Luísa Diogo

Mitochondrial dysfunction in autism spectrum disorders: a population-based study

A minority of cases of autism has been associated with several different organic conditions, including bioenergetic metabolism deficiency. In a population-based study, we screened associated medical conditions in a group of 120 children with autism (current age range 11y 5mo to 14y 4mo, mean age 12y 11mo [SD 9.6mo], male:female ratio 2.9:1). Children were diagnosed using Diagnostic and Statistical Manual of Mental Disorders criteria, the Autism Diagnostic Interview--Revised, and the Childhood Autism Rating Scale; 76% were diagnosed with typical autism and 24% with atypical autism. Cognitive functional level was assessed with the Griffiths scale and the Wechsler Intelligence Scale for Children and was in the normal range in 17%. Epilepsy was present in 19 patients. Plasma lactate levels were measured in 69 patients, and in 14 we found hyperlactacidemia. Five of 11 patients studied were classified with definite mitochondrial respiratory chain disorder, suggesting that this might be one of the most common disorders associated with autism (5 of 69; 7.2%) and warranting further investigation.

l-2-Hydroxyglutaric aciduria: clinical, biochemical and magnetic resonance imaging in six Portuguese pediatric patients

We present clinical, biochemical and cranial magnetic resonance imaging data of six pediatric patients with L-2-hydroxyglutaric aciduria. All the children have the same ethic origin and lived in the northern area of Portugal. Our findings reinforce the described phenotype of this rare metabolic disease with mental deficiency, severe cerebellar dysfunction, mild extrapyramidal and pyramidal symptoms, progressive macrocephaly and seizures. Magnetic resonance imaging revealed subcortical leukoencephalopathy, cerebellar atrophy and signal changes in the putamina and dentate nuclei. These were similar to those of the previous reports in all patients. The urinary excretion of L-2-hydroxyglutaric acid was variably increased in all patients. The other persistent biochemical abnormality was hyperlysinemia. We have found a strong correlation between the severity of the clinical manifestations and the extension of the lesions in the neuroimaging studies. There was no correlation between the clinical findings and the amount of urinary excretion of L-2-hydroxyglutaric acid. We report the second case in the literature of a cerebral thalamic tumor in L-2-hydroxyglutaric aciduria; neuropathological examination of the surgical biopsy demonstrated a diffuse fibrillary astrocytoma.

Spectrum of MMACHC mutations in Italian and Portuguese patients with combined methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria (MMA) and homocystinuria, cblC type (MIM 277400) is the most frequent inborn error of vitamin B(12). The recent identification of the disease gene, MMACHC, has permitted preliminary genotype-phenotype correlations. We studied 24 Italian and 17 Portuguese patients with cblC defect to illustrate the spectrum of mutations in a southern European population and discuss the impact that mutation identification has on routine diagnostic procedures. Since the metabolic defect raises the serum levels of homocysteine, we also tested if variants in MTHFR-playing a key role in homocysteine remethylation pathway-could act as genetic modifier in cblC defect. We found that the c.271dupA (accounting for 55% of the MMACH alleles in our cohort) followed by c.394C>T (16%) and c.331C>T (9%) were the most frequent mutations. In our study we also identified a novel mutation (c.544T>C). On the other hand, the MTHFR genotype did not appear to influence age at onset, the clinical phenotype and outcome of patients with cblC defect. This study shows that mutation screening for the most common MMACH mutations occurring in early-onset forms (c.271dupA and c.331C>T) seems to have a high diagnostic yield in a southern European population with cblC defect. Although the identification of the gene defect per se does not predict completely time and severity of disease appearance, our data corroborate the importance of a molecular testing to offer accurate prenatal diagnosis to couples at high risk of having affected children.

Phenotype and genotype in 101 males with X-linked creatine transporter deficiency

Background Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and phenotype–genotype correlation has been lacking. Methods We performed a retrospective study of clinical, biochemical and molecular genetic data of 101 males with X-linked creatine transporter deficiency from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). Results and conclusions Most patients developed moderate to severe intellectual disability; mild intellectual disability was rare in adult patients. Speech language development was especially delayed but almost a third of the patients were able to speak in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine to creatinine ratio proved to be a reliable screening method besides MR spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. A third of patients had a de novo mutation in the SLC6A8 gene. Mothers with an affected son with a de novo mutation should be counselled about a recurrence risk in further pregnancies due to the possibility of low level somatic or germline mosaicism. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3′ end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesise creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurones.

