Neuromuscular Disorders 12 (2002) 501–505 www.elsevier.com/locate/nmd
Rapid scanning of myotubularin (MTM1) gene by denaturing highperformance liquid chromatography (DHPLC) Elisabetta Flex a, Alessandro De Luca a, Maria Rosaria D’Apice b, Anna Buccino a, Bruno Dallapiccola a,c, Giuseppe Novelli b,* a CSS Mendel Institute, Rome, Italy Department of Biopathology and Diagnostic Imaging, Tor Vergata University of Rome, Rome, Italy c Department of Experimental Medicine and Pathology, La Sapienza University of Rome, Rome, Italy b
Received 9 August 2001; received in revised form 27 October 2001; accepted 14 November 2001
Abstract X-linked myotubular myopathy (XLMTM; OMIM# 310400) is a severe congenital muscle disease caused by mutations in the myotubularin (MTM1) gene. This gene encodes for a lipid phosphatase belonging to a large gene family involved in the regulation of phosphatidylinositide-3-kinase (PI 3-kinase) pathway and membrane trafficking. To date, more than 130 different mutations, distributed in all exons, have been identified in a large number of families. The majority of MTM1 mutations are private and rare, generating high allelic diversity, with a restricted number of recurrent mutations. We set up and formatted a denaturing high performance liquid chromatography (DHPLC) method to allow high throughput, greater accuracy and high resolution in detecting myotubularin mutations. The entire coding sequence of the gene was screened in 10 XLMTM patients using this technique. We identified seven mutated alleles [R37X, (137-11) A, (592-593) insA, T197I, R253X, G378R, G402R] previously characterised by SSCP and DNA sequencing, plus two novel mutations which are reported here [P199S, (1644 1 2) insG]. In addition we detected a common polymorphism within intron 11 (1314 1 3A/G). Our results suggest that denaturing high-performance liquid chromatography provides an accurate method for the rapid identification of MTM1 mutations. q 2002 Elsevier Science B.V. All rights reserved. Keywords: X-linked myotubular myopathy; Myotubularin; MTM1 gene; Denaturing high-performance liquid chromatography; Mutation detection
1. Introduction X-linked recessive myotubular myopathy (XLMTM; MIM# 310400) is a congenital muscle disorder characterised by severe hypotonia and generalised muscle weakness in affected males. Most patients die within the first year of life from respiratory failure. The characteristic muscle histopathology consists of small rounded muscle fibres with centrally located nuclei surrounded by a halo devoid of contractile elements but containing mitochondria . These resemble foetal myotubes, and it has been suggested that the disorder results either from an arrest in the normal development of muscle fibres or more likely from a defect in the structural organisation of fibres. The MTM1 gene was isolated by positional cloning  and found to be mutated
* Corresponding author. Fax: 139-06-20427313. E-mail address: [email protected]
in the vast majority of the XLMTM patients [3–5]. This gene is composed of 15 exons encompassing 100 Kb of genomic DNA and encodes for a 603-amino acid long protein named MTM1. Although MTM1 was thought to be a dual-specificity protein phosphatase, recent results indicate that it is primarily a lipid phosphatase, acting on phosphatidylinositol-3-monophosphate, and might be involved in the regulation of phosphatidylinositide-3-kinase (PI 3-kinase) pathway and membrane trafficking [6,7]. To date, more than 130 different disease MTM1 mutations have been found in 198 unrelated families . A list of MTM1 mutations is maintained on the web at the Human Gene Mutation Database (HGMD; entry for XLMTM: http://www.uwcm.ac.uk/ uwcm/mg/search/119439.html). The majority of mutations are family-specific and widespread throughout the gene. This generates a high mutation heterogeneity that in turn makes difficult their identification and therefore the molecular diagnosis of the disease. The most widely used protocol for MTM1 gene mutational analysis is based on PCR amplification of single
0960-8966/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0960-896 6(01)00328-5
E. Flex et al. / Neuromuscular Disorders 12 (2002) 501–505
exons, single-strand conformation polymorphism analysis (SSCP) and/or heteroduplexes analysis (HA), followed by sequencing of abnormal bands . Alternatively, a direct sequencing approach of all exons has been used . Denaturing high performance liquid chromatography (DHPLC) is a rapid and accurate method for detecting DNA sequence alterations . We have assessed the efficacy of this method as a screening assay for detecting mutation in the MTM1 gene. We established theoretical conditions for DHPLC analysis of all coding exons of the MTM1 gene and carried out a mutational analysis of MTM1 gene in ten unrelated sporadic patients (eight males and two females). Noncoding exon 1 has been excluded from the analysis because no exon 1 point mutations have been identified to date.
