Clinical and molecular studies in three portuguese mtdna t8993g families

Clinical and Molecular Studies in Three Portuguese mtDNA T8993G Families Laura Vilarinho, MSc*, Elisa Lea˜o, MD†, Clara Barbot, MD‡, Manuela Santos, MD‡, Hugo Rocha, MSc*, and Filippo M. Santorelli, MD§ The T8993G mutation in the mitochondrial DNA adenosine triphosphatase 6 gene represents an important cause of maternally inherited Leigh’s syndrome. Reported are the clinical findings and mutational loads in three Portuguese T8993G pedigrees. Polymerase chain reactionrestriction fragment length polymorphism analyses demonstrated the T8993G mutation in a high percentage of tissues from all patients (97% ⴞ 2.3%), but it was less abundant in the blood from 14 maternal relatives. The disease progressed severely in the probands but did not have the fatal course reported by others. To test whether this prolonged course was related to the presence of a specific, disease-associated haplogroup the origin of the mutational event in Portugal was traced. Haplotype investigation revealed an independent occurrence of the mutation in the three probands. These analyses represent the first molecular characterization of Portuguese patients with Leigh’s syndrome. © 2000 by Elsevier Science Inc. All rights reserved. Vilarinho L, Lea˜o E, Barbot C, Santos M, Rocha H, Santorelli FM. Clinical and molecular studies in three Portuguese mtDNA T8993G families. Pediatr Neurol 2000;22:29-32.

Introduction Leigh’s syndrome (LS) is a genetically determined neurodegenerative disorder of infancy or childhood characterized by developmental delay with psychomotor regression, signs of brainstem dysfunction, and lactic acidosis [1]. The diagnosis of LS rests on neuroradiologic or postmortem examination evidence of bilateral and symmetric necrotic lesions in the basal ganglia, thalamus, brainstem, and spinal cord. The pathophysiology is related to an altered energy metabolism. Deficiencies of cyto-

From the *Department of Clinical Biology; Instituto de Gene´tica Me´dica; †Department of Pediatrics; Hospital S. Joa˜o; and ‡Department of Neuropediatrics; Hospital Maria Pia, Porto, Portugal; and §Departments of Molecular Medicine and Neurology; IRCCS-Bambino Gesu`; Rome, Italy.

© 2000 by Elsevier Science Inc. All rights reserved. PII S0887-8994(99)00113-7 ● 0887-8994/00/$20.00

chrome-c oxidase, an autosomal-recessive condition, and of pyruvate dehydrogenase complex E1␣ subunit, inherited as an X-linked trait, have been associated with LS [2]. More recently, a mitochondrial DNA (mtDNA) point mutation at nucleotide 8993 (T8993G) has been demonstrated to be a common cause of maternally inherited LS [3]. This mutation may impair adenosine triphosphate (ATP) production by altering the electric charge in the proton channel of the F1F0-ATP synthase complex [4]. To determine the relative frequency of LS/T8993G in Portugal the levels of mutated mtDNA were assessed in three patients and 14 maternal relatives, and the origin of the mutational event was traced.

Case Reports Patient 1. A 3-year-old male born to unrelated parents presented with generalized hypotonia, psychomotor regression, and myoclonic seizures. From 3 months of age, he had experienced recurrent vomiting, bronchiolitis, and delayed development. A cranial computed tomography scan revealed bilateral hypodensities in the basal ganglia. The family history was unremarkable (Family A, Fig 1). Patient 2. A female was born to nonconsanguineous parents and suffered from an acute febrile illness at 2 years of age. Shortly after, she complained of lower limb weakness and was hospitalized. She became progressively comatose and apneic and required tracheal intubation. Electroencephalography revealed diffuse delta activity. An initial cranial computed tomographic scan was normal. Elevated levels of aldolase, creatine kinase, and carnitine, but not lactate and pyruvate, were detected in blood samples. At a recent examination, she had dystonic postures; a hypokinetic face; and choreic movements of hands, tongue, and lips. Cranial magnetic resonance imaging disclosed extensive bilateral lesions in the basal ganglia (Fig 2). The family history was remarkable for an 8-year-old sister who had mental retardation, spastic paraparesis, and elevated lactic acid levels (3.1 mmol/L) (Family B, Fig 1). Patient 3. A 7-year-old female had normal development until 5 months of age when her parents observed psychomotor regression with loss of contact and lethargy. At 8 months of age, she suffered from generalized seizures. At that time the physical examination also disclosed squinted eyes, hypotonia, brisk tendon reflexes, and choreiform move-

Communications should be addressed to: Dr. Vilarinho; Instituto de Gene´tica Me´ica Jacinto de Magalha˜es; Prac¸a Pedro Nunes, 88; 4050 Porto, Portugal. Received February 18, 1999; accepted September 3, 1999.

