Estelle Colin

Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing

Background Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation. Methods We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases. Results We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients’ clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders. Conclusions With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism.

Mutation screening of the EYA1, SIX1, and SIX5 genes in a large cohort of patients harboring branchio-oto-renal syndrome calls into question the pathogenic role of SIX5 mutations†

Branchio‐oto‐renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial, ear, and renal anomalies. Over 80 mutations in EYA1 have been reported in BOR. Mutations in SIX1, a DNA binding protein that associates with EYA1, have been reported less frequently. One group has recently described four missense mutations in SIX5 in five unrelated patients with BOR. Here, we report a screening of these three genes in a cohort of 140 patients from 124 families with BOR. We identified 36 EYA1 mutations in 42 unrelated patients, 2 mutations, and 1 change of unknown significance in SIX1 in 3 unrelated patients, but no mutation in SIX5. We did not find correlation between genotype and phenotype, and observed a high phenotypic variability between and within BOR families. We show the difficulty in establishing a molecular diagnosis strategy in BOR syndrome: the screening focusing on patients with typical BOR would detect a mutation rate of 76%, but would also miss mutations in 9% of patients with atypical BOR. We detected a deletion removing three EYA1 exons in a patient who was previously reported to carry the SIX5 Thr552Met mutation. This led us to reconsider the role of SIX5 in the development of BOR. Hum Mutat 32:183–190, 2011.

Mutations in WNT10A are frequently involved in oligodontia associated with minor signs of ectodermal dysplasia

Ectodermal dysplasias (ED) are a clinically and genetically heterogeneous group of hereditary disorders that have in common abnormal development of ectodermal derivatives. Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of eccrine sweat glands, hair, and teeth. The X‐linked form of the disease, caused by mutations in the EDA gene, represents the majority of patients with the hypohidrotic form. Autosomal dominant and autosomal recessive forms are occasionally seen, and result from mutations in at least three genes (WNT10A, EDAR, or more rarely EDARADD). We have screened for mutations in EDAR (commonly involved in the hypohidrotic form) and WNT10A (involved in a wide spectrum of ED and in isolated hypodontia) in a cohort of 36 patients referred for EDA molecular screening, which failed to identify any mutation. We identified eight EDAR mutations in five patients (two with homozygous mutations, one with compound heterozygous mutations, and two with heterozygous mutation), four of which were novel variants. We identified 28 WNT10A mutations in 16 patients (5 with homozygous mutations, 7 with compound heterozygous mutations, and 4 with heterozygous mutations), seven of which were novel variants. Our study allows a more precise definition of the phenotypic spectrum associated with EDAR and WNT10A mutations and underlines the importance of the implication of WNT10A among patients with ED.

Sensorineural hearing loss in OPA1-linked disorders

ARTICLE Sir, We have read with interest the article by Yu-Wai-Man et al. (2010) reporting heterogeneous multi-system manifestations in 104 patients affected with autosomal dominant optic atrophy (DOA) caused by mutations in the nuclear gene encoding the mitochondrial OPA1 protein. Yu-Wai-Man et al. (2010) found that ∼20% of the OPA1 mutation carriers had complicated forms of dominant optic atrophy, the so-called ‘DOA+’ forms, associating extra-ocular features such as hearing loss or neuromuscular disorders similar to those observed in mitochondrial oxidative phosphorylation defects or in syndromes with mitochondrial DNA instability (Amati-Bonneau et al. , 2008; Hudson et al. , 2008; Yu-Wai-Man et al. , 2010). Interestingly, Yu-Wai-Man et al. (2010) report that sensorineural hearing loss, the most frequent extra-ocular manifestation in ‘DOA+’, occurred in nearly two-thirds of their patients. To characterize the prevalence and the type of hearing loss in OPA1 mutation carriers, we retrospectively reviewed the files of 1380 patients affected with hereditary optic neuropathies referred to our laboratory from 2003 to 2011 for molecular diagnosis. During this period, an OPA1 mutation was identified in 327 patients (24%), 21 of whom (6.4%) had hearing impairment. Clinical information about these 21 patients was obtained from 13 departments of ophthalmology, ENT and medical genetics (12 in France, one in Denmark) (Table 1). All patients had undergone standard pure-tone air and bone conduction audiometry (125–8000 Hz), and speech audiometry had been performed in eight patients. Results of auditory brainstem responses and otoacoustic emission testing were available for only eight patients (Table 2). View this table: Table 1 Clinical and molecular findings in 21 OPA1 mutation carriers affected with optic atrophy and hearing loss View this table: Table 2 Audiological data from 10 OPA1 mutation carriers The 21 OPA1 mutation carriers with hearing loss (11 females and 10 males) belonged to 13 families and were aged from 12 to 73 years …

