Broadening the phenotypic spectrum of TUBA1A tubulinopathy to syndromic arthrogryposis multiplex congenita.

The recent finding that some patients with fetal akinesia deformation sequence (FADS) carry variants in the TUBB2B gene has prompted us to add to the existing literature a first description of two fetal FADS cases carrying TUBA1A variants. Hitherto, only isolated cortical malformations have been described with TUBA1A mutation, including microlissencephaly, lissencephaly, central pachygyria and polymicrogyria-like cortical dysplasia, generalized polymicrogyria cortical dysplasia, and/or the "simplified" gyral pattern. The neuropathology of our fetal cases shows several common features of tubulinopathies, in particular, the dysmorphism of the basal ganglia, as the most pathognomonic sign. The cortical ribbon anomalies were extremely severe and concordant with the complex cortical malformation. In conclusion, we broaden the phenotypic spectrum of TUBA1A variants, to include FADS.

Postnatal Diagnostic Workup in Children With Arthrogryposis: A Series of 82 Patients.

OBJECTIVE: To describe a postnatal series of patients with arthrogryposis multiplex congenita by the causal mechanisms involved. METHODS: In this single-center study, the local data warehouse was used to identify patients with arthrogryposis multiplex congenita. Patients were classified into different etiologic groups. RESULTS: Of 82 patients included, the most frequent cause of arthrogryposis multiplex congenita was a neuromuscular disorder (39%), including skeletal muscle (n = 19), neuromuscular junction (n = 3), and peripheral nerve (n = 11) involvement. In other subgroups, 19 patients (23%) were classified by disorders in the central nervous system, 5 (6%) in connective tissue, 7 (8.5%) had mixed mechanisms, and 18 (22%) could not be classified. Contractures topography was not associated with a causal mechanism. Cerebral magnetic resonance imaging (MRI), electroneuromyography, and muscle biopsy were the most conclusive investigations. Metabolic investigations were normal in all the patients tested. Targeted or whole exome sequencing diagnostic rates were 51% and 71%, respectively. Thirty-three percent of patients died (early death occurred in patients with polyhydramnios, prematurity, and ventilatory dependency). DISCUSSION: The benefits of a precise diagnosis in the neonatal period include more tailored management of arthrogryposis multiplex congenita and better genetic information.

Loss of function mutations in EPHB4 are responsible for vein of Galen aneurysmal malformation.

See Meschia (doi:10.1093/brain/awy066) for a scientific commentary on this article.Vein of Galen aneurysmal malformation is a congenital anomaly of the cerebral vasculature representing 30% of all paediatric vascular malformations. We conducted whole exome sequencing in 19 unrelated patients presenting this malformation and subsequently screened candidate genes in a cohort of 32 additional patients using either targeted exome or Sanger sequencing. In a cohort of 51 patients, we found five affected individuals with heterozygous mutations in EPHB4 including de novo frameshift (p.His191Alafs*32) or inherited deleterious splice or missense mutations predicted to be pathogenic by in silico tools. Knockdown of ephb4 in zebrafish embryos leads to specific anomalies of dorsal cranial vessels including the dorsal longitudinal vein, which is the orthologue of the median prosencephalic vein and the embryonic precursor of the vein of Galen. This model allowed us to investigate EPHB4 loss-of-function mutations in this disease by the ability to rescue the brain vascular defect in knockdown zebrafish co-injected with wild-type, but not truncated EPHB4, mimicking the p.His191Alafs mutation. Our data showed that in both species, loss of function mutations of EPHB4 result in specific and similar brain vascular development anomalies. Recently, EPHB4 germline mutations have been reported in non-immune hydrops fetalis and in cutaneous capillary malformation-arteriovenous malformation. Here, we show that EPHB4 mutations are also responsible for vein of Galen aneurysmal malformation, indicating that heterozygous germline mutations of EPHB4 result in a large clinical spectrum. The identification of EPHB4 pathogenic mutations in patients presenting capillary malformation or vein of Galen aneurysmal malformation should lead to careful follow-up of pregnancy of carriers for early detection of anomaly of the cerebral vasculature in order to propose optimal neonatal care. Endovascular embolization indeed greatly improved the prognosis of patients.

Loss-of-Function Mutations in LGI4, a Secreted Ligand Involved in Schwann Cell Myelination, Are Responsible for Arthrogryposis Multiplex Congenita.

