89 research outputs found
Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik
Defects of N-linked glycosylation represent diseases with multiple organ involvements that are classified as congenital disorders of glycosylation (CDG). In recent years, several CDG types have been attributed to defects of dolichol-linked oligosaccharide assembly in the endoplasmic reticulum. The profiling of [3H]mannose-labeled lipid-linked oligosaccharides was instrumental in identifying most of these glycosylation disorders. However, this method is poorly suited for the identification of short lipid-linked oligosaccharide biosynthesis defects. To adequately resolve deficiencies affecting the first steps of lipid-linked oligosaccharide formation, we have used a non-radioactive procedure employing the fluorescence detection of 2-aminobenzamide-coupled oligosaccharides after HPLC separation. By applying this method, we have detected the accumulation of dolichylpyrophosphate-GlcNAc2 in a previously untyped CDG patient. The accumulation pattern suggested a deficiency of the ALG1 β1,4 mannosyltransferase, which adds the first mannose residue to lipid-linked oligosaccharides. This was supported by the finding that this CDG patient was compound heterozygous for three mutations in the ALG1 gene, leading to the amino acid substitutions S150R and D429E on one allele and S258L on the other. The detrimental effect of these mutations on ALG1 protein function was demonstrated in a complementation assay using alg1 Saccharomyces cerevisiae yeast mutants. The ALG1 mannosyltransferase defect described here represents a novel type of CDG, which should be referred to as CDG-I
PTPN11 mutations are not responsible for the Cardiofaciocutaneous (CFC) syndrome
Cardiofaciocutaneous (CFC) syndrome is a multiple congenital anomalies/mental retardation syndrome characterized by congenital heart defects, characteristic facial appearance, short stature, ectodermal abnormalities and mental retardation. It was described in 1986, and to date is of unknown genetic etiology. All reported cases are sporadic, born to non-consanguineous parents and have apparently normal chromosomes. Noonan and Costello syndromes remain its main differential diagnosis. the recent finding of PTPN11 missense mutations in 45-50% of the Noonan patients studied with penetrance of almost 100% and the fact that in animals mutations of this gene cause defects of semilunar valvulogenesis, made PTPN11 mutation screening in CFC patients a matter of interest. We sequenced the entire coding region of the PTPN11 gene in ten well-characterised CFC patients and found no base changes. We also studied PTPN11 cDNA in our patients and demonstrated that there are no interstitial deletions either. the genetic cause of CFC syndrome remains unknown, and PTPN11 can be reasonably excluded as a candidate gene for the CFC syndrome, which we regard as molecular evidence that CFC and Noonan syndromes are distinct genetic entities.Univ Sacred Heart, Ist Genet Med, I-00168 Rome, ItalyUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Med Genet, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Dermatol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Med Genet, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Dermatol, São Paulo, BrazilWeb of Scienc
A colorimetric strategy based on dynamic chemistry for direct detection of Trypanosomatid species
Leishmaniasis and Chagas disease are endemic in many countries, and re-emerging in the developed
countries. A rapid and accurate diagnosis is important for early treatment for reducing the duration
of infection as well as for preventing further potential health complications. In this work, we have
developed a novel colorimetric molecular assay that integrates nucleic acid analysis by dynamic
chemistry (ChemNAT) with reverse dot-blot hybridization in an array format for a rapid and easy
discrimination of Leishmania major and Trypanosoma cruzi. The assay consists of a singleplex PCR
step that amplifies a highly homologous DNA sequence which encodes for the RNA component of the
large ribosome subunit. The amplicons of the two different parasites differ between them by single
nucleotide variations, known as “Single Nucleotide Fingerprint” (SNF) markers. The SNF markers can
be easily identified by naked eye using a novel micro Spin-Tube device "Spin-Tube", as each of them
creates a specific spot pattern. Moreover, the direct use of ribosomal RNA without requiring the PCR
pre-amplification step is also feasible, further increasing the simplicity of the assay. The molecular
assay delivers sensitivity capable of identifying up to 8.7 copies per μL with single mismatch specificity.
