- Email: [email protected]
Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion
Molecular Genetics and Metabolism 99 (2010) 53–57
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion Shulin Zhang a, Fang-yuan Li a, Harold N. Bass b, Amber Pursley a, Eric S. Schmitt a, Blaire L. Brown b, Ellen K. Brundage a, Rebecca Mardach b, Lee-Jun Wong a,* a b
Medical Genetics Laboratories, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States Department of Genetics, Kaiser Permanente Medical Program, Bakersfield, Los Angeles and Panorama City, CA, United States
a r t i c l e
i n f o
Article history: Received 3 August 2009 Received in revised form 9 September 2009 Accepted 9 September 2009 Available online 13 September 2009 Keywords: TK2 deletion Array CGH mtDNA depletion
a b s t r a c t Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T > A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 50 UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1–495 to c.283–2899 of the TK2 gene (nucleotide 65,136,256–65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6 months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects. Ó 2009 Elsevier Inc. All rights reserved.
Introduction The mitochondrial deoxyribonucleotide (dNTP) pool is separated from the cytosolic pool due to impermeability of the mitochondrial inner-membrane to charged molecules. The mitochondrial dNTP pool is maintained by either the import of cytosolic dNTPs through dedicated transporters or through salvaging the deoxynucleosides within mitochondria [1]. Two of the four human deoxyribonucleoside kinases, deoxyguanosine kinase (DGK) and thymidine kinase 2 (TK2), are expressed in mitochondria. Human DGK, encoded by the DGUOK gene, efficiently phosphorylates deoxyguanosine and deoxyadenosine, whereas TK2 phosphorylates deoxythymidine, deoxycytidine, and deoxyuridine.
* Corresponding author. Address: Department of Molecular and Human Genetics, Mitochondrial Diagnostic Laboratory, Baylor College of Medicine, One Baylor Plaza, NAB 2015, Houston, TX 77030, United States. Fax: +1 713 798 8937. E-mail address: [email protected] (L.-J. Wong). 1096-7192/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2009.09.003
Mitochondrial DNA (mtDNA) depletion syndrome (MDDS, OMIM 251880) is a clinically heterogeneous group of hereditary disorders characterized by a decreased quantity of mtDNA in affected tissues, which in turn impairs the synthesis of mtDNA-encoded respiratory chain components [2,3]. Depending on the tissues involved, clinical MDDS can be classified as myopathic, hepatocerebral or encephalomyopathic [4]. Defects of genes responsible for either mtDNA replication or the maintenance of mitochondrial dNTP pools can cause mtDNA depletion syndrome. Currently, defects in nine genes, including POLG, TYMP, DGUOK, TK2, SUCLA2, SULG1, RRM2B, MPV17, and C10ORF2, have been identified in patients with MDDS [5]. This list is likely to expand with greater understanding of mtDNA biosynthesis. TK2 deficiency has mainly been reported in the myopathic form of MDDS, including severe infantile myopathy, a spinal muscular atrophy type 3-like presentation, rigid spine syndrome, and subacute myopathy without motor regression [6]. To date, 23 mutations have been reported in TK2-deficient patients (HGMDÒ
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S. Zhang et al. / Molecular Genetics and Metabolism 99 (2010) 53–57
B
A
1
I
II
1
2
2
3
Fig. 1. (A) A non-consanguineous family with three children deceased due to mitochondrial depletion syndrome. The third child was diagnosed prenatally. (B) Sequence analysis revealed a heterozygous c.761T > C (p.I254N) mutation of TK2 gene in I-1, II-2 and II-3 (top) but negative in I-2 (bottom).
Professional Release 2009.1; https://portal.biobase-international.com/hgmd/pro/start.php). These consist of 18 point mutations with the remainders being are small (1–4 nucleotides) insertions or deletions. In this report, a 5.8-kb deletion in the TK2 gene was identified using a novel custom oligonucleotide array comparative genomic hybridization (CGH1). Depletion of mtDNA in both muscle and liver tissues was also detected by this assay. Methods Sanger sequencing was carried out using a 3730XL DNA analyzer from Applied Biosystems Inc. according to manufacturer’s instructions (Applied Biosystems Inc. Foster City, CA). Gene-specific primers linked to M13 universal primers were custom designed and validated for clinical use. A custom designed clinical oligonucleotide array CGH was manufactured using Agilent microarray platform (Agilent Technologies, Santa Clara, CA). This is 44K oligonucleotide array with a complete coverage of mitochondrial genome (6400 probes for the 16.6-kb genome) and about 130 nuclear genes related to mitochondrial functions and metabolic diseases at an average spacing of approximately 250 bp/ oligonucleotide probe [7,8]. The mtDNA content was confirmed by real-time quantitative PCR using age- and tissue-matched controls [9,10]. Case report This is a non-consanguineous Jordanian family (Fig. 1A); both I1 and I-2 are in good health except I-2 is a thalassemia carrier. II-1 and II-2 were born after an unremarkable full term pregnancy and uncomplicated delivery. The infants developed progressive hypotonia and weakness accompanied by loss of deep-tendon reflexes at the age of 3–4 months and gradually deteriorated until death from pneumonia at 8 months. II-2 had elevated creatine kinase activity of 3600 U/L. SMN1 analysis for possible spinal muscular atrophy type I and mitochondrial genome sequencing were negative. Muscle tissue from the proband, II-1, who received all of his care in Jordan, revealed modest fiber-size variation with minimal endomysial and perimysial fibrosis. Increased lipid was present. Succinate dehydrogenase, Gomori trichrome staining, myophosphorylase, NADH tetrazolium reductase, ATPase, and glycogen were appropriate. On electron microscopy, disrupted myofilaments were noted, some of which were associated with abnormal 1 Abbreviations used: CGH, comparative genomic hybridization; mtDNA, mitochondrial DNA; MDDS, mitochondrial DNA depletion syndrome.
