Nathalie Drouot

ADCK3, an Ancestral Kinase, Is Mutated in a Form of Recessive Ataxia Associated with Coenzyme Q10 Deficiency

Muscle coenzyme Q(10) (CoQ(10) or ubiquinone) deficiency has been identified in more than 20 patients with presumed autosomal-recessive ataxia. However, mutations in genes required for CoQ(10) biosynthetic pathway have been identified only in patients with infantile-onset multisystemic diseases or isolated nephropathy. Our SNP-based genome-wide scan in a large consanguineous family revealed a locus for autosomal-recessive ataxia at chromosome 1q41. The causative mutation is a homozygous splice-site mutation in the aarF-domain-containing kinase 3 gene (ADCK3). Five additional mutations in ADCK3 were found in three patients with sporadic ataxia, including one known to have CoQ(10) deficiency in muscle. All of the patients have childhood-onset cerebellar ataxia with slow progression, and three of six have mildly elevated lactate levels. ADCK3 is a mitochondrial protein homologous to the yeast COQ8 and the bacterial UbiB proteins, which are required for CoQ biosynthesis. Three out of four patients tested showed a low endogenous pool of CoQ(10) in their fibroblasts or lymphoblasts, and two out of three patients showed impaired ubiquinone synthesis, strongly suggesting that ADCK3 is also involved in CoQ(10) biosynthesis. The deleterious nature of the three identified missense changes was confirmed by the introduction of them at the corresponding positions of the yeast COQ8 gene. Finally, a phylogenetic analysis shows that ADCK3 belongs to the family of atypical kinases, which includes phosphoinositide and choline kinases, suggesting that ADCK3 plays an indirect regulatory role in ubiquinone biosynthesis possibly as part of a feedback loop that regulates ATP production.

Mutations in ABHD12 Cause the Neurodegenerative Disease PHARC: An Inborn Error of Endocannabinoid Metabolism

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a neurodegenerative disease marked by early-onset cataract and hearing loss, retinitis pigmentosa, and involvement of both the central and peripheral nervous systems, including demyelinating sensorimotor polyneuropathy and cerebellar ataxia. Previously, we mapped this Refsum-like disorder to a 16 Mb region on chromosome 20. Here we report that mutations in the ABHD12 gene cause PHARC disease and we describe the clinical manifestations in a total of 19 patients from four different countries. The ABHD12 enzyme was recently shown to hydrolyze 2-arachidonoyl glycerol (2-AG), the main endocannabinoid lipid transmitter that acts on cannabinoid receptors CB1 and CB2. Our data therefore represent an example of an inherited disorder related to endocannabinoid metabolism. The endocannabinoid system is involved in a wide range of physiological processes including neurotransmission, mood, appetite, pain appreciation, addiction behavior, and inflammation, and several potential drugs targeting these pathways are in development for clinical applications. Our findings show that ABHD12 performs essential functions in both the central and peripheral nervous systems and the eye. Any future drug-mediated interference with this enzyme should consider the potential risk of long-term adverse effects.