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through genetic mapping of disease loci and whole-exome sequencing in four unrelated multiplex families presenting with severe AMC, we identified biallelic loss-of-function mutations in LGI4 (leucine-rich glioma-inactivated 4). LGI4 is a ligand secreted by Schwann cells that regulates peripheral nerve myelination via its cognate receptor ADAM22 expressed by neurons. Immunolabeling experiments and transmission electron microscopy of the sciatic nerve from one of the affected individuals revealed a lack of myelin. Functional tests using affected individual-derived iPSCs showed that these germline mutations caused aberrant splicing of the endogenous LGI4 transcript and in a cell-based assay impaired the secretion of truncated LGI4 protein. This is consistent with previous studies reporting arthrogryposis in Lgi4-deficient mice due to peripheral hypomyelination. This study adds to the recent reports implicating defective axoglial function as a key cause of AMC.

Application of whole-exome sequencing to unravel the molecular basis of undiagnosed syndromic congenital neutropenia with intellectual disability.

Neutropenia can be qualified as congenital when of neonatal onset or when associated with extra-hematopoietic manifestations. Overall, 30% of patients with congenital neutropenia (CN) remain without a molecular diagnosis after a multidisciplinary consultation and tedious diagnostic strategy. In the rare situations when neutropenia is identified and associated with intellectual disability (ID), there are few diagnostic hypotheses to test. This retrospective multicenter study reports on a clinically heterogeneous cohort of 10 unrelated patients with CN associated with ID and no molecular diagnosis prior to whole-exome sequencing (WES). WES provided a diagnostic yield of 40% (4/10). The results suggested that in many cases neutropenia and syndromic manifestations could not be assigned to the same molecular alteration. Three sub-groups of patients were highlighted: (i) severe, symptomatic chronic neutropenia, detected early in life, and related to a known mutation in the CN spectrum (ELANE); (ii) mild to moderate benign intermittent neutropenia, detected later, and associated with mutations in genes implicated in neurodevelopmental disorders (CHD2, HUWE1); and (iii) moderate to severe intermittent neutropenia as a probably undiagnosed feature of a newly reported syndrome (KAT6A). Unlike KAT6A, which seems to be associated with a syndromic form of CN, the other reported mutations may not explain the entire clinical picture. Although targeted gene sequencing can be discussed for the primary diagnosis of severe CN, we suggest that performing WES for the diagnosis of disorders associating CN with ID will not only provide the etiological diagnosis but will also pave the way towards personalized care and follow-up. © 2016 Wiley Periodicals, Inc.

The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype.

Heterozygous COL2A1 variants cause a wide spectrum of skeletal dysplasia termed type II collagenopathies. We assessed the impact of this gene in our French series. A decision tree was applied to select 136 probands (71 Stickler cases, 21 Spondyloepiphyseal dysplasia congenita cases, 11 Kniest dysplasia cases, and 34 other dysplasia cases) before molecular diagnosis by Sanger sequencing. We identified 66 different variants among the 71 positive patients. Among those patients, 18 belonged to multiplex families and 53 were sporadic. Most variants (38/44, 86%) were located in the triple helical domain of the collagen chain and glycine substitutions were mainly observed in severe phenotypes, whereas arginine to cysteine changes were more often encountered in moderate phenotypes. This series of skeletal dysplasia is one of the largest reported so far, adding 44 novel variants (15%) to published data. We have confirmed that about half of our Stickler patients (46%) carried a COL2A1 variant, and that the molecular spectrum was different across the phenotypes. To further address the question of genotype-phenotype correlation, we plan to screen our patients for other candidate genes using a targeted next-generation sequencing approach.

An atypical human induced pluripotent stem cell line with a complex, stable, and balanced genomic rearrangement including a large de novo 1q uniparental disomy.

Human induced pluripotent stem cells (hiPSCs) hold great promise for cell therapy through their use as vital tools for regenerative and personalized medicine. However, the genomic integrity of hiPSCs still raises some concern and is one of the barriers limiting their use in clinical applications. Numerous articles have reported the occurrence of aneuploidies, copy number variations, or single point mutations in hiPSCs, and nonintegrative reprogramming strategies have been developed to minimize the impact of the reprogramming process on the hiPSC genome. Here, we report the characterization of an hiPSC line generated by daily transfections of modified messenger RNAs, displaying several genomic abnormalities. Karyotype analysis showed a complex genomic rearrangement, which remained stable during long-term culture. Fluorescent in situ hybridization analyses were performed on the hiPSC line showing that this karyotype is balanced. Interestingly, single-nucleotide polymorphism analysis revealed the presence of a large 1q region of uniparental disomy (UPD), demonstrating for the first time that UPD can occur in a noncompensatory context during nonintegrative reprogramming of normal fibroblasts.