Vilarinho et al: T8993G Mutation 29

Figure 2. Cranial magnetic resonance image of Patient 2 disclosed extensive lesions in the basal ganglia and brain atrophy.

enzymatic activities—all normal values. Electroencephalography revealed disturbed cerebral electric activity, with bursts of multifocal activity. In the following years, she gradually deteriorated, with several hospitalizations because of febrile seizures. Recent magnetic resonance imaging revealed diffuse brain atrophy and bilateral lesions in the putamen and caudate nucleus. The family history was not contributory (Family C, Fig 1).

Methods

Figure 1. Pedigrees of three families harboring the T8993G mutation. Dark symbols indicate subjects with detectable levels of mutated genomes in muscle or blood. Arrows indicate the probands, and crossed symbols indicate individuals not tested.

ments of her hands. Biochemical screening for inborn errors of metabolism included gas chromatography of urinary organic acids, lactate and pyruvate levels in the blood and cerebrospinal fluid, and lysosomal

Table 1.

Metabolic investigations in Patients 1-3

Plasma lactate (mmol/L) Plasma pyruvate (mmol/L) Lactate/pyruvate CSF lactate (mmol/L) Plasma citrulline (␮mol/dL) Respiratory chain enzyme activities

Patient 1

Patient 2

Patient 3

Control Patients

3.1/3.7 249/235 12/16 NP 6 Normal

1.7 117 14 2.6 1 Partial deficiency of complex IV (33% residual activity)

2.3 95 24 3.3 24 Normal

0.63-2.44 0.047-0.083 10-25 1.2-2.1 15-30

Abbreviations: CSF ⫽ Cerebrospinal fluid NP ⫽ Not performed

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Plasma amino acids were measured by ion exchange chromatography on a Pharmacia (Piscataway, NJ), Biochrome 20 amino acid analyzer. Muscle morphologic and biochemical investigations directed to unveil typical mitochondrial abnormalities were performed as previously described [5]. Extraction of total DNA from tissues, Southern blot analysis, and testing for the most commonly encountered mtDNA point mutations used reported methods [3,6]. Quantitative analysis of the T8993G point mutation in tissues was performed as previously described [4]. Sequencing of the hypervariable region I, between nucleotide positions 16090 and 16365, used described oligonucleotide primers [7]. Sequencing was performed in an ABI PRISM 310 (Perkin Elmer Biosystems, Foster City, CA) automatic sequencer.

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Table 2.

Percentage of mtDNA deleted in the families studied

Probands % M/B Mother % B Grandmother % B Siblings % B

Table 3.

Family A

Family B

Family C

Patient

95/NP 21 Negative 49 (sister)

⬎ 99/⬎ 99 76 NP ⬎ 99 (sister)

