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Erschienen in: BMC Medical Genetics 1/2018

Open Access 01.12.2018 | Research article

Mis-splicing of the GALNS gene resulting from deep intronic mutations as a cause of Morquio a disease

verfasst von: Anna Caciotti, Rodolfo Tonin, Matthew Mort, David N. Cooper, Serena Gasperini, Miriam Rigoldi, Rossella Parini, Federica Deodato, Roberta Taurisano, Michelina Sibilio, Giancarlo Parenti, Renzo Guerrini, Amelia Morrone

Erschienen in: BMC Medical Genetics | Ausgabe 1/2018

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.
Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12881-018-0694-6) contains supplementary material, which is available to authorized users.
Anna Caciotti and Rodolfo Tonin contributed equally to this work.
Abkürzungen
ERT
Enzyme replacement therapy
ESE
Exonic splicing enhancer
ESS
Exonic splicing silencer
GAGs
Glycosaminoglycans
GALNS
N-acetylgalactosamine-6-sulfatase
ISE
Intronic splicing enhancer
ISS
Intronic splicing silencers
MAF
Minor allele frequency
NGS
Next generation sequencing
NMD
Nonsense-mediated decay

Background

Mucopolysaccharidosis IV-A or Morquio A disease (MIM #253000] is an autosomal recessive lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), the lysosomal enzyme responsible for the hydrolytic degradation of keratan sulfate and chondroitin-6-sulfate [1]. GALNS is encoded by the GALNS gene (NM_000512.4) which is located on chromosome 16q24.3; the gene has a length of about 50 kb and is organized into 14 exons [2]. The GALNS gene is alternatively spliced, with two other reported protein coding transcripts in the RefSeq database (NM_001323543.1 and NM_001323544.1) [3]. In Ensembl, 13 transcripts are reported of which four are protein coding [4].
In excess of 330 different mutations have been reported in the GALNS gene causing Morquio A disease (Human Gene Mutation Database; http://​www.​hgmd.​org). Among them, only one solitary deep intronic mutation which created a cryptic donor splice site was previously reported [5]. Empirically, standard sequencing procedures, covering all GALNS exons and intron/exon boundaries, have failed to identify ~ 16% of mutant alleles in patients affected by the disease [6, 7]. However, this percentage falls to 5% when gross DNA rearrangements are also screened for [8].
Morquio A disease affects multiple organ systems but its principal features are the cartilage defects, caused by keratan sulfate accumulation, that are responsible for the typical skeletal complications including coxa valga, scoliosis, short trunk dwarfism and cervical instability [1, 9]. The diagnosis tends to be particularly challenging in attenuated Morquio A patients, with consequent increased risk for missing the correct diagnosis [1].
Several therapeutic approaches including hematopoietic stem cell transplantation, gene therapy, substrate reduction therapy, and enzyme replacement therapy (ERT) have been developed in order to try to ameliorate the disease [1014].
Newborn screening, whether by tandem mass spectrometry on dried blood spots and/or by fluorimetric assays, has proved to be both reliable and effective in identifying most mucopolysaccharidoses, including Morquio A disease [15, 16]. It is likely that the widespread adoption of this methodology will have a significant impact on the diagnosis of Morquio A disease [9, 17, 18].
Here we delineate the criteria that we have found efficacious in making an early diagnosis of Morquio A disease, together with a novel screening strategy that we have devised in order to optimize the probability of obtaining a molecular diagnosis in each case. Adoption of this strategy allowed the identification of novel splicing defects in three individuals in whom only one GALNS coding region mutation had originally been found.

Methods

Patients

The clinical features, and biochemical [urinary excretion of glycosaminoglycans (GAGs) and GALNS enzyme activity] and molecular analyses of these three Morquio A disease patients are summarized in Table 1. None of these patients has been previously reported. Based upon various empirical measurements to establish the severity of the MPS IVA phenotype [i.e. age at onset, growth/height (based on gender) and life span; [9, 19]], Pt1 and Pt3 are affected by the severe form of the disease, whereas Pt2 may be defined as mild.
Table 1
Clinical biochemical and molecular features of MPSIVA patients
Patient
1
2
3
Phenotype
severe
mild
severe
Age
14 y 6 m
16y
14y
Age at diagnosis
3y 5 m
8y
3y
Age at onset
3 m
3y
2-3y
GALNS activity (nmol/17 h/mg protein)
0.07 (nv 40–170)
0.2 (nv 40–170)
0 (nv 70–180)
Genotype
c.463G > A (ex 5)/ c.899-167A > G
c.463 G > A (ex 5)/ c.1002 + 307G > C^
c.697G > A (ex 7)/ c.759-67G > A^
mRNA alteration
−/ r.898_899ins53
−/ r.1003_1119del
−/ r.456_916del
Protein alteration
p.Gly155Arg / p.Gly300Valfs*37
p.Gly155Arg/ p.(Val335_Leu373del)
p.Asp233Asn/ p.(Lys153_Phe306del)
Sex
female
female
male
Parental consanguinity
no
no
no
Height (cm) at diagnosis
89.5
121
Height (cm) at last observation
108.6
136.3
114.5
Weight (kg)
18.2
26.6
27.5
Spondyloepiphyseal dysplasia
yes
yes
yes
Chest deformity
yes
yes
yes
Hearing loss
no
yes
Corneal clouding
no
mild
no
Coxa valga
yes
yes
yes
Odontoid hypoplasia
yes
yes
no
Hepatomegaly
no
yes
mild
Cardiomyopathy
yes
no
no
Valvular regurgitation
no
mitral/ tricuspid
Mental retardation
no
no
WISC-III TIQ 89
Surgery (type and age)
odontoid hypoplasia (7y), coxa valga (13y), cardiomyopathy (9 m)
right hip (6y), left hip (7y), coxa valga (10y)
coxa valga (10y)
Urine GAGs (mg/g creatinine)
92 (nv 48–82)
52 (nv 10–63)
6.6 (nv 2.1–23.2)
The patients’ parents gave their written consent for genetic testing to be performed on a local consent form, in accordance with the Declaration of Helsinki.

