Bruno Maranda

Excess of De Novo Deleterious Mutations in Genes Associated with Glutamatergic Systems in Nonsyndromic Intellectual Disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.

Molecular and clinical genetics of mitochondrial diseases due to POLG mutations.

Mutations in the POLG gene have emerged as one of the most common causes of inherited mitochondrial disease in children and adults. They are responsible for a heterogeneous group of at least 6 major phenotypes of neurodegenerative disease that include: 1) childhood Myocerebrohepatopathy Spectrum disorders (MCHS), 2) Alpers syndrome, 3) Ataxia Neuropathy Spectrum (ANS) disorders, 4) Myoclonus Epilepsy Myopathy Sensory Ataxia (MEMSA), 5) autosomal recessive Progressive External Ophthalmoplegia (arPEO), and 6) autosomal dominant Progressive External Ophthalmoplegia (adPEO). Due to the clinical heterogeneity, time‐dependent evolution of symptoms, overlapping phenotypes, and inconsistencies in muscle pathology findings, definitive diagnosis relies on the molecular finding of deleterious mutations. We sequenced the exons and flanking intron region from approximately 350 patients displaying a phenotype consistent with POLG related mitochondrial disease and found informative mutations in 61 (17%). Two mutant alleles were identified in 31 unrelated index patients with autosomal recessive POLG‐related disorders. Among them, 20 (67%) had Alpers syndrome, 4 (13%) had arPEO, and 3 (10%) had ANS. In addition, 30 patients carrying one altered POLG allele were found. A total of 25 novel alterations were identified, including 6 null mutations. We describe the predicted structural/functional and clinical importance of the previously unreported missense variants and discuss their likelihood of being pathogenic. In conclusion, sequence analysis allows the identification of mutations responsible for POLG‐related disorders and, in most of the autosomal recessive cases where two mutant alleles are found in trans, finding deleterious mutations can provide an unequivocal diagnosis of the disease. Published 2008 Wiley‐Liss, Inc.

Mutations in C5ORF42 Cause Joubert Syndrome in the French Canadian Population

Joubert syndrome (JBTS) is an autosomal-recessive disorder characterized by a distinctive mid-hindbrain malformation, developmental delay with hypotonia, ocular-motor apraxia, and breathing abnormalities. Although JBTS was first described more than 40 years ago in French Canadian siblings, the causal mutations have not yet been identified in this family nor in most French Canadian individuals subsequently described. We ascertained a cluster of 16 JBTS-affected individuals from 11 families living in the Lower St. Lawrence region. SNP genotyping excluded the presence of a common homozygous mutation that would explain the clustering of these individuals. Exome sequencing performed on 15 subjects showed that nine affected individuals from seven families (including the original JBTS family) carried rare compound-heterozygous mutations in C5ORF42. Two missense variants (c.4006C>T [p.Arg1336Trp] and c.4690G>A [p.Ala1564Thr]) and a splicing mutation (c.7400+1G>A), which causes exon skipping, were found in multiple subjects that were not known to be related, whereas three other truncating mutations (c.6407del [p.Pro2136Hisfs*31], c.4804C>T [p.Arg1602*], and c.7477C>T [p.Arg2493*]) were identified in single individuals. None of the unaffected first-degree relatives were compound heterozygous for these mutations. Moreover, none of the six putative mutations were detected among 477 French Canadian controls. Our data suggest that mutations in C5ORF42 explain a large portion of French Canadian individuals with JBTS.

Exome sequencing identifies mutations in the gene TTC7A in French-Canadian cases with hereditary multiple intestinal atresia

Background Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort. Methods We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. Results Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes. Conclusions Based on our genetic results, TTC7A is the likely causal gene for MIA.

Effect of nitisinone (NTBC) treatment on the clinical course of hepatorenal tyrosinemia in Québec.

