Surveyor Nuclease Detection of Mutations and Polymorphisms of mtDNA in Children

Surveyor Nuclease Detection of Mutations and Polymorphisms of mtDNA in Children Jacek Pilch, MD, PhD*, Marek Asman, MSc†, Ewa Jamroz, MD, PhD*, Maciej Kajor, MD, PhD‡, El_zbieta Kotrys-Puchalska, PhD§, Ma1gorzata Goss, PhD§, Maria Krzak, MSck, Joanna Witecka, MSck, Jan Gmin´ski, MD, PhD§, and Aleksander L. Sieron´, PhDk Mitochondrial encephalomyopathies are complex disorders with wide range of clinical manifestations. Particularly time-consuming is the identification of mutations in mitochondrial DNA. A group of 20 children with clinical manifestations of mitochondrial encephalomyopathies was selected for molecular studies. The aims were (a) to identify mutations in mtDNA isolated from muscle and (b) to verify detected mutations in DNA isolated from blood, in order to assess the utility of a Surveyor nuclease assay kit for patient screening. The most common changes found were polymorphisms, including a few missense mutations altering the amino acid sequence of mitochondrial proteins. In two boys with MELAS (i.e., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), a mutation A/G3243 was detected in the tRNALeu gene of mtDNA isolated from muscle and blood. In one boy, the carrier status of his mother was confirmed, based on molecular analysis of DNA isolated from blood. A method using Surveyor nuclease allows systematic screening for small mutations in mtDNA, using as its source blood of the patients and asymptomatic carriers. The method still requires confirmation studying a larger group. In some patients, the use of this method should precede and might limit indications for traumatic muscle and skin biopsy. Ó 2010 by Elsevier Inc. All rights reserved. Pilch J, Asman M, Jamroz E, Kajor M, Kotrys-Puchalska E, Goss M, Krzak M, Witecka J, Gmin´ski J, Sieron´ AL. Surveyor nuclease detection of mutations and polymorphisms of mtDNA in children. Pediatr Neurol 2010;43:325-330.

From the Departments of *Child Neurology, ‡Anatomopathology, § Biochemistry, and kGeneral & Molecular Biology & Genetics, Medical University of Silesia, Katowice, Poland; and the †Department of Parasitology, Medical University of Silesia, Sosnowiec, Poland.

Ó 2010 by Elsevier Inc. All rights reserved. doi:10.1016/j.pediatrneurol.2010.05.023  0887-8994/$—see front matter

Introduction Mitochondrial diseases make up an enormously heterogeneous group of disorders, occurring at a frequency of approximately 1 per 5000 births [1-3]; they probably are the most frequent group of neurometabolic diseases in children [4]. Approximately 20% of mitochondrial disorders in childhood are caused by mutations in mtDNA [5]. The majority of cases with clinical suspicion of mitochondrial encephalomyopathies require broad differential diagnosis to exclude myopathies and a number of various metabolic diseases. Clinical symptoms and biochemical, histopathologic, and molecular assays are included in the diagnostic procedures. The diagnosis is particularly difficult in children, because of the varied clinical manifestation of specific mtDNA mutations. Sometimes there is a lack of characteristic clinical symptoms, as well as lack of histopathologic or biochemical changes specific to a given syndrome [5-8]. A pattern of neurogenic muscle atrophy is also found in some patients with mitochondrial disorders [9,10]. Normal results of these assays, however, do not exclude mitochondrial disease [11]. Because the final diagnosis may be confirmed by molecular analyses, in dubious cases identification of a mutation may often be helpful [8,12]. In groups of patients with confirmed mitochondrial disease, analyses of clinical symptoms are conducted together with other laboratory tests, such as biochemical, histopathologic, and molecular studies. Together, the tests help establish diagnostic criteria and scoring systems to facilitate diagnosis of respiratory chain disorders [13-16]. Identification of mitochondrial DNA mutations is particularly difficult because of the wide variation of the

Communications should be addressed to: Dr. Pilch; Department of Child Neurology; Medical University of Silesia; ul. Medyko´w 16; 40-752 Katowice, Poland. E-mail: [email protected] Received November 15, 2009; accepted May 26, 2010.

