ALG11-CDG: Three novel mutations and further characterization of the phenotype.

We report on two novel patients with ALG11-CDG. The phenotype was characterized by severe psychomotor disability, progressive microcephaly, sensorineural hearing loss, therapy-resistant epilepsy with burst suppression EEG, cerebral atrophy with, in one of them, neuronal heterotopia, and early lethality. Analysis of ALG11 revealed compound heterozygosity involving three novel mutations: the splice site mutation c.45-2A > T, the c.36dupG duplication, and the missense mutation c.479G > T (p.G160V) that was present in both.

Clinical onset and course, response to treatment and outcome in 24 patients with the cblE or cblG remethylation defect complemented by genetic and in vitro enzyme study data.

BACKGROUND: The cobalamin E (cblE) (MTRR, methionine synthase reductase) and cobalamin G (cblG) (MTR, methionine synthase) defects are rare inborn errors of cobalamin metabolism leading to impairment of the remethylation of homocysteine to methionine. METHODS: Information on clinical and laboratory data at initial full assessment and during the course of the disease, treatment, outcome and quality of life was obtained in a survey-based, retrospective study from physicians caring for patients with the CblE or CblG defect. In addition, data on enzyme studies in cultured skin fibroblasts and mutations in the MTRR and MTR gene were analysed. RESULTS: In 11 cblE and 13 cblG patients, failure to thrive, feeding problems, delayed milestones, muscular hypotonia, cognitive impairment and macrocytic anaemia were the most frequent symptoms. Delay in diagnosis depended on age at first symptom and clinical pattern at presentation and correlated significantly with impaired communication abilities at follow-up. Eighteen/22 patients presented with brain atrophy or white matter disease. Biochemical response to treatment with variable combinations of betaine, cobalamin, folate was significant. The overall course was considered improving (n = 8) or stable (n = 15) in 96% of patients, however the average number of CNS symptoms per patient increased significantly over time and 16 of 23 patients were classified as developmentally delayed or severely handicapped. In vitro enzyme analysis data showed no correlation with outcome. Predominantly private mutations were detected and no genotype- phenotype correlations evident. CONCLUSIONS: The majority of patients with the cblE and cblG defect show limited clinical response to treatment and have neurocognitive impairment.

Prevalence and impact on FEV(1) decline of chronic methicillin-resistant Staphylococcus aureus (MRSA) colonization in patients with cystic fibrosis. A single-center, case control study of 165 patients.

BACKGROUND: Risk factors for methicillin-resistant Staphylococcus aureus (MRSA) in Cystic Fibrosis (CF) and the impact on CF disease progression are still under debate. The objectives of this study were to determine clinical variables associated with MRSA colonization and examine impact on FEV(1) evolution in CF patients. METHODS: A retrospective case-control study using the University Hospital of Brussels CF clinic patient registry from 2002 to 2010, comparing clinical variables and decline of FEV(1) of MRSA positive patients with age and sex matched controls, chronically colonized with S. aureus. RESULTS: Thirty of the 165 CF patients, chronically colonized with S. aureus, had cultures positive for MRSA (18.2%). Excluding patients under 4 years, the prevalence became 15.2% (23/151). Chronic colonization (i.e., three or more consecutive positive cultures) was found in 19/151 (12.6%). The MRSA positive group showed a higher proportion of patients with genotype F508del, less pancreas sufficient patients, more bronchiectasis and more frequent hospitalization. The FEV(1) recorded one year prior to, and at the moment of MRSA acquisition, was lower but not significantly different from that obtained in controls (72.9%±26.6 vs 84.3±21.8 and 68.2%±27.1 vs 81.4%±24.3 respectively, p>0.1). However, FEV(1) decline over 2- and 6-year periods, were significantly greater in the chronic MRSA group than in the controls (-5%±5.5 vs -2.5±2.3 over 2 years (p=0.043) and -1.8%±4.6 vs -1.0%±1.9 over a 6-year period (p=0.026)). CONCLUSION: In our center the prevalence of MRSA in CF patients, chronically colonized with S. aureus and over the age of 4 years, was 15.2% (12.6% chronic infection). MRSA colonization was shown to be associated with a genotype F508del, presence of bronchiectasis and hospitalization. Our spirometric data also show that a MRSA episode entails an FEV(1) decline that is almost double that predicted for CF patients who can remain unaffected by MRSA.

