ClinVar Genomic variation as it relates to human health

Low-set ears (present) , Bilateral single transverse palmar creases (present) , 2-3 toe syndactyly (present) , Epicanthus (present) , Hepatosplenomegaly (present) , Hypochromic microcytic anemia (present) , Macrocephaly (present) , Febrile seizure (within the age range of 3 months to 6 years) (present) , Hypotonia (present) , Periventricular leukomalacia (present) , Clinodactyly of the 5th toe (present) , Umbilical hernia (present) (less)

Holt et al. (1990) found a heteroplasmic T-to-G transversion at nucleotide pair 8993 in a maternal pedigree which resulted in the change of a hydrophobic leucine to a hydrophilic arginine at position 156 in subunit 6 of mitochondrial H(+)-ATPase. The clinical symptoms varied in proportion to the percentage of mutant mtDNAs but the most common clinical presentation included neurogenic muscle weakness, ataxia, and retinitis pigmentosa, leading to the designation of NARP syndrome (551500). The insertion of an arginine in the hydrophobic sequence of ATPase 6 probably interferes with the hydrogen ion channel formed by subunits 6 and 9 of the ATPase, thus causing failure of ATP synthesis. Harding et al. (1992) demonstrated that prenatal diagnosis was possible, although the approach was hampered by incomplete knowledge concerning the proportion of mutant mtDNA and its relationship to disease severity, how it may change during fetal and postnatal development, and its tissue distribution. In families with mitochondrial complex V (ATP synthase) deficiency mitochondrial type 1 (MC5DM1; 500015) resulting in Leigh syndrome (see 256000), Tatuch et al. (1992) and Shoffner et al. (1992) identified a nucleotide 8993 mutation in the MTAPT6 gene. Tatuch et al. (1992) found the heteroplasmic mtDNA mutation in a female infant showing lactic acidemia, hypotonia, and neurodegenerative disease leading to death at the age of 7 months. Autopsy revealed lesions typical of Leigh disease, both in the basal ganglia and in the brainstem. A maternal uncle and aunt died 5 months and 1 year, respectively, after a similar clinical course, while another maternal uncle, 33 years of age, had retinitis pigmentosa, ataxia, and mental retardation. The index patient had more than 95% abnormal mtDNA in her skin fibroblasts, brain, kidney, and liver tissues, as measured by laser densitometry. The maternal aunt who died at 1 year likewise had more than 95% abnormal mtDNA in her lymphoblasts. The uncle with retinitis pigmentosa had 78% and 79% abnormal mtDNA in his skin fibroblasts and lymphoblasts, respectively, while an asymptomatic maternal aunt and her son had no trace of the mutation. The mother of the index case had 71% and 39% abnormal mtDNA in her skin fibroblasts and lymphoblasts, respectively. Shoffner et al. (1992) reported a family which was heteroplasmic for the ATPase 6 nucleotide 8993 mutation in which 2 daughters died at ages 2.5 years and 14 months. Pathologic analyses showed classic basal ganglial lesions, vascular proliferation, and glioses. Two brothers manifested psychomotor retardation, ataxia, hypotonia, and retinal degeneration. The mother had retinal degeneration and experienced migraine headaches. The mother's 2 sisters were normal. The 4 affected children had high levels of mutant mtDNA, in excess of 95% by Southern blot. The mother had a 78% level of mutant mtDNA while her 2 sisters had 100% normal mtDNA. Ciafaloni et al. (1993) described 2 sisters with Leigh syndrome who had a T-to-G transversion at nucleotide 8993 in the MTATP6 gene. The asymptomatic mother had the same mutation. All 3 were heteroplasmic. The proportion of mutant genomes was lower in the mother's blood than in the blood of the more mildly affected sister, whereas all tissues from the other sister were almost homoplasmic for the mutation. Santorelli et al. (1993) found the T-to-G point mutation at nucleotide 8993 in 12 patients with Leigh syndrome from 10 unrelated families. Pastores et al. (1994) expanded the clinical phenotype of the nucleotide 8993 mtDNA mutations to include hypertrophic cardiomyopathy and confirmed its role in producing Leigh syndrome. The patient was a boy of Chinese descent who presented at the age of 6 months with a history of developmental delay and hypotonia and who had recurrent lactic acidosis. The mother's first pregnancy resulted in the birth of a stillborn female; an apparently healthy older brother had died suddenly at age 2 months. The 8993T-G mutation was heteroplasmic in the patient's skeletal muscle (90%) and fibroblasts (90%). The identical mutation was present