Biochemical assays

Total urinary glycosaminoglycans (GAGs) were assayed as previously described [20]. Qualitative GAGs assay was performed by thin layer chromatography [21]. GALNS enzyme activity was measured from leukocytes/lymphocytes employing the fluorogenic method [22]. Beta-galactosidase (GLB1) activity was assayed in all patient samples as previously reported [23, 24] in order to allow exclusion of Morquio B syndrome.
It should be noted that control values for total urinary GAG evaluation can differ quite markedly between samples when the assays are performed in different centres, even though all such centres are qualified to perform the diagnostic assays. Urine keratan sulfate was found to be abundant in all samples from the three analysed patients.

T-lymphocyte cell culture and treatment with cycloheximide to rescue aberrant transcripts from nonsense-mediated mRNA decay (NMD)

Patient and control T-lymphocytes were cultured in RPMI medium supplemented with fetal bovine serum (heat inactivated for 30 mins at 56 °C), interleukin 2 (800 U/ml), phytohemagglutinin (2.5 μg/ml) and antibiotics. The pool of control T-lymphocytes was separated from blood derived from 10 normal individuals. For each patient and control, lymphocytes were treated with (or without) 100 mg/ml cycloheximide (Sigma-Aldrich, Saint Louis, Missouri, USA) for 16 h. Total mRNA extraction and RT-PCR analysis are described below. Cycloheximide is used to identify cases of abnormal mRNA processing caused by mutations that generate premature termination codons (PTCs) that are then subject to NMD [2527]. PTCs can result either from nonsense or frameshift mutations or from errors that occur during transcription or mRNA splicing [28]. Cycloheximide, being a translation elongation inhibitor, acts as a potent NMD inhibitor [26].

Analysis of GALNS genomic DNA, total mRNA and cDNA synthesis

Genomic DNA was isolated from the patients’ peripheral blood lymphocytes. GALNS exons were PCR amplified using oligonucleotides and reaction conditions reported previously [8].
Isolation of total mRNA from cultured lymphocytes (with and without the addition of cycloheximide) was performed with the RNeasy Mini Kit (Qiagen, Hilden, Germany) for cells and tissues and the QIAamp RNA Blood Mini kit (Qiagen, Hilden, Germany) for blood samples. RNA concentrations were determined with a Nanodrop® ND-1000 Spectrophotometer (Nanodrop technologies, Wilmington, USA). RNA integrity was checked on a 1% agarose gel. GALNS mRNA reverse transcription was carried out as previously described [29]. Nucleotide numbering of the GALNS gene corresponded to the GenBank reference sequence, NM_000512.4. Patient mutational homozygosity and/or parental carrier status were verified by targeted DNA sequence analysis of the GALNS gene in each patient’s parents.

Quantitative fluorescent PCR (QF-PCR) analysis and copy number variation (CNV) assays

QF-PCR fragments corresponding to all GALNS exons were obtained both by simplex and multiplex amplifications on genomic DNAs. CNV assays were performed combining TaqMan® MGB probe chemistry with Real Time PCR instruments (Applied Biosystems® 7500 Real-Time PCR) (Life Technologies Italia, Monza, Italy). The methods employed were as previously described [8].

Whole GALNS gene sequencing and filtering of genetic variants

DNA samples from patients Pt2 and Pt3 were prepared using the TruSeq DNA PCR-Free protocol (Illumina Inc., San Diego, CA, USA) and sequenced with an Illumina HiSeqX Ten generating 150 bp paired-end reads. All fragments were mapped to the hg19 human genome reference sequence using iSAAC align version 03.16.12.05. Genetic variants identified within the GALNS gene region (NM_000512.4, chr16:88880141–88,923,374) in both patients (Pt2; 144 variants and Pt3; 73 variants) were filtered in an attempt to identify the variant(s) ultimately responsible for the observed splicing defects. The first filter was employed to eliminate variants called with low confidence (< 20 Phred quality score). A second filter was then applied to remove those variants which had been found to occur with a minor allele frequency of > 1% in the 1000 Genomes Project data (http://​www.​internationalgen​ome.​org/​). A location-based filter was also applied to remove those variants that were considered unlikely, based on their location, to be responsible for the splicing defect (thus, for example, the partial deletion of exon 10 in the aberrant mRNA transcript of Pt2 was deemed most unlikely to have been caused by a variant in exon 1). Therefore, for Pt2, this location-based filter was applied to the range chr16:88898406–88,891,277 (hg19), starting at the first base of GALNS IVS9 and extending to the last base of IVS10 (NM_000512.4). With patient Pt3, the location filter was applied to the range chr16: 88907400–88,893,246 (hg19) spanning the first base of GALNS IVS4 to the last base of IVS9 [the aberrant mRNA splicing phenotype in Pt3 involved the skipping of half of exon 5, all of exons 6, 7 and 8, plus half of exon 9]. Manual review of the remaining variants employed Human Splicing Finder 3.0 (HSF; http://​www.​umd.​be/​HSF3/​) to assess the potential impact of these variants on the mRNA splicing phenotype. Zygosity in the patients’ parents was also employed to filter out likely non-pathogenic GALNS variants e.g. those variants found in the homozygous state in a clinically unaffected parent. The Combined Annotation-Dependent Depletion (CADD) method [30] was also used under manual review to rank the variants in terms of their potential pathogenicity; the higher the CADD score, the higher the probability that the variant is disease-causing.