TUBA1A mutations: from isolated lissencephaly to familial polymicrogyria.

BACKGROUND: Mutations in the TUBA1A gene have been reported in patients with lissencephaly and perisylvian pachygyria. METHODS: Twenty-five patients with malformations of cortical development ranging from lissencephaly to polymicrogyria were screened for mutations in TUBA1A. RESULTS: Two novel heterozygous missense mutations in TUBA1A were identified: c.629A>G (p.Tyr210Cys) occurring de novo in a boy with lissencephaly, and c.13A>C (p.Ile5Leu) affecting 2 sisters with polymicrogyria whose mother presented somatic mosaicism for the mutation. CONCLUSIONS: Mutations in TUBA1A have been described in patients with lissencephaly and pachygyria. We report a mutation in TUBA1A as a cause of polymicrogyria. So far, all mutations in TUBA1A have occurred de novo, resulting in isolated cases. This article describes familial recurrence of TUBA1A mutations due to somatic mosaicism in a parent. These findings broaden the phenotypic spectrum associated with TUBA1A mutations and have implications for genetic counseling.

Mutations in SACS cause atypical and late-onset forms of ARSACS.

BACKGROUND: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a complex neurodegenerative disorder caused by mutations in SACS. The phenotype consists of a childhood-onset triad of cerebellar ataxia, peripheral neuropathy, and pyramidal tract signs. OBJECTIVE: To provide more insight into the prevalence of SACS mutations and the variability of the associated phenotype. METHODS: Mutation screening of SACS by direct sequencing and multiplex amplicon quantification for detection of intragenic copy number variations in a cohort of 85 index patients with phenotypes suggestive for ARSACS. Additional short tandem repeat (STR) marker analysis was performed for haplotype sharing. RESULTS: In 11 families,18 new SACS mutations were found (12.9% of total cohort). Five patients displayed onset ages in adulthood, a feature not known to be associated with ARSACS. The remaining index patients displayed a classic early onset phenotype. Initial phenotypic presentation was atypical in several patients, obscuring the clinical diagnosis. A founder mutation in SACS was identified in 3 Belgian families. In one isolated patient, an intragenic SACS deletion of exons 3-5 was detected. Partial SACS deletions were not previously described. CONCLUSIONS: In this study, we enlarge the ARSACS phenotype and the underlying genetic spectrum of SACS mutations. Patients with ARSACS are more common than previously known and risk underdiagnosis due to late onset age and unusual presentation.

Progressive myoclonic epilepsy as an adult-onset manifestation of Leigh syndrome due to m.14487T>C.

BACKGROUND: m.14487T>C, a missense mutation (p.M63V) affecting the ND6 subunit of complex I of the mitochondrial respiratory chain, has been reported in isolated childhood cases with Leigh syndrome (LS) and progressive dystonia. Adult-onset phenotypes have not been reported. OBJECTIVES: To determine the clinical-neurological spectrum and associated mutation loads in an extended m.14487T>C family. METHODS: A genotype-phenotype correlation study of a Belgian five-generation family with 12 affected family members segregating m.14487T>C was carried out. Clinical and mutation load data were available for nine family members. Biochemical analysis of the respiratory chain was performed in three muscle biopsies. RESULTS: Heteroplasmic m.14487T>C levels (36-52% in leucocytes, 97-99% in muscle) were found in patients with progressive myoclonic epilepsy (PME) and dystonia or progressive hypokinetic-rigid syndrome. Patients with infantile LS were homoplasmic (99-100% in leucocytes, 100% in muscle). We found lower mutation loads (between 8 and 35% in blood) in adult patients with clinical features including migraine with aura, Leber hereditary optic neuropathy, sensorineural hearing loss and diabetes mellitus type 2. Despite homoplasmic mutation loads, complex I catalytic activity was only moderately decreased in muscle tissue. INTERPRETATION: m.14487T>C resulted in a broad spectrum of phenotypes in our family. Depending on the mutation load, it caused severe encephalopathies ranging from infantile LS to adult-onset PME with dystonia. This is the first report of PME as an important neurological manifestation of an isolated mitochondrial complex I defect.

Mutational study in the PDHA1 gene of 40 patients suspected of pyruvate dehydrogenase complex deficiency.

