthyosis (ARCI) is a group of epidermal keratinization disorders. One of the disease-associated proteins, patatin-like phospholipase domain-containing protein-1 (PNPLA1), plays a key role in the epidermal omega-O-acylceramide synthesis and localizes on the surface of lipid droplets (LDs). OBJECTIVE: Previously, routine clinical test results showed abnormal LD accumulation in blood smear samples of our ARCI patients with PNPLA1 mutations. To investigate the abnormal accumulation of LDs, we analyzed primary fibroblast cells of ARCI patients with PNPLA1 mutations (p.Y245del and p.D172N). We hypothesized that PNPLA1 mutations might affect lipophagy-mediated regulation of LDs and cause intracellular lipid accumulation in ARCI patients. METHODS: LD accumulation was analyzed by fluorescence staining with BODIPY®493/503 in the fibroblasts of patient cells and PNPLA1 siRNA transfected control fibroblast cells. The expression of PNPLA1 and its effects on the lipophagy-mediated degradation of LDs were analyzed by immunocytochemistry and immunoblotting. RESULTS: Our results showed that mutant or downregulated PNPLA1 protein causes abnormal intracellular LD accumulation. We found that PNPLA1 mutations affect neither the cellular localization nor the expression levels of the protein in fibroblast cells. When we analyzed lipophagic degradation process, LC3 expression and the number of autophagosomes were significantly decreased in fibroblast cells of the patients. In addition, co-localization of LDs with autophagosomes and lysosomes were markedly less than that of the control group. CONCLUSION: PNPLA1 mutations caused disturbances in both autophagosome formation and fusion of autophagosomes with lysosomes. Our results indicate a possible role for PNPLA1 protein in LD regulation via lipophagy-mediated degradation.

Application of Fourier transform infrared spectroscopy to biomolecular profiling of cultured fibroblast cells from Gaucher disease patients: A preliminary investigation.

BACKGROUND: Gaucher disease (GD) is defined as an autosomal recessive disorder resulting from the deficiency of glucocerebrosidase (E.C. 3.2.1.45). Glucocerebrosidase is responsible for the degradation of glucosylceramide into ceramide and glucose. The deficiency of this enzyme results in the accumulation of undegraded glucosylceramide, almost exclusively in macrophages. With Fourier transform infrared (FTIR) spectroscopy, the complete molecular diversity of the samples can be studied comparatively and the amount of the particular materials can be determined. Also, the secondary structure ratios of proteins can be determined by analysing the amide peaks. OBJECTIVES: The primary aim of this study is to introduce FTIR-ATR spectroscopy technique to GD research for the first time in the literature and to assess its potential as a new molecular method. MATERIAL AND METHODS: Primary fibroblast cell cultures obtained from biopsy samples were used, since this material is widely used for the diagnosis of GD. Intact cells were placed onto a FTIR-ATR crystal and dried by purging nitrogen gas. Spectra were recorded in the mid-infrared region between 4500-850 cm-1 wavenumbers. Each peak in the spectra was assigned to as organic biomolecules according to their chemical bond information. A quantitative analysis was performed using peak areas and we also used a hierarchical cluster analysis as a multivariate spectral analysis. RESULTS: We obtained FTIR spectra of fibroblast samples and assigned the biomolecule origins of the peaks. We observed individual heterogeneity in FTIR spectra of GD fibroblast samples, confirming the well-known phenotypic heterogeneity in GD at the molecular level. Significant alterations in protein, lipid and carbohydrate levels related to the enzyme replacement therapy were also observed, which is also supported by cluster analysis. CONCLUSIONS: Our results showed that the application of FTIR spectroscopy to GD research deserves more attention and detailed studies with an increased sample size in order to evaluate its potential in the diagnosis and follow-up of GD patients.

Lipid Droplets in Health and Disease.