Messenger RNA- versus retrovirus-based induced pluripotent stem cell reprogramming strategies: analysis of genomic integrity.

The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications.

Germline and somatic genetic variations of TNFAIP3 in lymphoma complicating primary Sjogren's syndrome.

Several autoimmune diseases, including primary Sjögren's syndrome (pSS), are associated with an increased risk for lymphoma. Polymorphisms of TNFAIP3, which encodes the A20 protein that plays a key role in controlling nuclear factor κB activation, have been associated with several autoimmune diseases. Somatic mutations of TNFAIP3 have been observed in the mucosa-associated lymphoid tissue lymphoma subtype frequently associated with pSS. We studied germline and somatic abnormalities of TNFAIP3 in 574 patients with pSS, including 25 with lymphoma. Nineteen additional patients with pSS and lymphoma were available for exome sequence analysis. Functional abnormalities of A20 were assessed by gene reporter assays. The rs2230926 exonic variant was associated with an increased risk for pSS complicated by lymphoma (odds ratio, 3.36 [95% confidence interval, 1.34-8.42], and odds ratio, 3.26 [95% confidence interval, 1.31-8.12], vs controls and pSS patients without lymphoma, respectively; P = .011). Twelve (60%) of the 20 patients with paired germline and lymphoma TNFAIP3 sequence data had functional abnormalities of A20: 6 in germline DNA, 5 in lymphoma DNA, and 1 in both. The frequency was even higher (77%) among pSS patients with mucosa-associated lymphoid tissue lymphoma. Some of these variants showed impaired control of nuclear factor κB activation. These results support a key role for germline and somatic variations of A20 in the transformation between autoimmunity and lymphoma.

Microtubule-targeting drugs rescue axonal swellings in cortical neurons from spastin knockout mice.

Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP), a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.

Amniotic fluid stem cells restore the muscle cell niche in a HSA-Cre, Smn(F7/F7) mouse model.

Mutations in the survival of motor neuron gene (SMN1) are responsible for spinal muscular atrophy, a fatal neuromuscular disorder. Mice carrying a homozygous deletion of Smn exon 7 directed to skeletal muscle (HSA-Cre, Smn(F7/F7) mice) present clinical features of human muscular dystrophies for which new therapeutic approaches are highly warranted. Herein we demonstrate that tail vein transplantation of mouse amniotic fluid stem (AFS) cells enhances the muscle strength and improves the survival rate of the affected animals. Second, after cardiotoxin injury of the Tibialis Anterior, only AFS-transplanted mice efficiently regenerate. Most importantly, secondary transplants of satellite cells (SCs) derived from treated mice show that AFS cells integrate into the muscle stem cell compartment and have long-term muscle regeneration capacity indistinguishable from that of wild-type-derived SC. This is the first study demonstrating the functional and stable integration of AFS cells into the skeletal muscle, highlighting their value as cell source for the treatment of muscular dystrophies.

Mapping of gene expression reveals CYP27A1 as a susceptibility gene for sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease characterized by loss of upper and lower motor neurons. ALS is considered to be a complex trait and genome-wide association studies (GWAS) have implicated a few susceptibility loci. However, many more causal loci remain to be discovered. Since it has been shown that genetic variants associated with complex traits are more likely to be eQTLs than frequency-matched variants from GWAS platforms, we conducted a two-stage genome-wide screening for eQTLs associated with ALS. In addition, we applied an eQTL analysis to finemap association loci. Expression profiles using peripheral blood of 323 sporadic ALS patients and 413 controls were mapped to genome-wide genotyping data. Subsequently, data from a two-stage GWAS (3,568 patients and 10,163 controls) were used to prioritize eQTLs identified in the first stage (162 ALS, 207 controls). These prioritized eQTLs were carried forward to the second sample with both gene-expression and genotyping data (161 ALS, 206 controls). Replicated eQTL SNPs were then tested for association in the second-stage GWAS data to find SNPs associated with disease, that survived correction for multiple testing. We thus identified twelve cis eQTLs with nominally significant associations in the second-stage GWAS data. Eight SNP-transcript pairs of highest significance (lowest p = 1.27 × 10(-51)) withstood multiple-testing correction in the second stage and modulated CYP27A1 gene expression. Additionally, we show that C9orf72 appears to be the only gene in the 9p21.2 locus that is regulated in cis, showing the potential of this approach in identifying causative genes in association loci in ALS. This study has identified candidate genes for sporadic ALS, most notably CYP27A1. Mutations in CYP27A1 are causal to cerebrotendinous xanthomatosis which can present as a clinical mimic of ALS with progressive upper motor neuron loss, making it a plausible susceptibility gene for ALS.