in leukocytes (38%) isolated from the mother, but not from the father or maternal grandmother. Degoul et al. (1995) found the 8993T-G in a family with Leigh syndrome. The proband, who died at 9 years of age, developed hypotonia in the first 6 months of life and developmental retardation was noted. At 3 years of age he showed ataxia, dysmetria, myopathic weakness, nystagmus and ptosis. The electroretinogram was altered. She became deaf and developed progressive spasticity. Blood lactate concentration was normal. In contrast, lactate concentration in the CSF was always elevated. Two brothers died with acute apnea during infectious episodes, before the end of their first year. An older sister was mentally retarded, with ataxia, dysarthria, dystonia, and pes cavus, and had retinal degeneration. The mother's brother was mentally retarded and severely handicapped. Except for the father, all members of the family showed the mutation in all tissues studied, with high percentages in the 2 symptomatic sisters and even in 1 asymptomatic boy. Ferlin et al. (1997) reported a child with Leigh syndrome who died at age 14 months. Genetic analysis identified the 8993T-G mutation in 3 generations of the family and showed that the percentage of mutant mtDNA increased through each generation. The maternal grandmother of the proband, the mother, and the eldest aunt had 10%, 52%, and 50% mutant mtDNA in lymphocytes, respectively. The proband's mother and the proband had 84% and 90% mutant mtDNA in skin fibroblasts, respectively. The eldest aunt terminated a pregnancy when the 8993T-G mutation was identified in chorionic villi. In fetal tissues, the mutation load ranged from 91 to 96%. Ferlin et al. (1997) concluded that the findings in this family were consistent with a threshold effect, in which over 90% mutant mtDNA load results in clinical disease, and noted that prenatal diagnosis is feasible. Blok et al. (1997) analyzed mtDNA in oocytes from an asymptomatic mother of 3 children exhibiting heteroplasmic expression of the 8993T-G mutation associated with Leigh syndrome. The mother had 50% mutant mtDNA in her blood. One of the 7 oocytes analyzed showed no evidence of the mutation, while the remaining 6 had a mutant load of more than 95%. Blok et al. (1997) suggested that this observation reflected preferential amplification of the mtDNA variant during oogenesis. During formation of the zygote, mtDNA is derived exclusively from the oocyte; thus, it is possible that a de novo mutation may arise during oogenesis. A first carrier of a de novo mutation may be a mother who exhibits mosaicism for the mutation restricted to oocytes. However, the usual finding is that mothers of patients with Leigh syndrome and the 8993T-G mutation have substantial levels of the mutant mtDNA (38 to 76%). Takahashi et al. (1998) reported the case of a 1-year-old boy with Leigh syndrome associated with the 8993T-G mutation whose mother did not have the mutant mtDNA in her blood or urine sediment cells. Thus, a de novo mutation had occurred at a high level in oocytes, thereby causing Leigh syndrome in the boy. Generalized hypotonia was noted at birth. He developed apnea attacks and altered consciousness after upper respiratory infections at the ages of 2 and 4 months. At the age of 7 months, he showed symptoms of brainstem dysfunction, such as irregular respiration and swallowing difficulty. At the age of 9 months, growth retardation and microcephaly were obvious. Laboratory examinations showed increased lactate and pyruvate levels in blood and cerebrospinal fluid. In plants, cytoplasmic male sterility (CMS) is a mitochondrially inherited inability to produce viable pollen, and has been observed in more than 150 different plant species. Kempken et al. (1998) pointed out that in sorghum RNA editing is required to generate codons that encode leucine residues at positions equivalent to human 156 and 217. Loss of ATP6 RNA editing, as it occurs in sorghum, thus mimics mutations in human mitochondrial diseases. In all ATP6 protein sequences found in databases, including protists, plants (edited sequence), fungi, and animals, both amino acid positions are completely conserved. White et al. (1999) performed prenatal diagnosis in 2 mothers at risk of having affected children. One was the sister of a severely affected individual, and had previously had an unaffected child and a stillborn child. The other mother had 2 unaffected children and 2 affected children. The 8993T-G transversion was not found in the chorionic villus sample from 1 fetus or in the amniocytes from the other fetus. Both