De Novo Truncating Mutations in the Kinetochore-Microtubules Attachment Gene CHAMP1 Cause Syndromic Intellectual Disability

A rare syndromic form of intellectual disability with impaired speech was recently found associated with mutations in CHAMP1 (chromosome alignment‐maintaining phosphoprotein 1), the protein product of which is directly involved in microtubule‐kinetochore attachment. Through whole‐exome sequencing in six unrelated nonconsanguineous families having a sporadic case of intellectual disability, we identified six novel de novo truncating mutations in CHAMP1: c.1880C>G p.(Ser627*), c.1489C>T; p.(Arg497*), c.1876_1877delAG; p.(Ser626Leufs*4), c.1043G>A; p.(Trp348*), c.1002G>A; p.(Trp334*), and c.958_959delCC; p.(Pro320*). Our clinical observations confirm the phenotypic homogeneity of the syndrome, which represents therefore a distinct clinical entity. Besides, our functional studies show that CHAMP1 protein variants are delocalized from chromatin and are unable to bind to two of its direct partners, POGZ and HP1. These data suggest a pathogenic mechanism of the CHAMP1‐associated intellectual disability syndrome mediated by direct interacting partners of CHAMP1, several of which are involved in chromo/kinetochore‐related disorders.

SLC24A5 Mutations Are Associated with Non-Syndromic Oculocutaneous Albinism

Cite this article as: Fanny Morice-Picard, Eulalie Lasseaux, Stéphane François, Delphine Simon, Caroline Rooryck, Eric Bieth, Estelle Colin, Dominique Bonneau, Hubert Journel, Sophie Walraedt, Bart P Leroy, Francoise Meire, Didier Lacombe, Benoit Arveiler, SLC24A5 Mutations are Associated with NonSyndromic Oculocutaneous Albinism, Journal of Investigative Dermatology accepted article preview 28 August 2013; doi: 10.1038/jid.2013.360.

Biallelic Variants in UBA5 Reveal that Disruption of the UFM1 Cascade Can Result in Early-Onset Encephalopathy

Via whole-exome sequencing, we identified rare autosomal-recessive variants in UBA5 in five children from four unrelated families affected with a similar pattern of severe intellectual deficiency, microcephaly, movement disorders, and/or early-onset intractable epilepsy. UBA5 encodes the E1-activating enzyme of ubiquitin-fold modifier 1 (UFM1), a recently identified ubiquitin-like protein. Biochemical studies of mutant UBA5 proteins and studies in fibroblasts from affected individuals revealed that UBA5 mutations impair the process of ufmylation, resulting in an abnormal endoplasmic reticulum structure. In Caenorhabditis elegans, knockout of uba-5 and of human orthologous genes in the UFM1 cascade alter cholinergic, but not glutamatergic, neurotransmission. In addition, uba5 silencing in zebrafish decreased motility while inducing abnormal movements suggestive of seizures. These clinical, biochemical, and experimental findings support our finding of UBA5 mutations as a pathophysiological cause for early-onset encephalopathies due to abnormal protein ufmylation.