Lipids are essential building blocks synthesized by complex molecular pathways and deposited as lipid droplets (LDs) in cells. LDs are evolutionary conserved organelles found in almost all organisms, from bacteria to mammals. They are composed of a hydrophobic neutral lipid core surrounding by a phospholipid monolayer membrane with various decorating proteins. Degradation of LDs provide metabolic energy for divergent cellular processes such as membrane synthesis and molecular signaling. Lipolysis and autophagy are two main catabolic pathways of LDs, which regulate lipid metabolism and, thereby, closely engaged in many pathological conditons. In this review, we first provide an overview of the current knowledge on the structural properties and the biogenesis of LDs. We further focus on the recent findings of their catabolic mechanism by lipolysis and autophagy as well as their connection ragarding the regulation and function. Moreover, we discuss the relevance of LDs and their catabolism-dependent pathophysiological conditions.

Identification of two novel PNPLA1 mutations in Turkish families with autosomal recessive congenital ichthyosis.

Dökmeci-Emre S, Taskiran ZE, Yüzbasioglu A, Önal G, Akarsu AN, Karaduman A, Özgüç M. Identification of two novel PNPLA1 mutations in Turkish families with autosomal recessive congenital ichthyosis. Turk J Pediatr 2017; 59: 475-482. Autosomal recessive congenital ichthyosis (ARCI) is a group of inherited keratinization disorders that are characterized by abnormal epidermal keratinization. ARCI patients generally represent serious symptoms including collodion baby phenotype accompanied by dehydration, heat loss, electrolytic imbalance, and sepsis. ARCI shows high degree of clinical and genetic heterogeneity. To date, nine genes were shown to be responsible for ARCI phenotype. One of these genes, patatin-like phospholipase domain containing protein-1 (PNPLA1) was suggested to be involved in the synthesis of ω-O-acylceramides related to epidermal cornified lipid envelope organization. In addition to previously reported PNPLA1 mutations, we report two novel PNPLA1 mutations including one novel missense mutation c.335C > A (p.Ser112Tyr) and one novel deletion mutation c.733_735delTAC (p.Tyr245del) in Turkish ARCI patients from unrelated consanguineous families. We also report previously reported missense mutation c.514G > A (p.Asp172Asn) in Turkish ARCI patients. Novel PNPLA1 mutations were shown to be located in the catalytic patatin domain of PNPLA1 gene. Identification of novel mutations in PNPLA1 gene expands the mutational spectrum in the causative gene. Increase in the total number of cases has high diagnostic value in terms of genotype-phenotype correlation in ARCI patients.

Chanarin-Dorfman syndrome: Genotype-Phenotype Correlation.

Chanarin-Dorfman syndrome is an autosomal recessive lipid storage disease characterized by non-bullous congenital ichthyosiform erythroderma, and involvement of the liver, muscles and central nervous system due to a multisystemic accumulation of neutral lipids in various types of cells. Less than 100 affected individuals have been reported worldwide, the majority from the Mediterranean and Middle-East countries, especially Turkey. We present clinical and molecular data of four affected relatives with Chanarin-Dorfman syndrome homozygous for a N209X mutation in ABHD5, and provide a short review by comparing patients with N209X homozygous mutations to patients with other ABHD5 mutations. No major clinical differences exist between individuals with an N209X mutation and those with other mutations, which argues against a genotype/phenotype correlation.

Outcome of enzyme replacement therapy in Turkish patients with Gaucher disease: does late intervention affect the response?

We aimed to evaluate the outcome of enzyme replacement therapy (ERT) in Turkish Gaucher patients since it first became available in our country. Eleven patients with type I and one patient with type III Gaucher disease (GD) received therapy as 30-60 U/kg and 120 U/kg every two weeks, respectively, for at least six months, starting a mean period of 4.2 years after the diagnosis. Assessment of response included serial measurements of hematological and biochemical parameters and liver and spleen volumes. Symptoms and signs of bone disease, growth and severity scores were also evaluated. ERT in Turkish patients led to marked improvement in hematological parameters and organomegaly in the majority of them. Patients with growth failure demonstrated catch-up growth. Progression of bone disease was not observed except in two patients who experienced a delay of 15 and 8.6 years, respectively, between the diagnosis and the start of ERT.