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nucleotide sequence and the heteroplasmy, and because of a lack of close genotype-phenotype correlation. Skin and muscle biopsy for biochemical analysis and both qualitative and quantitative analyses of muscle mtDNA are of great value. Muscle biopsy is traumatic, however, and in some cases children and their parents find it difficult to accept. There is, therefore, a need for alternative methods enabling the identification of mutations. Various methods are used in the quest for point mutations in the mtDNA, including denaturing high performance liquid chromatography, single-strand conformational polymorphism, temperature gradient gel electrophoresis, denaturing gradient gel electrophoresis, restriction fragment length polymorphism, and allele-specific oligonucleotide screening. Subsequently, if mismatch is detected, the fragment of mtDNA with potential mutation is subjected to DNA sequencing [17,18]. Another method is a direct sequencing of whole mtDNA. The study objective was to identify small mutations in the mtDNA of children with clinical symptoms of mitochondrial encephalomyopathies using their blood for testing. In the screening, the Surveyor endonuclease recognizing single-stranded structures resulting from nucleotide mismatches was used [19]. In particular, two aims of the present study were (a) to identify mutations in mitochondrial DNA isolated from muscle of affected children and (b) to verify the detected mutations in the DNA isolated from blood samples in order to assess the utility of the Surveyor nuclease assay kit to screen the patients for mutations in their mtDNA. Patients and Methods Patients Diagnostic procedures were performed on a group of children with clinical suspicion of a mitochondrial disorder. In all cases, inborn errors of metabolism were excluded. Based on clinical symptoms and on laboratory and electrophysiology test results, including neuroimaging, a group of 20 children was selected for further molecular analyses. In this group, a mixed clinical spectrum was found (Table 1). In the majority of patients (15/20), the disease first manifested prior to or during the child’s 3rd year; in 11 cases, there was clinical suspicion of mitochondrial encephalomyopathy in infancy. Muscle dysfunction was found in most of the children, together with disturbances of the central nervous system. Lactic acidosis was detected in 17/20 cases. Electromyographic examination indicated a myogenic pattern in eight children and neurogenic pattern in one. In most cases, cranial magnetic resonance imaging revealed some abnormality, such as cortical atrophy, demyelination, abnormalities in subcortical nuclei, and infarct-like lesions. In one boy, computed tomography additionally revealed calcifications in basal ganglia. Muscle biopsy was performed in all cases, and histopathologic, biochemical, and ultrastructural analyses were conducted. Histopathologic abnormalities were present in 11/20 children (Table 2). Respiratory chain enzyme activities were assayed in muscles and skin fibroblasts according to standard procedures [20]. These analyses revealed aberrant values in nine cases, mostly for complexes IV and I. In two cases, Leigh syndrome was suspected, but mutations in the SURF1 gene were excluded. In four children with neurogenic muscle atrophy, deletion of exon 7 in the SMN1 gene was excluded. Analysis of the present patients according to mitochondrial disease criteria established by Morava et al. [16] revealed 8 patients has having

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definite, 10 having probable, and 2 having possible mitochondrial disorder (data not shown). In all patients, therefore, clinical suspicion of various mitochondrial syndromes was postulated. Eight of the children died during the follow-up period.

Molecular Analyses In all 20 patients, DNA was isolated from muscle and blood samples. Molecular analyses were performed in two steps for entire mitochondrial DNA. The first step was to screen mitochondrial DNA for any mismatches by heteroduplex analysis using the Surveyor endonuclease (Transgenomic, Omaha, NE) and a set of 19 primers provided in the kit for heteroduplex analysis. Surveyor nuclease, which is a member of a class of plant DNA endonucleases, cleaves DNA with high specificity at sites of base substitution mismatch and DNA distortion [21,22]. Heteroduplexes are formed during hybridization of polymerase chain reaction products derived from wild-type and mutant mtDNA. The Surveyor mutation detection kits allow analysis of all polymerase chain reaction fragments, including long and highly polymorphic amplicons. DNA fragments showing mismatches were subjected to DNA sequencing using an ABI PRISM 310 DNA analyzer (Applied Biosystems, Foster City, CA). The sequence obtained was aligned each time to the reference mtDNA sequence [23]. The differences found in the analyzed mtDNA sequence were verified using two public databases [23,24]. In the individuals with identified mutations in mtDNA isolated from muscle biopsies, the same analytical strategy was repeated on DNA purified from peripheral blood.