Variable clinical expression in patients with mosaicism for KCNQ2 mutations

Mutations in the KCNQ2 gene, encoding a potassium channel subunit, were reported in patients presenting epileptic phenotypes of varying severity. Patients affected by benign familial neonatal epilepsy (BFNE) are at the milder end of the spectrum, they are affected by early onset epilepsy but their subsequent neurological development is usually normal. Mutations causing BFNE are often inherited from affected parents. Early infantile epileptic encephalopathy type 7 (EIEE7) is at the other end of the severity spectrum and, although EIEE7 patients have early onset epilepsy too, their neurological development is impaired and they will present motor and intellectual deficiency. EIEE7 mutations occur de novo. Electrophysiological experiments suggested a correlation between the type of mutation and the severity of the disease but intra and interfamilial heterogeneity exist. Here, we describe the identification of KCNQ2 mutation carriers who had children affected with a severe epileptic phenotype, and found that these individuals were mosaic for the KCNQ2 mutation. These findings have important consequences for genetic counseling and indicate that neurological development can be normal in the presence of somatic mosaicism for a KCNQ2 mutation.

Hematopoietic stem cell transplantation in 29 patients hemizygous for hypomorphic IKBKG/NEMO mutations

X-linked recessive ectodermal dysplasia with immunodeficiency is a rare primary immunodeficiency caused by hypomorphic mutations of the IKBKG gene encoding the nuclear factor κB essential modulator (NEMO) protein. This condition displays enormous allelic, immunological, and clinical heterogeneity, and therapeutic decisions are difficult because NEMO operates in both hematopoietic and nonhematopoietic cells. Hematopoietic stem cell transplantation (HSCT) is potentially life-saving, but the small number of case reports available suggests it has been reserved for only the most severe cases. Here, we report the health status before HSCT, transplantation outcome, and clinical follow-up for a series of 29 patients from unrelated kindreds from 11 countries. Between them, these patients carry 23 different hypomorphic IKBKG mutations. HSCT was performed from HLA-identical related donors (n = 7), HLA-matched unrelated donors (n = 12), HLA-mismatched unrelated donors (n = 8), and HLA-haploidentical related donors (n = 2). Engraftment was documented in 24 patients, and graft-versus-host disease in 13 patients. Up to 7 patients died 0.2 to 12 months after HSCT. The global survival rate after HSCT among NEMO-deficient children was 74% at a median follow-up after HSCT of 57 months (range, 4-108 months). Preexisting mycobacterial infection and colitis were associated with poor HSCT outcome. The underlying mutation does not appear to have any influence, as patients with the same mutation had different outcomes. Transplantation did not appear to cure colitis, possibly as a result of cell-intrinsic disorders of the epithelial barrier. Overall, HSCT can cure most clinical features of patients with a variety of IKBKG mutations.

Gain-of-Function Mutation in Filamin A Potentiates Platelet Integrin α IIb β 3 ActivationHighlights

Objective— Dominant mutations of the X-linked filamin A (FLNA) gene are responsible for filaminopathies A, which are rare disorders including brain periventricular nodular heterotopia, congenital intestinal pseudo-obstruction, cardiac valves or skeleton malformations, and often macrothrombocytopenia. Approach and Results— We studied a male patient with periventricular nodular heterotopia and congenital intestinal pseudo-obstruction, his unique X-linked FLNA allele carrying a stop codon mutation resulting in a 100–amino acid–long FLNa C-terminal extension (NP_001447.2: p.Ter2648SerextTer101). Platelet counts were normal, with few enlarged platelets. FLNa was detectable in all platelets but at 30% of control levels. Surprisingly, all platelet functions were significantly upregulated, including platelet aggregation and secretion, as induced by ADP, collagen, or von Willebrand factor in the presence of ristocetin, as well as thrombus formation in blood flow on a collagen or on a von Willebrand factor matrix. Most importantly, patient platelets stimulated with ADP exhibited a marked increase in &agr;IIb&bgr;3 integrin activation and a parallel increase in talin recruitment to &bgr;3, contrasting with normal Rap1 activation. These results are consistent with the mutant FLNa affecting the last step of &agr;IIb&bgr;3 activation. Overexpression of mutant FLNa in the HEL megakaryocytic cell line correlated with an increase (compared with wild-type FLNa) in PMA-induced fibrinogen binding to and in talin and kindlin-3 recruitment by &agr;IIb&bgr;3. Conclusions— Altogether, our results are consistent with a less binding of mutant FLNa to &bgr;3 and the facilitated recruitment of talin by &bgr;3 on platelet stimulation, explaining the increased &agr;IIb&bgr;3 activation and the ensuing gain-of-platelet functions.