Molecular analysis of Chanarin-Dorfman syndrome (CDS) patients: Identification of novel mutations in the ABHD5 gene.

Chanarin-Dorfman syndrome (CDS) is an autosomal recessive metabolic disorder associated with congenital ichthyosis and a multisystemic accumulation of neutral lipids in various types of cells. Recently, mutations of the ABHD5 gene were identified as the cause of CDS. In this work, we carried out molecular analysis of the ABHD5 gene in 6 unrelated patients. We identified one previously reported mutation, N209X and two novel genetic alterations; a nonsense mutation (p.Y50X) and missense mutation (p.S73A).

Molecular analysis of Turkish Gaucher disease patients: identification of novel mutations in glucocerebrosidase (GBA) gene.

Gaucher disease (GD) is the most frequent lysosomal glycolipid storage disorder due to autosomal recessive deficiency of acid beta-glucosidase and is characterized by the accumulation of glucocerebroside. In this work we carried out molecular analysis of the glucocerebrosidase gene (GBA) in 57 unrelated patients and the alleleic frequencies of gene mutations in Turkish patients are reported. The most prevalent are L444P and N370S accounting for 42% and 30% in our patients. We identified three novel genetic alterations: two missense changes S356F, L296V that are associated with the severe phenotype of type 1 GD. 303-305delCAC was identified in a homozygous state in one patient type 1 or type 3.

Multiple endocrine neoplasia type 2b associated with lichen nitidus.

Multiple endocrine neoplasia (MEN) type 2B syndrome is an autosomal dominantly inherited endocrine disorder with rare skin manifestations. We report the case of a 19-year-old Turkish girl who presented with skin-colored flat papules scattered all over the trunk and extremities. Additionally, she had marfanoid habitus, thick lips, and multiple flesh-colored papules over the inner eyelids and oral mucosa. Histopathological examination of one of the trunk lesions was consistent with lichen nitidus. Her past medical history was significant for medullary thyroid carcinoma. Genetic testing showed a point mutation in exon 16 at codon 918 (M918T) in the RET proto-oncogene. Based on all these findings, MEN type 2B was diagnosed. To the best of our knowledge we report the first case of MEN type 2B associated with lichen nitidus.

Novel mutations in ALOX12B in patients with autosomal recessive congenital ichthyosis and evidence for genetic heterogeneity on chromosome 17p13.

We report clinical and molecular findings in 20 patients from 11 families with autosomal recessive congenital ichthyosis (ARCI) linked to chromosome 17p13, and attributed to mutations in the ALOX gene cluster, which includes three lipoxygenase genes, ALOXE3, ALOX12B, and ALOX15B. We identified six novel missense mutations and one novel deletion leading to a premature stop codon in ALOX12B in only six out of the 11 families which led us to investigate a possible implication of ALOX15B. Mutation analysis of this gene, as well as ALOXE3, which is known to be mutated in some cases of ARCI, failed to reveal causative mutations in the five remaining ARCI families, indicating that other genes on chromosome 17p13 may be involved in this disease. However, by adding new variants to the repertoire of ALOX12B mutations in non-bullous congenital ichthyosiform erythroderma, our data contribute to an enlargement of the spectrum of mutations for the development of efficient molecular genetic tests for analysis of at risk individuals whose carrier status is unknown.

Molecular analysis of Turkish mucopolysaccharidosis IVA (Morquio A) patients: identification of novel mutations in the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS; EC 3.1.6.4). The deficiency of N-acetylgalactosamine-6-sulfate sulfatase leads to lysosomal accumulation of undegraded glycosaminoglycans, keratan sulfate and chondroitin-6-sulfate. Mutation screening of the GALNS gene was performed by SSCP and direct sequence analyses using genomic DNA samples from 10 Morquio A patients. By nonradioactive SSCP screening, 6 different gene mutations and 2 polymorphisms were identified in 10 severely affected MPS IVA patients. Five of the mutations and one of the polymorphisms are novel. The vast majority of the gene alterations were found to be single nucleotide deletions (389delG, 929delG, and 763delT) or insertions (1232-1233insT). The other two mutations were one previously identified missense mutation (Q473X) and one novel nonsense (P179S) mutation. Together they account for 95% of the disease alleles of the patients investigated. Beside mutations, one previously identified E477 polymorphism and one novel W520 polymorphism were found among Turkish MPS IVA patients.

