Phenotypic spectrum and genomics of undiagnosed arthrogryposis multiplex congenita.

BACKGROUND: Arthrogryposis multiplex congenita (AMC) is characterised by congenital joint contractures in two or more body areas. AMC exhibits wide phenotypic and genetic heterogeneity. Our goals were to improve the genetic diagnosis rates of AMC, to evaluate the added value of whole exome sequencing (WES) compared with targeted exome sequencing (TES) and to identify new genes in 315 unrelated undiagnosed AMC families. METHODS: Several genomic approaches were used including genetic mapping of disease loci in multiplex or consanguineous families, TES then WES. Sanger sequencing was performed to identify or validate variants. RESULTS: We achieved disease gene identification in 52.7% of AMC index patients including nine recently identified genes (CNTNAP1, MAGEL2, ADGRG6, ADCY6, GLDN, LGI4, LMOD3, UNC50 and SCN1A). Moreover, we identified pathogenic variants in ASXL3 and STAC3 expanding the phenotypes associated with these genes. The most frequent cause of AMC was a primary involvement of skeletal muscle (40%) followed by brain (22%). The most frequent mode of inheritance is autosomal recessive (66.3% of patients). In sporadic patients born to non-consanguineous parents (n=60), de novo dominant autosomal or X linked variants were observed in 30 of them (50%). CONCLUSION: New genes recently identified in AMC represent 21% of causing genes in our cohort. A high proportion of de novo variants were observed indicating that this mechanism plays a prominent part in this developmental disease. Our data showed the added value of WES when compared with TES due to the larger clinical spectrum of some disease genes than initially described and the identification of novel genes.

Distal duplication of chromosome 16q22.1q23.1 in a Vietnamese patient with midface hypoplasia and intellectual disability.

The clinical presentation of distal duplications of the long arm of chromosome (chr) 16 is currently not well described. Only one case of microduplication of chr16q22.1 and another involving the chr16q22.1q23.1 region have been reported so far. Here, using array comparative genomic hybridization, we identified a second case of chr16q22.1q23.1 duplication in a Vietnamese boy, who shares significant clinical phenotype with the previously described case. Aside from developmental delay, intellectual disability and midface hypoplasia, our patient also displays a forked tongue, visual impairment and external ptosis. Our report further expands the clinical spectrum associated with duplication of this region.

Clinical, Biomarker, and Molecular Delineations and Genotype-Phenotype Correlations of Ataxia With Oculomotor Apraxia Type 1.

Importance: Ataxia with oculomotor apraxia type 1 (AOA1) is an autosomal recessive cerebellar ataxia due to mutations in the aprataxin gene (APTX) that is characterized by early-onset cerebellar ataxia, oculomotor apraxia, axonal motor neuropathy, and eventual decrease of albumin serum levels. Objectives: To improve the clinical, biomarker, and molecular delineation of AOA1 and provide genotype-phenotype correlations. Design, Setting, and Participants: This retrospective analysis included the clinical, biological (especially regarding biomarkers of the disease), electrophysiologic, imaging, and molecular data of all patients consecutively diagnosed with AOA1 in a single genetics laboratory from January 1, 2002, through December 31, 2014. Data were analyzed from January 1, 2015, through January 31, 2016. Main Outcomes and Measures: The clinical, biological, and molecular spectrum of AOA1 and genotype-phenotype correlations. Results: The diagnosis of AOA1 was confirmed in 80 patients (46 men [58%] and 34 women [42%]; mean [SD] age at onset, 7.7 [7.4] years) from 51 families, including 57 new (with 8 new mutations) and 23 previously described patients. Elevated levels of α-fetoprotein (AFP) were found in 33 patients (41%); hypoalbuminemia, in 50 (63%). Median AFP level was higher in patients with AOA1 (6.0 ng/mL; range, 1.1-17.0 ng/mL) than in patients without ataxia (3.4 ng/mL; range, 0.8-17.2 ng/mL; P < .01). Decreased albumin levels (ρ = -0.532) and elevated AFP levels (ρ = 0.637) were correlated with disease duration. The p.Trp279* mutation, initially reported as restricted to the Portuguese founder haplotype, was discovered in 53 patients with AOA1 (66%) with broad white racial origins. Oculomotor apraxia was found in 49 patients (61%); polyneuropathy, in 74 (93%); and cerebellar atrophy, in 78 (98%). Oculomotor apraxia correlated with the severity of ataxia and mutation type, being more frequent with deletion or truncating mutations (83%) than with presence of at least 1 missense variant (17%; P < .01). Mean (SD) age at onset was higher for patients with at least 1 missense mutation (17.7 [11.4] vs 5.2 [2.6] years; P < .001). Conclusions and Relevance: The AFP level, slightly elevated in a substantial fraction of patients, may constitute a new biomarker for AOA1. Oculomotor apraxia may be an optional finding in AOA1 and correlates with more severe disease. The p.Trp279* mutation is the most frequent APTX mutation in the white population. APTX missense mutations may be associated with a milder phenotype.

Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts.

Although ribosomes are ubiquitous and essential for life, recent data indicate that monogenic causes of ribosomal dysfunction can confer a remarkable degree of specificity in terms of human disease phenotype. Box C/D small nucleolar RNAs (snoRNAs) are evolutionarily conserved non-protein-coding RNAs involved in ribosome biogenesis. Here we show that biallelic mutations in the gene SNORD118, encoding the box C/D snoRNA U8, cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts (LCC), presenting at any age from early childhood to late adulthood. These mutations affect U8 expression, processing and protein binding and thus implicate U8 as essential in cerebral vascular homeostasis.

MR angiography findings in infants with neonatal arterial ischemic stroke in the middle cerebral artery territory: A prospective study using circle of Willis MR angiography.

AIM: Neonatal arterial ischemic stroke (NAIS) results from a focal disruption of the blood flow in a cerebral artery by a not well understood mechanism. Our objective is to describe the acute MRangiography (MRA) findings in infants with an NAIS in the middle cerebral artery (MCA) territory and correlate them with early parenchymal infarcts and motor outcome. METHODS: Among one hundred prospectively followed neonates with NAIS, we studied thirty-seven patients with an MCA infarct explored with circle of Willis MRA. MCA flow characteristics were documented, along with infarct location/extent and motor outcome at age 7 years. RESULTS: Twenty-three (62%) of the children showed arterial changes, all ipsilateral to the NAIS, with occlusion in six, thrombus-type flow defect in nine, and unilateral increased flow in enlarged insular arteries in the remaining eight. There was a statistically significant correlation between parenchymal and arterial MR findings (p=0.0002). A normal MRA had a negative predictive value of 100% (95% CI: 76.8-100) in ruling out a main branch infarct. Patients with abnormal MRA tended to be at increased risk for cerebral palsy (OR=3.1). Occlusion was associated with a worse outcome (p=0.04). INTERPRETATION: MRangiography shows arterial abnormalities suggesting that embolism is a frequent cause of NAIS.

Unusual clinical expression and long survival of a pseudouridylate synthase (PUS1) mutation into adulthood.

A homozygote missense mutation of the pseudouridylate synthase gene was found in an adult patient with chronic sideroblastic anemia, diarrhea, microcephaly and failure to thrive. Moderate muscle weakness occurred in adulthood (6-min walk distance at 26 years: 240 m, control range 380-782 m) but a profound deficiency of mitochondrial respiratory chain complexes I and IV were found in her skeletal muscle. This, to our knowledge, is the first example of long survival of this usually fatal mitochondrial deficiency into adulthood. We suggest giving consideration to mitochondrial translation deficiency in unexplained syndromic sideroblastic anemia in adulthood.

Mutations in CNTNAP1 and ADCY6 are responsible for severe arthrogryposis multiplex congenita with axoglial defects.