pregnancies were continued, and the resulting children were healthy at 2 years and 5 years of age. In 3 patients from 2 unrelated families, Baracca et al. (2000) investigated the biochemical phenotype associated with the 8993T-G mutation in the MTATP6 gene. All 3 carried more than 80% mutant genome in platelets and were manifesting clinically various degrees of the NARP syndrome phenotype. Their results suggested that the 8993T-G mutation induces a structural defect in F1F0-ATPase that causes a severe impairment of ATP synthesis. Kerrison et al. (2000) described the progression of retinopathy in NARP syndrome due to the T-to-G point mutation at the mtDNA nucleotide position 8993 in the MTATP6 gene. Prior to the onset of visual field constriction, ophthalmoscopy revealed salt-and-pepper retinopathy. After the visual fields had become constricted, fundus examination showed diffuse peripheral bone spicule formation, optic nerve pallor, and arteriolar attenuation consistent with retinitis pigmentosa. The authors stressed that mild mottling of the peripheral retinal pigment epithelium (salt-and-pepper retinopathy or retinitis pigmentosa sine pigmento) does not represent a specific entity but is an early stage of the retinitis pigmentosa, whether the patient has NARP or isolated retinitis pigmentosa. Hayashi et al. (2000) reported the histopathologic findings in the eyes from a patient with Leigh syndrome associated with the 8993T-G point mutation in mtDNA. Ophthalmologic signs and symptoms of Leigh syndrome include nystagmus, ophthalmoplegia, strabismus, optic nerve atrophy, and loss of the foveal reflex. A child with hypotonia, developmental delay, persistent lactic acidosis, seizures, and ataxia died of aspiration pneumonia at age 15 months. Analysis of mtDNA was positive for the 8993T-G mutation. The proportion of mutant genomes was estimated at approximately 95%. Light microscopic examination of the left eye revealed thinning of the nerve fiber and ganglion cell layers in the nasal macula and mild atrophy of the temporal optic nerve. Electron microscopy of the right eye showed numerous distended mitochondria in all cells, particularly in the retinal pigment epithelium, nonpigmented ciliary epithelium, and corneal endothelium. By transferring NARP mutant mtDNA (8993G-T in the MTATP6 gene) from fetal fibroblasts to lung carcinoma and osteosarcoma cells lacking endogenous mtDNA by cell-cytoplast fusion, Nijtmans et al. (2001) created mitochondrial transformant cells, or cybrids, able to grow in the absence of uridine. Immunoblot analysis revealed an abnormal amount of subcomplexes, F1-ATPase and V*, of mitochondrial ATP synthase. The cybrids had decreased subcomplex V assembly and decreased ATP synthesis capacity. However, the cells had no marked phenotype, suggesting that the effects of this mutation are subtle and have no effect on cell viability. Geromel et al. (2001) investigated the oxidative stress resulting from the NARP mutation in MTATP6, using cultured skin fibroblasts from 2 NARP patients presenting with an isolated complex V deficiency. A huge induction of the superoxide dismutase (147450 and 147460) activity was observed in these fibroblasts harboring more than 90% of mutant mitochondrial DNA. The oxidative stress denoted by the high SOD activity was associated with increased cell death. In glucose-rich medium, apoptosis appeared as the main cell death process associated with complex V deficiency. Complex V-deficient fibroblasts were successfully rescued by perfluoro-tris-phenyl nitrone, an antioxidant spin-trap molecule. The authors hypothesized that the superoxide production associated with the ATPase deficiency triggered by the NARP mutation could be sufficient to override cell antioxidant defenses and to result in cell commitment to die. Porto et al. (2001) reported an otherwise healthy 42-year-old woman with isolated late-onset cone-rod dystrophy characterized by difficulty driving at night beginning at age 40 years with deterioration of central and color vision due to the T8993G mitochondrial mutation. Two of her sons had NARP syndrome. A third son who was clinically diagnosed with Leigh syndrome died at age 4 years, prior to the recognition of the T8993G mutation in this family. The mother's mutation load was 50% T8993G mtDNA, while her sons with NARP had 75% T8993G mtDNA. The authors stated that Leigh disease is related to extreme heteroplasmy (more than 90%). They felt this family illustrated the remarkably variable expression of retinal and systemic manifestations related to the T8993G mutation, ranging from an isolated