mitochondrial architecture, including megaconial and concentric ringed forms. A liver biopsy performed at age 7 months revealed patchy periportal inflammation. II-2, who received his care in the United States, had a course similar to II-1, with onset of neurologic symptoms at 3 months and death from pneumonia at 8 months. He had a mild lactic acidosis of 2.5 mmol/L (normal range 0.6–2.2 mmol/L) and elevated liver enzymes. ALT was 217 IU/L (normal <35 IU/L) and AST, 304 IU/L (normal range 10–40 IU/L). Muscle and liver biopsies sent to Columbia University in New York revealed marked mtDNA depletion, compatible with MDDS. MDDS in III-3 was diagnosed prenatally through amniocentesis and confirmed postnatally in cord blood. Following a normal delivery, the infant began to show signs of hypotonia and weakness by age of 3 months and died of respiratory failure at 6 months. Serum ALT and AST were 96 IU/L (normal <35 IU/L) and 84 IU/L (normal range 17–63 IU/L), respectively. At the parents’ request, no biopsy or autopsy was performed. Results Since the proband and his sibling were deceased, sequence analysis of genes responsible for mtDNA depletion was performed on the parents. Mutations were not detected in the POLG, DGUOK, and SUCLA2 genes. Sequence analysis of the TK2 gene detected a c.761T > A (p.I254N) mutation in the father but was negative for the mother (Fig. 1B). The c.761T > A (p.I254N) mutation has been reported in three patients with the myopathic form of MDDS [11]. Since the myopathic form of the mtDNA depletion syndrome is an autosomal recessive disorder, and only one mutant allele was detected, other genetic mechanisms possibly underlying TK2 deficiency were pursued. An oligonucleotide array CGH analysis was used to evaluate for large intragenic deletions [7], and a deletion of approximately 5.8 kb was detected in the TK2 gene (Fig. 2A). The deletion extended from 50 UTR to intron 2 of the TK2 gene. Subsequent sequence analysis of the deletion junction confirmed that the deletion spans from c.1–495 to c.283–2899 of the TK2 gene (nucleotide 65,136,256–65,142,086 of chromosome 16) (Fig. 3). A 49-bp intervening sequence was located between these two junctions, although the origin of this sequence is unknown. Direct repeats of the flanking sequences of the break points were not detected. Targeted mutation analysis of muscle and liver specimens from patient II-2 revealed compound heterozygosity for the c.761T > A (p.I254N) mutation and 5.8 kb deletion (Figs. 1B and 2A). The same oligonucleotide array CGH analysis also detected a significant reduction in mtDNA content in muscle and liver specimens of II-2, but not in blood specimens from the parents (Fig. 2B). Real-time quantitative PCR analysis revealed the mtDNA content to
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Fig. 2. (A) Nuclear gene profile of array CGH, an approximately 5.8 Kb intragenic deletion of the TK2 gene in the blood specimen of I-2, muscle and liver of II-2, amniocytes of II-3 were detected. No TK2 copy number abnormalities were detected in I-1. (B) Mitochondrial genome profile obtained from the same array. A significant reduction of mtDNA copy number in the muscle and liver specimens of II-2 was observed (mtDNA profile shifted from central line to 2 on log 2 scale. mtDNA contents are estimated to be approximately 16% and 20% of controls by this assay). The mtDNA content of I-2 and I-1 appears to be at the similar level as normal controls (central line), whereas the mtDNA content is not informative for amniocytes of II-3 due to the unavailability of appropriately matched control tissue (blood was used in this case).
be about 20% and 10% of age- and tissue-matched controls in muscle and liver specimens, respectively (Table 1). Discussion The mtDNA depletion syndrome is characterized by quantitative insufficiency of mtDNA in affected tissues [3]. Most of the patients
present in the neonatal period with muscle weakness, hepatic failure, or renal tubulopathy associated with lactic academia, and die during the first year of life; others may present with isolated myopathy associated with motor function regression or a slowly progressive encephalomyopathy that begins in early childhood [12–16]. Deficiency of TK2 in humans has been reported mainly in the myopathic form of mtDNA depletion syndrome [11]. To date, 23 muta-
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S. Zhang et al. / Molecular Genetics and Metabolism 99 (2010) 53–57
E1
E2
E6
E5
E4
E3
E7
E8
E10
E9
Red are existing probes
19probes
R-primer F-primer
E1
E3
E2
65,142,086
65,136,256 c.1-495 ((ch.16:65,142,086) , , ) 49bp inserted sequence
c.283-2899 c.283 2899 (ch.16: 65,136,256)
Fig. 3. Determination of the deletion junction. The upper part shows the genomic structure of TK2 gene and the PCR primer positions. Middle part is the schematic array CGH profile which shows the deleted probes (black). The lower section shows the sequencing results of deletion junction.