Non-syndromic arthrogryposis multiplex congenita (AMC) is characterized by multiple congenital contractures resulting from reduced fetal mobility. Genetic mapping and whole exome sequencing (WES) were performed in 31 multiplex and/or consanguineous undiagnosed AMC families. Although this approach identified known AMC genes, we here report pathogenic mutations in two new genes. Homozygous frameshift mutations in CNTNAP1 were found in four unrelated families. Patients showed a marked reduction in motor nerve conduction velocity (<10 m/s) and transmission electron microscopy (TEM) of sciatic nerve in the index cases revealed severe abnormalities of both nodes of Ranvier width and myelinated axons. CNTNAP1 encodes CASPR, an essential component of node of Ranvier domains which underlies saltatory conduction of action potentials along the myelinated axons, an important process for neuronal function. A homozygous missense mutation in adenylate cyclase 6 gene (ADCY6) was found in another family characterized by a lack of myelin in the peripheral nervous system (PNS) as determined by TEM. Morpholino knockdown of the zebrafish orthologs led to severe and specific defects in peripheral myelin in spite of the presence of Schwann cells. ADCY6 encodes a protein that belongs to the adenylate cyclase family responsible for the synthesis of cAMP. Elevation of cAMP can mimic axonal contact in vitro and upregulates myelinating signals. Our data indicate an essential and so far unknown role of ADCY6 in PNS myelination likely through the cAMP pathway. Mutations of genes encoding proteins of Ranvier domains or involved in myelination of Schwann cells are responsible for novel and severe human axoglial diseases.

Early onset (childhood) monogenic neuropathies.

Hereditary neuropathies (HN) with onset in childhood are categorized according to clinical presentation, pathogenic mechanism based on electrophysiology, genetic transmission and, in selected cases, pathological findings. Especially relevant to pediatrics are the items "secondary" versus "primary" neuropathy, "syndromic versus nonsyndromic," and "period of life." Different combinations of these parameters frequently point toward specific monogenic disorders. Ruling out a neuropathy secondary to a generalized metabolic disorder remains the first concern in pediatrics. As a rule, metabolic diseases include additional, orienting symptoms or signs, and their biochemical diagnosis is based on logical algorithms. Primary, motor sensory are the most frequent HN and are dominated by demyelinating autosomal dominant (AD) forms (CMT1). Other forms include demyelinating autosomal recessive (AR) forms, axonal AD/AR forms, and forms with "intermediate" electrophysiological phenotype. Peripheral motor neuron disorders are dominated by AR SMN-linked spinal muscular atrophies. (Distal) hereditary motor neuropathies represent <10% of HN but exhibit large clinical and genetic heterogeneity. Sensory/dysautonomic HN involves five classic subtypes, each one related to specific genes. However, genetic heterogeneity is larger than initially suspected. Syndromic HN distinguish "purely neurological syndromes", which are multisystemic, such as spinocerebellar atrophies +, spastic paraplegias +, etc. Peripheral neuropathy is possibly the presenting feature, including in childhood. Autosomal recessive forms, on average, start more frequently in childhood. "Multiorgan syndromes", on the other hand, are more specific to Pediatrics. AR forms, which are clearly degenerative, prompt the investigation of a large set of pleiotropic genes. Other syndromes expressed in the perinatal period are mainly developmental disorders, and can sometimes be related to specific transcription factors. Systematic malformative workup and ethical considerations are necessary. Altogether, >40 genes with various biological functions have been found to be responsible for primary HN. Many are responsible for various phenotypes, including some without the polyneuropathic trait, and some for various types of transmission.

Hereditary motor-sensory, motor, and sensory neuropathies in childhood.