Guanidinoacetate methyltransferase (GAMT) deficiency : Outcomes in 48 individuals and recommendations for diagnosis, treatment and monitoring

We collected data on 48 patients from 38 families with guanidinoacetate methyltransferase (GAMT) deficiency. Global developmental delay/intellectual disability (DD/ID) with speech/language delay and behavioral problems as the most affected domains was present in 44 participants, with additional epilepsy present in 35 and movement disorder in 13. Treatment regimens included various combinations/dosages of creatine-monohydrate, l-ornithine, sodium benzoate and protein/arginine restricted diets. The median age at treatment initiation was 25.5 and 39 months in patients with mild and moderate DD/ID, respectively, and 11 years in patients with severe DD/ID. Increase of cerebral creatine and decrease of plasma/CSF guanidinoacetate levels were achieved by supplementation with creatine-monohydrate combined with high dosages of l-ornithine and/or an arginine-restricted diet (250 mg/kg/d l-arginine). Therapy was associated with improvement or stabilization of symptoms in all of the symptomatic cases. The 4 patients treated younger than 9 months had normal or almost normal developmental outcomes. One with inconsistent compliance had a borderline IQ at age 8.6 years. An observational GAMT database will be essential to identify the best treatment to reduce plasma guanidinoacetate levels and improve long-term outcomes.

NMR Derivatives for Quantification of 2H and 13C‐Enrichment of Human Glucuronide from Metabolic Tracers

Quantification of 2H and 13C enrichment distributions in human urinary glucuronide following ingestion of 2H2O and 13C gluconeogenic tracers was achieved by NMR spectroscopy of the 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone and 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivatives. The derivatization process is simple and can be applied to any glucuronide species. The derivatives are highly soluble in acetonitrile and generate well‐resolved and narrow 2H and 13C NMR signals. The 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided resolution of the six glucuronide 13C signals and numerous 13C isotopomer populations through one‐ and two‐bond 13C‐13C‐coupling, while the 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided complete resolution of the 2H NMR signals for the five glucuronide hydrogens. The isopropylidene methyl signals were also resolved and provided an internal 2H enrichment standard following the acetonation of glucuronolactone with deuterated acetone.

CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.

Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes

We evaluated coenzyme Q₁₀ (CoQ) levels in patients studied under suspicion of mitochondrial DNA depletion syndromes (MDS) (n=39). CoQ levels were quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A high percentage of MDS patients presented with CoQ deficiency as compared to other mitochondrial patients (Mann-Whitney-U test: p=0.001). Our findings suggest that MDS are frequently associated with CoQ deficiency, as a possible secondary consequence of disease pathophysiology. Assessment of muscle CoQ status seems advisable in MDS patients since the possibility of CoQ supplementation may then be considered as a candidate therapy.

Molecular and structural analyses of maple syrup urine disease and identification of a founder mutation in a Portuguese Gypsy community

Maple syrup urine disease (MSUD) is an autosomal recessive disorder, caused by the defective function of the branched-chain alpha-ketoacid dehydrogenase complex (BCKD). BCKD is a mitochondrial complex, encoded by four nuclear genes (BCKDHA, BCKDHB, DBT and DLD), involved in the metabolism of branched-chain amino acids (BCAAs). Since the MSUD mutational spectrum has not been previously assessed in Portugal, in this study we present the molecular characterization of 30 MSUD Portuguese patients. Seventeen putative mutations have been identified (six in BCKDHA, five in BCKDHB and six in DBT); seven of them are here described for the first time. The most common mutation identified was a C deletion in BCKDHA gene (c.117delC; p.R40GfsX23), already reported in the Spanish population. Interestingly, it was found in all patients of a Gypsy community from South of the country, so a founder effect is probably responsible for the high incidence of the disease in this community. Structural models of MSUD missense mutations have been performed to understand their pathogenic effect, in order to elucidate and often to predict the severity of a mutation clinical consequence.

Molecular mechanisms underlying large genomic deletions in ornithine transcarbamylase (OTC) gene.

Ornithine transcarbamylase deficiency (OTCD) is an X‐linked urea cycle error causing hyperammonemia and orotic aciduria. Clinical diagnosis is generally confirmed by mutation detection. However, in ∼20% of the patients, no mutation is found by conventional mutation‐searching strategies, which fail to detect deletions spanning at least a whole exon, large rearrangements, or mutations at non‐coding regions. To detect large deletions or duplications, we have applied the multiplex ligation‐dependent probe amplification (MLPA) methodology to three OTCD patients (two females and one male). MLPA revealed copy number alterations of OTC exons in all of them. The two females were found to be heterozygous for deletions of either exon 2 or exons 6–9, and the male was confirmed to lack all OTC exons. Females’ characterization of the deletion breakpoints by long polymerase chain reaction and sequencing revealed the mutations c.78‐3544_217‐129del5921 and c.541‐600_1005 + 1880del10862 corresponding to exon 2 and exon 6–9 deletions, respectively. Examination of the deletion‐flanking regions suggests that exon 2 deletion probably resulted from replication slippage facilitated by a secondary structure formed by two inverted Alu repeats, whereas an Alu–Alu homologous recombination was probably responsible for the exon 6–9 deletion. This work contributes to the identification of novel disease‐causing mutations in OTCD and increases the knowledge on possible mutational mechanisms generating deletions in OTC.