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Journal ArticleDOI

GALNS gene expression profiling in Morquio A patients' fibroblasts.

TL;DR: The development of a real-time RT-PCR assay allows to absolutely quantify the GALNS mRNAs carrying mutations that lead to PTCs bearing transcripts, which escape the NMD process and are potentially suitable for the new therapeutic approach.
About: This article is published in Clinica Chimica Acta.The article was published on 2008-11-01 and is currently open access. It has received 7 citations till now. The article focuses on the topics: Stop codon.

Summary (2 min read)

1. Introduction

  • Classical forms are characterised by a lifespan of 20–30 years, short trunk dwarfism, spondyloepiphyseal dysplasia, coxa valga, odontoid hypo- ate sulfatase; MPS IVA, mucone-6-sulfate; KS, keratan sulain reaction; RT-PCR, reverse ure termination codon; NMD, se. 9 55 570380.
  • About 150 mutations have so far been identified, revealing a high degree of genetic heterogeneity that is probably responsible for the clinical variability in MPS IVA patients.
  • These mutations can cause very unstable mRNA transcripts or transcripts capable of escaping the nonsense-mediated decay (NMD) pathway, a molecular mechanism that reduces the amount of transcripts carrying premature termination codons (PTCs).

2.1. Patients

  • The clinical and biochemical findings of the two patients (Pt1 and Pt2) are shown in Table 1.
  • Deficiency of GALNS enzyme activity was confirmed on fibroblasts by the fluorogenic method previously reported by Van Diggelen et al. [13].
  • The molecular study was performed after informed consent, for genetic testing from patients' parents, was obtained.

2.2. Analysis of genomic DNA

  • To identify genetic lesions in the GALNS gene, genomic DNA was isolated from peripheral blood lymphocytes and fibroblasts.
  • The GALNS exons were amplified with the primers reported in Table 2. PCR products were visualized on a 2% agarose gel, excised and purified using Nucleospin Extract II extraction kit (MACHEREY-NAGEL, Duren, Germany).
  • About 100 ng of purified DNA was analyzed for mutation detection by nucleotide sequencing on ABI PRISM 310 Genetic Analyzer using BigDye terminator chemicals (Applied Biosystems, Foster City, CA).

2.3. RNA isolation and retrotranscription

  • Isolation of total RNA from cultured skin fibroblasts was performed with the TRIzol reagent (Life Technologies, Rockville, MD).
  • RNA integrity and concentrations were both checked by 1% agarose gel and Nanodrop® ND-1000 Spectrophotometer (Nanodrop technologies, Wilmington, USA).
  • RNA reverse transcription was carried out as follows: 1. 1–7 μg of total RNAs were reverse transcribed with Display THERMO-RT (Eppendorf, Hamburg, Germany) using the specific 3′ UTR primer 5′ GGAGGGTCCTGAAATCTGAGG 3′, according to the manufacturer.
  • The reverse transcripts obtained from the second method were used for quantitative real-time analysis.

2.4. RT-PCR analysis

  • Nucleotide numbers are derived from cDNA GALNS sequence (EMBL/Gen Bank/ DDBJ; accession number NM_000512).
  • The measurement of GALNS gene mRNA was performed using a quantitative realtime RT-PCR method, based on TaqMan™ technology.
  • Probe and primers were selected by the computer program “Primer Express” (Applied Biosystems, Foster City, USA).
  • Plasmid vector, carrying GALNS gene transcript (pCXN-GALNS), was tenfold serially diluted from a starting quantity of 11×106 plasmid copies to 11 plasmid copies and used as standard curve.

2.8. Statistical analysis

  • Statistical analysis of different real-time assay measurement was carried out using the SPSS software package (SPSS INC, Chicago, IL).
  • Statistical differences between I° PCR GALNSc1F 5′ CAGCCCAGCCGGAAGGGCC.
  • B. Schematic representation of the aberrant transcripts detected in MPS IVA Pt1.
  • Black boxes mark the exons, white boxes indicate exonic sequence loss.
  • Differences with pb0.05 were considered statistically significant.

3. Results

  • The fibroblasts from patients with enzymatic diagnosis of MPS IVA disease underwent molecular characterisation (Table 1).
  • The new c.385ANT leading to the premature stop codon p. K129X and the c.899−1GNC splicing mutations were identified in Pt1 and the reported c.120+1GNA mutation was identified in a homozygous state in Pt2.
  • Quantitative analysis of GALNS gene mRNA was performed by absolute real-time PCR, using probe and primers encompassing exon 6–7 junction, present in all Pt1's GALNS transcripts.
  • Results, reported in Fig. 2, indicated that this assay effectively distinguished 10-fold differences in concentration from about 11 to 11×106 vectors' molecules per reaction mixture.
  • The authors analyzed total RNA from the fibroblasts of two MPS IVA patients and 15 normal controls.