late-onset cone-rod dystrophy to a severe neurodegenerative process with a dramatic outcome. Porto et al. (2001) recommended genetic counseling for retinal dystrophy patients and emphasized careful evaluation of the family medical history. The 8993T-G mutation in MTATP6 impairs mitochondrial ATP synthesis. To overcome the biochemical defect, Manfredi et al. (2002) expressed wildtype ATPase 6 protein allotopically. The protein was derived from nucleus-transfected constructs encoding an amino-terminal mitochondrial targeting signal appended to a recoded ATPase 6 gene (made compatible with the universal genetic code) that also contained a carboxy-terminal fluorescent epitope tag. After transfection of human cells, the precursor polypeptide was expressed, imported into and processed within mitochondria, and incorporated into complex V. Allotopic expression of stably transfected constructs in cytoplasmic hybrids (cybrids) homoplasmic with respect to the 8993T-G mutation showed a significantly improved recovery after growth in selective medium as well as a significant increase in ATP synthesis. This was said to be the first successful demonstration of allotopic expression of an mtDNA-encoded polypeptide in mammalian cells and could form the basis of a genetic approach to treatment of a number of human mitochondrial disorders. Srivastava and Moraes (2001) showed that a mitochondrially targeted PstI restriction endonuclease degraded mtDNA harboring PstI sites, in some cases leading to a complete loss of mitochondrial genomes. When expressed in a heteroplasmic rodent cell line, containing 1 mtDNA haplotype with 2 sites for PstI and another haplotype having none, the mitochondrial PstI caused a significant shift in heteroplasmy, with an accumulation of the mtDNA haplotype lacking PstI sites. These experiments provided proof of the principle that restriction endonucleases may be feasible tools for genetic therapy of a subgroup of mitochondrial disorders. Patients harboring the T8993G mutation are potential candidates, since the mutation creates a novel PstI site which is not present in wildtype human mtDNA. In 6 individuals from 3 unrelated Italian families who had the 8993T-G mutation, Carelli et al. (2002) showed a close relationship between extent of tissue heteroplasmy, expression of the biochemical defect in platelets, and clinical involvement. A defect of ATP synthesis was evident even at low levels of mutant heteroplasmy (10 to 34% of normal) in the absence of clinical symptoms. ATP synthesis was severely decreased (4 to 9% of control values) in patients with high levels of mutation (greater than 80%), who showed the more severe clinical phenotypes of NARP and Leigh syndromes. No biochemical threshold effect was found. Carelli et al. (2002) noted that the combined effect of decreased ATP synthesis and increased reactive oxygen species production underlie the pathophysiology of mitochondrial diseases. Mattiazzi et al. (2004) showed that the 8993T-G mutation inhibits oxidative phosphorylation and results in enhanced free radical production. Antioxidants restored respiration and partially rescued ATP synthesis in cells harboring the T8993G mutation. The authors hypothesized that free radicals may play an important role in the pathogenesis of NARP/MILS and that antioxidants may be considered as a potentially useful tool in its treatment. Jung et al. (2007) reported a family with the 8993T-G mutation in which adult-onset progressive myoclonic epilepsy was a prominent feature. The proband was a 46-year-old woman with myoclonus, epilepsy, ataxia, and peripheral neuropathy. She had onset of myoclonus at age 19, which progressively worsened over her life. She did not have retinitis pigmentosa. Her mother had possible epilepsy, and 1 of her daughters developed epilepsy and ataxia in her late teens. There was a history of 3 infantile deaths on the maternal side, resulting from seizures in 2. Genetic analysis identified heteroplasmy for the 8993T-G mutation in the proband (80% in fibroblasts) and her daughter (60% in lymphocytes). Sgarbi et al. (2009) demonstrated that human fibroblasts containing the NARP-associated 8993T-G mutation could be protected from cell death when treated with alpha-ketoglutarate/aspartate to boost mitochondrial substrate-level phosphorylation (70% vs 5%; treated vs untreated survival after 72 hours). Homoplasmic 8993T-G cybrids showed similar results (75% vs 15%; treated vs untreated survival after 72 hours). In untreated fibroblasts and cybrids, the decrease in ATP content paralleled cell death, but ATP content returned to control levels after treatment. The findings indicated that ATP synthase-deficient cells can be rescued by increasing mitochondrial substrate-level phosphorylation, suggesting a potential therapeutic option for patients with such disorders. (less)