2. Patients and methods 2.1. Patients Ten Italian patients, eight males and two females, were diagnosed with XLMTM based on clinical features and histological examinations. Two male patients (M2 and M3) had positive family histories. There was no consanguinity in the families. All male patients satisfied the diagnostic criteria of the International Consortium of Myotubular Myopathy (http://www.enmc.spc.oxac.uk/DC/Myotucrit). Differential diagnosis from myotonic dystrophy and/or spinal muscular atrophy was performed by direct gene analysis. All patients showed the common clinical manifestations since birth: generalised muscle weakness and hypotonia, involving facial and respiratory muscle. Some needed ventilator support at birth, to sustain their respiratory function, and their mothers noticed diminished foetal movements in utero and polyhydramnios. Muscle biopsies revealed in all studied cases, a wide variation of myofibre diameter. Approximately 40% of fibres contained internal nuclei, surrounded by an area devoid of myofibrils. The female patients showed similar features to the male patients with an onset at about 5–10 years when gait difficulty was first noticed. Physical examination revealed an elongated face, prognathism, and crowded teeth. The proximal upper limb muscles and the distal muscles were weak and wasted, whereas the forearm muscles showed almost normal strength. One of these patients, F2, was never able to run and could not play sports. Muscle biopsies in both cases, showed almost 30% of fibres with centrally placed nuclei and central aggregation of oxidative enzyme activity, indicating muscle fibre immaturity. Five patients, three males (M1, M5 and M8), and the two females are alive (ages 3–10 years), although present gait difficulty. The other patients died in the first months of life mainly due to respiratory failure. Genomic DNA was extracted using standard procedures  from muscle biopsy and/or peripheral blood leukocytes of ten unrelated patients, eight males and two females.
2.2. Polymerase chain reaction Genomic polymerase chain reaction (PCR) was carried out in 50 ml reaction volume containing 50 ng genomic DNA, 0.2 mM primers, 100 mM dNTPs, 5 ml reaction buffer (100 mM TRIS pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin) and 2.5 U Ampli Taq Gold Polymerase (Applied Biosystems, Foster City, CA). PCR primers and conditions for the amplification of MTM1 exons were as previously described . Exon 11 was also amplified using the antisense primer (11a) 5 0 -CTTTTTCTACCAGTATTTCG-3 0 . One primer for each couple was modified at 5 0 by adding the universal forward M13 sequence 5 0 GTAAAACGACGGCCAG-3 0 . Amplicons were checked by agarose gel electrophoresis before DHPLC analysis, to make sure that only the specific product was amplified and that not additional band occurred that could lead to artificial heteroduplex conformation. 2.3. DHPLC analysis DHPLC was carried out on a WAVE DNA fragment analysis system (Transgenomic, Crewe, UK) equipped with a DNASepwcolumn (Transgenomic). PCR products were examined for heteroduplexes by subjecting 4–7 ml of each PCR product, containing ,50–100 ng DNA, that was denatured for 5 min at 958C and then gradually reannealed by decreasing sample temperature from 95 to 658C over a period of 30 min. In males, duplexes for DHPLC analysis were created by mixing, denaturing, and reannealing a PCR product from a normal subject with the corresponding amplicon of the MTM1 patient. The PCR products were then separated (flow rate of 0.9 ml/min) through a 2% linear acetonitrile gradient. The start and end points of the gradients were adjusted according to the size of each PCR product (Table 1). Analysis per amplified sample took 9.2 min including column regeneration and equilibration. Samples were analysed at the melt temperature (Tm) deterTable 1 DHPLC conditions for the mutational analysis of MTM1 gene Exon
Size of PCR fragment (bp)
DHPLC temperature (8C)
Acetonitrile gradient (%B)
2 3 4 5 6 7 8 9 10 11 11(a) 12 13 14 15
288 188 271 250 191 204 255 260 267 308 226 211 227 264 272
55 59 54 56 60 56 58 57 55 55 55 56 55 56 56–61
53/63 41/51 54/64 52/62 41/51 51/61 52/62 52/62 52/62 52/62 52/62 51/61 53/63 52/62 51/61–52/62
E. Flex et al. / Neuromuscular Disorders 12 (2002) 501–505
Figure 1. Specimen chromograms produced by DHPLC analysis.
mined by using the DHPLCMelt software (http://insertion. stanford.edu/melt.html) (Table 1). Where recommended by the software, two different temperatures were used. 2.4. Sequence analysis The PCR product that showed heteroduplex by DHPLC analysis was sequenced with the Thermo Sequenase cycle sequencing Kit (Amersham Pharmacia Biotech, Uppsala, Sweden), using 2 pmol of the M13 forward primer labelled with infrared dye IRD41 (LI-COR Lincoln, NE). The nucleotide sequence was established using a LI-COR 4000L automated DNA sequencer.
forward primer. PCR products were analysed on a model ABI 310 automated fluorescent DNA sequencer (Applied Biosystems). The fluorescent data collected were analysed automatically by the Genescan Analysis program (Applied Biosystems) at the end of each run. The X-inactivation pattern was given as the ratio between the amount of PCR products, with smaller allele being indicated first, and were classified as random (ratios 50:50 to ,65:35), moderately skewed (ratios 65:35 to ,80:20), skewed (ratios 80:20 to ,95:5), and extremely skewed (ratio 95:5 higher).
2.5. X-inactivation assay 3.1. DHPLC analysis Using the DNA methylation assay we have examined the X-chromosome inactivation patterns in muscle biopsy and peripheral blood from female patients, in order quantify the X-inactivation rate in different tissues. X-chromosome inactivation was studied by PCR analysis of a polymorphic CAG repeat in the first exon the human androgen receptor (AR) gene on genomic DNA . In brief, the genomic DNA of female patients was digested with HpaII, and then the digested samples were amplified using a FAM-labelled
Molecular analysis of the 14 exons (2–15) of the MTM1 gene by DHPLC in ten unrelated patients (eight males and two females) and 20 normal control samples revealed variant elution profiles in all MTM1 patients examined (Fig. 1). Sequencing identified seven previously described mutations [R37X, (137-11) T/A, 592-593insA, T197I, R253X, G378R, G402R], two novel mutations, one in exon 8 (P199S) and one in the donor splice site of exon
E. Flex et al. / Neuromuscular Disorders 12 (2002) 501–505
Table 2 MTM 1 gene mutations identified by DHPLC Patient a
Predicted protein or splicing alteration
Type of mutation
M1 M2 M3 M4 M5 M6a b M6b c M8 M9 F1 F2
Exon 8 Exon 8 Exon 8 Exon 9 Exon 11 Exon 11 Intron 11 Intron 14 Exon 3 Exon 8 Intron 3
590C/T 592-593insA 592-593insA 757C/T 1132G/A 1252C/G (1314 1 3)A/G (1644 1 2)insG 109C/T 649T/C (137-11)T/A
T197I Frameshift 198 Frameshift 198 R253X G378R G420R (1314 1 3)A/G Donor splice site R37X P199S Frameshift 46 (ex04 skipping)
Missense Nonsense Nonsense Nonsense Missense Missense Polymorphism Splicing Nonsense Missense Splicing
       This study  This study 
a b c
M, male; F, female. M6a, mutation in M6 patient. M6b, polymorphism in M6 patient.
14 (1644 1 2)insG and one polymorphism [(1314 1 3)A/ G], found to be common in the Italian population [12–15] (Table 2).
both patients has allowed us to demonstrate the presence of an extremely skewed inactivation (ratio 95:5 higher) (data not shown) both in blood and muscle.
3.2. Mutation identification in male patients
3.4. Characterisation of (1314 1 3)A/G polymorphism
Mutation T197I, found in patient M1, was associated with a severe clinical course showing typical muscle disease and in addition birth length and head circumference were greater than the 90th percentile. Abnormalities in growth is frequently observed in XLMTM patients, probably as a pleoiotropic effect of the MTMT1 gene . T197I were not detected in the parents of patient M1 suggesting a de novo origin. The mutation 592-593insA, that introduces a single base between nucleotides 592-593, has been identified in two non-consanguineous patients (M2 and M3) originating in the same geographic region (Trentino Alto Adige). This mutation causes the introduction of a stop codon in position 198 of the coding sequence and in both cases is associated with a severe phenotype of the disease. Both patients died during the first three months of life. Mutation R253X, identified in exon 9, consists in a C ! T transition at nucleotide 757 that introduces a stop codon in place of an arginine in position 253. The patient died at birth of respiratory failure. In exon 11 we detected two missense mutations, G378R and G402R, respectively, that replace two amino acids placed within the consensus sequence for the active site for the tyrosine phosphatase activity. It is therefore plausible that these two amino acid substitutions cause the loss of function of the catalytic site of the MTM1. The nonsense R37X mutation was identified in exon 3 and introduces a stop codon in a CpG site.
The sequence analysis of exon 11 variants has allowed to identification of the previously reported (1314 1 3)A/G polymorphism . This gene variant shows a frequency of 0.50 in the Italian population. This polymorphism interfered with the identification of the G402R mutation, present in the same amplified fragment. We were able to identify the G402R mutation during DHPLC analysis, only when a different antisense primer (11a), designed upstream the splicing site, was used for amplification.
3.3. Mutation identification in female patients The mutations P199S and (137-11)T/A has been identified in two female patients (F1 and F2) affected by a mild phenotype of the disease. X-inactivation assay performed of
4. Discussion XLMTM is a severe congenital muscle disorder characterised by severe hypotonia and generalised muscle weakness. Most patients are sporadic cases and die within the first year of life from respiratory failure. The clinical similarities to other disease and the early death of the majority of patients, makes the diagnosis difficult. Differential diagnosis with severe neonatal form of myotonic dystrophy and spinal muscular atrophy should be considered in some cases. Direct mutation analysis is therefore useful to confirm the clinical diagnosis and to offer appropriate genetic counselling to families with a history of miscarriages or neonatal deaths of male infants in the maternal line. Mutation scanning in MTM1 gene is particularly tedious, considering: (i) the large size and exonic fragmentation of the gene; and (ii) the allelic heterogeneity of the disease. The currently used screening protocols are based SSCP analysis and sequencing of anomalous conformers . However, this method seems do not displays high sensitivity and selectivity . We evaluated an alternative method based on DHPLC: a
E. Flex et al. / Neuromuscular Disorders 12 (2002) 501–505
novel automated technique based on the detection of heteroduplexes of PCR products by ion pair reverse-phase HPLC under partially denaturing conditions . We therefore carried out a mutational screening of MTM1 gene in ten unrelated sporadic patients (eight males and two females) and identified the causative mutation in all examined cases. The majority of detected mutations were distributed within exons 8 and 11, affecting residues conserved in a Drosophila ortholog that shares 54% amino acid identity with the human protein . DHPLC mutation analysis did not reveal any false positive results. In fact, direct sequencing showed the presence of a mutation in all the ambiguous elution profiles. A false negative was instead detected during exon 11 analysis. The electropherogram of this exon in patient M6 was similar to that observed in the presence of common polymorphism (1314 1 3)A/G mapping in the donor splice site. The sequence analysis identified in this patient the G402R mutation in addition to the (1314 1 3)A/G polymorphism. Only by using an antisense primer (11a) appositely designed upstream the splicing site, we were able to detect by DHPLC the G402R mutation. The presence of the common (1314 1 3)A/G polymorphism could therefore hide the G402R mutation during standard exon 11 profile elution. Since each amplified fragment could contain more than one melting domain, the mutation detections have been performed at two temperatures, as recommended by the DHPLCMelt software (see exon 15). However, the flexibility of the DHPLC procedure easily overwhelms this drawback. Nevertheless, any deviation from the control elution profile should be considered as a potential mutation. This methodology promises to improve mutational screening of MTM1 mutations and turnaround time in the diagnostic laboratory. We envisage that further refinement of the primers selection and run conditions may expand the capacity in discriminating elution profiles. Acknowledgements We thank the patients with XLMTM, as well as their parents, for their participation; E. Bertini, C. Angelini and G. Castellan for the clinical assessment of patients; and Graziano Bonelli for his expert technical assistance. The Italian Ministry of Health supported this work.
References  Fardeau M, et al. Congenital myopathies. In: Mastaglia, editor. Skeletal muscle pathology, Edinburgh: Churchill Livingstone, 1992.  Laporte J, Hu LJ, Kretz C, et al. A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast. Nat Genet 1996;13(2):175–182.  Laporte J, Guiraud-Chaumeil C, Vincent MC, et al. Mutations in the MTM1 gene implicated in X-linked myotubular myopathy ENMC International Consortium on Myotubular Myopathy European Neuro-Muscular Center. Hum Mol Genet 1997;6(9):1505–1511.  De Gouyon BM, Zhao W, Laporte J, et al. Characterization of mutations in the myotubularin gene in twenty six patients with X-linked myotubular myopathy. Hum Mol Genet 1997;6:1499–1504.  Tanner SM, Schneider V, Thomas NS, et al. Characterization of 34 novel and six known MTM1 gene mutations in 47 unrelated X-linked myotubular myopathy patients. Neuromuscul Disord 1999;9:41–49.  Blondeau F, Laporte J, Bodin S, et al. Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3kinase and phosphatidylinositol 3-phosphate pathway. Hum Mol Genet 2000;9:2223–2229.  Laporte J, Blondeau F, Buj-Bello A, et al. The myotubularin family: from genetic disease to phosphoinositide metabolism. Trends Genet 2001;17:221–228.  Laporte J, Biancalana V, Tanner SM, et al. MTM1 mutations in Xlinked myotubular myopathy. Hum Mutat 2000;15:393–409.  Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography: a review. Hum Mutat 2001;17:439–474.  Sambroock J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1992.  Allen RC, Zoghbi HY, Moseley AB, et al. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgenreceptor gene correlates with X chromosome inactivation. Am J Hum Genet 1992;51:1229–1239.  Novelli G, De Luca A, Torrente I, et al. Mutation Acc H971495. Hum Genet 1999;373.  Novelli G, De Luca A, Torrente I, et al. Mutation Acc H971496. Hum Genet 1999;374.  De Luca A, Torrente I, Mangino M, et al. A novel mutation (R271X) in the myotubularin gene causes a severe miotubular myopathy. Hum Hered 1999;49:59–60.  Nishino I, Minami N, Kobayashi O, et al. MTM1 gene mutations in Japanese patients with the severe infantile form of myotubular myopathy. Neuromuscul Disord 1998;8:453–458.  Herman GE, Finegold M, Zhao W, et al. Medical complications in long-term survivors with X-linked myotubular myopathy. J Pediatr 1999;134(2):206–214.  Laporte J, Blondeau F, Buj-Bello A, et al. Characterization of the myotubularin dual specificity phosphatase gene family from yeast to human. Hum Mol Genet 1998;7:1703–1712.