We screened for PDHA1 mutations in 40 patients with biochemically demonstrated PDHc deficiency or strong clinical suspicion, and found changes with probable pathological significance in 20. Five patients presented new mutations: p.A169V, c.932_938del, c.1143_1144 ins24, c.1146_1159dup and c.510-30G> A, this latter is a new undescribed cause of exon 6 skipping. Another four mutations have been found, and previously reported, in our patients: p.H113D, p.P172L, p.Y243del and p.Y369Q. Eleven patients presented seven known mutations: p.R127Q, p.I166I, p.A198T, p.R263G, p.R302C, p.R378C and c.1142_1145dup. The latter three were found in more than one unrelated patient: p.R302C was detected in a heterozygous girl and a mosaic male, p.R378C in two males and finally, c.1142_1145dup in three females; only one in 20 mothers was found to be a carrier (p.R263G). Apart from those 20 patients, the only alteration detected in one girl with clear PDHc and PDH-E1 deficiency was the silent change c.396A> C (p.R132R), and other eight PDHc deficient patients carry combinations of known infrequent polymorphisms that are overrepresented among our 20 unsolved patients. The importance of these changes on PDH activity is unclear. Investigations in the other PDHc genes are in course in order to elucidate the genetic defect in the unresolved patients.

Abdominal pain and vomiting as first sign of mitochondrial disease.

We describe a patient in whom abdominal pain and vomiting were the presenting symptoms of Mitochondrial Myopathy Encephalopathy, Lactic Acidosis with Stroke-like episodes syndrome (MELAS). Mitochondrial disorders usually present with neurological symptoms or with myopathic features at any age. Although many patients develop visceral symptoms at a certain moment during the course of the disease, only in a minority of patients these symptoms are the unique presenting ones. The proband was initially diagnosed as having gastro-oesophageal reflux and it was only after detailed clinical history that an underlying metabolic defect was suspected and the molecular defect identified.

Immunohistochemical analysis of the oxidative phosphorylation complexes in skeletal muscle from patients with mitochondrial DNA encoded tRNA gene defects.

BACKGROUND: Mitochondrial diseases display a heterogeneous spectrum of clinical phenotypes and therefore the identification of the underlying gene defect is often a difficult task. AIMS: To develop an immunohistochemical approach to stain skeletal muscle for the five multi-protein complexes that organise the oxidative phosphorylation (OXPHOS) in order to improve the diagnostic workup of mitochondrial defects. METHODS: OXPHOS complexes were visualised in skeletal muscle tissue using antibodies directed against different subunits. The staining patterns of patients with heteroplasmic defects in mtDNA tRNA genes were compared with those of normal and disease controls. RESULTS: Normal skeletal muscle displayed a checkerboard staining pattern for complexes I to V due to the higher mitochondrial content of slow muscle fibres versus fast fibres. In patients with tRNA defects, a much more heterogeneous staining pattern was observed for complex I (all six patients) and complex IV (4 of 6 patients): a mosaic staining pattern in which individual fibres displayed staining intensities that ranged from strong to negative. Ragged red fibres (RRFs) in patients with MERRF (myoclonic epilepsy and ragged red fibres) were all complex I and IV negative, while in patient with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) the majority of RRFs were complex I negative and complex IV positive. CONCLUSION: Immunohistochemical detection of OXPHOS complexes could represent a valuable additional diagnostic tool for the evaluation of mitochondrial cytopathy. The technique helps to detect heteroplasmic mtDNA defects. Staining for complex I in particular was able to identify two tRNA patients that stayed undetected with routine histochemical evaluation.

Defining the phenotype in an autosomal recessive cutis laxa syndrome with a combined congenital defect of glycosylation.

Autosomal recessive cutis laxa is a genetically heterogeneous condition. Its molecular basis is largely unknown. Recently, a combined disorder of N- and O-linked glycosylation was described in children with congenital cutis laxa in association with severe central nervous system involvement, brain migration defects, seizures and hearing loss. We report on seven additional patients with similar clinical features in combination with congenital disorder of glycosylation type IIx. On the basis of phenotype in 10 patients, we define an autosomal recessive cutis laxa syndrome. The patients have a complex phenotype of neonatal cutis laxa, transient feeding intolerance, late closure of the fontanel, characteristic facial features including down-slanting palpebral fissures, short nose and small mouth, and developmental delay. There is a variable degree of the central nervous system involvement and variable systemic presentation. The biochemical analysis using transferrin isoelectric focusing gives false negative results in some of the youngest patients. Analysis of the apolipoprotein C-III isoelectric focusing, however, is diagnostic in all cases.