Early-Onset Brain Tumor and Lymphoma in MSH2-Deficient Children

To the Editor: Homozygous germline mutations of MLH1 have been reported so far in three families with hereditary nonpolyposis colorectal cancer (HNPCC [MIM 114500]) and have been shown to be associated with leukemia or lymphoma, CNS tumors, and the neurofibromatosis type I phenotype (Ricciardone et al. 1999; Wang et al. 1999; Vilkki et al. 2001). More recently, the first case of a homozygous germline mutation of MSH2 was described in a child with leukemia and multiple cafe-au-lait spots (Whiteside et al. 2002). We report here the incidental discovery of a new case of MSH2 deficiency, which is remarkable because of the presentation of the family and because of the association with an early-onset brain tumor. The proband (III.2) and her husband (III.1), of French origin, were seen for genetic counseling in the dramatic context of the death of their two children (fig. 1). Individual IV.1 died at age 15 mo from a T mediastinal lymphoma; her brother (IV.2) died at age 4 years from a temporal glioblastoma. Their mother and father, age 29 years and 32 years, respectively, had no personal history of cancer. Although several second-degree relatives of the parents had developed cancers (fig. 1), the presentation of the family did not fulfill the criteria for a Mendelian genetic predisposition to cancer. The development of a CNS tumor and lymphoma in two sibs led us to consider initially the hypothesis of Li-Fraumeni syndrome (LFS) in this family. Since no DNA was available from the affected children, we analyzed the TP53 gene in both unaffected parents. Sequencing analysis of TP53 revealed no mutation. Stimulated by our recent finding of a family with LFS with complete heterozygous germline deletion of TP53 (unpublished data), we completed the analysis of TP53 by searching for a similar defect using quantitative multiplex PCR of short fluorescent fragments (QMPSF) (Charbonnier et al. 2000, 2002). QMPSF analysis of TP53 performed in the father (III.1) demonstrated that the TP53 gene was not affected, but, to our surprise, revealed a heterozygous deletion of MSH2 exon 3 corresponding to the control amplicon. Therefore, we analyzed, by QMPSF, the 16 exons of MSH2, and this analysis showed the presence, in the unaffected father, of a heterozygous genomic deletion of MSH2 removing exons 1–6 (fig. 2A). We then sequenced the MSH2 gene in the unaffected mother (III.2) and identified a 1-bp heterozygous deletion at codon 153 within exon 3 (fig. 2B). In the absence of constitutional DNA from the affected children, we sequenced MSH2 exon 3 from the glioblastoma DNA of individual IV.2. As shown in figure 2C, we detected only the mutant maternal allele, which strongly suggested that individual IV.2 had received from his father the mutant allele harboring the exons 1–6 deletion. Haplotype analysis at the MSH2 locus confirmed the presence of two parental MSH2 alleles within the tumor, ruling out a somatic loss of heterozygosity (data not shown). Screening for microsatellite instability (MSI), as recommended by Boland et al. (1998), revealed no replication error (RER) phenotype within the glioblastoma. Figure 1 Partial pedigree of the family. Filled symbols denote affected subjects; open symbols denote asymptomatic subjects; oblique line denote deceased. Numbers beside symbols are subject identifiers. The ages of unaffected individuals are indicated. For affected ... Figure 2 Detection of the MSH2 alterations. A, Heterozygous deletion of MSH2 exons 1–6 in the father (individual III.1) detected by QMPSF. The electropherogram of the father (red) was superposed on that of a control individual (blue). The Y-axis displays ... This case report shows that MSH2 deficiency in humans can result in early-onset CNS tumors. Homozygous MLH1 mutations have been detected in two children who had developed a medulloblastoma (Wang et al. 1999) and a glioma (Vilkki et al. 2001). Compound heterozygous mutations of the PMS2 gene, which is rarely involved in HNPCC, have been identified in two sisters with early-onset brain and colorectal tumors (De Rosa et al. 2000). These studies, together with the present report, indicate that germline MMR deficiency predisposes to primary early-onset neuroepithelial tumors. Turcot syndrome (MIM 276300) was originally defined by the association of CNS malignant tumors with familial polyposis of the colon, but molecular studies have subsequently distinguished two entities, resulting from APC and MMR gene mutations, respectively (Hamilton et al. 1995; Paraf et al. 1997). It is tempting to speculate, as suggested elsewhere (De Rosa et al. 2000), that, in some families with Turcot syndrome, the association of colorectal neoplasms with childhood brain tumors may be due to a complete MMR deficiency. We were surprised that we could not detect an RER phenotype in the MSH2-deficient brain tumor. It is interesting that MSI was not also detected in the nontumoral DNA of the MSH2-deficient patient reported by Whiteside et al. (2002), in contrast to the case of the two MLH1-deficient subjects analyzed by Wang et al. (1999) and Vilkki et al. (2001). This result could suggest that, at least in certain tissues, MSH2 deficiency could lead to tumorigenesis through a mechanism distinct from a defect in the repair of postreplicative mismatches affecting repetitive sequences. Indeed, MSH2, like its bacterial homolog, MutS, has been shown to play additional roles in genetic recombination, since these proteins prevent exchange between divergent DNA sequences (Modrich and Lahue 1996). Furthermore, MSH2 was recently shown to be associated with TP53 within recombinative repair complexes during S phase (Zink et al. 2002). As in the previous report of a MSH2 deficiency (Whiteside et al. 2002), the familial history presented in this study was not strongly suggestive of HNPCC, although the young age of the parents and their sibs could explain the absence of cancer within this generation. Therefore, as shown in this report, the presence of homozygous mutations of the different MMR genes must be considered in families with early-onset CNS tumors and hematological malignancies, even in the absence of a familial history of HNPCC.

Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.

The tumour suppressor gene WWOX is mutated in autosomal recessive cerebellar ataxia with epilepsy and mental retardation

We previously localized a new form of recessive ataxia with generalized tonic-clonic epilepsy and mental retardation to a 19 Mb interval in 16q21-q23 by homozygosity mapping of a large consanguineous Saudi Arabian family. We now report the identification by whole exome sequencing of the missense mutation changing proline 47 into threonine in the first WW domain of the WW domain containing oxidoreductase gene, WWOX, located in the linkage interval. Proline 47 is a highly conserved residue that is part of the WW motif consensus sequence and is part of the hydrophobic core that stabilizes the WW fold. We demonstrate that proline 47 is a key amino acid essential for maintaining the WWOX protein fully functional, with its mutation into a threonine resulting in a loss of peptide interaction for the first WW domain. We also identified another highly conserved homozygous WWOX mutation changing glycine 372 to arginine in a second consanguineous family. The phenotype closely resembled the index family, presenting with generalized tonic-clonic epilepsy, mental retardation and ataxia, but also included prominent upper motor neuron disease. Moreover, we observed that the short-lived Wwox knock-out mouse display spontaneous and audiogenic seizures, a phenotype previously observed in the spontaneous Wwox mutant rat presenting with ataxia and epilepsy, indicating that homozygous WWOX mutations in different species causes cerebellar ataxia associated with epilepsy.

Simple detection of genomic microdeletions and microduplications using QMPSF in patients with idiopathic mental retardation

In contrast to the numerous well-known microdeletion syndromes, only a few microduplications have been described, and this discrepancy may be due in part to methodological bias. In order to facilitate the detection of genomic microdeletions and microduplications, we developed a new assay based on QMPSF (Quantitative Multiplex PCR of Short fluorescent Fragments) able to explore simultaneously 12 candidate loci involved in mental retardation (MR) and known to be the target of genomic rearrangements. We first screened 153 patients with MR and facial dysmorphism associated with malformations, or growth anomalies, or familial history, with cytogenetically normal chromosomes, and the absence of FRAXA mutation and subtelomeric rearrangements. In this series, we found a 5q35 deletion removing the NSD1 gene in a patient with severe epilepsy, profound MR and, retrospectively, craniofacial features of Sotos syndrome. In a second series, we screened 140 patients with MR and behaviour disturbance who did not fulfil the de Vries criteria for subtelomeric rearrangements and who had a normal karyotype and no detectable FRAXA mutation. We detected a 22q11 deletion in a patient with moderate MR, obesity, and facial dysmorphism and a 4 Mb 17p11 duplication in a patient with moderate MR, behaviour disturbance, strabismus, and aspecific facial features. This new QMPSF assay can be gradually upgraded to include additional loci involved in newly recognised microduplication/microdeletion syndromes, and should facilitate wide screenings of patients with idiopathic MR and provide better estimates of the microduplication frequency in the MR population.

Rundataxin, a novel protein with RUN and diacylglycerol binding domains, is mutant in a new recessive ataxia

We have identified a novel form of recessive ataxia that segregates in three children of a large consanguineous Saudi Arabian family. The three patients presented with childhood onset gait and limb ataxia, dysarthria and had limited walking without aid into their teenage years. Two patients developed epilepsy at 7 months without relapse after treatment, and mental retardation. Linkage studies allowed us to identify a single locus that segregated with the disease on chromosome 3q28-qter. Mutation screening of all coding sequences revealed a single nucleotide deletion, 2927delC, in exon 19 of the KIAA0226 gene, which results in a frame shift of the C-terminal domain (p.Ala943ValfsX146). The KIAA0226 gene encodes a protein that we named rundataxin, with two conserved domains: an N-terminal RUN domain and a C-terminal domain containing a diacylglycerol binding-like motif. The closest paralogue of rundataxin, the plekstrin homology domain family member M1, has been shown to colocalize with Rab7, a small GTPase associated with late endosomes/lysosomes, suggesting that rundataxin may also be associated with vesicular trafficking and signalling pathways through its RUN and diacylglycerol binding-like domains. The rundataxin pathway appears therefore distinct from the ataxia pathways involving deficiency in mitochondrial or nuclear proteins and broadens the range of mechanisms leading to recessive ataxias.