Results

GALNS gene and mRNA molecular analyses

In our cohort of about 40 Morquio A patients [8, 29, 31], the second disease-causing mutation remained uncharacterized in only three patients; the genetic analysis of these patients is reported here. Although heterozygous GALNS mutations were identified in all three patients (1, 2 and 3), the second anticipated pathogenic GALNS variant was not found in any of them by standard sequencing procedures. As depicted in the flowchart (Fig. 1), the presence of large deletions/duplications was excluded by means of quantitative fluorescent-PCR and copy number variation analysis as previously described [8].

GALNS gene and mRNA molecular analyses in Pt1

Initial GALNS gene sequencing analysis of Pt1 revealed a paternally inherited heterozygous c.463G > A (p.Gly155Arg) mutation, previously described as being disease-causing [32], but no evidence of a maternally inherited lesion.
GALNS mRNA analysis, performed by RT-PCR on lymphocytes from Pt1, revealed two distinct RT-PCR products, one corresponding to the wild-type GALNS transcript, the other slightly larger. The normal RT-PCR product harboured the c.463G > A (p.Gly155Arg) mutation whereas the aberrant RT-PCR product contained a 53-nucleotide sequence insertion between exons 8 and 9. When this inserted sequence was aligned to the GALNS gene sequence, it was identified as DNA sequence originating from intron 8, which had been included as a consequence of the activation of a cryptic donor splice site. Hence, the added DNA sequence constitutes, in effect, a cryptic exon (Fig. 2f).
The targeted sequencing of GALNS intron 8 from Pt1 then revealed the mRNA defect to be due to a novel heterozygous c.899–167 A > G transition that serves to create an additional donor splice site (Human Splicing Finder; http://​www.​umd.​be/​HSF3/​). The upstream splice acceptor site that was co-activated, thereby allowing inclusion of the 53 bp cryptic exon, was an AG dinucleotide flanked by a stretch of pyrimidines on its 5′ side (Additional file 1: Figure S3). The sequence of the aberrant GALNS mRNA transcript predicted a frameshift ending in a premature stop codon located at position c.953 within exon 9 (Gly300Valfs*37) (Fig. 2f; Additional file 1: Figure S3). The c.899–167 A > G lesion was also detected in the heterozygous state in the mother of Pt1.

The GALNS gene and mRNA molecular analyses in Pt2 and Pt3

Standard sequencing of the GALNS gene identified a single missense mutation in both Pt2 and Pt3 [p.Gly155Arg and p.Asp233Asn, respectively], but failed to identify the second disease-causing mutation in either patient. Further, RT-PCR analysis of the GALNS gene initially failed to reveal any additional RT-PCR products in Pt2 and Pt3 as compared to normal controls (data not shown). However, the same analysis performed on T-lymphocytes grown in the presence of cycloheximide revealed the presence of aberrant RT-PCR products in both patients (Fig. 2a-e).
The aberrant in-frame RT-PCR product detected in Pt2 would predict the omission of 117 nt from exon 10 of the GALNS transcript [r.1003_1119del, p.(Val335_Leu373del)] (Fig. 2e). The aberrant splicing product detected in lymphocyte cDNA samples from Pt3 was sequenced and shown to have resulted from the skipping of half of exon 5, all of exons 6, 7 and 8, plus half of exon 9 (r.456_916del; Fig. 2a) thereby generating a frameshift.
The GALNS mRNA evaluation performed in the father of Pt2 identified the same deletion, r.1003_1119del, that had been detected in the proband. By contrast, RT-PCR analysis revealed that the r.456_916del lesion detected in Pt3 was absent from his parents whilst two aberrant transcripts, characterized by different (albeit similar) deleted portions of GALNS exons, were detected in the mother of Pt3 (Fig. 2b and c). In addition, in both Pt3 and his father, an aberrant GALNS splicing product, that harboured the deletion of exon 9, was identified (Fig. 2d). In silico analysis suggested that both the c.697G > A (p.Asp233Asn) and c.775C > A (p.Arg259Arg) nucleotide changes could alter splicing (see HSF predictions given in Table 2); c.697G > A (p.Asp233Asn) in Pt3 corresponds to the pathological lesion harboured by the paternal GALNS allele. Since c.775C > A (p.Arg259Arg) was found in cis with the c.697G > A (p.Asp233Asn) mutation in Pt3 (i.e. it also has a paternal origin), it cannot correspond to the second pathological lesion anticipated in the GALNS gene of Pt3, despite its potential impact on splicing (Table 2). The c.697G > A (p.Asp233Asn) and c.775C > A (p.Arg259Arg) substitutions were nevertheless both identified, apparently in the homozygous state, in the aberrant splicing product lacking exon 9 that was detected in both Pt3 and his father (Fig. 2d), thereby confirming their likely role in generating this aberrant splicing product.
Table 2
GALNS nucleotide sequence variants with potential significance in the generation of the GALNS aberrant splicing products detected in Pt2 and Pt3
Chr: Pos (GRCh37.p13)
Zygosity
Parental Zygosity
Variant Type
Identifier (dbSNP 149, NCBI)
Exon/Intron
MAF
HSF Summary/CADD score
Pt2
16:88898099
Heterozygous
Paternal (heterozygous)
c.1002 + 307G > C
rs866140272
INTRON 9
ESE gain and alterations to various ESRs - CADD 3.002
Pt3
16:88901744
Heterozygous
Paternal (heterozygous)
c.775C > A; p.Arg259Arg
rs61742258
EXON 8
0.002
Cryptic donor splice site created, ESE loss and ESS gain - CADD 14.49
16:88901827
Heterozygous
Maternal (heterozygous)
c.759-67G > A
rs565875595
INTRON 7
0.001
ESE gain and ESS loss - CADD 6.652
16:88902194
Heterozygous
Paternal (heterozygous)
c.697G > A; p.Asp233Asn
rs753051547
EXON 7
Alteration of an exonic ESE site - CADD 24.1
16:88905035
Heterozygous
Maternal (heterozygous)
c.423-862C > T
INTRON 4
Cryptic donor splice site created - CADD 0.094
- = not present in gnomAD browser beta (http://​gnomad.​broadinstitute.​org/​); MAF = minor allele frequency; ESE = exonic splicing enhancer; ESS = exonic splicing silencer; ESRs = exonic splicing regulatory sequences; HSF (Human Splicing Finder) predictions = a tool which assesses the potential impact of these variants on the mRNA splicing phenotype (http://​www.​umd.​be/​HSF3/​); CADD = Combined Annotation Dependent Depletion = a tool for scoring the deleteriousness of single nucleotide variants as well as insertion/deletion variants in the human genome. A CADD score of > 10 is applied as a threshold to identify high-confidence disease-causing mutations [27]