Results There were numerous mismatches in the assays of mtDNA obtained from muscle biopsy and performed with the use of Surveyor endonuclease kit. Highly variable regions and regions with low variability or even conserved sequences in the mtDNA were found. All the fragments with mismatches were subjected to DNA sequencing. The most common changes found in all analyzed fragments of mitochondrial DNA were polymorphisms (data not shown). In two boys with MELAS (i.e., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) (cases 4 and 5), an A/G3243 mutation in the gene encoding tRNALeu was detected, which is typical for this syndrome (Table 2). In eight patients, additional deletion or insertion in the MT-TFH region, changes in the MT-RNR2 gene, and in the mtDNA sequence encoding MT-ND2, MT-ND5, and MT-ND6 subunits leading to amino acid substitution were found. In two children (cases 4 and 5) with MELAS and detected mutation in muscle mtDNA, the same analyses were performed in the mtDNA purified from peripheral blood. The assays with the use of Surveyor endonuclease also revealed mismatches. Subsequent sequencing of mtDNA confirmed presence of the same mutation. Additionally, the same analyses were performed on the DNA obtained from peripheral blood of the mother of patient 5, and the A/G 3243 mutation was identified in her mtDNA. Discussion Mitochondrial dysfunction may manifest as abnormal function of most organs and systems of the body [25-27].

Table 1. Clinical spectrum of 20 patients with clinical suspicion of mitochondrial dysfunction Age at Case Sex Onset (at Dx) 1 M

2 M

3 M

4 M

Main Clinical Features

10 F 11 M 12 M

13 M 14 F 15 M

16 F 17 F

Magnetic Resonance Imaging

ENG

Status

Clinical Syndrome

7 mo (19 mo) hypotonia; muscle weakness; muscle atrophy; developmental delay; optic atrophy; excessive sweating; hirsutism; LA 1 mo (2 mo) hypotonia; muscle weakness; myoclonus; atrioventricular hemiblock; dysmorphic features; growth retardation; LA 1 mo (1 mo) hypotonia; epilepsy; cardiomyopathy; LA

myogenic normal

—

died

mitochondrial myopathy

myogenic normal

—

alive

mitochondrial myopathy

—

—

—

died

12 yr (14 yr)

normal

normal

normal

normal

basal ganglia calcifications; died cortical atrophy; infarct-like lesions infarct-like lesions alive

lethal infantile mitochondrial myopathy MELAS

MELAS

demyelination

died

MELAS

died

Leigh-like syndrome

died

Leigh-like syndrome

alive

MERRF

headache; vomitus; muscle weakness; exercise intolerance; stroke-like episodes; mental retardation; LA 5 M 8 yr (9 yr) microcephaly; headache; vomitus; epilepsy; ataxia; stroke-like episodes; mental retardation; LA 6 F 17 mo (21 mo) hemiparesis; retinitis pigmentosa; developmental delay; LA 7 F 2 yr 8 mo progressive encephalopathy; vomitus; (2 yr 10 mo) hypotonia; ataxia; dystonia; epilepsy; LA 8 F 3 mo (6 mo) developmental delay; dystonia; epilepsy; microcephaly; LA

9 F

EMG

3 yr (18 yr)

myoclonic epilepsy; hypoacusis; gastrointestinal dysmotility osteoporosis; LA 16 yr (16 yr) myoclonic epilepsy; LA 9 yr (14 yr) CPEO; exercise intolerance; LA 1 mo (6 mo) hypotonia; muscle weakness; stridor; developmental delay; cardiomyopathy; arrhythmia; LA 1 mo (2 yr) hypotonia; developmental delay; epilepsy; microcephaly; LA 11 mo (2.5 yr) progressive encephalopathy; hypotonia; epilepsy; intermittent course 10 mo (5 yr) alternating hemiplegia; ataxia; epilepsy; retinitis pigmentosa; optic atrophy; developmental delay; LA 2 mo (10 mo) hypotonia; epilepsy; macrocephaly; developmental delay 6 mo (2.5 yr) hypotonia; gastrointestinal dysmotility; renal tubular acidosis; LA

18 F

12 yr (17 yr)

19 F

3 yr (5.5 yr)

20 M

5 mo (4 yr)

respiratory insufficiency; cardiomyopathy; growth retardation; obesity, hypoacusis; endocrine dysfunction; LA hypotonia; exercise intolerance; vomitus; epilepsy; diabetes mellitus; bronchial asthma; hypoacusis; LA epilepsy; developmental delay; ataxia; gastrointestinal dysmotility; endocrine dysfunction; hirsutism

neurogenic normal normal

normal

normal

normal

basal ganglia abnormalities; demyelination normal basal ganglia abnormalities; corpus callosum agenesis poly-phasic demyelination responses