The Spin-Tube thus represents an innovative solution providing benefits in terms of time, cost, and
simplicity, all of which are crucial for the diagnosis of infectious disease in developing countries.This research work has received funding from Junta de Andalucía, Consejería de Economía e Innovación (project
number 2012-BIO1778), the Spanish Ministerio de Economía y Competitividad (Grants CTQ2012-34778,
BIO2016-80519-R, FPI Grant BES-2013- 063020). This research was partially supported by the 7th European
Community Framework Program (FP7-PEOPLE-2012-CIG-Project Number 322276)
Involvement of the Modifier Gene of a Human Mendelian Disorder in a Negative Selection Process
BACKGROUND:Identification of modifier genes and characterization of their effects represent major challenges in human genetics. SAA1 is one of the few modifiers identified in humans: this gene influences the risk of renal amyloidosis (RA) in patients with familial Mediterranean fever (FMF), a Mendelian autoinflammatory disorder associated with mutations in MEFV. Indeed, the SAA1 alpha homozygous genotype and the p.Met694Val homozygous genotype at the MEFV locus are two main risk factors for RA. METHODOLOGY/PRINCIPAL FINDINGS:HERE, WE INVESTIGATED ARMENIAN FMF PATIENTS AND CONTROLS FROM TWO NEIGHBORING COUNTRIES: Armenia, where RA is frequent (24%), and Karabakh, where RA is rare (2.5%). Sequencing of MEFV revealed similar frequencies of p.Met694Val homozygotes in the two groups of patients. However, a major deficit of SAA1 alpha homozygotes was found among Karabakhian patients (4%) as compared to Armenian patients (24%) (p = 5.10(-5)). Most importantly, we observed deviations from Hardy-Weinberg equilibrium (HWE) in the two groups of patients, and unexpectedly, in opposite directions, whereas, in the two control populations, genotype distributions at this locus were similar and complied with (HWE). CONCLUSIONS/SIGNIFICANCE:The excess of SAA1alpha homozygotes among Armenian patients could be explained by the recruitment of patients with severe phenotypes. In contrast, a population-based study revealed that the deficit of alpha/alpha among Karabakhian patients would result from a negative selection against carriers of this genotype. This study, which provides new insights into the role of SAA1 in the pathophysiology of FMF, represents the first example of deviations from HWE and selection involving the modifier gene of a Mendelian disorder
Congenital disorder of glycosylation type Ia: a non-progressive encephalopathy associated with multisystemic involvement
Esterase. Xxi. Es-9, a possibly new polymorphic esterase in mus musculus genetically linked to es-2.
Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type 1A.
Carbohydrate-deficient-glycoprotein syndrome type 1 (CDG1; also known as "Jaeken syndrome") is an autosomal recessive disorder characterized by defective glycosylation. Most patients show a deficiency of phosphomannomutase (PMM), the enzyme that converts mannose 6-phosphate to mannose 1-phosphate in the synthesis of GDP-mannose. The disease is linked to chromosome 16p13, and mutations have recently been identified in the PMM2 gene in CDG1 patients with a PMM deficiency (CDG1A). The availability of the genomic sequences of PMM2 allowed us to screen for mutations in 56 CDG1 patients from different geographic origins. By SSCP analysis and by sequencing, we identified 23 different missense mutations and 1 single-base-pair deletion. In total, mutations were found on 99% of the disease chromosomes in CDG1A patients. The R141H substitution is present on 43 of the 112 disease alleles. However, this mutation was never observed in the homozygous state, suggesting that homozygosity for these alterations is incompatible with life. On the other hand, patients were found homozygous for the D65Y and F119L mutations, which must therefore be mild mutations. One particular genotype, R141H/D188G, which is prevalent in Belgium and the Netherlands, is associated with a severe phenotype and a high mortality. Apart from this, there is only a limited relation between the genotype and the clinical phenotype
Exhaustive mutation analysis of the PMM2 gene in patients with the carbohydrate-deficient glycoprotein syndrome type I (CDG1 or Jaeken syndrome) and cloning of the mouse Pmm1 and Pmm2 genes.
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