Whole GALNS gene sequence analysis

Among the list of GALNS gene variants identified in genomic DNA samples from Pt2 and Pt3 [143 variants in Pt2 and 69 variants in Pt3, data not shown], in silico analyses identified several potentially pathogenic variants (summarised in Table 2).
The c.1002 + 307G > C variant detected in Pt2 was predicted to result in mis-splicing (Table 2) and represents a highly plausible candidate pathological lesion (i) on the basis of its location within intron 9, (ii) because the mutant allele creates an exonic splicing enhancer (ESE) site and (iii) because it is predicted to create an exon-identity element (ccgcct) [33] and may possibly also create or abolish splice silencer motifs [34].
The c.759-67G > A variant, identified in both patient Pt3 and his mother, represents the most plausible candidate for the observed splicing defect in these individuals. Although it should be appreciated that the consequences for mRNA splicing of changes in exonic splicing regulatory elements are often unpredictable, this deep intronic variant (c.759-67G > A) is predicted to abolish an existing exonic splicing silencer (ESS) motif while creating a novel ESE motif.
Intriguingly, the observed RNA splicing phenotypes differed between patient Pt3 and his mother. Thus, whereas the GALNS mRNA transcript in Pt3 harboured a 461 bp deletion (Fig. 2a), his mother exhibited two quite distinct mis-spliced transcripts, the first harbouring a 521 bp deletion, the second an indel comprising a 385 bp deletion together with a 21 bp insertion (Fig. 2b-c).

Discussion

The spectrum of pathological mutations and benign polymorphisms in the GALNS gene displays considerable allelic heterogeneity [HGMD Professional (Stenson et al., 2017), Exome Variant Server (http://​evs.​gs.​washington.​edu/​EVS/​), GALNS Mutation Database (http://​galns.​mutdb.​org/​database) etc.]. However, in approximately 16% of patients, the anticipated second disease-causing GALNS mutation cannot be unequivocally identified within the gene coding region or at the exon-intron boundaries [6, 7].
Establishing a diagnostic plan, including the requisite genetic analyses, is essential to distinguish between bona fide Morquio A disease patients and individuals with other disorders presenting with similar clinical and radiological findings [6, 9, 12]. In addition, early diagnosis is crucial for the prompt deployment of available therapies before permanent systemic lesions occur. Here, we have integrated a series of clinical and analytical tools to provide a diagnostic flow chart for Morquio A disease (Fig. 1). In the algorithm we propose, next generation sequencing (NGS) procedures may be employed in two distinct analytical steps (Fig. 1). In the first, the exonic sequences and exon-intron boundaries of the gene in question are sequenced by NGS methodology (whole exome); alternatively, whole genome sequencing can be employed to sequence the entire gene region (~ 50 kb). In a second step, high-throughput sequencing-based methods can be used to perform transcriptome analysis (RNA-Seq) [35], including of course, in our case, GALNS transcripts.
We have presented here the cases of three Morquio A patients in whom the second disease-causing GALNS mutation was not initially identifiable either by standard sequencing procedures or by the analysis of gross DNA rearrangements and instead had to be determined by means of RT-PCR. Aberrant GALNS mRNA splicing products were noted in all three patients. In Pt1, the deep intronic mutation c.899–167 A > G was unequivocally identified as the lesion responsible for the aberrant mRNA (including an elongated exon) and a prematurely truncated GALNS protein. Hence, this aberrant splicing event can be directly and unambiguously related to the severe clinical phenotype observed in this patient.
Mutations located within deep intronic regions, that appear capable of promoting the use of alternative natural or non-natural splicing sites, were identified by GALNS whole gene sequencing analyses of Pt2 and Pt3 samples. After following the variant prioritisation protocol described above, specific GALNS variants in Pt2 (c.1002 + 307G > C) and Pt3 (c.759-67G > A) were predicted to make a contribution to the clinical phenotype in these individuals by impacting mRNA splicing. These variants exhibited a very low Minor allele frequency (MAF), and were predicted to modulate splicing, particularly with respect to potential ESE and ESS sequences.
Since both of the aberrantly spliced products detected in Pt2 and Pt3 disrupt exons, the mechanism responsible for these splicing alterations cannot be precisely ascertained. Indeed, it remains possible that a particular combination of variants could have been responsible for the observed splicing defects, rather than one variant on its own. Consequently, it may be that any of the other variants detected in the patients, including the putatively non-pathogenic GALNS variants (143 variants in Pt2 and 69 variants in Pt3, post-prioritization), may have contributed to the generation of the non-physiological splicing events detected e.g. c.423-862C > T identified in Pt3 and his mother. It should also be appreciated that variants such as c.423-862C > T may disrupt canonical splice junction sequences, i.e. cryptic acceptor and donor splice sites. Thus, it may well be that it is the combination of altered canonical and non-canonical splice sites in both patients that gives rise to these unique splicing alterations.
The GALNS gene is known to be alternatively spliced, with at least three known protein coding transcripts currently annotated (NM_00512.4, NM_001323544.1 and NM_00132354.1). It is possible that these alternative transcripts were differentially expressed between Pt3 and his mother; if so, this might have led to changes in splicing factor supply and demand, which could in turn account for the differences in the observed mRNA splicing phenotype between the patient and his mother.
Owing to the difficulties inherent in interpreting this atypical type of splicing event and our veritable ignorance of splicing regulatory regions, our approach did not unequivocally identify disease-causing variants at the DNA level for Pt2 and Pt3 even after intensive DNA sequence analysis of the GALNS gene region. Additional functional studies, including the construction of expression systems that variously combine the identified candidate mis-spliced variants, would be necessary to formally confirm our hypotheses. However, for diagnostic purposes, the pathogenicity of the observed mRNA splicing defects is evidenced by: a) the absence of RT-PCR amplification, corresponding to the aberrant mRNA transcripts detected in Pt2 and Pt3 samples, in a pool of 10 normal controls both treated and untreated with cycloheximide; b) the splicing products detected in Pt2 and Pt3 differing from the 13 known GALNS physiological mRNAs, collected in the Ensembl Human Genome browser (http://​www.​ensembl.​org/​index.​html). These considerations imply that the alternative GALNS mRNA splicing products detected in Pt2 and Pt3 are non-physiological, and are therefore likely to be consequent to the (hitherto unidentified) second disease-causing GALNS alleles in these Morquio A patients.

