ABSTRACT
A personalized treatment decision for Gaucher disease (GD) patients should be based on relevant markers that are specific to GD, play a direct role in GD pathophysiology, exhibit low genetic variation, reflect the therapy, and can be used for all patients. Thirty-four GD patients treated with enzyme replacement therapy (ERT) or substrate reduction therapy (SRT) were analyzed for platelet count, chitotriosidase, and tartrate-resistant acid phosphatase activity in plasma samples, and quantitative measurement of Lyso-Gb1 was performed in dried blood spots. In our ERT and SRT study cohorts, plasma lyso-GL1 correlated significantly with chito-triosidase (ERT: r = 0.55, p < 0.001; SRT: r = 0.83, p < 0.001) and TRAP (ERT: r = 0.34, p < 0.001; SRT: r = 0.88, p < 0.001), irrespective of treatment method. A platelet count increase was associated with a Lyso-Gb1 decrease in both treatment groups (ERT: p = 0.021; SRT: p = 0.028). The association of Lyso-Gb1 with evaluated markers was stronger in the SRT cohort. Our results indicate that ERT and SRT in combination or in a switch manner could offer the potential of individual drug effectiveness for particular GD symptoms. Combination of the key biomarker of GD, Lyso-Gb1, with other biomarkers can offer improved response assessment to long-term therapy.
Subject(s)
Gaucher Disease , Humans , Czech Republic , Gaucher Disease/diagnosis , Gaucher Disease/drug therapy , Biomarkers , Enzyme Replacement Therapy , Platelet CountABSTRACT
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding alpha-galactosidase A (AGAL). The impact of X-chromosome inactivation (XCI) on the phenotype of female FD patients remains unclear. In this study we aimed to determine pitfalls of XCI testing in a cohort of 35 female FD patients. XCI was assessed by two methylation-based and two allele-specific expression assays. The results correlated, although some variance among the four assays was observed. GLA transcript analyses identified crossing-over in three patients and detected mRNA instability in three out of four analyzed null alleles. AGAL activity correlated with XCI pattern and was not influenced by the mutation type or by reduced mRNA stability. Therefore, AGAL activity may help to detect crossing-over in patients with unstable GLA alleles. Tissue-specific XCI patterns in six patients, and age-related changes in two patients were observed. To avoid misinterpretation of XCI results in female FD patients we show that (i) a combination of several XCI assays generates more reliable results and minimizes possible biases; (ii) correlating XCI to GLA expression and AGAL activity facilitates identification of cross-over events; (iii) age- and tissue-related XCI specificities of XCI patterning should be considered.
Subject(s)
Fabry Disease , Chromosomes , Fabry Disease/diagnosis , Fabry Disease/genetics , Female , Humans , Mutation , Phenotype , X Chromosome Inactivation/genetics , alpha-Galactosidase/geneticsABSTRACT
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.
Subject(s)
Cullin Proteins/genetics , Glycogen Storage Disease Type IIb/genetics , Lysosomal-Associated Membrane Protein 2/genetics , Mental Retardation, X-Linked/genetics , Adult , Alu Elements/genetics , Cardiomyopathies/genetics , Cardiomyopathies/physiopathology , Chromosome Deletion , DNA Copy Number Variations/genetics , Exons/genetics , Female , Glycogen Storage Disease Type IIb/diagnosis , Glycogen Storage Disease Type IIb/physiopathology , Humans , Loss of Function Mutation/genetics , Male , Mental Retardation, X-Linked/physiopathology , Myocardium/metabolism , Sodium-Potassium-Exchanging ATPase/genetics , Transcription Factors/genetics , X Chromosome Inactivation/geneticsABSTRACT
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.