Three independent mutations in the TSC2 gene in a family with tuberous sclerosis.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder characterized by hamartomas and hamartias in multiple organs. TSC is caused by a wide spectrum of mutations within the TSC1 and TSC2 genes. Here, we report a unique family with three independent pathological mutations in TSC2. A c.1322G>A mutation in exon 12 created a stop codon, whereas a second mutation in exon 23 (c.2713C>T) was a missense change. The third mutation was a 4 base pair deletion in intron 20 of TSC2. We showed that this mutation was responsible for abnormal splicing. The three mutations were most likely de novo, as parents of affected patients did not present any features of TSC. In addition, we showed gonadal mosaicism in a branch of the family. To our knowledge, several independent mutations in TSC2 have never been observed in a single family. The probability of finding a family with three different pathological TSC2 mutations is extremely low. We discuss two main hypotheses that may be raised to explain this recurrence: (i) the TSC2 mutation rate is underestimated. In such a case, the likelihood of finding a family with three independent mutations in TSC2 may not be dramatically low; (ii) a heritable defect in a DNA repair gene (eg, mismatch repair gene) segregating in the family that is unlinked to the TSC2 gene might predispose to the occurrence of multiple TSC2 gene mutations, used as a specific target during embryogenesis.

Mutations in DDR2 gene cause SMED with short limbs and abnormal calcifications.

The spondylo-meta-epiphyseal dysplasia [SMED] short limb-hand type [SMED-SL] is a rare autosomal-recessive disease, first reported by Borochowitz et al. in 1993.(1) Since then, 14 affected patients have been reported.(2-5) We diagnosed 6 patients from 5 different consanguineous Arab Muslim families from the Jerusalem area with SMED-SL. Additionally, we studied two patients from Algerian and Pakistani ancestry and the parents of the first Jewish patients reported.(1) Using a homozygosity mapping strategy, we located a candidate region on chromosome 1q23 spanning 2.4 Mb. The position of the Discoidin Domain Receptor 2 (DDR2) gene within the candidate region and the similarity of the ddr2 knockout mouse to the SMED patients' phenotype prompted us to study this gene(6). We identified three missense mutations c.2254 C > T [R752C], c. 2177 T > G [I726R], c.2138C > T [T713I] and one splice site mutation [IVS17+1g > a] in the conserved sequence encoding the tyrosine kinase domain of the DDR2 gene. The results of this study will permit an accurate early prenatal diagnosis and carrier screening for families at risk.

A mutation of spastin is responsible for swellings and impairment of transport in a region of axon characterized by changes in microtubule composition.

Mutations of the spastin gene (Sp) are responsible for the most frequent autosomal dominant form of spastic paraplegia, a disease characterized by the degeneration of corticospinal tracts. We show that a deletion in the mouse Sp gene, generating a premature stop codon, is responsible for progressive axonal degeneration, restricted to the central nervous system, leading to a late and mild motor defect. The degenerative process is characterized by focal axonal swellings, associated with abnormal accumulation of organelles and cytoskeletal components. In culture, mutant cortical neurons showed normal viability and neurite density. However, they develop neurite swellings associated with focal impairment of retrograde transport. These defects occur near the growth cone, in a region characterized by the transition between stable microtubules rich in detyrosinated alpha-tubulin and dynamic microtubules composed almost exclusively of tyrosinated alpha-tubulin. Here, we show that the Sp mutation has a major impact on neurite maintenance and transport both in vivo and in vitro. These results highlight the link between spastin and microtubule dynamics in axons, but not in other neuronal compartments. In addition, it is the first description of a human neurodegenerative disease which involves this specialized region of the axon.

Bone marrow transplantation attenuates the myopathic phenotype of a muscular mouse model of spinal muscular atrophy.