Conclusions

Morquio A disease is particularly prone to delayed diagnoses and/or misdiagnoses, owing to the difficulties inherent in the differential diagnosis of this rheumatic disease that requires specialist metabolic expertise. The addition of mRNA analysis and whole GALNS gene sequencing to this flowchart promises to help to identify those molecular causes of Morquio A disease which until now have been refractory to analysis. These analyses are likely to be particularly important for Morquio A screening programs in which the drawing up of a general diagnostic molecular plan is key to distinguishing between newborns who are carrying mutations associated with severe forms of the disease and those who are carrying mutations that are likely to give rise to milder or asymptomatic forms.
Our sequence analysis of the GALNS gene, involving gene level, genomic and RT-PCR analyses, suggests that although deep intronic mutations may be individually infrequent, they may be largely responsible for our occasional failure to identify GALNS disease alleles in Morquio A disease; it follows that marked improvements in our knowledge of the splicing machinery will be required before any diagnostic workflow can be regarded as being 100% effective.

Acknowledgements

We gratefully thank the AMMeC (Associazione Malattie Metaboliche e Congenite, Italia) for technical assistance. We also thank Dr. Marzia Rossato, Functional Genomic Laboratory, Department of Biotechnology, University of Verona, for the whole genome sequencing service.

Funding

BioMarin Pharmaceutical Inc. (USA) grants (2052 and 2616) funded the GALNS whole gene sequencing.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on request.
According to local ethical guidelines, all nucleic acid samples were obtained for storage and analysis only after the patients’ family members’ written informed consent had been obtained, using a form approved by the local Ethics Committee (Ethics Committee of the Meyer Hospital, Florence, Italy).
The patients’ family members’ written informed consents contain their permission to publish patient data.

Competing interests

The authors declare that they have no competing interests.