normal normal myogenic normal — —

normal demyelination —

alive alive died

MERRF CPEO encephalomyopathy

myogenic normal

normal

alive

encephalomyopathy

myogenic normal

cortical atrophy

alive

encephalomyopathy

myogenic normal

cortical atrophy

alive

encephalomyopathy

myogenic normal

cortical atrophy

alive

encephalomyopathy

normal

normal

alive

multisystem disorder

normal

normal

cortical atrophy; Dandy-Walker anomaly normal

died

multisystem disorder

myogenic normal

normal

alive

multisystem disorder

normal

cortical atrophy

alive

multisystem disorder

normal

Abbreviations: CPEO = Chronic progressive external ophthalmoplegia Dx = Diagnosis EMG = Electromyography ENG = Electroneurography LA = Lactic acidosis MELAS = Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes MERRF = Myoclonic epilepsy with ragged-red fibers

Pilch et al: mtDNA Assay 327

Table 2. Histopathology, enzymology, and molecular investigations in 20 patients with clinical suspicion of mitochondrial dysfunction

Case

Sex

Muscle Biopsy

Cox

EM

Rare Polymorphisms, Selected/Gene

RCC Defect

1 2 3 4

M M M M

myogenic a. normal normal RRF

() 80% (+) () ()

AM normal AM AM

normal normal normal C1 + C4

— A/G2765/MT-RNR2* — InsAC525-526/MT-TFH

5

M

RRF

() 20%

AM

C1 + C4

—

6 7 8 9

F F F F

normal neurogenic a. normal neurogenic a.

(+) () 20% (+) () 20%

normal normal normal not done

normal C1 + C4 C4 C1 + C4

10 11 12

F M M

normal RRF neurogenic a.

() 20% () (+)

normal AM normal

C4 C4 C4

13 14 15 16 17 18 19 20

M F M F F F F M

normal normal normal neurogenic a. normal normal normal normal

(+) (+) (+) () () (+) (+) (+)

normal normal normal normal normal normal normal normal

normal normal normal normal normal normal normal C2 + C4

— C/C/A2077/MT-RNR2* DelAC523-524/MT-TFH DelAC523-524/MT-TFH; T/C5442; Phe/Leu/ND2 A/G13780 Ile/Val/ND5 — A/G2757/MT-RNR2; T/C14182; Tyr/His/ND6 — — — — — — G/A13708; Ala/Thr/ND5 —

Mutation/ Gene normal normal normal A/G3243/ tRNALeu A/G3243/ tRNALeu normal normal normal normal normal normal normal normal normal normal normal normal normal normal normal

* Asterisk indicates changes not reported as polymorphisms or mutations in the available databases. Abbreviations: a. = Atrophy AM = Abnormal mitochondria = Cytochrome c oxidase Cox EM = Electron microscopy MT-RNR2 = Mitochondrially encoded 16S RNA MT-TFH = Mitochondrially encoded transcription factor binding site H RCC = Respiratory chain complex RRF = Ragged red fibers

Variable clinical patterns reflect heterogeneity of mitochondrial diseases. Very similar clinical manifestations may result from either nuclear or mitochondrial DNA mutations, such as Leigh or maternally inherited Leigh’s syndrome [25,28]. In the group of children studied here, it was supposed that the clinical syndromes are due rather to mutations in mtDNA. Variability of syndromes in this study may be due to the small number of patients. Mitochondrial diseases with mutations in mtDNA manifest more clearly later in life, and in childhood they may have nonspecific initial presentations or may be oligosymptomatic [29-31]. In the present study, only in 5/20 children did histopathologic investigations reveal typical changes for mitochondrial disorders. According to the literature, in patients with confirmed mutations in mtDNA or alterations in oxidative phosphorylation the histopathologic pattern may not show changes specific for mitochondrial encephalomyopathies [11,16,32-38]. It seems that the diagnostic extension to molecular analyses in patients with clinical suspicion of mitochondrial disease is important [18,39].

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The gold standard in point mutation screening of mtDNA is sequencing. This is time-consuming, however, and it cannot detect mutations present at a low heteroplasmic level (<20%) [40,41]. Surveyor nuclease is used for mapping of known and unknown variations in genomes, whether these are base substitutions or small insertions or deletions up to at least 12 nucleotides [19]. It is a highly sensitive method, allowing detection of the changes in just a few copies of the template, as little as 1 in 32 copies (3%) [42]. The method provides for a fast and systematic analysis of the entire mtDNA, and seems to be particularly useful for screening of mutations in mtDNA because of the presence of heteroplasmy. Surveyor nuclease was applied for the first time for mtDNA search by Bannwarth et al. [42] in 2005. Subsequently, the utility of this method has been reported by the same research group in other studies [43,44]. This technique cannot detect homoplasmic mutations; however, pathogenic mutations are most frequently found in a heteroplasmic state [44]. Although in some of the children in the present study there was a lack of strong evidence indicating