Table 1 Quantitative PCR analysis of the mtDNA contents of muscle and liver tissues of II-2.
Muscle Liver a b
Ct of nuclear ß2M gene
Average Cta
Ct of mt tRNA Leu (UUR) gene
Average Ct
Difference of Ct value between ß2M gene and tRNA gene (dCt)
mtDNA content in this patient (2 2dCt)
mtDNA content in controls (2 2dCt)b
Percentage of mtDNA content of the patient relative to controls (%)
28.7 28.6
28.3 ± 0.6 28.4 ± 0.3
20.7 21.3
20.9 ± 0.2 21.5 ± 0.4
7.4 6.9
345 235
1746 ± 361 2252 ± 620
20 10
27.8 28.2
21.0 21.8
Ct, threshold cycle number. The mtDNA content in controls were determined based on 20 and 10 pooled age-matched muscle and liver samples, respectively.
tions have been uncovered in TK2-deficient patients. All have either been point mutations or small insertions or deletions. In this paper, we report a novel intragenic 5.8-kb deletion, which is usually not detectable by conventional sequence analysis. This result thus expands the mutational spectrum of the TK2 gene. Sanger sequencing is the ‘‘gold standard” for detecting point mutations and small insertions and deletions. This technique does not detect intragenic deletions. The technical limitations of Sanger sequencing may sometimes cause diagnostic difficulties for autosomal recessive disorders when only one mutant allele was detected. Therefore, when clinically indicated, dosage analysis is a necessary diagnostic component. Different molecular platforms have been developed to address gene dosage abnormalities, including MLPA and real-time PCR. Limitations of these platforms include product availability of specific genes, limited number of probes per exon, time-consuming probe design and inability to accurately define the deletion/duplication boundary. Recently, customized oligonucleotide array CGH has been developed in a clinical setting to address copy number abnormalities at the whole genome, as well as at the exonic level [17–20]. Our custom oligonucleotide array CGH not only assays genes involved in mitochondrial function, but assays the entire mitochondrial genome [7,8]. This oligonucleotide array CGH simultaneously detects copy number changes in both mitochondrial and nuclear genes involved in mitochondrial biogenesis and function. In addition, by using tissue-matched controls, mtDNA content can be estimated. In this report, the simultaneous detection of an intragenic deletion in the TK2 gene and mtDNA depletion demonstrated the utility of this oligonucleotide array CGH. The dense coverage of the oligonucleotide probes also can provide an estimate of the deletion break points. Subsequent PCR of the deletion junction followed by Sanger sequencing can then precisely determine the deletion junction. This custom aCGH
has been applied to detect copy number changes in a patient with hepatocerebral form of mtDNA depletion syndrome [10]. While TK2 is broadly expressed in various tissues [21,22], myopathy seems to be the major phenotype of TK2 deficiency. In our report, a significant mtDNA copy number decrease in the liver, along with increased hepatic transaminases, suggests that liver may also be affected by TK2 deficiency. In conclusion, this report demonstrates that TK2 deficiency can result from both point mutations and intragenic deletions; that hepatic involvement may occur in addition to myopathy; and that custom oligonucleotide array CGH is a valuable tool for the simultaneous detection of gene copy number abnormalities in both nuclear and mitochondrial genomes. Disclaimer Medical Genetics Laboratories at Baylor College of Medicine provide fee-based service for the TK2 gene sequencing and array CGH analysis. References [1] C.K. Mathews, S. Song, Maintaining precursor pools for mitochondrial DNA replication, FASEB J. 21 (2007) 2294–2303. [2] S. Dimauro, C.T. Moraes, S. Shanske, A. Lombes, H. Nakase, S. Mita, H.J. Tritschler, E. Bonilla, A.F. Miranda, E.A. Schon, Mitochondrial encephalomyopathies: biochemical approach, Rev. Neurol. (Paris) 147 (1991) 443–449. [3] C.T. Moraes, S. Shanske, H.J. Tritschler, J.R. Aprille, F. Andreetta, E. Bonilla, E.A. Schon, S. DiMauro, MtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases, Am. J. Hum. Genet. 48 (1991) 492–501. [4] A. Spinazzola, F. Invernizzi, F. Carrara, E. Lamantea, A. Donati, M. Dirocco, I. Giordano, M. Meznaric-Petrusa, E. Baruffini, I. Ferrero, Clinical and molecular
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[5] [6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
features of mitochondrial DNA depletion syndromes, J. Inherit. Metab. Dis. 32 (2009) 143–158. S. Rahman, J. Poulton, Diagnosis of mitochondrial DNA depletion syndromes, Arch. Dis. Child. 94 (2009) 3–5. M. Oskoui, G. Davidzon, J. Pascual, R. Erazo, J. Gurgel-Giannetti, S. Krishna, E. Bonilla, D.C. De Vivo, S. Shanske, S. DiMauro, Clinical spectrum of mitochondrial DNA depletion due to mutations in the thymidine kinase 2 gene, Arch. Neurol. 63 (2006) 1122–1126. L.J. Wong, D. Dimmock, M.T. Geraghty, R. Quan, U. Lichter-Konecki, J. Wang, E.K. Brundage, F. Scaglia, A.C. Chinault, Utility of oligonucleotide array-based comparative genomic hybridization for detection of target gene deletions, Clin. Chem. 54 (2008) 1141–1148. A.C. Chinault, C.A. Shaw, E.K. Brundage, L.Y. Tang, L.J. Wong, Application of dual-genome oligonucleotide array-based comparative genomic hybridization to the molecular diagnosis of mitochondrial DNA deletion and depletion syndromes, Genet. Med. 11 (2009) 518–526. R.K. Bai, L.J. Wong, Simultaneous detection and quantification of mitochondrial DNA deletion(s), depletion, and over-replication in patients with mitochondrial disease, J. Mol. Diagn. 7 (2005) 613–622. N.C. Lee, D. Dimmock, W.L. Hwu, L.Y. Tang, W.C. Huang, A.C. Chinault, L.J. Wong, Simultaneous detection of mitochondrial DNA depletion and singleexon deletion in the deoxyguanosine gene using array-based comparative genomic hybridisation, Arch. Dis. Child. 94 (2009) 55–58. A. Saada, A. Shaag, H. Mandel, Y. Nevo, S. Eriksson, O. Elpeleg, Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy, Nat. Genet. 29 (2001) 342–344. J.C. Blake, J.W. Taanman, A.M. Morris, R.G. Gray, J.M. Cooper, P.J. McKiernan, J.V. Leonard, A.H. Schapira, Mitochondrial DNA depletion syndrome is expressed in amniotic fluid cell cultures, Am. J. Pathol. 155 (1999) 67–70. P.H. Ducluzeau, A. Lachaux, R. Bouvier, N. Streichenberger, G. Stepien, B. Mousson, Depletion of mitochondrial DNA associated with infantile cholestasis and progressive liver fibrosis, J. Hepatol. 30 (1999) 149–155.
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[14] J.W. Taanman, A.G. Bodnar, J.M. Cooper, A.A. Morris, P.T. Clayton, J.V. Leonard, A.H. Schapira, Molecular mechanisms in mitochondrial DNA depletion syndrome, Hum. Mol. Genet. 6 (1997) 935–942. [15] H.J. Tritschler, F. Andreetta, C.T. Moraes, E. Bonilla, E. Arnaudo, M.J. Danon, S. Glass, B.M. Zelaya, E. Vamos, N. Telerman-Toppet, Mitochondrial myopathy of childhood associated with depletion of mitochondrial DNA, Neurology 42 (1992) 209–217. [16] T.H. Vu, M. Sciacco, K. Tanji, C. Nichter, E. Bonilla, S. Chatkupt, P. Maertens, S. Shanske, J. Mendell, M.R. Koenigsberger, Clinical manifestations of mitochondrial DNA depletion, Neurology 50 (1998) 1783–1790. [17] A.L. Beaudet, J.W. Belmont, Array-based DNA diagnostics: let the revolution begin, Annu. Rev. Med. 59 (2008) 113–129. [18] S.W. Cheung, C.A. Shaw, D.A. Scott, A. Patel, T. Sahoo, C.A. Bacino, A. Pursley, J. Li, R. Erickson, A.L. Gropman, Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics, Am. J. Med. Genet. A 143A (2007) 1679–1686. [19] X. Lu, C.A. Shaw, A. Patel, J. Li, M.L. Cooper, W.R. Wells, C.M. Sullivan, T. Sahoo, S.A. Yatsenko, C.A. Bacino, Clinical implementation of chromosomal microarray analysis: summary of 2513 postnatal cases, PLoS ONE 2 (2007) e327. [20] P. Stankiewicz, A.L. Beaudet, Use of array CGH in the evaluation of dysmorphology, malformations, developmental delay, and idiopathic mental retardation, Curr. Opin. Genet. Dev. 17 (2007) 182–192. [21] L. Wang, B. Munch-Petersen, A. Herrstrom Sjoberg, U. Hellman, T. Bergman, H. Jornvall, S. Eriksson, Human thymidine kinase 2: molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates, FEBS Lett. 443 (1999) 170–174. [22] L. Wang, A. Saada, S. Eriksson, Kinetic properties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy, J. Biol. Chem. 278 (2003) 6963–6968.
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion Shulin Zhang a, Fang-yuan Li a, Harold N. Bass b, Amber Pursley a, Eric S. Schmitt a, Blaire L. Brown b, Ellen K. Brundage a, Rebecca Mardach b, Lee-Jun Wong a,* a b
Medical Genetics Laboratories, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States Department of Genetics, Kaiser Permanente Medical Program, Bakersfield, Los Angeles and Panorama City, CA, United States
a r t i c l e
i n f o
Article history: Received 3 August 2009 Received in revised form 9 September 2009 Accepted 9 September 2009 Available online 13 September 2009 Keywords: TK2 deletion Array CGH mtDNA depletion
a b s t r a c t Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T > A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 50 UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1–495 to c.283–2899 of the TK2 gene (nucleotide 65,136,256–65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6 months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects. Ó 2009 Elsevier Inc. All rights reserved.
Introduction The mitochondrial deoxyribonucleotide (dNTP) pool is separated from the cytosolic pool due to impermeability of the mitochondrial inner-membrane to charged molecules. The mitochondrial dNTP pool is maintained by either the import of cytosolic dNTPs through dedicated transporters or through salvaging the deoxynucleosides within mitochondria [1]. Two of the four human deoxyribonucleoside kinases, deoxyguanosine kinase (DGK) and thymidine kinase 2 (TK2), are expressed in mitochondria. Human DGK, encoded by the DGUOK gene, efficiently phosphorylates deoxyguanosine and deoxyadenosine, whereas TK2 phosphorylates deoxythymidine, deoxycytidine, and deoxyuridine.
* Corresponding author. Address: Department of Molecular and Human Genetics, Mitochondrial Diagnostic Laboratory, Baylor College of Medicine, One Baylor Plaza, NAB 2015, Houston, TX 77030, United States. Fax: +1 713 798 8937. E-mail address: [email protected] (L.-J. Wong). 1096-7192/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2009.09.003
Mitochondrial DNA (mtDNA) depletion syndrome (MDDS, OMIM 251880) is a clinically heterogeneous group of hereditary disorders characterized by a decreased quantity of mtDNA in affected tissues, which in turn impairs the synthesis of mtDNA-encoded respiratory chain components [2,3]. Depending on the tissues involved, clinical MDDS can be classified as myopathic, hepatocerebral or encephalomyopathic [4]. Defects of genes responsible for either mtDNA replication or the maintenance of mitochondrial dNTP pools can cause mtDNA depletion syndrome. Currently, defects in nine genes, including POLG, TYMP, DGUOK, TK2, SUCLA2, SULG1, RRM2B, MPV17, and C10ORF2, have been identified in patients with MDDS [5]. This list is likely to expand with greater understanding of mtDNA biosynthesis. TK2 deficiency has mainly been reported in the myopathic form of MDDS, including severe infantile myopathy, a spinal muscular atrophy type 3-like presentation, rigid spine syndrome, and subacute myopathy without motor regression [6]. To date, 23 mutations have been reported in TK2-deficient patients (HGMDÒ
54
S. Zhang et al. / Molecular Genetics and Metabolism 99 (2010) 53–57
B
A
1
I
II
1
2
2
3
Fig. 1. (A) A non-consanguineous family with three children deceased due to mitochondrial depletion syndrome. The third child was diagnosed prenatally. (B) Sequence analysis revealed a heterozygous c.761T > C (p.I254N) mutation of TK2 gene in I-1, II-2 and II-3 (top) but negative in I-2 (bottom).
Professional Release 2009.1; https://portal.biobase-international.com/hgmd/pro/start.php). These consist of 18 point mutations with the remainders being are small (1–4 nucleotides) insertions or deletions. In this report, a 5.8-kb deletion in the TK2 gene was identified using a novel custom oligonucleotide array comparative genomic hybridization (CGH1). Depletion of mtDNA in both muscle and liver tissues was also detected by this assay. Methods Sanger sequencing was carried out using a 3730XL DNA analyzer from Applied Biosystems Inc. according to manufacturer’s instructions (Applied Biosystems Inc. Foster City, CA). Gene-specific primers linked to M13 universal primers were custom designed and validated for clinical use. A custom designed clinical oligonucleotide array CGH was manufactured using Agilent microarray platform (Agilent Technologies, Santa Clara, CA). This is 44K oligonucleotide array with a complete coverage of mitochondrial genome (6400 probes for the 16.6-kb genome) and about 130 nuclear genes related to mitochondrial functions and metabolic diseases at an average spacing of approximately 250 bp/ oligonucleotide probe [7,8]. The mtDNA content was confirmed by real-time quantitative PCR using age- and tissue-matched controls [9,10]. Case report This is a non-consanguineous Jordanian family (Fig. 1A); both I1 and I-2 are in good health except I-2 is a thalassemia carrier. II-1 and II-2 were born after an unremarkable full term pregnancy and uncomplicated delivery. The infants developed progressive hypotonia and weakness accompanied by loss of deep-tendon reflexes at the age of 3–4 months and gradually deteriorated until death from pneumonia at 8 months. II-2 had elevated creatine kinase activity of 3600 U/L. SMN1 analysis for possible spinal muscular atrophy type I and mitochondrial genome sequencing were negative. Muscle tissue from the proband, II-1, who received all of his care in Jordan, revealed modest fiber-size variation with minimal endomysial and perimysial fibrosis. Increased lipid was present. Succinate dehydrogenase, Gomori trichrome staining, myophosphorylase, NADH tetrazolium reductase, ATPase, and glycogen were appropriate. On electron microscopy, disrupted myofilaments were noted, some of which were associated with abnormal 1 Abbreviations used: CGH, comparative genomic hybridization; mtDNA, mitochondrial DNA; MDDS, mitochondrial DNA depletion syndrome.
mitochondrial architecture, including megaconial and concentric ringed forms. A liver biopsy performed at age 7 months revealed patchy periportal inflammation. II-2, who received his care in the United States, had a course similar to II-1, with onset of neurologic symptoms at 3 months and death from pneumonia at 8 months. He had a mild lactic acidosis of 2.5 mmol/L (normal range 0.6–2.2 mmol/L) and elevated liver enzymes. ALT was 217 IU/L (normal <35 IU/L) and AST, 304 IU/L (normal range 10–40 IU/L). Muscle and liver biopsies sent to Columbia University in New York revealed marked mtDNA depletion, compatible with MDDS. MDDS in III-3 was diagnosed prenatally through amniocentesis and confirmed postnatally in cord blood. Following a normal delivery, the infant began to show signs of hypotonia and weakness by age of 3 months and died of respiratory failure at 6 months. Serum ALT and AST were 96 IU/L (normal <35 IU/L) and 84 IU/L (normal range 17–63 IU/L), respectively. At the parents’ request, no biopsy or autopsy was performed. Results Since the proband and his sibling were deceased, sequence analysis of genes responsible for mtDNA depletion was performed on the parents. Mutations were not detected in the POLG, DGUOK, and SUCLA2 genes. Sequence analysis of the TK2 gene detected a c.761T > A (p.I254N) mutation in the father but was negative for the mother (Fig. 1B). The c.761T > A (p.I254N) mutation has been reported in three patients with the myopathic form of MDDS [11]. Since the myopathic form of the mtDNA depletion syndrome is an autosomal recessive disorder, and only one mutant allele was detected, other genetic mechanisms possibly underlying TK2 deficiency were pursued. An oligonucleotide array CGH analysis was used to evaluate for large intragenic deletions [7], and a deletion of approximately 5.8 kb was detected in the TK2 gene (Fig. 2A). The deletion extended from 50 UTR to intron 2 of the TK2 gene. Subsequent sequence analysis of the deletion junction confirmed that the deletion spans from c.1–495 to c.283–2899 of the TK2 gene (nucleotide 65,136,256–65,142,086 of chromosome 16) (Fig. 3). A 49-bp intervening sequence was located between these two junctions, although the origin of this sequence is unknown. Direct repeats of the flanking sequences of the break points were not detected. Targeted mutation analysis of muscle and liver specimens from patient II-2 revealed compound heterozygosity for the c.761T > A (p.I254N) mutation and 5.8 kb deletion (Figs. 1B and 2A). The same oligonucleotide array CGH analysis also detected a significant reduction in mtDNA content in muscle and liver specimens of II-2, but not in blood specimens from the parents (Fig. 2B). Real-time quantitative PCR analysis revealed the mtDNA content to
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Fig. 2. (A) Nuclear gene profile of array CGH, an approximately 5.8 Kb intragenic deletion of the TK2 gene in the blood specimen of I-2, muscle and liver of II-2, amniocytes of II-3 were detected. No TK2 copy number abnormalities were detected in I-1. (B) Mitochondrial genome profile obtained from the same array. A significant reduction of mtDNA copy number in the muscle and liver specimens of II-2 was observed (mtDNA profile shifted from central line to 2 on log 2 scale. mtDNA contents are estimated to be approximately 16% and 20% of controls by this assay). The mtDNA content of I-2 and I-1 appears to be at the similar level as normal controls (central line), whereas the mtDNA content is not informative for amniocytes of II-3 due to the unavailability of appropriately matched control tissue (blood was used in this case).
be about 20% and 10% of age- and tissue-matched controls in muscle and liver specimens, respectively (Table 1). Discussion The mtDNA depletion syndrome is characterized by quantitative insufficiency of mtDNA in affected tissues [3]. Most of the patients
present in the neonatal period with muscle weakness, hepatic failure, or renal tubulopathy associated with lactic academia, and die during the first year of life; others may present with isolated myopathy associated with motor function regression or a slowly progressive encephalomyopathy that begins in early childhood [12–16]. Deficiency of TK2 in humans has been reported mainly in the myopathic form of mtDNA depletion syndrome [11]. To date, 23 muta-
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S. Zhang et al. / Molecular Genetics and Metabolism 99 (2010) 53–57
E1
E2
E6
E5
E4
E3
E7
E8
E10
E9
Red are existing probes
19probes
R-primer F-primer
E1
E3
E2
65,142,086
65,136,256 c.1-495 ((ch.16:65,142,086) , , ) 49bp inserted sequence
c.283-2899 c.283 2899 (ch.16: 65,136,256)
Fig. 3. Determination of the deletion junction. The upper part shows the genomic structure of TK2 gene and the PCR primer positions. Middle part is the schematic array CGH profile which shows the deleted probes (black). The lower section shows the sequencing results of deletion junction.
Table 1 Quantitative PCR analysis of the mtDNA contents of muscle and liver tissues of II-2.
Muscle Liver a b
Ct of nuclear ß2M gene
Average Cta
Ct of mt tRNA Leu (UUR) gene
Average Ct
Difference of Ct value between ß2M gene and tRNA gene (dCt)
mtDNA content in this patient (2 2dCt)
mtDNA content in controls (2 2dCt)b
Percentage of mtDNA content of the patient relative to controls (%)
28.7 28.6
28.3 ± 0.6 28.4 ± 0.3
20.7 21.3
20.9 ± 0.2 21.5 ± 0.4
7.4 6.9
345 235
1746 ± 361 2252 ± 620
20 10
27.8 28.2
21.0 21.8
Ct, threshold cycle number. The mtDNA content in controls were determined based on 20 and 10 pooled age-matched muscle and liver samples, respectively.
tions have been uncovered in TK2-deficient patients. All have either been point mutations or small insertions or deletions. In this paper, we report a novel intragenic 5.8-kb deletion, which is usually not detectable by conventional sequence analysis. This result thus expands the mutational spectrum of the TK2 gene. Sanger sequencing is the ‘‘gold standard” for detecting point mutations and small insertions and deletions. This technique does not detect intragenic deletions. The technical limitations of Sanger sequencing may sometimes cause diagnostic difficulties for autosomal recessive disorders when only one mutant allele was detected. Therefore, when clinically indicated, dosage analysis is a necessary diagnostic component. Different molecular platforms have been developed to address gene dosage abnormalities, including MLPA and real-time PCR. Limitations of these platforms include product availability of specific genes, limited number of probes per exon, time-consuming probe design and inability to accurately define the deletion/duplication boundary. Recently, customized oligonucleotide array CGH has been developed in a clinical setting to address copy number abnormalities at the whole genome, as well as at the exonic level [17–20]. Our custom oligonucleotide array CGH not only assays genes involved in mitochondrial function, but assays the entire mitochondrial genome [7,8]. This oligonucleotide array CGH simultaneously detects copy number changes in both mitochondrial and nuclear genes involved in mitochondrial biogenesis and function. In addition, by using tissue-matched controls, mtDNA content can be estimated. In this report, the simultaneous detection of an intragenic deletion in the TK2 gene and mtDNA depletion demonstrated the utility of this oligonucleotide array CGH. The dense coverage of the oligonucleotide probes also can provide an estimate of the deletion break points. Subsequent PCR of the deletion junction followed by Sanger sequencing can then precisely determine the deletion junction. This custom aCGH
has been applied to detect copy number changes in a patient with hepatocerebral form of mtDNA depletion syndrome [10]. While TK2 is broadly expressed in various tissues [21,22], myopathy seems to be the major phenotype of TK2 deficiency. In our report, a significant mtDNA copy number decrease in the liver, along with increased hepatic transaminases, suggests that liver may also be affected by TK2 deficiency. In conclusion, this report demonstrates that TK2 deficiency can result from both point mutations and intragenic deletions; that hepatic involvement may occur in addition to myopathy; and that custom oligonucleotide array CGH is a valuable tool for the simultaneous detection of gene copy number abnormalities in both nuclear and mitochondrial genomes. Disclaimer Medical Genetics Laboratories at Baylor College of Medicine provide fee-based service for the TK2 gene sequencing and array CGH analysis. References [1] C.K. Mathews, S. Song, Maintaining precursor pools for mitochondrial DNA replication, FASEB J. 21 (2007) 2294–2303. [2] S. Dimauro, C.T. Moraes, S. Shanske, A. Lombes, H. Nakase, S. Mita, H.J. Tritschler, E. Bonilla, A.F. Miranda, E.A. Schon, Mitochondrial encephalomyopathies: biochemical approach, Rev. Neurol. (Paris) 147 (1991) 443–449. [3] C.T. Moraes, S. Shanske, H.J. Tritschler, J.R. Aprille, F. Andreetta, E. Bonilla, E.A. Schon, S. DiMauro, MtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases, Am. J. Hum. Genet. 48 (1991) 492–501. [4] A. Spinazzola, F. Invernizzi, F. Carrara, E. Lamantea, A. Donati, M. Dirocco, I. Giordano, M. Meznaric-Petrusa, E. Baruffini, I. Ferrero, Clinical and molecular