Glomerular filtration rate in patients with Cushing's disease: a matched case-control study.

BACKGROUND: Patients with Cushing's disease have a high prevalence of atherosclerosis and maintain an increased cardiovascular risk even after cure of the disease. However, the impact of Cushing's disease on renal function remains to be quantified. OBJECTIVES: To evaluate glomerular filtration rate (GFR) and to identify predictors of GFR in patients with Cushing's disease. DESIGN AND METHODS: We conducted a matched case-control study: 18 patients with active or cured Cushing's disease were compared with healthy population controls matched for age and sex. The main outcome measures were GFR and micro-albuminuria. RESULTS: Patients with Cushing's disease had a lower GFR, as measured by 24-h creatinine clearance (79 versus 95 ml/min per 1.73 m2, P = 0.005) and estimated by the MDRD2 formula (75 versus 88 ml/min per 1.73 m2, P = 0.008). Multiple regression analyses indicated that disease duration was the strongest predictor for a worse GFR. The prevalence of micro-albuminuria was low (5.5% in both groups). CONCLUSIONS: Patients with Cushing's disease have a decreased GFR. Even if they are cured, close follow-up with strict control of cardiovascular risk factors and monitoring of GFR seems mandatory. Furthermore, the dosage of certain drugs should be adapted to the individual GFR.

Aminoacylase I deficiency: a novel inborn error of metabolism.

This is the first report of a patient with aminoacylase I deficiency. High amounts of N-acetylated amino acids were detected by gas chromatography-mass spectrometry in the urine, including the derivatives of serine, glutamic acid, alanine, methionine, glycine, and smaller amounts of threonine, leucine, valine, and isoleucine. NMR spectroscopy confirmed these findings and, in addition, showed the presence of N-acetylglutamine and N-acetylasparagine. In EBV transformed lymphoblasts, aminoacylase I activity was deficient. Loss of activity was due to decreased amounts of aminoacylase I protein. The amount of mRNA for the aminoacylase I was decreased. DNA sequencing of the encoding ACY1 gene showed a homozygous c.1057 C>T transition, predicting a p.Arg353Cys substitution. Both parents were heterozygous for the mutation. The mutation was also detected in 5/161 controls. To exclude the possibility of a genetic polymorphism, protein expression studies were performed showing that the mutant protein had lost catalytic activity.

Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP12.

In patients with mitochondrial encephalomyopathies an increasing number of causative gene defects have been detected. The number of identified pathogenic mitochondrial DNA mutations has largely increased over the past 15 years. Recently, much attention has turned to the investigation of nuclear oxidative phosphorylation (OXPHOS) gene defects. Within the OXPHOS defects, complex V deficiency is rarely found and, so far, these defects have only been attributed to mutations in the mitochondrial MTATP6 gene. Mutation analysis of the complete coding regions at the cDNA level of the nuclear ATP11, ATP12, ATPalpha, ATPbeta and ATPgamma genes and the mitochondrial MTATP6 and MTAT8 genes was undertaken in two unrelated patients. Blue Native polyacrylamide gel electrophoresis followed by catalytic staining had already documented their complex V decreased activity. Extensive molecular analysis of five nuclear and two mitochondrial genes revealed a mutation in the ATP12 assembly gene in one patient. This mutation is believed to be the cause of the impaired complex V activity. To our knowledge, this is the first report of a pathogenic mutation in a human nuclear encoded ATPase assembly gene.

Anti-epileptogenesis research: the clinical relevance.

In recent years, different research lines have examined the epileptogenic process in order to understand the different stages in this process, and with the hope that early recognition and intervention could prevent chronic epilepsy in patients with epileptic seizures. In animals, acquired epilepsy is studied most commonly with kindling models, status epilepticus models and traumatic brain injury models. Molecular genetic studies substantially help to understand age-specific channel and receptor abnormalities. Major progress has been made in recent years and we are now waiting for the first large scale multi-center clinical trials that test the possible anti-epileptogenic properties of anti-epileptic drugs or other compounds in well defined patient groups. In clinical practice, a structured diagnostic work-up in all patients with recurrent seizures is a first and necessary step in the recognition of patients at risk for developing chronic and refractory epilepsy.