Subject(s)
DNA Copy Number Variations/genetics , Exons/genetics , Glycogen Storage Disease Type IIb/genetics , Lysosomal-Associated Membrane Protein 2/genetics , Adolescent , Adult , Base Sequence , Child , Family , Female , Heterozygote , Humans , Male , Pedigree , Young AdultABSTRACT
BACKGROUND: Liver enzymes are released from hepatocytes into circulation and their activity can be measured in the blood. We examined whether the plasma activity of the liver enzyme ornithine carbamoyltransferase, determined by a novel liquid chromatography-mass spectrometry (LC-MS/MS) assay, could be utilized for the detection of OTC deficiency (OTCD), an X-linked inborn error of the urea cycle. METHODS: The plasma ornithine carbamoyltransferase (OTC) activity was assayed in the reverse reaction using isotopically labeled citrulline-d4 as a substrate and by determination of the product, ornithine-d4, by LC-MS/MS analysis. RESULTS: The plasma OTC activity in the controls was in the range of 111-658 pkat/L (n=49, median 272 pkat/L), and the activity increased linearly with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in patients with hepatopathy. The OTC activity was subsequently determined in 32 individuals carrying mutations in the OTC gene, and OTC/ALT and OTC/AST ratios were calculated to account for the degree of hepatopathy, which is a common finding in OTCD. The OTC/ALT ratio enabled clear differentiation of OTCD hemizygotes (n=11, range 0-69×10-6) from controls (504-3440×10-6). This ratio also enabled the detection of 11 of 12 symptomatic heterozygotes (range 38-794×10-6), while this marker did not allow for reliable differentiation of asymptomatic heterozygotes (n=9) from controls. CONCLUSIONS: LC-MS/MS assay of plasma OTC activity enabled the detection of all hemizygous and the majority of symptomatic heterozygous OTCD patients in the tested cohort. This study demonstrates that non-invasive assay of enzymes expressed predominantly in the liver could be used as an alternative approach for diagnosing inborn errors of metabolism.
Subject(s)
Enzyme Assays/methods , Liver/enzymology , Ornithine Carbamoyltransferase Deficiency Disease/blood , Ornithine Carbamoyltransferase Deficiency Disease/diagnosis , Ornithine Carbamoyltransferase/blood , Calibration , Chromatography, Liquid , Chromosomes, Human, X/genetics , Cohort Studies , Enzyme Stability , Female , Heterozygote , Humans , Linear Models , Male , Mutation , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase/metabolism , Ornithine Carbamoyltransferase Deficiency Disease/enzymology , Tandem Mass SpectrometryABSTRACT
OBJECTIVES: X-linked adrenoleukodystrophy (X-ALD) causes cerebral adrenoleukodystrophy (cALD), myelopathy and/or adrenal insufficiency in males, and myelopathy/peripheral neuropathy in females. These distinct phenotypes are scarcely linked to a specific mutations. The objective herein was to find a link between the phenotype with the genotype mutation, serum very long-chain fatty acids (VLCFA), and the diet with Lorenzo´s and GTO oils in hemizygous males and heterozygous females. METHODS: A retrospective study design with follow-up of 45 hemizygous males and 50 heterozygous females carrying mutations in ABCD1 from 35 unrelated families with X-ALD. Mutation analysis was performed by Sanger sequencing of PCR and/or RT-PCR and the severity of missense mutations was evaluated using GERP++ score and CADD score. RESULTS: Twenty-five described and eight novel ABCD1 mutations were identified. Fifteen males and 23 females had severe mutations while 30 males and 27 females had less detrimental ones. cALD developed in 25 males (56%) including nine boys with severe mutations, 10 boys with less detrimental mutations and 6 adults with adrenomyelopathy. Myelopathy and/or adrenal insufficiency developed in 14 males (31%), six were asymptomatic. Adrenal insufficiency developed in two of five boys treated with hematopoietic stem cell transplantation (HSCT). Myelopathy/peripheral neuropathy developed in 26% of females. No correlation was found between the disease severity and the genotype, GERP++ and CADD scores, presence/absence of aberrant ALDP protein or X-inactivation. VLCFA were higher in males than heterozygous females and decreased during Lorenzo´s and GTO oils diet without a clear clinical impact on the disease. CONCLUSION: The prognosis was unfavourable in most males and significant part of females. Therapy with early HSCT is effective. Thus, the need for early diagnosis with the neonatal screening is crucial.
ABSTRACT
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.
Subject(s)
Carrier Proteins , Membrane Glycoproteins , Adolescent , Carrier Proteins/genetics , Fibroblasts/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mutation/genetics , Niemann-Pick C1 ProteinABSTRACT
Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder that causes recurrent and life-threatening episodes of hyperammonemia. The clinical picture in heterozygous females is highly diverse and derives from the genotype and the degree of inactivation of the mutated X chromosome in hepatocytes. Here, we describe molecular genetic, biochemical, and histopathological findings in the livers explanted from two female patients with late-onset OTC deficiency. Analysis of X-inactivation ratios by DNA methylation-based assays showed remarkable intra-organ variation ranging from 46:54 to 82:18 (average 70:30, n = 37), in favor of the active X chromosome carrying the mutation c.583G>C (p.G195R), in the first patient and from 75:25 to 90:10 (average 82:18, n = 20) in favor of the active X chromosome carrying the splicing mutation c.663+1G>A in the second patient. The X-inactivation ratios in liver samples correlated highly with the proportions of OTC-positive hepatocytes calculated from high-resolution image analyses of the immunohistochemically detected OTC in frozen sections that was performed on total area > 5 cm2. X-inactivation ratios in blood in both female patients corresponded to the lower limit of the liver values. Our data indicate that the proportion of about 20-30% of hepatocytes expressing the functional OTC protein is not sufficient to maintain metabolic stability. X-inactivation ratios assessed in liver biopsies taken from heterozygous females with X-linked disorders should not be considered representative of the whole liver.
Subject(s)
Chromosomes, Human, X/genetics , Glutamate-Ammonia Ligase/metabolism , Liver/enzymology , Ornithine Carbamoyltransferase Deficiency Disease/genetics , Ornithine Carbamoyltransferase/metabolism , X Chromosome Inactivation , Biopsy , Female , Genotype , Glutamate-Ammonia Ligase/genetics , Heterozygote , Humans , Male , Ornithine Carbamoyltransferase/genetics , Sex CharacteristicsABSTRACT
BACKGROUND: Poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) are very rare tumors with extremely aggressive behavior. Their comprehensive genetic background is still unclear. Some of the main genetic changes of differentiated thyroid carcinomas, such as mutations in BRAF and RAS genes, as well as changes in CTNNB1, PIK3CA, TP53, AXIN1, PTEN or APC genes leading to the dedifferentiation of the tumors, are described. MATERIALS AND METHODS: DNAs from fresh frozen thyroid tissues of 3 PDTCs and 5 ATCs were extracted. The next-generation sequencing (NGS) approach was used to target 94 genes involved in cancer. The samples were prepared using a TruSight Cancer panel and sequenced with a MiSeq sequencer. Analysis of variants was performed by the MiSeq Reporter and NextGENe software and stringent criteria for prioritization of the variants were used in the Illumina VariantStudio software. RESULTS: Using NGS, we identified 26 genetic changes in 18 genes, novel variants included. CONCLUSION: NGS is a useful tool for searching for new variants and genes involved in PDTC and ATC. It seems that each of these rare tumor types has its own specific genetic background. These data could be helpful for recognizing new genetic markers and targets for future personalized therapy.
Subject(s)
Neoplasm Proteins/genetics , Proto-Oncogene Proteins B-raf/genetics , Thyroid Carcinoma, Anaplastic/genetics , Thyroid Neoplasms/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/isolation & purification , Cell Differentiation/genetics , Class I Phosphatidylinositol 3-Kinases , High-Throughput Nucleotide Sequencing , Humans , Mutation , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Thyroid Carcinoma, Anaplastic/pathology , Thyroid Neoplasms/pathologyABSTRACT
Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer. Its development is often caused by the formation of RET/PTC fused genes. RET/PTC1 is the most prevalent form, where exon 1 of CCDC6 gene is fused with the intracellular portion of RET protooncogene starting with exon 12. We have discovered a novel RET/PTC1 variant which we have named RET/PTC1ex9 in metastatic PTC of 8-year-old boy. RET/PTC1ex9 detection was performed by real-time polymerase chain reaction with melting curve analysis and subsequent Sanger and next-generation sequencing. A fusion of exon 1 of CCDC6 with exon 9 of extracellular domain of RET followed by exon 12 of RET was revealed. This is the first RET/PTC variant among PTC cases that contain the extracellular part of RET. This observation could be probably explained by incorrect splicing of RET due to the somatic 32-bp deletion in exon-intron 11 boundary of RET.