Bone marrow (BM) transplantation was performed on a muscular mouse model of spinal muscular atrophy that had been created by mutating the survival of motor neuron gene (Smn) in myofibers only. This model is characterized by a severe myopathy and progressive loss of muscle fibers leading to paralysis. Transplantation of wild-type BM cells following irradiation at a low dose (6 Gy) improved motor capacity (+85%). This correlated with a normalization of myofiber number associated with a higher number of regenerating myofibers (1.6-fold increase) and an activation of CD34 and Pax7 satellite cells. However, BM cells had a very limited capacity to replace or fuse to mutant myofibers (2%). These data suggest that BM transplantation was able to attenuate the myopathic phenotype through an improvement of skeletal muscle regeneration of recipient mutant mice, a process likely mediated by a biological activity of BM-derived cells. This hypothesis was further supported by the capacity of muscle protein extracts from transplanted mutant mice to promote myoblast proliferation in vitro (1.6-fold increase). In addition, a tremendous upregulation of hepatocyte growth factor (HGF), which activates quiescent satellite cells, was found in skeletal muscle of transplanted mutants compared with nontransplanted mutants. Eventually, thanks to the Cre-loxP system, we show that BM-derived muscle cells were strong candidates harboring this biological activity. Taken together, our data suggest that a biological activity is likely involved in muscle regeneration improvement mediated by BM transplantation. HGF may represent an attractive paracrine mechanism to support this activity.

Deletion of murine Smn exon 7 directed to liver leads to severe defect of liver development associated with iron overload.

Spinal muscular atrophy (SMA) is characterized by degeneration of lower motor neurons caused by mutations of the survival motor neuron 1 gene (SMN1). SMN is involved in various processes including the formation of the spliceosome, pre-mRNA splicing and transcription. To know whether SMN has an essential role in all mammalian cell types or an as yet unknown specific function in the neuromuscular system, deletion of murine Smn exon 7, the most frequent mutation found among SMA patients, has been restricted to liver. Homozygous mutation results in severe impairment of liver development associated with iron overload and lack of regeneration leading to dramatic liver atrophy and late embryonic lethality of mutant mice. These data strongly suggest an ubiquitous and essential role of full-length SMN protein in various mammalian cell types. In SMA patients, the residual amount of SMN allows normal function of various organs except motor neurons. However, data from mouse and human suggest that other tissues might be involved in severe form of SMA or during prolonged disease course which reinforce the need of therapeutic approaches targeted to all tissues. In addition, liver function of patients should be carefully investigated and followed up before and during therapeutic trials.

Therapeutic benefits of cardiotrophin-1 gene transfer in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a recessive autosomal disorder characterized by degeneration of lower motor neurons caused by mutations of the survival motor neuron gene (SMN1). No curative treatment is known so far. Mutant mice carrying homozygous deletion of Smn exon 7 directed to neurons display skeletal muscle denervation, moderate loss of motor neuron cell bodies and severe axonal degeneration. These features, similar to those found in human SMA, strongly suggest the involvement of a dying back process of motor neurons and led us to test whether neurotrophic factors might have a protective role in SMA. We report here the therapeutic benefits of systemic delivery of cardiotrophin-1 (CT-1), a neurotrophic factor belonging to the IL-6 cytokine family. Intra-muscular injection of adenoviral vector expressing CT-1, even at very low dose, improves median survival, delays motor defect of mutant mice and exerts protective effect against loss of proximal motor axons and aberrant cytoskeletal organization of motor synaptic terminals. In spite of the severity of SMA phenotype in mutant mice, CT-1 is able to slow down disease progression. Neuroprotection could be regarded as valuable therapeutic approach in SMA.

Mutations of SPG4 are responsible for a loss of function of spastin, an abundant neuronal protein localized in the nucleus.

Mutations of spastin are responsible for the most common autosomal dominant form of hereditary spastic paraplegia (AD-HSP), a disease characterized by axonal degeneration of corticospinal tracts and posterior columns. Generation of polyclonal antibodies specific to spastin has revealed two isoforms of 75 and 80 kDa in both human and mouse tissues with a tissue-specific variability of the isoform ratio. Spastin is an abundant protein in neural tissues and immunolabeling experiments have shown that spastin is expressed in neurons but not in glial cells. These data indicate that axonal degeneration linked to spastin mutations is caused by a primary defect of neurons. Protein and transcript analyses of patients carrying either nonsense or frameshift spastin mutations revealed neither truncated protein nor mutated transcripts, providing evidence that these mutations are responsible for a loss of spastin function. Identifying agents able to induce the expression of the non-mutated spastin allele should represent an attractive therapeutic strategy in this disease.