Management of Leigh syndrome: Current status and new insights.

Leigh syndrome (LS) is an inherited mitochondrial encephalopathy associated with gene mutations of oxidative phosphorylation pathway that result in early disability and death in affected young children. Currently, LS is incurable and unresponsive to many treatments, although some case reports indicate that supplements can improve the condition. Many novel therapies are being continuously tested in pre-clinical studies. In this review, we summarize the genetic basis of LS, current treatment, pre-clinical studies in animal models and the management of other mitochondrial diseases. Future therapeutical strategies and challenges are also discussed.

Experimental treatment of bilateral fetal chylothorax using in-utero pleurodesis.

OBJECTIVE: To assess the use and efficacy of in-utero pleurodesis for experimental treatment of bilateral fetal chylothorax. METHODS: This was a study of 78 fetuses with bilateral pleural effusion referred to three tertiary referral centers in Taiwan between 2005 and 2009. Fetuses were karyotyped following amniocentesis and the lymphocyte ratio in the pleural effusion was determined following thoracocentesis. Forty-nine (62.8%) fetuses had a normal karyotype and were recognized to have fetal chylothorax; of these, 45 underwent intrapleural injection of 0.1KE OK-432 per side per treatment. We evaluated clinical (hydrops vs. no hydrops) and genetic (mutations in the reported lymphedema-associated loci: VEGFR3, PTPN11, FOXC2, ITGA9) parameters, as well as treatment outcome. Long-term survival was defined as survival to 1 year of age. RESULTS: The overall long-term survival rate (LSR) was 35.6% (16/45); the LSR for non-hydropic fetuses was 66.7% (12/18) and for hydropic fetuses it was 14.8% (4/27). If we included only fetuses with onset of the condition in the second trimester, excluding those with onset in the third trimester, the LSR decreased to 29.4% (10/34). Notably, 29.6% (8/27) of hydropic fetuses had mutations in three of the four loci examined. CONCLUSIONS: OK-432 pleurodesis appeared to be an experimental alternative to the gold-standard technique of thoracoamniotic shunting in non-hydropic fetal chylothorax. In hydropic fetuses, pleurodesis appeared less effective.

Miglustat in patients with Niemann-Pick disease Type C (NP-C): a multicenter observational retrospective cohort study.

Miglustat has been shown to stabilize disease progression in children, juveniles and adults with Niemann-Pick disease type C (NP-C), a rare genetic disorder characterized by progressive neurological deterioration. We report findings from a retrospective observational cohort study assessing the effects of miglustat on neurological disease progression in patients treated in the clinical practice setting. Data from all NP-C patients prescribed miglustat at 25 expert centers were evaluated using a disease disability scale. The scale analyzed four key parameters of neurological disease progression in NP-C (ambulation, manipulation, language, swallowing). Mean individual parameter scores and a composite score were calculated at baseline (time of diagnosis) and up to 4 follow-up visits. Overall, 66 patients were included (mean [SD] age at diagnosis, 9.7 [7.6] years, and at treatment start, 12.8 [9.5] years). The median (range) miglustat exposure was 1.46 (0.05-4.51) years. Mean annual progression was +0.11 score units/year from diagnosis to treatment start, indicating disease progression prior to therapy, and decreasing to -0.01 score units/year from treatment start to last clinic visit, indicating stabilization. Stabilization of neurological disease on miglustat was observed in all age groups, but the magnitude of the effect was greater in patients diagnosed in late childhood and in juveniles and adults. Stabilization of neurological disease was also observed in a subset of 19 patients with extended pre-treatment information. Overall, these data support previous clinical trial findings indicating clinically relevant beneficial effects of miglustat on neurological disease progression in patients with NP-C.

A longitudinal study of Taiwanese sialidosis type 1: an insight into the concept of cherry-red spot myoclonus syndrome.

