Novel 5.712 kb mitochondrial DNA deletion in a patient with Pearson syndrome: A case report
- Authors:
- Published online on: December 22, 2014 https://doi.org/10.3892/mmr.2014.3127
- Pages: 3741-3745
Abstract
Introduction
Pearson marrow-pancreas syndrome (PS; MIM #557000), was initially described in 1979, and is a progressive multi-organ disorder caused by deletions and duplications of mitochondrial DNA (mtDNA) (1). The presenting symptoms include hematological features, and/or growth retardation, secondary to pancreatic exocrine dysfunction (1). The onset of PS generally occurs in early infancy. The hematological features include macrocytic sideroblastic anemia and vacuolization of bone marrow (BM) precursors, which are sometimes associated with neutropenia or thrombocytopenia (1). Other symptoms include tubulopathy and aminoaciduria, hepatomegaly, cytolysis and cholestasis, endocrine gland disturbances, neuromuscular manifestations, cardiac involvement or splenic atrophy, which may develop simultaneously or during the course of the disease (2). PS is often fatal in infancy, and the majority of the reported patients with PS succumb to the disease before reaching three-years-of-age, due to septicemia, metabolic acidosis or hepatocellular insufficiency (2). Previous studies have demonstrated that surviving infants exhibit hematological improvement; however, they were eventually shown to develop the typical features of Kearns-Sayre syndrome, which is characterized by a triad of symptoms: Progressive external ophthalmoplegia, pigmentary degeneration of the retina and cardiomyopathy (3–5). Numerous studies have identified genetic defects associated with PS, which include the presence of the common 4.977 kb deletion, de novo large-scale deletions or rare duplications in the mitochondrial genome (6–8), which lead to impaired production of ATP (9).
The present report describes the case of a four-month-old infant who presented with the clinical features of PS. Molecular analysis identified a novel 5.712 kb deletion between nucleotides 8,011 and 13,722 in mtDNA.
Case report
Patient and clinical findings
The female patient was the first-born child of healthy non-consanguineous Korean parents. She was born following an uncomplicated pregnancy and was delivered at term (40+4 weeks). Her birth weight was 2,400 g and the first months of her life were uneventful. Family members typically did not show signs and symptoms associated with PS. At the age of four months, the patient visited Seoul St. Mary’s Hospital (Seoul, South Korea) presenting with severe pallor. Mucocutaneous pallor was observed upon physical examination. Physical and psychomotor development was shown to be within the normal range and no clinical signs of neuromuscular disease were present. Blood examination detected severe normocytic normochromic anemia. Complete blood count data were as follows: White blood cell count, 4,390/mm3; hemoglobin, 5.0 g/dl; platelets, 180,000/mm3; mean corpuscular volume, 95.7 fl; mean corpuscular hemoglobin concentration, 33.1%; and reticulocytes 0.79%. The results of other routine laboratory tests, including blood glucose, pH, blood gas analysis, electrolytes, blood urea nitrogen, creatinine, aspartate transaminase, alanine transaminase, creatine kinase, lactate dehydrogenase, prothrombin time, activated partial thromboplastin time and urinalysis were all within the normal ranges. No viral pathogens were detected, in particular, infections with parvovirus B19, Epstein-Barr virus or cytomegalovirus were excluded. A BM aspirate was performed at 4.5 months. Erythroid and myeloid precursors exhibited numerous cytoplasmic vacuoles (Fig. 1A and B). Histiocytes exhibited erythrophagocytosis (Fig. 1C) and ringed sideroblasts were observed following Prussian blue staining (Fig. 1D). Karyotyping and a chromosome instability assay revealed normal results. Echocardiography demonstrated normal ventricular systolic and diastolic function, without any definite structural anomalies. Abdominal ultrasonography demonstrated mild pelviectasia in both kidneys. Brain magnetic resonance imaging detected no abnormal signal in the brain parenchyma.