Hereditary neuropathies (HN) are categorized according to clinical presentation, pathogenic mechanism based on electrophysiology, genetic transmission, age of occurrence, and, in selected cases, pathological findings. The combination of these parameters frequently orients towards specific genetic disorders. Ruling out a neuropathy secondary to a generalized metabolic disorder remains the first pediatric concern. Primary, motor-sensory are the most frequent HN and are dominated by demyelinating AD forms (CMT1). Others are demyelinating AR forms, axonal AD/AR forms, and forms with "intermediate" electrophysiological phenotype. Pure motor HN represent<10% of HN but exhibit large clinical and genetic heterogeneity. Sensory/dysautonomic HN cover five classical subtypes, each one related to specific genes. However, genetic heterogeneity is largly greater than initially suspected. Syndromic HN distinguish: "purely neurological syndromes", which are multisystemic, usually AD disorders, such as spinocerebellar atrophies +, spastic paraplegias +, etc. Peripheral Neuropathy may be the presenting feature, including in childhood. Clearly degenerative, AR forms prompt to investigate a large set of pleiotropic genes. Other syndromes, expressed in the perinatal period and comprising malformative features, are mainly developmental disorders, sometimes related to specific transcription factors. Altogether, >40 genes with various biological functions have been found responsible for HN. Many are responsible for various phenotypes, including some without the polyneuropathic trait: for the pediatric neurologist, phenotype/genotype correlations constitute a permanent bidirectional exercise.

Mechanisms underlying a life-threatening skeletal muscle Na+ channel disorder.

Myotonia is an intrinsic muscular disorder caused by muscle fibre hyperexcitability, which produces a prolonged time for relaxation after voluntary muscle contraction or internal mechanical stimulation. Missense mutations in skeletal muscle genes encoding Cl− or Na+ channels cause non-dystrophic myotonias.Mutations of the SCN4A gene that encodes the skeletal voltage-gated Na+ channel Nav1.4 can produce opposing phenotypes leading to hyperexcitable or inexcitable muscle fibres. Nav1.4 mutations result in different forms of myotonias that can be found in adults. However, the recently reported myotonic manifestations in infants have been shown to be lethal. This was typically the case for children suffering from severe neonatal episodic laryngospasm (SNEL). A novel Nav1.4 channel missense mutation was found in these children that has not yet been analysed. In this study, we characterize the functional consequences of the new A799S Na+ channel mutation that is associated with sodium channel myotonia in newborn babies. We have used mammalian cell expression and patch-clamp techniques to monitor the channel properties.We found that the A799S substitution changes several biophysical properties of the channel by causing a hyperpolarizing shift of the steady-state activation, and slowing the kinetics of fast inactivation and deactivation. In addition, the single channel open probability was dramatically increased, contributing hence to a severe phenotype. We showed that substitutions at position 799 of the Nav1.4 channel favoured the channel open state with sustained activity leading to hyperexcitability of laryngeal muscles that could be lethal during infancy.

New insights (and new interrogations) in perinatal arterial ischemic stroke.

With an incidence of 1/2800 to 1/5000 live-births, perinatal arterial ischemic stroke is the most frequent form of cerebral infarction in children. About 40% of the children do not have specific symptoms in the neonatal period, and are only recognized later with the emergence of motor impairment, developmental delay, specific cognitive deficiency or seizures. In the remaining 60%, children present with early symptoms, mostly recurrent focal seizures in the first 3 days of life. The diagnosis is easily confirmed by cranial ultrasounds and MRI. Early MRI has both a key role in the diagnosis, dating the injury, but also an important prognostic value to predict the motor outcome of the child. Indeed, although the infarct does not recur, the majority of children show subsequent sequels: cerebral palsy, epilepsy, cognitive or behavioural problems. Finding predictors of outcome regarding these latter concerns (and the way to prevent or alleviate them) is of major interest. The main etiological hypothesis for perinatal AIS is a cerebral embolus, originating from the placenta through the foramen ovale. Most of the established risk factors are indeed either determinants or biomarkers of vasculo-placental pathology. Injury to the cervico-cerebral arteries, giving rise to thrombus/embolus during the birthing process is also suggested. Both placento-embolic and traumatic theories are supported by a few, but well-analysed pathological or arteriographic reports. Nevertheless, their relative frequency, the implication of other mechanisms, and their repercussions to evidence-based preventive strategies remain to be determined. Moreover, the mechanism of stroke in the different groups of newborns with stroke (term vs. preterm; symptomatic neonates vs. those with a delayed presentation) is likely to be different, and there is a need for future studies to assess all populations as different entities. Neonatal supportive care remains important for all infants while there is no evidence for preventive anticoagulant use at present. In an effort to reduce neurological dysfunction, and in adjunction with ongoing physical therapy and pharmacological treatment, new rehabilitative interventions, such as constraint-induced movement therapy and mirror therapy, are increasingly being used.