Holt et al. (1990) found a heteroplasmic T-to-G transversion at nucleotide pair 8993 in a maternal pedigree which resulted in the change of a hydrophobic leucine to a hydrophilic arginine at position 156 in subunit 6 of mitochondrial H(+)-ATPase. The clinical symptoms varied in proportion to the percentage of mutant mtDNAs but the most common clinical presentation included neurogenic muscle weakness, ataxia, and retinitis pigmentosa, leading to the designation of NARP syndrome (551500). The insertion of an arginine in the hydrophobic sequence of ATPase 6 probably interferes with the hydrogen ion channel formed by subunits 6 and 9 of the ATPase, thus causing failure of ATP synthesis. Harding et al. (1992) demonstrated that prenatal diagnosis was possible, although the approach was hampered by incomplete knowledge concerning the proportion of mutant mtDNA and its relationship to disease severity, how it may change during fetal and postnatal development, and its tissue distribution. In families with mitochondrial complex V (ATP synthase) deficiency mitochondrial type 1 (MC5DM1; 500015) resulting in Leigh syndrome (see 256000), Tatuch et al. (1992) and Shoffner et al. (1992) identified a nucleotide 8993 mutation in the MTAPT6 gene. Tatuch et al. (1992) found the heteroplasmic mtDNA mutation in a female infant showing lactic acidemia, hypotonia, and neurodegenerative disease leading to death at the age of 7 months. Autopsy revealed lesions typical of Leigh disease, both in the basal ganglia and in the brainstem. A maternal uncle and aunt died 5 months and 1 year, respectively, after a similar clinical course, while another maternal uncle, 33 years of age, had retinitis pigmentosa, ataxia, and mental retardation. The index patient had more than 95% abnormal mtDNA in her skin fibroblasts, brain, kidney, and liver tissues, as measured by laser densitometry. The maternal aunt who died at 1 year likewise had more than 95% abnormal mtDNA in her lymphoblasts. The uncle with retinitis pigmentosa had 78% and 79% abnormal mtDNA in his skin fibroblasts and lymphoblasts, respectively, while an asymptomatic maternal aunt and her son had no trace of the mutation. The mother of the index case had 71% and 39% abnormal mtDNA in her skin fibroblasts and lymphoblasts, respectively. Shoffner et al. (1992) reported a family which was heteroplasmic for the ATPase 6 nucleotide 8993 mutation in which 2 daughters died at ages 2.5 years and 14 months. Pathologic analyses showed classic basal ganglial lesions, vascular proliferation, and glioses. Two brothers manifested psychomotor retardation, ataxia, hypotonia, and retinal degeneration. The mother had retinal degeneration and experienced migraine headaches. The mother's 2 sisters were normal. The 4 affected children had high levels of mutant mtDNA, in excess of 95% by Southern blot. The mother had a 78% level of mutant mtDNA while her 2 sisters had 100% normal mtDNA. Ciafaloni et al. (1993) described 2 sisters with Leigh syndrome who had a T-to-G transversion at nucleotide 8993 in the MTATP6 gene. The asymptomatic mother had the same mutation. All 3 were heteroplasmic. The proportion of mutant genomes was lower in the mother's blood than in the blood of the more mildly affected sister, whereas all tissues from the other sister were almost homoplasmic for the mutation. Santorelli et al. (1993) found the T-to-G point mutation at nucleotide 8993 in 12 patients with Leigh syndrome from 10 unrelated families. Pastores et al. (1994) expanded the clinical phenotype of the nucleotide 8993 mtDNA mutations to include hypertrophic cardiomyopathy and confirmed its role in producing Leigh syndrome. The patient was a boy of Chinese descent who presented at the age of 6 months with a history of developmental delay and hypotonia and who had recurrent lactic acidosis. The mother's first pregnancy resulted in the birth of a stillborn female; an apparently healthy older brother had died suddenly at age 2 months. The 8993T-G mutation was heteroplasmic in the patient's skeletal muscle (90%) and fibroblasts (90%). The identical mutation was present in leukocytes (38%) isolated from the mother, but not from the father or maternal grandmother. Degoul et al. (1995) found the 8993T-G in a family with Leigh syndrome. The proband, who died at 9 years of age, developed hypotonia in the first 6 