At the age of 23 months, biochemical analyses detected metabolic acidosis with a high pyruvic acid concentration of 1.07 mg/dl (reference range 0.3–0.9). A blood organic acid profile revealed increased 3-hydroxybutyric acid (512.3 μmol/l; reference range, 22–213 μmol/l) and palmitic acid (267.9 μmol/l; reference range, 27–222 μmol/l) levels. A urinary organic acid profile revealed an elevated excretion of 3-hydroxybutyric acid (224.7 mmol/mol creatinine; normal <62.6 mmol/mol), 3-methylglutaconic acid (61.3 mmol/mol creatinine; normal <6.0 mmol/mol), 3-hydroxyglutaric acid (11.6 mmol/mol creatinine; normal: Not detected), 4-hydroxyphenylacetic acid (105.7 mmol/mol creatinine; normal <69.9 mmol/mol), and 4-hydroxyphenyllactic acid (73.3 mmol/mol creatinine; normal <19.6 mmol/mol). These clinical features indicated the presence of a mitochondrial disease, such as PS.
mtDNA deletion analysis
A molecular analysis was performed in order to confirm the diagnosis of PS. Written informed consent was obtained and mtDNA was extracted from the peripheral blood of the patient using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany). Amplification of mtDNA was performed using long-distance polymerase chain reaction (PCR) (Expand Long Template PCR system; Roche Diagnostics, Basel, Switzerland) with various combinations of primers, as previously described (10) (Table I). Direct sequencing of the PCR products was performed using the BigDye® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems Life Technologies, Foster City, CA, USA) and the products were resolved on an ABI PRISM 3130 Genetic analyzer (Applied Biosystems). Following long-distance PCR amplification using M12F and M20R primers a large-scale mtDNA deletion was detected in the patient, as compared with the normal control (Fig. 2A). Sanger sequencing identified the breakpoints of the 5.712 kb deletion, and the deletion was shown to span between the nucleotide positions 8,011 and 13,722 of mtDNA (Fig. 2B). No perfect or imperfect repeats were detected at the boundaries of the mtDNA deletion (Fig. 2C). This heteroplasmic deletion removes part of the COXII gene, the entire tRNALys, ATPase 8, ATPase 6, COXIII, tRNAGly, ND3, tRNAArg, ND4L, ND4, tRNAHis, tRNASer(AGY), tRNALeu(CUN) genes and part of the ND5 gene (Fig. 3). The sequence homology in the 49 nt region (24 nt on each side of the breakpoint nucleotide) of the proximal and distal sequences was analyzed using Clustal Omega software (11), with default parameters (http://www.ebi.ac.uk/Tools/msa/clustalo/), and was compared with the updated consensus Cambridge sequence (GenBank Accession no. NC_012920.1).
Table IList and sequence of primers used for the long-distance polymerase chain reaction of mitochondrial DNA. |
At the time of initial organic acid analysis and mtDNA evaluation, the patient had been admitted to hospital with a fever, poor oral intake and decreased activity, which persisted despite administration of fluids and broad spectrum antibiotics. Blood culture was negative and urine culture was positive for Pseudomonas aeruginosa infection, which was treated with ceftazidime. The patient’s fever did not remit and her overall condition failed to show improvement, therefore she was transferred to another institution for further care.
Discussion
The present report describes the case of a female patient with PS harboring a large-scale mitochondrial deletion, which is to the best of our knowledge, the first case reported in South Korea. Molecular investigation of mtDNA extracted from a peripheral blood sample detected a novel large-scale heteroplasmic mitochondrial deletion without direct repeats. This deletion was shown to affect numerous tRNA and protein-coding genes, which may lead to defects in mitochondrial polypeptide synthesis, and impaired oxidative phosphorylation and energy metabolism in the respiratory chain.
Usually, mtDNA in humans is ~16.6 kb in length, and encodes numerous enzymes of the respiratory chain and oxidative phosphorylation system, ribosomal RNAs, and various tRNAs (12). Impairment of the mitochondrial respiratory chain may lead to lactic acidemia, high plasma lactate/pyruvate molar ratios, and even fatal metabolic acidosis (13). Previous studies have reported various mitochondrial deletions in patients with PS (4–10). A common 4.9 kb deletion spanning the ATPase 8 gene to the ND5 gene, between 13 bp direct repeats has previously been frequently observed (14). This deletion has been identified in >80% of affected children; however, numerous other mtDNA deletions have also been detected (www.mitomap.org). Mitochondrial deletions differ in size and location; however, they are confined to a region delineated by the heavy- and light-strand origins of replication, which is the major arc of mtDNA (14). The known deletions include numerous tRNA and protein-coding genes (15), and are often flanked by direct repeats (16). In the majority of cases, mtDNA deletions are spontaneous events that occur either in the oocyte or during early stages of embryonic development (17).
The underlying mechanisms regarding the regulation of tissue distribution of deleted mtDNA molecules, or why the rate of disease progression and the degree of disease severity is variable, even for the same deletions, remains to be elucidated. No strict correlations have been observed between heteroplasmy in blood cells and the severity of hematopoietic features. Furthermore, no obvious genotype-phenotype correlation has been identified regarding the appearance of hematological manifestations in PS and the mtDNA deletion (18). The different phenotypic expression of the same mtDNA defects may be associated with nuclear modifier genes, polymorphisms and environmental factors (19).
No specific treatment is currently available for patients with PS, therefore awareness of possible complications, and early intervention may minimize PS-associated mortality and morbidity. Red blood cell transfusions are often required to manage macrocytic anemia, and patients may be dependent on these transfusions (20). Pancreatic enzyme replacement is required for patients with malabsorption due to exocrine pancreatic dysfunction (20). Intermittent metabolic crises are managed with hydration, correction of electrolyte abnormalities, and correction of acidosis (20). Bicarbonate supplementation and dichloroacetic acid have also been used to treat persistent metabolic acidosis. Previous studies have reported the case of two patients with PS, who were treated with allogeneic hematopietic stem cell transplantation (HSCT) (21,22). The procedure was shown to correct the hematological and metabolic abnormalities in the two patients; therefore, allogeneic HSCT may be a viable treatment option for patients with PS whose prognosis is uniformly fatal if left untreated.
In conclusion, to the best of our knowledge, the present study was the first to report a novel mtDNA deletion causing Korean PS. It is necessary to consider mitochondrial disorder in infants who present with persistent hematological abnormalities, such as hypoplastic anemia. Due to the often fatal course of PS, molecular analysis of possible mtDNA deletions may be beneficial for the diagnosis and prognosis of PS, and may also aid in the genetic counseling of relatives.
Acknowledgements
The present study was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (grant no. A120175).
References
Pearson HA, Lobel JS, Kocoshis SA, et al: A new syndrome of refractory sideroblastic anemia with vacuolization of marrow precursors and exocrine pancreatic dysfunction. J Pediatr. 95:976–984. 1979. View Article : Google Scholar : PubMed/NCBI | |
Rötig A, Cormier V, Blanche S, et al: Pearson’s marrow-pancreas syndrome. A multisystem mitochondrial disorder in infancy. J Clin Invest. 86:1601–1608. 1990. View Article : Google Scholar | |
Holt IJ, Harding AE and Morgan-Hughes JA: Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature. 331:717–719. 1988. View Article : Google Scholar : PubMed/NCBI | |
McShane MA, Hammans SR, Sweeney M, et al: Pearson syndrome and mitochondrial encephalomyopathy in a patient with a deletion of mtDNA. Am J Hum Genet. 48:39–42. 1991.PubMed/NCBI | |
Rötig A, Bourgeron T, Chretien D, Rustin P and Munnich A: Spectrum of mitochondrial DNA rearrangements in the Pearson marrow-pancreas syndrome. Hum Mol Genet. 4:1327–1330. 1995. View Article : Google Scholar : PubMed/NCBI | |
Blaw ME and Mize CE: Juvenile Pearson syndrome. J Child Neurol. 5:187–190. 1990. View Article : Google Scholar : PubMed/NCBI | |
Rotig A, Colonna M, Bonnefont JP, et al: Mitochondrial DNA deletion in Pearson’s marrow/pancreas syndrome. Lancet. 1:902–903. 1989. View Article : Google Scholar : PubMed/NCBI | |
van den Ouweland JM, de Klerk JB, van de Corput MP, et al: Characterization of a novel mitochondrial DNA deletion in a patient with a variant of the Pearson marrow-pancreas syndrome. Eur J Hum Genet. 8:195–203. 2000. View Article : Google Scholar : PubMed/NCBI | |
Santorelli FM, Barmada MA, Pons R, Zhang LL and DiMauro S: Leigh-type neuropathology in Pearson syndrome associated with impaired ATP production and a novel mtDNA deletion. Neurology. 47:1320–1323. 1996. View Article : Google Scholar : PubMed/NCBI | |
Ayed IB, Chamkha I, Mkaouar-Rebai E, et al: A Tunisian patient with Pearson syndrome harboring the 4.977kb common deletion associated to two novel large-scale mitochondrial deletions. Biochem Biophys Res Commun. 411:381–386. 2011. View Article : Google Scholar : PubMed/NCBI | |
Sievers F, Wilm A, Dineen D, et al: Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 7:5392011. View Article : Google Scholar : PubMed/NCBI | |
Schapira AH: Primary and secondary defects of the mitochondrial respiratory chain. J Inherit Metab Dis. 25:207–214. 2002. View Article : Google Scholar : PubMed/NCBI | |
Gibson KM, Bennett MJ, Mize CE, et al: 3-Methylglutaconic aciduria associated with Pearson syndrome and respiratory chain defects. J Pediatr. 121:940–942. 1992. View Article : Google Scholar : PubMed/NCBI | |
Kleinle S, Wiesmann U, Superti-Furga A, et al: Detection and characterization of mitochondrial DNA rearrangements in Pearson and Kearns-Sayre syndromes by long PCR. Hum Genet. 100:643–650. 1997. View Article : Google Scholar : PubMed/NCBI | |
Wallace DC, Shoffner JM, Trounce I, et al: Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim Biophys Acta. 1271:141–151. 1995. View Article : Google Scholar : PubMed/NCBI | |
Schon EA, Rizzuto R, Moraes CT, Nakase H, Zeviani M and DiMauro S: A direct repeat is a hotspot for large-scale deletion of human mitochondrial DNA. Science. 244:346–349. 1989. View Article : Google Scholar : PubMed/NCBI | |
Harding AE and Hammans SR: Deletions of the mitochondrial genome. J Inherit Metab Dis. 15:480–486. 1992. View Article : Google Scholar : PubMed/NCBI | |
Binder V, Steenpass L, Laws HJ, Ruebo J and Borkhardt A: A novel mtDNA large-scale mutation clinically exclusively presenting with refractory anemia: is there a chance to predict disease progression? J Pediatr Hematol Oncol. 34:283–292. 2012. View Article : Google Scholar : PubMed/NCBI | |
Limongelli A, Schaefer J, Jackson S, et al: Variable penetrance of a familial progressive necrotising encephalopathy due to a novel tRNA(Ile) homoplasmic mutation in the mitochondrial genome. J Med Genet. 41:342–349. 2004. View Article : Google Scholar : PubMed/NCBI | |
Seneca S, De Meirleir L, De Schepper J, et al: Pearson marrow pancreas syndrome: a molecular study and clinical management. Clin Genet. 51:338–342. 1997. View Article : Google Scholar : PubMed/NCBI | |
Faraci M, Cuzzubbo D, Micalizzi C, et al: Allogeneic bone marrow transplantation for Pearson’s syndrome. Bone Marrow Transplant. 39:563–565. 2007. View Article : Google Scholar : PubMed/NCBI | |
Hoyoux C, Dresse MF, Robinet S, et al: Cord blood transplantation in a child with Pearson’s disease. Pediatr Blood Cancer. 51:5662008. View Article : Google Scholar |