Phenotypic variability in ARCA2 and identification of a core ataxic phenotype with slow progression

Autosomal recessive cerebellar ataxia 2 (ARCA2) is a recently identified recessive ataxia due to ubiquinone deficiency and biallelic mutations in the ADCK3 gene. The phenotype of the twenty-one patients reported worldwide varies greatly. Thus, it is difficult to decide which ataxic patients are good candidates for ADCK3 screening without evidence of ubiquinone deficiency. We report here the clinical and molecular data of 10 newly diagnosed patients from seven families and update the disease history of four additional patients reported in previous articles to delineate the clinical spectrum of ARCA2 phenotype and to provide a guide to the molecular diagnosis. First signs occurred before adulthood in all 14 patients. Cerebellar atrophy appeared in all instances. The progressivity and severity of ataxia varied greatly, but no patients had the typical inexorable ataxic course that characterizes other childhood-onset recessive ataxias. The ataxia was frequently associated with other neurological signs. Importantly, stroke-like episodes contributed to significant deterioration of the neurological status in two patients. Ubidecarenone therapy markedly improved the movement disorders, including ataxia, in two other patients. The 7 novel ADCK3 mutations found in the 10 new patients were two missense and five truncating mutations. There was no apparent correlation between the genotype and the phenotype. Our series reveals that the clinical spectrum of ARCA2 encompasses a range of ataxic phenotypes. On one end, it may manifest as a pure ataxia with very slow progressivity and, on the other end, as a severe infantile encephalopathy with cerebellar atrophy. The phenotype of most patients, however, lies in between. It is characterized by a very slowly progressive or apparently stable ataxia associated with other signs of central nervous system involvement. We suggest undergoing the molecular analysis of ADCK3 in patients with this phenotype and in those with cerebellar atrophy and a stroke-like episode. The diagnosis of patients with a severe ARCA2 phenotype may also be performed on the basis of biological data, i.e. low ubiquinone level or functional evidence of ubiquinone deficiency. This diagnosis is crucial since the neurological status of some patients may be improved by ubiquinone therapy.

Autosomal Recessive Cerebellar Ataxia Type 3 Due to ANO10 Mutations: Delineation and Genotype-Phenotype Correlation Study

IMPORTANCE ANO10 mutations have been reported to cause a novel form of autosomal recessive cerebellar ataxia (ARCA). Our objective was to report 9 ataxic patients carrying 8 novel ANO10 mutations to improve the delineation of this form of ARCA and provide genotype-phenotype correlation. OBSERVATIONS The ANO10 gene has been sequenced in 186 consecutive patients with ARCA. The detailed phenotype of patients with ANO10 mutations was investigated and compared with the 12 previously reported cases. The mean age at onset was 33 years (range, 17-43 years), and the disease progression was slow. Corticospinal tract signs were frequent, including extensor plantar reflexes and/or diffuse tendon reflexes and/or spasticity. No patient in our series had peripheral neuropathy. Magnetic resonance imaging of the brains of our patients revealed marked cerebellar atrophy. The most frequent mutation, a mononucleotide expansion from a polyA repeat tract (c.132dupA) that causes protein truncation, was never observed in homozygosity. Only 2 truncating mutations were reported in homozygosity, one of which (c.1150-1151del) was associated with juvenile or adolescent onset and mental retardation, whereas we show that the presence of at least 1 missense or in-frame mutation is associated with adult onset and slow progression. CONCLUSIONS AND RELEVANCE An ANO10 mutation is responsible for ARCA that is mainly characterized by cerebellar atrophy and lack of peripheral neuropathy. We therefore suggest naming this entity autosomal recessive cerebellar ataxia type 3 (ARCA3).

Mutations in the HECT domain of NEDD4L lead to AKT–mTOR pathway deregulation and cause periventricular nodular heterotopia

Neurodevelopmental disorders with periventricular nodular heterotopia (PNH) are etiologically heterogeneous, and their genetic causes remain in many cases unknown. Here we show that missense mutations in NEDD4L mapping to the HECT domain of the encoded E3 ubiquitin ligase lead to PNH associated with toe syndactyly, cleft palate and neurodevelopmental delay. Cellular and expression data showed sensitivity of PNH-associated mutants to proteasome degradation. Moreover, an in utero electroporation approach showed that PNH-related mutants and excess wild-type NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin-based experiments, found differential deregulation of pathways involved. Excess wild-type NEDD4L leads to disruption of Dab1 and mTORC1 pathways, while PNH-related mutations are associated with deregulation of mTORC1 and AKT activities. Altogether, these data provide insights into the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development.