BACKGROUND AND PURPOSE: Sialidosis type 1 (ST-1) is a neurodegenerative disorder with limited long-term follow-up report. This study is to document the chronological profile of ST-1. METHODS: We perform serial analysis of 17 Taiwanese patients with ST-1 focusing on evolution of clinical features, electrophysiological findings, genetic studies, and neuroimage examinations. RESULTS: All patients had a mutation at 554A-->G in exon 3 of the NEU1 gene causing Ser182Gly substitution. Fifteen patients were homozygous. Two patients were heterozygous with novel mutations, 956C-->T causing Ala319Val in one and 163C-->T causing Gln55stop codon in the other. The neuraminidase activity was markedly decreased in all 11 available patients. Only three patients (17.6%) manifested the macular cherry-red spot. The majority of patients (82.3%) developed full-blown manifestation of myoclonus, ataxia, and seizures within 5 years. Abnormal somatosensory evoked potentials with giant cortical waves were found in all patients. Prolonged P100 peak latency of the visual evoked potentials (VEPs) were found in 16 patients (94.1%) in the early stage even without visual symptoms. CONCLUSION: ST-1 in Taiwanese population illustrates distinct characteristics of phenotype with infrequent cherry-red spot. We suggest to screen the NEU1 mutations in patients presenting action myoclonus with abnormal VEPs, even without macular cherry-red spots.

Simultaneous detection of mitochondrial DNA depletion and single-exon deletion in the deoxyguanosine gene using array-based comparative genomic hybridisation.

Intragenic exonic deletions, which cannot be detected by direct DNA sequencing, are a common cause of Mendelian disease. Array-based comparative genomic hybridisation (aCGH) is now widely used for the clinical diagnosis of large chromosomal deletions, but not small deletions or analysis of the mitochondrial genome. An oligonucleotide-based microarray that provides high-density coverage of the entire mitochondrial genome and nuclear genes related to mitochondrial disorders has been developed. In this report, the case of an infant referred with tyrosinaemia on newborn screening who developed liver failure is presented. DNA sequencing revealed a heterozygous missense mutation (c.679G>A, p.E227K) in the deoxyguanosine gene (DGUOK). Oligonucleotide aCGH allowed simultaneous detection of an intragenic heterozygous deletion of exon 4 of DGUOK and mitochondrial DNA depletion in blood and liver. Screening of the parents' DNA samples indicated that the patient was compound heterozygous for these mutations. An older sibling who had died from liver failure was then retrospectively diagnosed with the same mutations. This report shows the clinical utility of this oligoarray in the detection of changes in DNA copy number in both the mitochondrial and nuclear genomes, thus greatly improving the molecular diagnosis of mitochondrial disorders caused by nuclear genes involved in mitochondrial DNA biosynthesis.

Identification of eight novel mutations of the acid alpha-glucosidase gene causing the infantile or juvenile form of glycogen storage disease type II.

Glycogen-storage disease type II (GSDII; OMIM #232300), an autosomal recessive disorder caused by a deficiency of the glycogen hydrolysis enzyme acid alpha-glucosidase (acid GAA; acid maltase, EC. 3.2.10.20), results in the accumulation of glycogen in the lysosome. We performed a molecular genetic study on 29 patients with infantile-onset glycogen-storage disease type II (GSDII), 6 with juvenile-onset GSDII and one carrier for GSDII. Seventeen different mutations were identified among them; 8 were novel mutations: c.421C > A (p.L141M), c.872T > C (p.L291P), c.893A > C (p.Y298S), c.1375G > A (p.D459N), c.1437G > C (p.K479N), c.1509_1511del (p.A504del), c.1960T > C (p.S654P), and c.2174G > C (p.R725P). One of the mutations identified, c.2238G > C (p.W746C), which was a sequence change of unknown pathogenic significance causing diminished enzyme activity,was found homozygously in a juvenile-onset patient. We also found a juvenile-onset patient with homozygote c.1935C > A mutation which was frequently found in infantile-onset patients. In addition to mutations, we also identified 14 new polymorphisms in the acid alpha-glucosidase gene. The genotype/phenotype correlations indicated that c.2238G > C (p.W746C) is correlated with juvenile- onset GSDII and that c.872T > C (p.L291P) and c.1411_1414del (p.E471fsX5) are correlated with infantile-onset GSDII. Mutational analysis of GAA is useful in genetic counseling and prenatal diagnosis of the disease.

Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence.

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.