Biochemical and molecular analysis of mucopolysaccharidoses in Turkey.

The mucopolysaccharidoses (MPSs) are a family of heritable disorders caused by deficiency of lysosomal enzymes needed to degrade glycosaminoglycans (GAGs). The undegraded or partially degraded GAGs are stored in lysosomes and/or excreted in urine. In our study, 118 patients seen over the past 20 years and suspected to have lysosomal storage disorders (LSDs) were subjected to clinical and biochemical analysis at Hacettepe University Children's Hospital. We analyzed urine and blood samples from 42 patients given a clinical MPS diagnosis. Using urine screening technique, we were able to show that 34 of the 42 patients had MPS condition. Further analysis of eight patients with normal urine MPS patterns revealed four patients as likely to have alpha-mannosidosis, fucosidosis, sialidosis, and aspartylglucosaminuria (one each). Four patients had normal oligosaccharide patterns. We were able to clearly identify 4 MPS I, 2 MPS II, 5 MPS IIIA, 8 MPS IIIB, 11 MPS IVA, 3 MPS VI, and 1 MPS IIIC patients. These results provided biochemical diagnosis for these 34 patients, and clearly show that Turkey has a higher incidence of MPS IVA, IIIB, and IIIA than of previously suspected MPS types. Molecular analysis of four MPS I patients revealed three polymorphisms which have been previously reported (A314, T388, and A461T). In MPS II patients, mutation analysis identified one previously detected (R172X) and one novel mutation (W109C).

Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1.

We report the identification of mutations in lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) genes in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17. Linkage disequilibrium analysis of six families affected by NCIE permitted us to reduce a recently reported interval of 8.4 cM on chromosome 17p13.1 to a 600 kb region around the marker D17S1796, which contains LOX genes. LOX products have long been implicated in skin disorders. Two point mutations and one deletion were found in ALOXE3 and three point mutations were found in ALOX12B in these consanguineous families from the Mediterranean basin. ALOXE3 and ALOX12B are two genes which are physically linked and functionally related. They are separated by 38 kb, have one more exon than the other LOX genes and are mainly expressed in epithelial cells including keratinocytes. Although the main substrate(s) of the two enzymes is (are) still unknown, the products of ALOX12B obtained in experimental systems have been demonstrated to be of R-chirality. It seems likely that the product of one of these enzymes may be the substrate of the other, and that they belong to the same metabolic pathway.

Sanfilippo syndrome in Turkey: Identification of novel mutations in subtypes A and B.

Sanfilippo syndrome (mucopolysaccharidosis type III, MPS III) is a progressive disorder in which patients are characterized by severe central nervous system degeneration together with mild somatic disease. MPS III results from a deficiency in one of the four enzymes involved in the degradation of heparan sulfate, with sulfamidase (SGSH) being deficient in MPS IIIA and a-N-acetylglucosaminidase (NAGLU) deficient in MPS IIIB. Mutation screening using SSCP/heteroduplex analysis on genomic DNA fragments was performed in five Turkish MPS IIIA and eight Turkish MPS IIIB patients. In this study two mutations of SGSH were identified in MPS IIIA patients: R74C and the novel mutation P288S, and one polymorphism (IVS1+23 C>G). Five different mutations of NAGLU were identified in MPS IIIB patients: L682R, H248R, E153K, g.17703 A>G (novel), and T437I (novel). The clinical data of all patients are reported in detail. A high degree of genetic heterogeneity was observed in the Turkish MPS IIIA and MPS IIIB patients.