months of life and developmental retardation was noted. At 3 years of age he showed ataxia, dysmetria, myopathic weakness, nystagmus and ptosis. The electroretinogram was altered. She became deaf and developed progressive spasticity. Blood lactate concentration was normal. In contrast, lactate concentration in the CSF was always elevated. Two brothers died with acute apnea during infectious episodes, before the end of their first year. An older sister was mentally retarded, with ataxia, dysarthria, dystonia, and pes cavus, and had retinal degeneration. The mother's brother was mentally retarded and severely handicapped. Except for the father, all members of the family showed the mutation in all tissues studied, with high percentages in the 2 symptomatic sisters and even in 1 asymptomatic boy. Ferlin et al. (1997) reported a child with Leigh syndrome who died at age 14 months. Genetic analysis identified the 8993T-G mutation in 3 generations of the family and showed that the percentage of mutant mtDNA increased through each generation. The maternal grandmother of the proband, the mother, and the eldest aunt had 10%, 52%, and 50% mutant mtDNA in lymphocytes, respectively. The proband's mother and the proband had 84% and 90% mutant mtDNA in skin fibroblasts, respectively. The eldest aunt terminated a pregnancy when the 8993T-G mutation was identified in chorionic villi. In fetal tissues, the mutation load ranged from 91 to 96%. Ferlin et al. (1997) concluded that the findings in this family were consistent with a threshold effect, in which over 90% mutant mtDNA load results in clinical disease, and noted that prenatal diagnosis is feasible. Blok et al. (1997) analyzed mtDNA in oocytes from an asymptomatic mother of 3 children exhibiting heteroplasmic expression of the 8993T-G mutation associated with Leigh syndrome. The mother had 50% mutant mtDNA in her blood. One of the 7 oocytes analyzed showed no evidence of the mutation, while the remaining 6 had a mutant load of more than 95%. Blok et al. (1997) suggested that this observation reflected preferential amplification of the mtDNA variant during oogenesis. During formation of the zygote, mtDNA is derived exclusively from the oocyte; thus, it is possible that a de novo mutation may arise during oogenesis. A first carrier of a de novo mutation may be a mother who exhibits mosaicism for the mutation restricted to oocytes. However, the usual finding is that mothers of patients with Leigh syndrome and the 8993T-G mutation have substantial levels of the mutant mtDNA (38 to 76%). Takahashi et al. (1998) reported the case of a 1-year-old boy with Leigh syndrome associated with the 8993T-G mutation whose mother did not have the mutant mtDNA in her blood or urine sediment cells. Thus, a de novo mutation had occurred at a high level in oocytes, thereby causing Leigh syndrome in the boy. Generalized hypotonia was noted at birth. He developed apnea attacks and altered consciousness after upper respiratory infections at the ages of 2 and 4 months. At the age of 7 months, he showed symptoms of brainstem dysfunction, such as irregular respiration and swallowing difficulty. At the age of 9 months, growth retardation and microcephaly were obvious. Laboratory examinations showed increased lactate and pyruvate levels in blood and cerebrospinal fluid. In plants, cytoplasmic male sterility (CMS) is a mitochondrially inherited inability to produce viable pollen, and has been observed in more than 150 different plant species. Kempken et al. (1998) pointed out that in sorghum RNA editing is required to generate codons that encode leucine residues at positions equivalent to human 156 and 217. Loss of ATP6 RNA editing, as it occurs in sorghum, thus mimics mutations in human mitochondrial diseases. In all ATP6 protein sequences found in databases, including protists, plants (edited sequence), fungi, and animals, both amino acid positions are completely conserved. White et al. (1999) performed prenatal diagnosis in 2 mothers at risk of having affected children. One was the sister of a severely affected individual, and had previously had an unaffected child and a stillborn child. The other mother had 2 unaffected children and 2 affected children. The 8993T-G transversion was not found in the chorionic villus sample from 1 fetus or in the amniocytes from the other fetus. Both pregnancies were continued, and the resulting children were healthy at 2 years and 5 years of age. In 3 patients from 2 unrelated families, Baracca et al. (2000) investigated the biochemical phenotype associated with the 8993T-G mutation in the MTATP6 