Homozygous Gly555Glu mutation in the nuclear-encoded 70 kDa flavoprotein gene causes instability of the respiratory chain complex II.

A homozygous mutation in the flavoprotein (Fp) gene associated with complex II deficiency was demonstrated in a patient with consanguineous parents. She succumbed at 5(1/2) months of age following a respiratory infection. The c1664G-->A transition detected, predicted the substitution of the small uncharged glycine at position 555 by glutamic acid. Her clinical course was at variance with the Leigh syndrome in three previously reported patients due to Fp gene mutations. In this proband, CRM for flavoprotein as well as iron-containing protein (Ip) was decreased, CRM for the entire complex II (130 kDa) being reduced even more. This observation prompts speculation of a labile interaction between Ip and Fp polypeptides and of a key role of the amino acid at position 555 in the interacting domain.

Blue native polyacrylamide gel electrophoresis: a powerful tool in diagnosis of oxidative phosphorylation defects.

Catalytic activity of oxidative phosphorylation complexes is maintained following separation by Blue Native polyacrylamide gel electrophoresis (BN-PAGE). In BN-PAGE gels, using histochemical staining methods, we have demonstrated enzymatic activity of the complexes I, II, IV, and V in heart and skeletal muscle, liver, and cultured skin fibroblasts. The combination of BN-PAGE and catalytic staining can be successfully applied for detection of complex deficiencies. Tissues from 18 patients with deficiency in the oxidative phosphorylation as detected by spectrophotometric assay were used (10 patients complex IV, three patients complex I, one patient complex II, one patient complex I+III, three patients complex I+IV). The gene defect was located in nuclear DNA in five patients and mitochondrial DNA in one patient. In samples from patients with a severe deficiency, almost complete absence of the corresponding enzyme band is observed after catalytic staining in the gel. In patients with known partial deficiency, a milder decrease of the corresponding enzyme band is demonstrated. The amount of protein in complexes I, V, and III can easily be evaluated in samples from heart and skeletal muscle after separation by BN-PAGE using silver or Coomassie staining. The protein amount in complex IV is difficult to visualize by silver staining but easier by the Coomassie technique. In samples from liver and cultured skin fibroblasts, evaluation of protein amount is more difficult due to high background staining. In these tissues, immunoblotting can be done after BN-PAGE and subsequent transfer to a nitrocellulose membrane.

A new mitochondrial point mutation in the transfer RNA(Leu) gene in a patient with a clinical phenotype resembling Kearns-Sayre syndrome.

OBJECTIVE: To report on the molecular identification of a novel heteroplasmic G-to-A transition at mitochondrial DNA position 3249 in transfer RNA(Leu) gene in a patient with a clinical phenotype resembling Kearns-Sayre syndrome. PATIENT AND METHODS: A 34-year-old patient had been suffering for more than 10 years from progressive visual failure, neurosensorial hearing loss, exercise intolerance, muscle weakness, paresthesia in the lower limbs, and difficulties swallowing. Clinical examination revealed generalized muscle wasting, ptosis, external ophthalmoplegia, and ataxia. Ophthalmologic examination showed dystrophic features in the cornea and retina. In skeletal muscle, morphologic and biochemical studies of the respiratory chain complexes were performed. Polymerase chain reaction, single-strand conformation polymorphism, and direct sequencing were used to screen for mutations in the 22 mitochondrial transfer RNA genes. RESULTS: In skeletal muscle, a significantly decreased catalytic activity of complex I was detected by spectrophotometric analysis and numerous cytochrome c oxidase-negative ragged-red fibers were seen on morphologic examination. A G-to-A substitution 3249 (G3249A) mutation was found in the transfer RNA(Leu) gene of the patient and mutant mitochondrial DNA represented 85% of the total in skeletal muscle but only 45% in leukocytes. The mutation was shown to be present in a small fraction in leukocytes from the unaffected mother and to be absent in leukocytes from the healthy sister. CONCLUSIONS: A causal relationship between a heteroplasmic G3249A transfer RNA(Leu) mutation in a patient suffering from progressive external ophthalmoplegia, retinal dystrophy, ataxia, neurosensorial hearing loss, and muscle wasting is postulated. To our knowledge, the G3249A mutation has never previously been described and was not detected in control subjects.