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Literatur
1.
Zurück zum Zitat Wood TC, Harvey K, Beck M, Burin MG, Chien YH, Church HJ, D’Almeida V, van Diggelen OP, Fietz M, Giugliani R et al: Diagnosing mucopolysaccharidosis IVA. J Inherit Metab Dis 2013, 36(2):293–307.CrossRef Wood TC, Harvey K, Beck M, Burin MG, Chien YH, Church HJ, D’Almeida V, van Diggelen OP, Fietz M, Giugliani R et al: Diagnosing mucopolysaccharidosis IVA. J Inherit Metab Dis 2013, 36(2):293–307.CrossRef
2.
Zurück zum Zitat Nakashima Y, Tomatsu S, Hori T, Fukuda S, Sukegawa K, Kondo N, Suzuki Y, Shimozawa N, Orii T. Mucopolysaccharidosis IV a: molecular cloning of the human N-acetylgalactosamine-6-sulfatase gene (GALNS) and analysis of the 5′-flanking region. Genomics. 1994;20(1):99–104.CrossRef Nakashima Y, Tomatsu S, Hori T, Fukuda S, Sukegawa K, Kondo N, Suzuki Y, Shimozawa N, Orii T. Mucopolysaccharidosis IV a: molecular cloning of the human N-acetylgalactosamine-6-sulfatase gene (GALNS) and analysis of the 5′-flanking region. Genomics. 1994;20(1):99–104.CrossRef
3.
Zurück zum Zitat O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse B, Smith-White B, Ako-Adjei D, et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016;44(D1):D733–45.CrossRef O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse B, Smith-White B, Ako-Adjei D, et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016;44(D1):D733–45.CrossRef
4.
Zurück zum Zitat Aken BL, Achuthan P, Akanni W, Amode MR, Bernsdorff F, Bhai J, Billis K, Carvalho-Silva D, Cummins C, Clapham P, et al. Ensembl 2017. Nucleic Acids Res. 2017;45(D1):D635–42.CrossRef Aken BL, Achuthan P, Akanni W, Amode MR, Bernsdorff F, Bhai J, Billis K, Carvalho-Silva D, Cummins C, Clapham P, et al. Ensembl 2017. Nucleic Acids Res. 2017;45(D1):D635–42.CrossRef
5.
Zurück zum Zitat Tomatsu S, Fukuda S, Cooper A, Wraith JE, Ferreira P, Di Natale P, Tortora P, Fujimoto A, Kato Z, Yamada N et al: Fourteen novel mucopolysaccharidosis IVA producing mutations in GALNS gene. Hum Mutat 1997, 10(5):368–375.CrossRef Tomatsu S, Fukuda S, Cooper A, Wraith JE, Ferreira P, Di Natale P, Tortora P, Fujimoto A, Kato Z, Yamada N et al: Fourteen novel mucopolysaccharidosis IVA producing mutations in GALNS gene. Hum Mutat 1997, 10(5):368–375.CrossRef
6.
Zurück zum Zitat Morrone A, Caciotti A, Atwood R, Davidson K, Du C, Francis-Lyon P, Harmatz P, Mealiffe M, Mooney S, Oron TR, et al. Morquio a syndrome-associated mutations: a review of alterations in the GALNS gene and a new locus-specific database. Hum Mutat. 2014;35(11):1271–9.PubMedPubMedCentral Morrone A, Caciotti A, Atwood R, Davidson K, Du C, Francis-Lyon P, Harmatz P, Mealiffe M, Mooney S, Oron TR, et al. Morquio a syndrome-associated mutations: a review of alterations in the GALNS gene and a new locus-specific database. Hum Mutat. 2014;35(11):1271–9.PubMedPubMedCentral
7.
Zurück zum Zitat Tomatsu S, Montano AM, Nishioka T, Gutierrez MA, Pena OM, Tranda Firescu GG, Lopez P, Yamaguchi S, Noguchi A, Orii T. Mutation and polymorphism spectrum of the GALNS gene in mucopolysaccharidosis IVA (Morquio a). Hum Mutat. 2005;26(6):500–12.CrossRef Tomatsu S, Montano AM, Nishioka T, Gutierrez MA, Pena OM, Tranda Firescu GG, Lopez P, Yamaguchi S, Noguchi A, Orii T. Mutation and polymorphism spectrum of the GALNS gene in mucopolysaccharidosis IVA (Morquio a). Hum Mutat. 2005;26(6):500–12.CrossRef
8.
Zurück zum Zitat Caciotti A, Tonin R, Rigoldi M, Ferri L, Catarzi S, Cavicchi C, Procopio E, Donati MA, Ficcadenti A, Fiumara A, et al. Optimizing the molecular diagnosis of GALNS: novel methods to define and characterize Morquio-a syndrome-associated mutations. Hum Mutat. 2015;36(3):357–68.CrossRef Caciotti A, Tonin R, Rigoldi M, Ferri L, Catarzi S, Cavicchi C, Procopio E, Donati MA, Ficcadenti A, Fiumara A, et al. Optimizing the molecular diagnosis of GALNS: novel methods to define and characterize Morquio-a syndrome-associated mutations. Hum Mutat. 2015;36(3):357–68.CrossRef
9.
Zurück zum Zitat Hendriksz CJ, Harmatz P, Beck M, Jones S, Wood T, Lachman R, Gravance CG, Orii T, Tomatsu S. Review of clinical presentation and diagnosis of mucopolysaccharidosis IVA. Mol Genet Metab. 2013;110(1–2):54–64.CrossRef Hendriksz CJ, Harmatz P, Beck M, Jones S, Wood T, Lachman R, Gravance CG, Orii T, Tomatsu S. Review of clinical presentation and diagnosis of mucopolysaccharidosis IVA. Mol Genet Metab. 2013;110(1–2):54–64.CrossRef
10.
Zurück zum Zitat Tomatsu S, Averill LW, Sawamoto K, Mackenzie WG, Bober MB, Pizarro C, Goff CJ, Xie L, Orii T, Theroux M. Obstructive airway in Morquio a syndrome, the past, the present and the future. Mol Genet Metab. 2016;117(2):150–6.CrossRef Tomatsu S, Averill LW, Sawamoto K, Mackenzie WG, Bober MB, Pizarro C, Goff CJ, Xie L, Orii T, Theroux M. Obstructive airway in Morquio a syndrome, the past, the present and the future. Mol Genet Metab. 2016;117(2):150–6.CrossRef
11.
Zurück zum Zitat Tomatsu S, Almeciga-Diaz CJ, Montano AM, Yabe H, Tanaka A, Dung VC, Giugliani R, Kubaski F, Mason RW, Yasuda E, et al. Therapies for the bone in mucopolysaccharidoses. Mol Genet Metab. 2015;114(2):94–109.CrossRef Tomatsu S, Almeciga-Diaz CJ, Montano AM, Yabe H, Tanaka A, Dung VC, Giugliani R, Kubaski F, Mason RW, Yasuda E, et al. Therapies for the bone in mucopolysaccharidoses. Mol Genet Metab. 2015;114(2):94–109.CrossRef