gene. All 3 carried more than 80% mutant genome in platelets and were manifesting clinically various degrees of the NARP syndrome phenotype. Their results suggested that the 8993T-G mutation induces a structural defect in F1F0-ATPase that causes a severe impairment of ATP synthesis. Kerrison et al. (2000) described the progression of retinopathy in NARP syndrome due to the T-to-G point mutation at the mtDNA nucleotide position 8993 in the MTATP6 gene. Prior to the onset of visual field constriction, ophthalmoscopy revealed salt-and-pepper retinopathy. After the visual fields had become constricted, fundus examination showed diffuse peripheral bone spicule formation, optic nerve pallor, and arteriolar attenuation consistent with retinitis pigmentosa. The authors stressed that mild mottling of the peripheral retinal pigment epithelium (salt-and-pepper retinopathy or retinitis pigmentosa sine pigmento) does not represent a specific entity but is an early stage of the retinitis pigmentosa, whether the patient has NARP or isolated retinitis pigmentosa. Hayashi et al. (2000) reported the histopathologic findings in the eyes from a patient with Leigh syndrome associated with the 8993T-G point mutation in mtDNA. Ophthalmologic signs and symptoms of Leigh syndrome include nystagmus, ophthalmoplegia, strabismus, optic nerve atrophy, and loss of the foveal reflex. A child with hypotonia, developmental delay, persistent lactic acidosis, seizures, and ataxia died of aspiration pneumonia at age 15 months. Analysis of mtDNA was positive for the 8993T-G mutation. The proportion of mutant genomes was estimated at approximately 95%. Light microscopic examination of the left eye revealed thinning of the nerve fiber and ganglion cell layers in the nasal macula and mild atrophy of the temporal optic nerve. Electron microscopy of the right eye showed numerous distended mitochondria in all cells, particularly in the retinal pigment epithelium, nonpigmented ciliary epithelium, and corneal endothelium. By transferring NARP mutant mtDNA (8993G-T in the MTATP6 gene) from fetal fibroblasts to lung carcinoma and osteosarcoma cells lacking endogenous mtDNA by cell-cytoplast fusion, Nijtmans et al. (2001) created mitochondrial transformant cells, or cybrids, able to grow in the absence of uridine. Immunoblot analysis revealed an abnormal amount of subcomplexes, F1-ATPase and V*, of mitochondrial ATP synthase. The cybrids had decreased subcomplex V assembly and decreased ATP synthesis capacity. However, the cells had no marked phenotype, suggesting that the effects of this mutation are subtle and have no effect on cell viability. Geromel et al. (2001) investigated the oxidative stress resulting from the NARP mutation in MTATP6, using cultured skin fibroblasts from 2 NARP patients presenting with an isolated complex V deficiency. A huge induction of the superoxide dismutase (147450 and 147460) activity was observed in these fibroblasts harboring more than 90% of mutant mitochondrial DNA. The oxidative stress denoted by the high SOD activity was associated with increased cell death. In glucose-rich medium, apoptosis appeared as the main cell death process associated with complex V deficiency. Complex V-deficient fibroblasts were successfully rescued by perfluoro-tris-phenyl nitrone, an antioxidant spin-trap molecule. The authors hypothesized that the superoxide production associated with the ATPase deficiency triggered by the NARP mutation could be sufficient to override cell antioxidant defenses and to result in cell commitment to die. Porto et al. (2001) reported an otherwise healthy 42-year-old woman with isolated late-onset cone-rod dystrophy characterized by difficulty driving at night beginning at age 40 years with deterioration of central and color vision due to the T8993G mitochondrial mutation. Two of her sons had NARP syndrome. A third son who was clinically diagnosed with Leigh syndrome died at age 4 years, prior to the recognition of the T8993G mutation in this family. The mother's mutation load was 50% T8993G mtDNA, while her sons with NARP had 75% T8993G mtDNA. The authors stated that Leigh disease is related to extreme heteroplasmy (more than 90%). They felt this family illustrated the remarkably variable expression of retinal and systemic manifestations related to the T8993G mutation, ranging from an isolated late-onset cone-rod dystrophy to a severe neurodegenerative process with a dramatic outcome. Porto et al. (2001) recommended genetic counseling for retinal dystrophy patients and emphasized careful evaluation of the family medical history. The 