Deficiency of the carnitine transporter (OCTN2) with partial N-acetylglutamate synthase (NAGS) deficiency.

A patient with recurrent episodes of hyperammonaemia (highest ammonia level recorded 229 micromol/L, normal 9-33) leading to altered levels of consciousness was diagnosed with partial N-acetylglutamate synthase (NAGS) deficiency (9% residual activity) at age 5 years and was treated with ammonia-conjugating agents (Ucephan 250 mg/kg per day and later sodium phenylbutyrate 200-250 mg/kg per day) for 15 years. A chronically low serum carnitine level (pretreatment plasma free carnitine 4 nmol/L, normal 37 +/- 8 nmol/L; total carnitine 8 nmol/L, normal 46 +/- 10) was assumed to be secondary and was treated with supplemental carnitine (30-50 mg/kg per day). Hypoglycaemia (blood sugar 35 mg/dl, normal 70-100), cardiomegaly, and fatty liver were also noted at diagnosis. The patient died unexpectedly at age 20 years. In retrospect, it was learned that the patient had stopped his carnitine without medical consultation several weeks prior to his death. Additional molecular investigations identified two mutations (R254X and IVS3 + 1G > A) in the patient's OCTN2 (SLC22A5) gene, consistent with a diagnosis of primary carnitine deficiency due to carnitine transporter defect. R245X is a founder mutation in Southern Chinese populations. It is unknown whether the original NAGS deficiency was primary or secondary, but molecular analysis of the NAGS gene failed to identify mutations. Urea cycle enzyme expression may be affected by fatty acid suppression of an AP-1 binding site in the promoter enhancer region of the urea cycle gene. Regardless, it is clear that the NAGS abnormality has led to delay of recognition of the OCTN2 defect, and modified the clinical course in this patient.

Treatment of Niemann-Pick disease type C in two children with miglustat: initial responses and maintenance of effects over 1 year.

Niemann-Pick disease type C (NP-C) is a lipid storage disorder characterized by the accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system of certain cells in the central nervous system (CNS) and visceral organs. Clinical symptoms include progressive neurological deterioration and visceral organomegaly. Miglustat, a small iminosugar molecule approved for the treatment of Gaucher disease, reversibly inhibits glucosylceramide synthase, which catalyses the first committed step in glycosphingolipid synthesis. The physicochemical properties of miglustat allow it to cross the blood-brain barrier and suggest possible benefits in lysosomal storage diseases affecting the CNS. Here, we present findings in two children with NP-C, aged 14 years (patient 1) and 9 years (patient 2), treated with miglustat for 1 year. Before treatment, patient 1 presented with severe difficulties in swallowing and walking, and patient 2 with problems mostly affecting communication and social interaction. Videofluoroscopic studies in patient 1 demonstrated a substantial improvement in swallowing by month 6 of treatment, and ambulation index measurements indicated improved walking. Mini Mental-State Examination (MMSE) assessments in patient 2 showed cognitive improvement by month 6, which was sustained up to month 12. Liver/spleen volume and plasma chitotriosidase activities were stabilized in both cases. There was no weight loss during treatment. Patient 1 experienced severe but self-limiting paresthesia, which was not associated with peripheral neuropathy. We conclude that miglustat can provide therapeutic benefits in CNS symptoms and allows stabilization of systemic disease in childhood-onset NP-C. Further follow-up is crucial to determine the long-term maintenance of these effects.

Subtelomeric rearrangements and 22q11.2 deletion syndrome in anomalous growth-restricted fetuses with normal or balanced G-banded karyotype.