BACKGROUND Hepatorenal tyrosinemia (HT1, fumarylacetoacetate hydrolase deficiency, MIM 276700) can cause severe hepatic, renal and peripheral nerve damage. In Québec, HT1 is frequent and neonatal HT1 screening is practiced. Nitisinone (NTBC, Orfadin ®) inhibits tyrosine degradation prior to the formation of toxic metabolites like succinylacetone and has been offered to HT1 patients in Québec since 1994. METHODS We recorded the clinical course of 78 Québec HT1 patients born between 1984 and 2004. There were three groups: those who never received nitisinone (28 patients), those who were first treated after 1 month of age (26 patients) and those treated before 1 month (24 patients). Retrospective chart review was performed for events before 1994, when nitisinone treatment began, and prospective data collection thereafter. FINDINGS No hospitalizations for acute complications of HT1 occurred during 5731 months of nitisinone treatment, versus 184 during 1312 months without treatment (p<0.001). Liver transplantation was performed in 20 non-nitisinone-treated patients (71%) at a median age of 26 months, versus 7 late-treated patients (26%, p<0.001), and no early-treated patient (p<0.001). No early-treated patient has developed detectable liver disease after more than 5 years. Ten deaths occurred in non-nitisinone treated patients versus two in treated patients (p<0.01). Both of the latter deaths were from complications of transplantation unrelated to HT1. One probable nitisinone-related event occurred, transient corneal crystals with photophobia. INTERPRETATION Nitisinone treatment abolishes the acute complications of HT1. Some patients with established liver disease before nitisinone treatment eventually require hepatic transplantation. Patients who receive nitisinone treatment before 1 month had no detectable liver disease after more than 5 years.

11p14.1 microdeletions associated with ADHD, autism, developmental delay, and obesity.

Genomic copy number imbalances are being increasingly identified as an important cause of intellectual disability and behavioral abnormalities. The typical deletion in WAGR syndrome encompasses the PAX6 and WT1 genes, but larger deletions have been associated with neurobehavioral abnormalities and obesity. We identified four patients with overlapping interstitial deletions on 11p14.1 and extending telomeric to the WAGR critical domain. The minimal overlapping critical chromosomal region was 2.3 Mb at 11p14.1. The deletions encompass the BDNF and LIN7C genes that are implicated in the regulation of development and differentiation of neurons and synaptic transmission. All patients with this deletion exhibit variable degrees of developmental delay, behavioral problems, and obesity. Our data show that ADHD, autism, developmental delay, and obesity are highly associated with deletion involving 11p14.1 and provide additional support for a significant role of BDNF in obesity and neurobehavioral problems.

Citrin deficiency, a perplexing global disorder

Citrin deficiency, caused by mutations in SLC25A13, can present with neonatal intrahepatic cholestasis or with adult onset neuropsychiatric, hepatic and pancreatic disease. Until recently, it had been thought to be found mostly in individuals of East Asian ancestry. A key diagnostic feature has been the deficient argininosuccinate synthetase (ASS) activity (E.C. 6.3.4.5) in liver, with normal activity in skin fibroblasts. In this series we describe the clinical presentation of 10 patients referred to our laboratories for sequence analysis of the SCL25A13 gene, including several patients who presented with elevated citrulline on newborn screening. In addition to sequence analysis performed on all patients, ASS enzyme activity, citrulline incorporation and Western blot analysis for ASS and citrin were performed on skin fibroblasts if available. We have found 5 unreported mutations including two apparent founder mutations in three unrelated French-Canadian patients. In marked contrast to previous cases, these patients have a markedly reduced ASS activity in skin fibroblasts. The presence of citrin protein on Western blot in three of our cases reduces the sensitivity of a screening test based on protein immunoblotting. The finding of citrin mutations in patients of Arabic, Pakistani, French Canadian and Northern European origins supports the concept that citrin deficiency is a panethnic disease.