mitochondrial encephalomyopathy, further screening at the molecular level was conducted, including mtDNA sequencing. The findings revealed presence of nucleotide changes causing heteroduplex formation in different regions of mtDNA of all patients. The most common changes were polymorphisms. In three patients (cases 4, 5, and 6), MELAS was postulated based on clinical manifestations; in two of them, two boys, a mutation A/G3243 in the tRNALeu gene was detected. Additionally, histopathologic and biochemical analyses in these boys revealed typical abnormalities, confirming mitochondrial disorder. One proband did have family history of the disease. His mother revealed first symptoms of MELAS when she was 30. She died during progress of disease, but molecular screening of the mtDNA isolated from blood few years ago did not confirm any mutation. Subsequent analysis of the muscle biopsy and blood in her son (case 4) revealed the mutation in mtDNA. In the other boy (case 5) the screening of mtDNA isolated from the blood detected abnormality confirmed by sequencing of DNA, too. Additionally, the carrier status of his mother was confirmed. In the genes ND2, ND5, and ND6, a few missense mutations were detected, altering the amino acid sequence of mitochondrial proteins; however, no negative effect of these mutations at the protein level has yet been confirmed. In the Mitomap and mtDB databases, the mutations reported here were categorized as polymorphisms [23,24]. Criteria for estimation of mtDNA mutation pathogenicity have been developed separately for tRNA and for proteinencoding genes [45,46]. Due to variability of the mtDNA, any change in its sequence is potentially pathogenic, including amino acid changes that require confirmation by other laboratory methods. In six children, deletion or insertion in the mitochondrially encoded transcription factor binding site region and nucleotide substitutions in the 16S ribosomal RNA gene were identified, but these changes also were determined in the databases to be polymorphisms [23,24]. In parallel with the search for mutations in mtDNA, analyses and assessment of detected polymorphisms, based on their effect on mitochondrial functions, are conducted. Kazuno et al. [47] reported that some polymorphisms may disturb matrix pH and movement of calcium ions. Some other polymorphisms may be linked to predisposition to disease, such as type 2 diabetes or obesity [48], and some may even affect longevity [49]. Further investigations to explain of the role of polymorphisms are merited. Mutations in mtDNA have been identified in only 20% of children with abnormalities in oxidative phosphorylation, as deletions, duplications, or other point mutations [5,14]. Qi et al. [6], using other molecular methods, investigated five of the most frequent mutations in mtDNA by screening a group of 552 patients with manifestations typical for mitochondrial myopathy, encephalopathy, lactic acidosis with stroke-like episodes, myoclonic epilepsy with ragged-red fibers, or Leigh syndrome; mutations were identified in 64/552 patients (11.6%). The present study, using

the Surveyor nuclease method with a more heterogeneous group of patients, achieved similar results: 2/20 patients (10%). Conclusions Molecular analysis with Surveyor nuclease is fast and sensitive, and can detect mutations located in any gene of mtDNA. In the present study, it allowed systematic screening for point mutations and small deletions or insertions in mtDNA extracted from blood; however, confirmation studying a larger group of patients is required. This method can detect carrier status of family members, because it can identify low percentage heteroplasmy, and it even could be useful in prenatal diagnostics. In some patients with a clinical phenotype suggestive of mitochondrial disease and suspected for mutations in mtDNA, the first step in diagnostic procedure should be use of the Surveyor nuclease method for mutation screening in DNA extracted from blood. In some patients, it should precede and could thereby limit indications for traumatic muscle and skin biopsy, especially in children. The authors thank Prof. El_zbieta Marsza1 for clinical support, Dr. Dorota Abramczuk (Department of Medical Genetics, Children’s Memorial Health Institute, Warsaw) for molecular analysis of the SURF1 gene in patients 7 and 8, and Dr. Janusz Zimowski (Genetics Department, Institute of Psychiatry and Neurology, Warsaw) for molecular analysis of SMN1 gene in patients 7, 9, 12, and 16. The authors are grateful also to the patients and their families for collaboration in the study. The study was supported in part from grants by the Ministry of Science and Higher Education 2PO5E 087 26, European Union Regional Structural Funds WKP1/1.4/3/2/2005/103/223/565/2007/U, and institutional funding NN-1-014/06.

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