OBJECTIVE: To determine the frequencies of cryptic subtelomeric rearrangements and 22q11.2 deletion in anomalous growth-restricted fetuses with normal or balanced G-banded karyotypes. METHODS: This was a study of 27 consecutive fetuses at a median gestational age of 26 (range, 19-33) weeks, that had intrauterine growth restriction (IUGR) as well as at least one major structural anomaly, and a normal or balanced G-banded karyotype. The median maternal age was 29 (range, 17-39) years. Fluorescence z in-situ hybridization (FISH) diagnosis of the cultured amniocytes with the probe TUPLE 1, and then the Chromoprobe Multiprobe-T system were used, respectively, to screen for the frequency of 22q11.2 deletion syndrome and subtelomeric rearrangements involving the 41 unique chromosome ends (i.e. excluding the five short arms of acrocentric autosomes (no. 13, 14, 15, 21 and 22)). Those that had suspected deleted subtelomeres were reanalyzed with a specific subtelomeric probe, TelVysion. RESULTS: Of the 27 fetuses, three (11%) were affected with 22q11.2 deletion syndrome and two (7.4%) had subtelomeric deletions (one monosomy 21q22.3, one monosomy 1p36.3). Of the 11 fetuses with congenital heart defects, three (27.3%) had 22q11.2 deletion syndrome and one (9.1%) had monosomy 1p36.3. In the remaining 16 fetuses without congenital heart defects, none had 22q11.2 deletion syndrome. However, one (6.3%) had cryptic rearrangement involving subtelomeres. CONCLUSION: Prenatal subtelomeric FISH screening is technically feasible using cultured amniocytes. We propose that 22q11.2 deletion syndrome and cryptic subtelomere rearrangements may be important etiologies of fetuses with IUGR and at least one structural anomaly, along with a normal karyotype or one that is balanced by traditional G-banding. Fetuses with congenital heart defects and IUGR should undergo FISH to exclude 22q11.2 deletion syndrome. In fetuses with IUGR and at least one major structural anomaly but without congenital heart defects, screening of subtelomeric rearrangements may contribute to further elucidation of the underlying etiology.

Six novel NPC1 mutations in Chinese patients with Niemann-Pick disease type C.

In patients with Niemann-Pick disease type C (NPC), an autosomal recessive lipid storage disorder, neurodegeneration can occur in early life. Vertical ophthalmoplegia and extrapyramidal signs may be seen. Cholestatic jaundice and hepatosplenomegaly occur frequently in patients with early onset disease, with bone marrow biopsies showing diffuse infiltration of foamy histiocytes. Cholesterol esterification in skin fibroblasts is reduced, resulting in intracellular accumulation of cholesterol. NPC1 mutations are responsible for the disease in approximately 95% of patients. NPC1 encodes a 1278 amino acid protein which contains 13 transmembrane domains. Over 130 mutations have been identified in NPC1, with over a third present within an NPC1 specific cysteine-rich domain positioned in a large extracellular loop. It has been proposed that the defect in cholesterol homoeostasis is the cause of neuronal apoptosis, but the precise role of the NPC1 protein and the functional implications of its mutations remain unknown. Although NPC is routinely diagnosed by biochemical analysis, identification of molecular defects helps confirm the diagnosis and enables family studies, and rapid, accurate prenatal diagnosis. This report describe the analysis of the NPC1 gene in five Taiwanese/Chinese patients with NPC. Six novel NPC1 mutations (N968S, G1015V, G1034R, V1212L, S738Stop, and I635fs) were identified of which three are missense mutations located in the cysteine-rich domain. These are the first NPC1 mutations reported from Chinese patients with NPC.

Two novel mutations in the alpha-galactosidase A gene in Chinese patients with Fabry disease.

Fabry disease is an X-linked disorder caused by a deficiency of the lysosomal alpha-galactosidase A [EC 3.2.1.22]. The molecular diagnosis of Fabry disease is important for genotype/phenotype correlation, pre-natal or early diagnosis, and detection of carrier status. Although more than 200 genotypes of the alpha-galactosidase A gene have been identified, mutation data on the Chinese population is sparse. We recently identified two unrelated Chinese families with Fabry disease. Mutation analysis was performed by polymerase chain reaction (PCR) sequencing of the seven exons and adjacent introns of the alpha-galactosidase A gene. Two novel mutations were identified: in family I, a C-to-A transversion resulted in an early termination at amino acid 222 (Y222X), while in family II, an A-to-G transition resulted in a substitution of alanine for threonine at amino acid 410 (T410A). Carrier status was identified in all four females in the two families. The genotype Y222X is associated with classic Fabry disease, with unexpectedly rapid deterioration of visual acuity, while T410A is associated with a milder Fabry disease, with ventricular hypertrophy and neuropathic pain.