4. Discussion

  • About 150 genetic lesions have been reported in the GALNS gene of mucopolysaccharidosis IVA (MPS IVA) patients, indicating remarkable genetic heterogeneity.
  • The real-time technology is now highly sensitive, accurate and simple enough to be adopted as a routine method for measuring gene levels.
  • The presence in Pt1 of normal GALNS transcripts levels, identified by the real-time assay technique the authors describe, suggests that such mRNAs are not sensitive to NMD, according to what conventional RTPCR analysis has indicated.
  • Such transcripts can be highly unstable and subject to NMD [12,21].
  • Pt 2, who was homozygous for the c.120+1GNA mutation, had consanguineous North African parents whowere heterozygous for the mutation.

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Citations
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Journal ArticleDOI
TL;DR: The three-dimensional structure of human GALNS is reported, which establishes the molecular basis for MPS IV A and for the larger MPS family of diseases.

112 citations

Journal ArticleDOI
TL;DR: A molecular testing algorithm designed to help diagnosing MPS IVA and foreseeing disease progression is defined and two new large deletions are characterized and their corresponding breakpoints are characterized.
Abstract: Morquio A syndrome (MPS IVA) is a systemic lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), encoded by the GALNS gene. We studied 37 MPS IV A patients and defined genotype-phenotype correlations based on clinical data, biochemical assays, molecular analyses, and in silico structural analyses of associated mutations. We found that standard sequencing procedures, albeit identifying 14 novel small GALNS genetic lesions, failed to characterize the second disease-causing mutation in the 16% of the patients' cohort. To address this drawback and uncover potential gross GALNS rearrangements, we developed molecular procedures (CNV [copy-number variation] assays, QF-PCRs [quantitative fluorescent-PCRs]), endorsed by CGH-arrays. Using this approach, we characterized two new large deletions and their corresponding breakpoints. Both deletions were heterozygous and included the first exon of the PIEZO1 gene, which is associated with dehydrated hereditary stomatocitosis, an autosomal-dominant syndrome. In addition, we characterized the new GALNS intronic lesion c.245-11C>G causing m-RNA defects, although identified outside the GT/AG splice pair. We estimated the occurrence of the disease in the Italian population to be approximately 1:300,000 live births and defined a molecular testing algorithm designed to help diagnosing MPS IVA and foreseeing disease progression.

27 citations


Cites background or methods from "GALNS gene expression profiling in ..."

  • ...RNA reverse transcription was carried out as previously described [Carraresi et al., 2008]....

    [...]

  • ...The normalizations were performed by relatively quantizations, determined by the Ct ([FAM Ct- VIC Ct] sample—[FAM Ct–VIC Ct] calibrator) method as previously reported [Livak and Schmittgen, 2001]. mRNA Analyses Total mRNA quantitation was performed as previously described [Carraresi et al., 2008]....

    [...]

  • ...Only a few studies to date have focused on characterizing human GALNS mRNA [Nakashima et al., 1994; Tomatsu et al., 2004] in the diagnosis of MPS IVA, and we reported the first study on mRNA quantification by real-time PCR [Carraresi et al., 2008]....

    [...]

Journal ArticleDOI
TL;DR: The LSD Morquio A syndrome is added for the first time to the list of conditions that can be caused by UPD, and the possibility of UPD is relevant when giving genetic counseling to couples since the recurrent risk in future pregnancies is dramatically reduced.

17 citations


Cites methods from "GALNS gene expression profiling in ..."

  • ...The entire GALNS coding region and exon/intron boundaries were amplified and directly sequenced using previously described primers and conditions [26]....

    [...]