12.
Zurück zum Zitat Tomatsu S, Sawamoto K, Almeciga-Diaz CJ, Shimada T, Bober MB, Chinen Y, Yabe H, Montano AM, Giugliani R, Kubaski F, et al. Impact of enzyme replacement therapy and hematopoietic stem cell transplantation in patients with Morquio a syndrome. Drug Des Devel Ther. 2015;9:1937–53.CrossRef Tomatsu S, Sawamoto K, Almeciga-Diaz CJ, Shimada T, Bober MB, Chinen Y, Yabe H, Montano AM, Giugliani R, Kubaski F, et al. Impact of enzyme replacement therapy and hematopoietic stem cell transplantation in patients with Morquio a syndrome. Drug Des Devel Ther. 2015;9:1937–53.CrossRef
13.
Zurück zum Zitat Yabe H, Tanaka A, Chinen Y, Kato S, Sawamoto K, Yasuda E, Shintaku H, Suzuki Y, Orii T, Tomatsu S. Hematopoietic stem cell transplantation for Morquio a syndrome. Mol Genet Metab. 2016;117(2):84–94.CrossRef Yabe H, Tanaka A, Chinen Y, Kato S, Sawamoto K, Yasuda E, Shintaku H, Suzuki Y, Orii T, Tomatsu S. Hematopoietic stem cell transplantation for Morquio a syndrome. Mol Genet Metab. 2016;117(2):84–94.CrossRef
14.
Zurück zum Zitat Hendriksz CJ, Parini R, AlSayed MD, Raiman J, Giugliani R, Mitchell JJ, Burton BK, Guelbert N, Stewart FJ, Hughes DA, et al. Impact of long-term elosulfase alfa on activities of daily living in patients with Morquio a syndrome in an open-label, multi-center, phase 3 extension study. Mol Genet Metab. 2018;123(2):127–34.CrossRef Hendriksz CJ, Parini R, AlSayed MD, Raiman J, Giugliani R, Mitchell JJ, Burton BK, Guelbert N, Stewart FJ, Hughes DA, et al. Impact of long-term elosulfase alfa on activities of daily living in patients with Morquio a syndrome in an open-label, multi-center, phase 3 extension study. Mol Genet Metab. 2018;123(2):127–34.CrossRef
15.
Zurück zum Zitat Khaliq T, Sadilek M, Scott CR, Turecek F, Gelb MH. Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis IVA. Clin Chem. 2011;57(1):128–31.CrossRef Khaliq T, Sadilek M, Scott CR, Turecek F, Gelb MH. Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis IVA. Clin Chem. 2011;57(1):128–31.CrossRef
16.
Zurück zum Zitat Tomatsu S, Fujii T, Fukushi M, Oguma T, Shimada T, Maeda M, Kida K, Shibata Y, Futatsumori H, Montano AM, et al. Newborn screening and diagnosis of mucopolysaccharidoses. Mol Genet Metab. 2013;110(1–2):42–53.CrossRef Tomatsu S, Fujii T, Fukushi M, Oguma T, Shimada T, Maeda M, Kida K, Shibata Y, Futatsumori H, Montano AM, et al. Newborn screening and diagnosis of mucopolysaccharidoses. Mol Genet Metab. 2013;110(1–2):42–53.CrossRef
17.
Zurück zum Zitat Kubaski F, Mason RW, Nakatomi A, Shintaku H, Xie L, van Vlies NN, Church H, Giugliani R, Kobayashi H, Yamaguchi S et al: Newborn screening for mucopolysaccharidoses: a pilot study of measurement of glycosaminoglycans by tandem mass spectrometry. J Inherit Metab Dis 2017, 40(1):151–158.CrossRef Kubaski F, Mason RW, Nakatomi A, Shintaku H, Xie L, van Vlies NN, Church H, Giugliani R, Kobayashi H, Yamaguchi S et al: Newborn screening for mucopolysaccharidoses: a pilot study of measurement of glycosaminoglycans by tandem mass spectrometry. J Inherit Metab Dis 2017, 40(1):151–158.CrossRef
18.
Zurück zum Zitat Kubaski F, Osago H, Mason RW, Yamaguchi S, Kobayashi H, Tsuchiya M, Orii T, Tomatsu S. Glycosaminoglycans detection methods: applications of mass spectrometry. Mol Genet Metab. 2016. Kubaski F, Osago H, Mason RW, Yamaguchi S, Kobayashi H, Tsuchiya M, Orii T, Tomatsu S. Glycosaminoglycans detection methods: applications of mass spectrometry. Mol Genet Metab. 2016.
19.
Zurück zum Zitat Montano AM, Tomatsu S, Brusius A, Smith M, Orii T. Growth charts for patients affected with Morquio a disease. Am J Med Genet A. 2008;146A(10):1286–95.CrossRef Montano AM, Tomatsu S, Brusius A, Smith M, Orii T. Growth charts for patients affected with Morquio a disease. Am J Med Genet A. 2008;146A(10):1286–95.CrossRef
20.
Zurück zum Zitat Whitley CB, Ridnour MD, Draper KA, Dutton CM, Neglia JP. Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion. Clin Chem. 1989;35(3):374–9.PubMed Whitley CB, Ridnour MD, Draper KA, Dutton CM, Neglia JP. Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion. Clin Chem. 1989;35(3):374–9.PubMed
21.
Zurück zum Zitat Piraud M, Boyer S, Mathieu M, Maire I. Diagnosis of mucopolysaccharidoses in a clinically selected population by urinary glycosaminoglycan analysis: a study of 2,000 urine samples. Clin Chim Acta. 1993;221(1–2):171–81.CrossRef Piraud M, Boyer S, Mathieu M, Maire I. Diagnosis of mucopolysaccharidoses in a clinically selected population by urinary glycosaminoglycan analysis: a study of 2,000 urine samples. Clin Chim Acta. 1993;221(1–2):171–81.CrossRef
22.
Zurück zum Zitat van Diggelen OP, Zhao H, Kleijer WJ, Janse HC, Poorthuis BJ, van Pelt J, Kamerling JP, Galjaard H: A fluorimetric enzyme assay for the diagnosis of Morquio disease type a (MPS IV a). Clin Chim Acta 1990, 187(2):131–139. van Diggelen OP, Zhao H, Kleijer WJ, Janse HC, Poorthuis BJ, van Pelt J, Kamerling JP, Galjaard H: A fluorimetric enzyme assay for the diagnosis of Morquio disease type a (MPS IV a). Clin Chim Acta 1990, 187(2):131–139.
23.