Hereditary pulmonary alveolar proteinosis caused by recessive CSF2RB mutations

To the Editors: Pulmonary alveolar proteinosis (PAP) is a syndrome characterised by accumulation of surfactant in alveoli resulting in respiratory insufficiency 1. Surfactant homeostasis is critical for lung function and is tightly regulated, in part, by pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF), which is required for surfactant clearance by alveolar macrophages 2 and alveolar macrophage maturation 1. The effects of GM-CSF are mediated by cell-surface receptors composed of GM-CSF-binding α-chains and affinity-enhancing β-chains (encoded by CSF2RA and CSF2RB , respectively) 3. Ligand binding activates signalling via multiple pathways including the signal transducer and activator of phosphorylation (STAT)5 4. Disruption of GM-CSF signalling causes PAP by impairing surfactant catabolism in alveolar macrophages 1. In 90% of patients, PAP is caused by neutralising GM-CSF auto-antibodies 5, 6. Through the Rare Lung Diseases Network global PAP detection programme, we identified PAP caused by recessive CSF2RA mutations and developed novel diagnostic methods to identify patients with PAP caused by GM-CSF receptor dysfunction 4, 7. Herein, we report a case of hereditary PAP caused by disruption of GM-CSF receptor β-chain function. A previously healthy 9-yr-old female presented with bilateral pneumonia, followed 3 months later by progressive dyspnoea of insidious onset. The diagnosis of PAP was suggested by chest radiograph findings, high-resolution computed tomography and bronchoalveolar cytology, and was confirmed by surgical lung biopsy. Pulmonary histopathology was typical of primary PAP (fig. 1) and she was successfully treated by serial whole lung lavage therapy. Details of the case history are included in the online supplement. A GM-CSF auto-antibody test was negative and the serum GM-CSF level was increased (25.9 pg·mL−1) suggesting GM-CSF receptor dysfunction as the molecular basis of PAP 4, 7. A molecular evaluation was undertaken and included GM-CSF receptor detection, STAT-5 phosphorylation, CSF2RA …

Mutations in TMEM231 cause Joubert syndrome in French Canadians

Background Joubert syndrome (JBTS) is a predominantly autosomal recessive disorder characterised by a distinctive midhindbrain malformation, oculomotor apraxia, breathing abnormalities and developmental delay. JBTS is genetically heterogeneous, involving genes required for formation and function of non-motile cilia. Here we investigate the genetic basis of JBTS in 12 French–Canadian (FC) individuals. Methods and results Exome sequencing in all subjects showed that six of them carried rare compound heterozygous mutations in CC2D2A or C5ORF42, known JBTS genes. In addition, three individuals (two families) were compound heterozygous for the same rare mutations in TMEM231(c.12T>A[p.Tyr4*]; c.625G>A[p.Asp209Asn]). All three subjects showed a severe neurological phenotype and variable presence of polydactyly, retinopathy and renal cysts. These mutations were not detected among 385 FC controls. TMEM231 has been previously shown to localise to the ciliary transition zone, and to interact with several JBTS gene products in a complex involved in the formation of the diffusion barrier between the cilia and plasma membrane. siRNA knockdown of TMEM231 was also shown to affect barrier integrity, resulting in a reduction of cilia formation and ciliary localisation of signalling receptors. Conclusions Our data suggest that mutations in TMEM231 cause JBTS, reinforcing the relationship between this condition and the disruption of the barrier at the ciliary transition zone.

Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1

Tissues with high metabolic rates often use lipids, as well as glucose, for energy, conferring a survival advantage during feast and famine. Current dogma suggests that high-energy–consuming photoreceptors depend on glucose. Here we show that the retina also uses fatty acid β-oxidation for energy. Moreover, we identify a lipid sensor, free fatty acid receptor 1 (Ffar1), that curbs glucose uptake when fatty acids are available. Very-low-density lipoprotein receptor (Vldlr), which is present in photoreceptors and is expressed in other tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acid. In the retinas of Vldlr−/− mice with low fatty acid uptake but high circulating lipid levels, we found that Ffar1 suppresses expression of the glucose transporter Glut1. Impaired glucose entry into photoreceptors results in a dual (lipid and glucose) fuel shortage and a reduction in the levels of the Krebs cycle intermediate α-ketoglutarate (α-KG). Low α-KG levels promotes stabilization of hypoxia-induced factor 1a (Hif1a) and secretion of vascular endothelial growth factor A (Vegfa) by starved Vldlr−/− photoreceptors, leading to neovascularization. The aberrant vessels in the Vldlr−/− retinas, which invade normally avascular photoreceptors, are reminiscent of the vascular defects in retinal angiomatous proliferation, a subset of neovascular age-related macular degeneration (AMD), which is associated with high vitreous VEGFA levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in macular telangiectasia, neovascular AMD and other retinal diseases.