S. Zhang et al. / Molecular Genetics and Metabolism 99 (2010) 53–57
[5] [6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
features of mitochondrial DNA depletion syndromes, J. Inherit. Metab. Dis. 32 (2009) 143–158. S. Rahman, J. Poulton, Diagnosis of mitochondrial DNA depletion syndromes, Arch. Dis. Child. 94 (2009) 3–5. M. Oskoui, G. Davidzon, J. Pascual, R. Erazo, J. Gurgel-Giannetti, S. Krishna, E. Bonilla, D.C. De Vivo, S. Shanske, S. DiMauro, Clinical spectrum of mitochondrial DNA depletion due to mutations in the thymidine kinase 2 gene, Arch. Neurol. 63 (2006) 1122–1126. L.J. Wong, D. Dimmock, M.T. Geraghty, R. Quan, U. Lichter-Konecki, J. Wang, E.K. Brundage, F. Scaglia, A.C. Chinault, Utility of oligonucleotide array-based comparative genomic hybridization for detection of target gene deletions, Clin. Chem. 54 (2008) 1141–1148. A.C. Chinault, C.A. Shaw, E.K. Brundage, L.Y. Tang, L.J. Wong, Application of dual-genome oligonucleotide array-based comparative genomic hybridization to the molecular diagnosis of mitochondrial DNA deletion and depletion syndromes, Genet. Med. 11 (2009) 518–526. R.K. Bai, L.J. Wong, Simultaneous detection and quantification of mitochondrial DNA deletion(s), depletion, and over-replication in patients with mitochondrial disease, J. Mol. Diagn. 7 (2005) 613–622. N.C. Lee, D. Dimmock, W.L. Hwu, L.Y. Tang, W.C. Huang, A.C. Chinault, L.J. Wong, Simultaneous detection of mitochondrial DNA depletion and singleexon deletion in the deoxyguanosine gene using array-based comparative genomic hybridisation, Arch. Dis. Child. 94 (2009) 55–58. A. Saada, A. Shaag, H. Mandel, Y. Nevo, S. Eriksson, O. Elpeleg, Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy, Nat. Genet. 29 (2001) 342–344. J.C. Blake, J.W. Taanman, A.M. Morris, R.G. Gray, J.M. Cooper, P.J. McKiernan, J.V. Leonard, A.H. Schapira, Mitochondrial DNA depletion syndrome is expressed in amniotic fluid cell cultures, Am. J. Pathol. 155 (1999) 67–70. P.H. Ducluzeau, A. Lachaux, R. Bouvier, N. Streichenberger, G. Stepien, B. Mousson, Depletion of mitochondrial DNA associated with infantile cholestasis and progressive liver fibrosis, J. Hepatol. 30 (1999) 149–155.
57
[14] J.W. Taanman, A.G. Bodnar, J.M. Cooper, A.A. Morris, P.T. Clayton, J.V. Leonard, A.H. Schapira, Molecular mechanisms in mitochondrial DNA depletion syndrome, Hum. Mol. Genet. 6 (1997) 935–942. [15] H.J. Tritschler, F. Andreetta, C.T. Moraes, E. Bonilla, E. Arnaudo, M.J. Danon, S. Glass, B.M. Zelaya, E. Vamos, N. Telerman-Toppet, Mitochondrial myopathy of childhood associated with depletion of mitochondrial DNA, Neurology 42 (1992) 209–217. [16] T.H. Vu, M. Sciacco, K. Tanji, C. Nichter, E. Bonilla, S. Chatkupt, P. Maertens, S. Shanske, J. Mendell, M.R. Koenigsberger, Clinical manifestations of mitochondrial DNA depletion, Neurology 50 (1998) 1783–1790. [17] A.L. Beaudet, J.W. Belmont, Array-based DNA diagnostics: let the revolution begin, Annu. Rev. Med. 59 (2008) 113–129. [18] S.W. Cheung, C.A. Shaw, D.A. Scott, A. Patel, T. Sahoo, C.A. Bacino, A. Pursley, J. Li, R. Erickson, A.L. Gropman, Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics, Am. J. Med. Genet. A 143A (2007) 1679–1686. [19] X. Lu, C.A. Shaw, A. Patel, J. Li, M.L. Cooper, W.R. Wells, C.M. Sullivan, T. Sahoo, S.A. Yatsenko, C.A. Bacino, Clinical implementation of chromosomal microarray analysis: summary of 2513 postnatal cases, PLoS ONE 2 (2007) e327. [20] P. Stankiewicz, A.L. Beaudet, Use of array CGH in the evaluation of dysmorphology, malformations, developmental delay, and idiopathic mental retardation, Curr. Opin. Genet. Dev. 17 (2007) 182–192. [21] L. Wang, B. Munch-Petersen, A. Herrstrom Sjoberg, U. Hellman, T. Bergman, H. Jornvall, S. Eriksson, Human thymidine kinase 2: molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates, FEBS Lett. 443 (1999) 170–174. [22] L. Wang, A. Saada, S. Eriksson, Kinetic properties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy, J. Biol. Chem. 278 (2003) 6963–6968.