Zurück zum Zitat Caciotti A, Garman SC, Rivera-Colon Y, Procopio E, Catarzi S, Ferri L, Guido C, Martelli P, Parini R, Antuzzi D, et al. GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings. Biochim Biophys Acta. 2011;1812(7):782–90.CrossRef Caciotti A, Garman SC, Rivera-Colon Y, Procopio E, Catarzi S, Ferri L, Guido C, Martelli P, Parini R, Antuzzi D, et al. GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings. Biochim Biophys Acta. 2011;1812(7):782–90.CrossRef
24.
Zurück zum Zitat Galjaard H. Genetic metabolic diseases early diagnosis and prenatal analysis, vol. 825. Amsterdam: Elsevier/North Holland Biochemical Press; 1980. Galjaard H. Genetic metabolic diseases early diagnosis and prenatal analysis, vol. 825. Amsterdam: Elsevier/North Holland Biochemical Press; 1980.
25.
Zurück zum Zitat Macias-Vidal J, Gort L, Lluch M, Pineda M, Coll MJ. Nonsense-mediated mRNA decay process in nine alleles of Niemann-pick type C patients from Spain. Mol Genet Metab. 2009;97(1):60–4.CrossRef Macias-Vidal J, Gort L, Lluch M, Pineda M, Coll MJ. Nonsense-mediated mRNA decay process in nine alleles of Niemann-pick type C patients from Spain. Mol Genet Metab. 2009;97(1):60–4.CrossRef
26.
Zurück zum Zitat Noensie EN, Dietz HC. A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition. Nat Biotechnol. 2001;19(5):434–9.CrossRef Noensie EN, Dietz HC. A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition. Nat Biotechnol. 2001;19(5):434–9.CrossRef
27.
Zurück zum Zitat Usuki F, Yamashita A, Higuchi I, Ohnishi T, Shiraishi T, Osame M, Ohno S. Inhibition of nonsense-mediated mRNA decay rescues the phenotype in Ullrich’s disease. Ann Neurol. 2004;55(5):740–4.CrossRef Usuki F, Yamashita A, Higuchi I, Ohnishi T, Shiraishi T, Osame M, Ohno S. Inhibition of nonsense-mediated mRNA decay rescues the phenotype in Ullrich’s disease. Ann Neurol. 2004;55(5):740–4.CrossRef
28.
Zurück zum Zitat Kervestin S, Jacobson A. NMD: a multifaceted response to premature translational termination. Nat Rev Mol Cell Biol. 2012;13(11):700–12.CrossRef Kervestin S, Jacobson A. NMD: a multifaceted response to premature translational termination. Nat Rev Mol Cell Biol. 2012;13(11):700–12.CrossRef
29.
Zurück zum Zitat Carraresi L, Parini R, Filoni C, Caciotti A, Sersale G, Tomatsu S, Orlando C, Zammarchi E, Guerrini R, Donati MA, et al. GALNS gene expression profiling in Morquio a patients’ fibroblasts. Clin Chim Acta. 2008;397(1–2):72–6.CrossRef Carraresi L, Parini R, Filoni C, Caciotti A, Sersale G, Tomatsu S, Orlando C, Zammarchi E, Guerrini R, Donati MA, et al. GALNS gene expression profiling in Morquio a patients’ fibroblasts. Clin Chim Acta. 2008;397(1–2):72–6.CrossRef
30.
Zurück zum Zitat Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014;46(3):310–5.CrossRef Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014;46(3):310–5.CrossRef
31.
Zurück zum Zitat Catarzi S, Giunti L, Papadia F, Gabrielli O, Guerrini R, Donati MA, Genuardi M, Morrone A. Morquio a syndrome due to maternal uniparental isodisomy of the telomeric end of chromosome 16. Mol Genet Metab. 2012;105(3):438–42.CrossRef Catarzi S, Giunti L, Papadia F, Gabrielli O, Guerrini R, Donati MA, Genuardi M, Morrone A. Morquio a syndrome due to maternal uniparental isodisomy of the telomeric end of chromosome 16. Mol Genet Metab. 2012;105(3):438–42.CrossRef
32.
Zurück zum Zitat Bunge S, Kleijer WJ, Tylki-Szymanska A, Steglich C, Beck M, Tomatsu S, Fukuda S, Poorthuis BJ, Czartoryska B, Orii T, et al. Identification of 31 novel mutations in the N-acetylgalactosamine-6-sulfatase gene reveals excessive allelic heterogeneity among patients with Morquio a syndrome. Hum Mutat. 1997;10(3):223–32.CrossRef Bunge S, Kleijer WJ, Tylki-Szymanska A, Steglich C, Beck M, Tomatsu S, Fukuda S, Poorthuis BJ, Czartoryska B, Orii T, et al. Identification of 31 novel mutations in the N-acetylgalactosamine-6-sulfatase gene reveals excessive allelic heterogeneity among patients with Morquio a syndrome. Hum Mutat. 1997;10(3):223–32.CrossRef
33.
Zurück zum Zitat Zhang C, Li WH, Krainer AR, Zhang MQ. RNA landscape of evolution for optimal exon and intron discrimination. Proc Natl Acad Sci U S A. 2008;105(15):5797–802.CrossRef Zhang C, Li WH, Krainer AR, Zhang MQ. RNA landscape of evolution for optimal exon and intron discrimination. Proc Natl Acad Sci U S A. 2008;105(15):5797–802.CrossRef
34.
Zurück zum Zitat Sironi M, Menozzi G, Riva L, Cagliani R, Comi GP, Bresolin N, Giorda R, Pozzoli U. Silencer elements as possible inhibitors of pseudoexon splicing. Nucleic Acids Res. 2004;32(5):1783–91.CrossRef Sironi M, Menozzi G, Riva L, Cagliani R, Comi GP, Bresolin N, Giorda R, Pozzoli U. Silencer elements as possible inhibitors of pseudoexon splicing. Nucleic Acids Res. 2004;32(5):1783–91.CrossRef
35.
Zurück zum Zitat Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10(1):57–63.CrossRef Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10(1):57–63.CrossRef
Metadaten
Titel
Mis-splicing of the GALNS gene resulting from deep intronic mutations as a cause of Morquio a disease
verfasst von
Anna Caciotti
Rodolfo Tonin
Matthew Mort
David N. Cooper
Serena Gasperini
Miriam Rigoldi
Rossella Parini
Federica Deodato
Roberta Taurisano
Michelina Sibilio
Giancarlo Parenti
Renzo Guerrini
Amelia Morrone
Publikationsdatum
01.12.2018
Verlag
BioMed Central
Erschienen in
BMC Medical Genetics / Ausgabe 1/2018
Elektronische ISSN: 1471-2350
DOI
https://doi.org/10.1186/s12881-018-0694-6

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