8993T-G mutation in MTATP6 impairs mitochondrial ATP synthesis. To overcome the biochemical defect, Manfredi et al. (2002) expressed wildtype ATPase 6 protein allotopically. The protein was derived from nucleus-transfected constructs encoding an amino-terminal mitochondrial targeting signal appended to a recoded ATPase 6 gene (made compatible with the universal genetic code) that also contained a carboxy-terminal fluorescent epitope tag. After transfection of human cells, the precursor polypeptide was expressed, imported into and processed within mitochondria, and incorporated into complex V. Allotopic expression of stably transfected constructs in cytoplasmic hybrids (cybrids) homoplasmic with respect to the 8993T-G mutation showed a significantly improved recovery after growth in selective medium as well as a significant increase in ATP synthesis. This was said to be the first successful demonstration of allotopic expression of an mtDNA-encoded polypeptide in mammalian cells and could form the basis of a genetic approach to treatment of a number of human mitochondrial disorders. Srivastava and Moraes (2001) showed that a mitochondrially targeted PstI restriction endonuclease degraded mtDNA harboring PstI sites, in some cases leading to a complete loss of mitochondrial genomes. When expressed in a heteroplasmic rodent cell line, containing 1 mtDNA haplotype with 2 sites for PstI and another haplotype having none, the mitochondrial PstI caused a significant shift in heteroplasmy, with an accumulation of the mtDNA haplotype lacking PstI sites. These experiments provided proof of the principle that restriction endonucleases may be feasible tools for genetic therapy of a subgroup of mitochondrial disorders. Patients harboring the T8993G mutation are potential candidates, since the mutation creates a novel PstI site which is not present in wildtype human mtDNA. In 6 individuals from 3 unrelated Italian families who had the 8993T-G mutation, Carelli et al. (2002) showed a close relationship between extent of tissue heteroplasmy, expression of the biochemical defect in platelets, and clinical involvement. A defect of ATP synthesis was evident even at low levels of mutant heteroplasmy (10 to 34% of normal) in the absence of clinical symptoms. ATP synthesis was severely decreased (4 to 9% of control values) in patients with high levels of mutation (greater than 80%), who showed the more severe clinical phenotypes of NARP and Leigh syndromes. No biochemical threshold effect was found. Carelli et al. (2002) noted that the combined effect of decreased ATP synthesis and increased reactive oxygen species production underlie the pathophysiology of mitochondrial diseases. Mattiazzi et al. (2004) showed that the 8993T-G mutation inhibits oxidative phosphorylation and results in enhanced free radical production. Antioxidants restored respiration and partially rescued ATP synthesis in cells harboring the T8993G mutation. The authors hypothesized that free radicals may play an important role in the pathogenesis of NARP/MILS and that antioxidants may be considered as a potentially useful tool in its treatment. Jung et al. (2007) reported a family with the 8993T-G mutation in which adult-onset progressive myoclonic epilepsy was a prominent feature. The proband was a 46-year-old woman with myoclonus, epilepsy, ataxia, and peripheral neuropathy. She had onset of myoclonus at age 19, which progressively worsened over her life. She did not have retinitis pigmentosa. Her mother had possible epilepsy, and 1 of her daughters developed epilepsy and ataxia in her late teens. There was a history of 3 infantile deaths on the maternal side, resulting from seizures in 2. Genetic analysis identified heteroplasmy for the 8993T-G mutation in the proband (80% in fibroblasts) and her daughter (60% in lymphocytes). Sgarbi et al. (2009) demonstrated that human fibroblasts containing the NARP-associated 8993T-G mutation could be protected from cell death when treated with alpha-ketoglutarate/aspartate to boost mitochondrial substrate-level phosphorylation (70% vs 5%; treated vs untreated survival after 72 hours). Homoplasmic 8993T-G cybrids showed similar results (75% vs 15%; treated vs untreated survival after 72 hours). In untreated fibroblasts and cybrids, the decrease in ATP content paralleled cell death, but ATP content returned to control levels after treatment. The findings indicated that ATP synthase-deficient cells can be rescued by increasing mitochondrial substrate-level phosphorylation, suggesting a potential therapeutic option for patients with such disorders. (less)