95/NP ⬍5 NP

1 2 3

Haplotype investigation in Patients 1-3 Haplotype T16209C G16129A T16311C

Haplogroup T16304C C16233T T16362C

1 1 1

Abbreviations: M/B ⫽ Muscle/Blood NP ⫽ Not performed

Results and Discussion The clinical and radiologic features of these patients were consistent with a diagnosis of LS. Because maternal transmission was not evident, the most common mtDNAdisease specific mutations were analyzed. We determined that all three probands harbored high levels (greater than 95%) of the T8993G mutation. Table 1 summarizes the results of the metabolic investigations. Hypocitrullinemia (less than 10 mmol/L) was evident in two patients, a finding not usually seen in mtDNA-related disorders. In our experience with more than 500 patients tested since respiratory chain enzyme assays and mtDNA analyses were available in Portugal, such a finding has never been detected. Hypocitrullinemia usually occurs in mitochondrial urea cycle disorders, including N-acetylglutamate synthase and ornithine carbamyl transferase. The ATP/ adenosine diphosphate ratio necessary for regulating the corresponding enzymatic activities could be impaired by the reduced ATP production caused by the T8993G mutation [4]. Alternatively, mutant mtDNA could severely affect the functioning of liver and gut mucosa, major sites of production of N-acetylglutamate synthase, hampering the whole pathway leading to citrulline. Further studies are necessary to investigate whether this finding is specific to the T8993G mutation [8,9]. These patients demonstrated normal muscle morphologic features, but, as a general rule, patients with LS do not have ragged-red fibers in muscle, a hallmark of mitochondrial encephalomyopathies. Biochemical assays of pyruvate dehydrogenase complex and respiratory complex activities were also normal (data not presented). A partial cytochrome-c oxidase deficiency (residual activity 33% of normal) was evident in Patient 2 but was probably secondary to negative feedback produced by lowered ATP synthesis, such as has been demonstrated in the skin fibroblasts from patients with LS/T8993G [4]. The genetic load was assessed in the blood from 14 individuals from the three pedigrees. The mutant load in the three LS/T8993G families is summarized in Table 2. An abundance of mutation correlated well with the severity of the clinical phenotypes: the severely affected patients harbored near homoplasmic levels of mutated genomes and oligosymptomatic relatives had fewer mutant

mtDNAs. Even lower levels were detected in asymptomatic relatives. Mutated genomes were not detected in the mother, maternal grandmother, or maternal aunts of Patient 1. In this family the mutation apparently arose de novo in the maternal germ line and rapidly segregated in the proband’s tissues. The poor survival rate usually observed in LS was not evident in the reported patients. Whether genetic factors other than the pathogenic mtDNA mutation were implicated in this event is still uncertain. To test whether this eventually is associated with a specific mitochondrial genetic background, we investigated whether these patients were the products of one or more independent mutational events. Haplotype analysis revealed that the three pedigrees belonged to three different groups, all related to the most common European haplogroup (Table 3). The simplest interpretation of these data is that there have been multiple origins of the T8993G mutation in Portugal, rather than a single mutational event transmitted by descendants. This result does not allow for conclusions on the possible role of the genetic background in the disease expression and survival rates of these patients. The ability to diagnose LS at the molecular level has only recently become possible in Portugal. With the lack of efficient therapies and a high recurrence rate, recognizing families at risk is crucial for genetic counseling.

The authors are indebted to Professor Salvatore DiMauro (Columbia University, New York) for his expert guidance and strenuous encouragement throughout this study. The authors thank Professor Antonio Torroni for his helpful comments.

References [1] Leigh D. Subacute necrotizing encephalomyelopathy in an infant. J Neurol Neurosurg Psychiatry 1951;14:216-21. [2] DiMauro S, De Vivo DC. Genetic heterogeneity in Leigh syndrome. Ann Neurol 1996;40:5-7. [3] Santorelli FM, Shanske S, Macaya A, De Vivo DC, DiMauro S. The mutation at nt 8993 of mitochondrial DNA is a common cause of Leigh’s syndrome. Ann Neurol 1993;34:827-34. [4] Va´zquez-Memije ME, Shanske S, Santorelli FM, et al. Comparative biochemical studies in fibroblasts from patients with different forms of Leigh syndrome. J Inher Metab Dis 1996;19:43-50. [5] Vilarinho L, Santorelli FM, Cardoso ML, Coelho T, Guimara˜es A, Coutinho P. Mitochondrial DNA analysis in ocular myopathy. Observations in 29 Portuguese patients. Eur Neurol 1998;39:148-53. [6] Vilarinho L, Santorelli FM, Coelho I, et al. The mitochondrial

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DNA A3243G mutation in Portugal: Clinical and molecular studies in 5 families. J Neurol Sci 1999;163:168-74. [7] Richards M, Coˆrte-Real H, Forster P, et al. Paleolithic and neolithic lineages in the European mitochondrial gene pool. Am J Hum Genet 1996;59:185-203. [8] Rabier D, Dircy C, Ro¨tig A, et al. Persistent hypocitrullinaemia

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as a marker for mtDNA NARP T8993G mutation? J Inher Metab Dis 1998;21:216-9. [9] Parfait B, de Lonlay P, von Kleist-Retzow JC, et al. The neurogenic weakness, ataxia and retinitis pigmentosa (NARP) syndrome mtDNA mutation (T8993G) triggers muscle ATPase deficiency and hypocitrullinaemia. Eur J Pediatr 1999;158:55-8.