Motor outcomes after neonatal arterial ischemic stroke related to early MRI data in a prospective study.

OBJECTIVE: We aimed to correlate early imaging data with motor outcomes in a large, homogeneous, cohort of infants with neonatal (diagnosed before 29 days of life) arterial ischemic stroke (AIS). METHODS: From a prospective cohort of 100 children with neonatal AIS, we analyzed the MRI studies performed within the 28 first days of life for 80 infants evaluated at 2 years of age. The relationships between infarction location and corticospinal tract (CST) involvement and motor outcomes were studied RESULTS: Seventy-three infarctions involved the middle cerebral artery (MCA) territory. Of those, 50 were superficial infarctions, 5 deep infarctions, and 18 mixed infarctions. The CST was involved in 24 cases. Nineteen patients with MCA infarctions (26% [95% confidence interval: 16%-34%]) developed hemiplegia. Mixed infarctions (P<.0001) and CST involvement (P<.0001) were highly predictive of hemiplegia. In contrast, 88% of children with isolated superficial MCA infarctions did not exhibit impairment. CONCLUSIONS: Accurate prediction of motor outcomes can be obtained from early MRI scans after neonatal AIS. The absence of involvement of the CST resulted in normal motor development in 94% of cases. CST involvement resulted in congenital hemiplegia in 66% of cases.

Obstetrical and neonatal characteristics vary with birthweight in a cohort of 100 term newborns with symptomatic arterial ischemic stroke.

OBJECTIVES: Many questions remain regarding the mechanism of perinatal stroke. METHODS: In a series of 100 prospectively enrolled term neonates with symptomatic arterial ischemic stroke, we explored family antecedents, pregnancy and delivery conditions and clinical presenting features and distinguished features of the 50 larger infants with the remainder. Cardiac and cervical arterial imaging were performed in 70 and 51 cases. RESULTS: Previous fetal loss, first pregnancy, primiparity, twin-gestation, cesarean and traumatic delivery, neonatal distress, male sex and premature rupture of membranes were statistically more common than in the general population. Normal pregnancy proportion and mean birthweight were in the normal range, arguing against a vasculo-placental origin in the majority. Furthermore, there was an excess of large babies. The larger infants were more subject to suffer from acute perinatal events, with a trend for an excess of neonatal distress (p=0.065) and for more severe presenting features (p=0.027), while the lighter were more likely to have experienced longstanding obstetrical risk factors such as complicated pregnancy (p=0.047) and tobacco exposure (p=0.028). Cervical MR angiography showed an internal carotid occlusion in two babies, whereas echo-Doppler was always normal; in one case the two methods were discordant. Echocardiography was non-informative. INTERPRETATION: The data from this prospective cohort of neonates with stroke confirm that many obstetrical and perinatal factors are risk determinants. They also suggest that birthweight and gender may be biomarkers of two populations of neonates with different pathological mechanisms. MR angiography appears more sensitive than echo-Doppler for the exploration of the neonatal cervical vasculature.

Developmental dilatation of Virchow-Robin spaces: a genetic disorder?

In childhood, widening of Virchow-Robin spaces is rarely secondary to specific progressive disorders, but more often appears in poorly characterized developmental conditions. From data collected in a neuropediatric department, we examined whether clinical data associated with "constitutional widening of Virchow-Robin spaces" allowed delineation of recognizable entities. