Marie Vincent

Mutational spectrum of the ED1 gene in X-linked hypohidrotic ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XLHED) is the most common form of the ectodermal dysplasias characterised by an abnormal development of eccrine sweat glands, hair and teeth. The ED1 gene responsible for the disorder undergoes extensive alternative splicing and to date few studies have concerned the full length transcript. We screened 52 unrelated families or sporadic cases for mutation in the full coding sequence of this gene. SSCA analysis or direct sequencing allowed identification of mutations in 34 families: one initiation defect, twenty-two missenses, two nonsense, eight insertions or deletions, and a large deletion encompassing all the ED1 gene. Fourteen of these mutations have not been previously described, including five missenses. One third of identified mutations were localised in codons 155 and 156, affecting CpG dinucleotides and nine of them correspond to the R156H missense. Hypothesis of a founder effect has been ruled out by haplotype analysis of flanking microsatellites. These recurrent mutations indicate the functional importance of the positively charged domain of the protein. Including our data, there are now 56 different mutations reported in 85 independent patients, that we have tabulated. Review of clinical features in the present series of affected males and female carriers showed no obvious correlation between the type of mutations, the phenotype and its severity. The X-chromosome pattern of inactivation in leucocytes showed little correlation with expressivity of the disease in female carriers. Finally this study is useful for functional studies of the protein and to define a diagnostic strategy for mutation screening of the ED1 gene.

Tumor targeting of the IL‐15 superagonist RLI by an anti‐GD2 antibody strongly enhances its antitumor potency

Immunocytokines (ICKs) targeting cytokines to the tumor environment using antibodies directed against a tumor‐associated antigen often have a higher therapeutic index than the corresponding unconjugated cytokines. Various ICKs displaying significant antitumoral effects in several murine tumor models have already been developed, and some of them, in particular interleukin (IL)‐2‐based ICKs, are in Phase II clinical trials. Although sharing common biological activities with IL‐2 in vitro, IL‐15 is now considered as having a better potential in antitumor immunotherapeutical strategies and has been shown to be less toxic than IL‐2 in preclinical studies. We previously developed the fusion protein RLI, linking a soluble form of human IL‐15Rα‐sushi+ domain to human IL‐15. RLI showed better biological activities than IL‐15 in vitro as well as higher antitumoral effects in vivo in murine and human cancer models. Here, we investigated, in the context of an ICK, the effect of associating RLI with an antibody targeting the GD2 ganglioside, a validated tumoral target expressed on many neurectodermal tumors. Anti‐GD2‐RLI fully retained the cytokine potential of RLI and the antibody effector functions (antibody‐dependent cellular cytotoxicity and complement‐dependent cytotoxicity). It displayed strong antitumor activities in two syngeneic cancer models in immunocompetent mice (subcutaneous EL4 and metastatic NXS2). Its therapeutic potency was higher than those of RLI and anti‐GD2 alone or in combination. We suggest that this is related to its bifunctional (cytokine and antibody) nature.

De Novo Disruption of the Proteasome Regulatory Subunit PSMD12 Causes a Syndromic Neurodevelopmental Disorder

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.

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.

Autosomal recessive POLR1D mutation with decrease of TCOF1 mRNA is responsible for Treacher Collins syndrome.

Purpose:Treacher Collins syndrome is a mandibulofacial dysostosis caused by mutations in genes involved in ribosome biogenesis and synthesis. TCOF1 mutations are observed in ~80% of the patients and are inherited in an autosomal dominant manner. Recently, two other genes have been reported in <2% of patients—POLR1D in patients with autosomal dominant inheritance, and POLR1C in patients with autosomal recessive inheritance.Methods:We performed direct sequencing of TCOF1, POLR1C, and POLR1D in two unrelated consanguineous families.Results:The four affected children shared the same homozygous mutation in POLR1D (c.163C>G, p.Leu55Val). This mutation is localized in a region encoding the dimerization domain of the RNA polymerase. It is supposed that this mutation impairs RNA polymerase, resulting in a lower amount of mature dimeric ribosomes. A functional analysis of the transcripts of TCOF1 by real-time quantitative reverse transcription–polymerase chain reaction was performed in the first family, demonstrating a 50% reduction in the index case, compatible with this hypothesis.Conclusion:This is the first report of POLR1D mutation being responsible for an autosomal recessive inherited Treacher Collins syndrome. These results reinforce the concept of genetic heterogeneity of Treacher Collins syndrome and underline the importance of combining clinical expertise and familial molecular analyses for appropriate genetic counseling.Genet Med 16 9, 720–724.

Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse

Rituximab (RTX), a chimeric IgG1 monoclonal antibody directed against the CD20 antigen, has revolutionized the treatment of B-cell malignancies. Nevertheless, the relapsed/refractory rates are still high. One strategy to increase the clinical effectiveness of RTX is based on antibody-cytokine fusion protein (immunocytokine; ICK) vectorizing together at the tumor site the antibody effector activities and the cytokine co-signal required for the generation of cytotoxic cellular immunity. Such ICKs linking various antibody formats to interleukin (IL)-2 are currently being investigated in clinical trials and have shown promising results in cancer therapies. IL-15, a structurally-related cytokine, is now considered as having a better potential than IL-2 in antitumor immunotherapeutic strategies. We have previously engineered the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. Compared with IL-15, RLI displayed better biological activities in vitro and higher antitumor effects in vivo in murine and human cancer models. In this study, we investigated the advantages of fusing RLI to RTX. Anti-CD20-RLI kept its binding capacity to CD20, CD16 and IL-15 receptor and therefore fully retained both antibody effector functions (ADCC and CDC), and the cytokine potential of RLI. In a severe combined immunodeficiency (SCID) mouse model of disseminated residual lymphoma, anti-CD20-RLI was found to induce long-term survival of 90% of mice up to at least 120 days whereas RLI and RTX, alone or in combination, just delayed the disease onset (100% of death at 28, 40 and 51 days respectively). These findings suggest that such ICK could improve the clinical efficacy of RTX, particularly in patients with refractory B-cell lymphoma.

Genetic and phenotypic dissection of 1q43q44 microdeletion syndrome and neurodevelopmental phenotypes associated with mutations in ZBTB18 and HNRNPU

Subtelomeric 1q43q44 microdeletions cause a syndrome associating intellectual disability, microcephaly, seizures and anomalies of the corpus callosum. Despite several previous studies assessing genotype-phenotype correlations, the contribution of genes located in this region to the specific features of this syndrome remains uncertain. Among those, three genes, AKT3, HNRNPU and ZBTB18 are highly expressed in the brain and point mutations in these genes have been recently identified in children with neurodevelopmental phenotypes. In this study, we report the clinical and molecular data from 17 patients with 1q43q44 microdeletions, four with ZBTB18 mutations and seven with HNRNPU mutations, and review additional data from 37 previously published patients with 1q43q44 microdeletions. We compare clinical data of patients with 1q43q44 microdeletions with those of patients with point mutations in HNRNPU and ZBTB18 to assess the contribution of each gene as well as the possibility of epistasis between genes. Our study demonstrates that AKT3 haploinsufficiency is the main driver for microcephaly, whereas HNRNPU alteration mostly drives epilepsy and determines the degree of intellectual disability. ZBTB18 deletions or mutations are associated with variable corpus callosum anomalies with an incomplete penetrance. ZBTB18 may also contribute to microcephaly and HNRNPU to thin corpus callosum, but with a lower penetrance. Co-deletion of contiguous genes has additive effects. Our results confirm and refine the complex genotype-phenotype correlations existing in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of AKT3, ZBTB18 and HNRNPU in humans.

Duplication 8q12: confirmation of a novel recognizable phenotype with duane retraction syndrome and developmental delay

Duane retraction syndrome (DRS) is a rare congenital strabismus condition with genetic heterogeneity. DRS associated with intellectual disability or developmental delay is observed in several genetic diseases: syndromes such as Goldenhar or Wildervanck syndrome and chromosomal anomalies such as 12q12 deletion. We report on the case of a patient with DRS, developmental delay and particular facial features (horizontal and flared eyebrows, long and smooth philtrum, thin upper lip, full lower lip and full cheeks). We identified a duplication of the long arm of chromosome 8 (8q12) with SNP-array. This is the third case of a patient with common clinical features and 8q12 duplication described in the literature. The minimal critical region is 1.2 Mb and encompasses four genes: CA8, RAB2, RLBP1L1 and CHD7. To our knowledge, no information is available in the literature regarding pathological effects caused by to overexpression of these genes. However, loss of function of the CHD7 gene leads to CHARGE syndrome, suggesting a possible role of the overexpression of this gene in the phenotype observed in 8q12 duplication patients. We have observed that patients with 8q12 duplication share a common recognizable phenotype characterized by DRS, developmental delay and facial features. Such data combined to the literature strongly suggest that this entity may define a novel syndrome. We hypothesize that CHD7 duplication is responsible for a part of the features observed in 8q12.2 duplication.

Haploinsufficiency of MeCP2-interacting transcriptional co-repressor SIN3A causes mild intellectual disability by affecting the development of cortical integrity

Numerous genes are associated with neurodevelopmental disorders such as intellectual disability and autism spectrum disorder (ASD), but their dysfunction is often poorly characterized. Here we identified dominant mutations in the gene encoding the transcriptional repressor and MeCP2 interactor switch-insensitive 3 family member A (SIN3A; chromosome 15q24.2) in individuals who, in addition to mild intellectual disability and ASD, share striking features, including facial dysmorphisms, microcephaly and short stature. This phenotype is highly related to that of individuals with atypical 15q24 microdeletions, linking SIN3A to this microdeletion syndrome. Brain magnetic resonance imaging showed subtle abnormalities, including corpus callosum hypoplasia and ventriculomegaly. Intriguingly, in vivo functional knockdown of Sin3a led to reduced cortical neurogenesis, altered neuronal identity and aberrant corticocortical projections in the developing mouse brain. Together, our data establish that haploinsufficiency of SIN3A is associated with mild syndromic intellectual disability and that SIN3A can be considered to be a key transcriptional regulator of cortical brain development.

Large deletions encompassing the TCOF1 and CAMK2A genes are responsible for Treacher Collins syndrome with intellectual disability

Mandibulofacial dysostosis is part of a clinically and genetically heterogeneous group of disorders of craniofacial development, which lead to malar and mandibular hypoplasia. Treacher Collins syndrome is the major cause of mandibulofacial dysostosis and is due to mutations in the TCOF1 gene. Usually patients with Treacher Collins syndrome do not present with intellectual disability. Recently, the EFTUD2 gene was identified in patients with mandibulofacial dysostosis associated with microcephaly, intellectual disability and esophageal atresia. We report on two patients presenting with mandibulofacial dysostosis characteristic of Treacher Collins syndrome, but associated with unexpected intellectual disability, due to a large deletion encompassing several genes including the TCOF1 gene. We discuss the involvement of the other deleted genes such as CAMK2A or SLC6A7 in the cognitive development delay of the patients reported, and we propose the systematic investigation for 5q32 deletion when intellectual disability is associated with Treacher Collins syndrome.