Journal ArticleDOI
TL;DR: GALNS variants located within deep intronic regions that have the potential to impact splicing machinery are identified and incorporated into the diagnostic flow procedure for the molecular analysis of Morquio A disease.
Abstract: Mucopolysaccharidosis-IVA (Morquio A disease) is a lysosomal disorder in which the abnormal accumulation of keratan sulfate and chondroitin-6-sulfate is consequent to mutations in the galactosamine-6-sulfatase (GALNS) gene. Since standard DNA sequencing analysis fails to detect about 16% of GALNS mutant alleles, gross DNA rearrangement screening and uniparental disomy evaluation are required to complete the molecular diagnosis. Despite this, the second pathogenic GALNS allele generally remains unidentified in ~ 5% of Morquio-A disease patients. In an attempt to bridge the residual gap between clinical and molecular diagnosis, we performed an mRNA-based evaluation of three Morquio-A disease patients in whom the second mutant GALNS allele had not been identified. We also performed sequence analysis of the entire GALNS gene in two patients. Different aberrant GALNS mRNA transcripts were characterized in each patient. Analysis of these transcripts then allowed the identification, in one patient, of a disease-causing deep intronic GALNS mutation. The aberrant mRNA products identified in the other two individuals resulted in partial exon loss. Despite sequencing the entire GALNS gene region in these patients, the identity of a single underlying pathological lesion could not be unequivocally determined. We postulate that a combination of multiple variants, acting in cis, may synergise in terms of their impact on the splicing machinery. We have identified GALNS variants located within deep intronic regions that have the potential to impact splicing. These findings have prompted us to incorporate mRNA analysis into our diagnostic flow procedure for the molecular analysis of Morquio A disease.

15 citations

Journal ArticleDOI
TL;DR: In this paper, the authors report the clinical-biochemical data of nine patients with Morquio B disease and propose a diagnostic plan, setting out the specific clinical biochemical and molecular features of the disease, in order to avoid misdiagnosis and improve patients' management.

6 citations

References
More filters
Journal ArticleDOI
TL;DR: Investigation of the enzymic basis of Morquio's syndrome indicates that it is a deficiency of chondroitin sulfate N-acetylhexosamine sulfate sulfatase, and skin fibroblasts are used as an enzyme source.

131 citations

Journal ArticleDOI
TL;DR: The results suggest that TCR‐β transcripts contain one or more sequence elements that elicit an unusual NMD response triggered by a novel second signal that ultimately causes boundary‐independent polar regulation.
Abstract: Nonsense‐mediated decay (NMD) is an RNA surveillance mechanism that degrades mRNAs containing premature termination (nonsense) codons. The second signal for this pathway in mammalian cells is an intron that must be at least ∼55 nucleotides downstream of the nonsense codon. Although the functional significance of this ‘−55 boundary rule’ is not known, it is widely thought to reflect the important role of an exon junction protein complex deposited just upstream of exon–exon junctions after RNA splicing. Here we report that a T‐cell receptor (TCR)‐β gene did not conform to this rule. Rather than a definitive boundary position, nonsense codons had a polar effect, such that nonsense codons distant from the terminal downstream intron triggered robust NMD and proximal nonsense codons caused modest NMD. We identified a region of the TCR‐β gene that conferred this boundary‐independent polar expression pattern on a heterologous gene. Collectively, our results suggest that TCR‐β transcripts contain one or more sequence elements that elicit an unusual NMD response triggered by a novel second signal that ultimately causes boundary‐independent polar regulation. TCR genes may have evolved this unique NMD response because they frequently acquire nonsense codons during normal development.

97 citations

Journal ArticleDOI
TL;DR: Human N-acetylgalactosamine-6-Sulfate sulfatase (6-sulfatase) activity is measured by using as a substrate a sulfated tetrasaccharide obtained by digesting purified chondroitin- 6-S with testicular hyaluronidase.
Abstract: Human N-acetylgalactosamine-6-sulfate sulfatase (6-sulfatase) activity is measured by using as a substrate a sulfated tetrasaccharide obtained by digesting purified chondroitin-6-sulfate (C-6-S) with testicular hyaluronidase. The amount of inorganic sulfate released is measured turbidimetrically. The enzyme from human kidney has a pH optimum of 4.8; its activity is augmented by low levels of NaCl and inhibited by phosphate and high levels of NaCl. Free glucuronate, acetylgalactosamine, inorganic sulfate, polymeric C-6-S, or tetrasaccharide obtained from chondroitin-4-sulfate do not affect the enzyme activity. The method may be used for the diagnosis of Morquio disease since extracts of Morquio fibroblasts are devoid of 6-sulfatase activity.

93 citations

Journal ArticleDOI
TL;DR: Preclinical studies have shown that gentamicin induced the read-through of premature stop codons, resulting in enzyme activity that reduced substrate storage in lysosomal-storage-disorder cells.

92 citations

Journal ArticleDOI
15 Oct 2000-Blood
TL;DR: Findings suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3'-most intron, and that other factors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells.

92 citations

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Frequently Asked Questions (1)
Q1. What are the contributions mentioned in the paper "Galns gene expression profiling in morquio a patients' fibroblasts" ?

In this paper, the authors report characterisation of GALNS genemutations andmRNA stability verifying a genotype/phenotype correlation in two MPS IVA patients with a severe form.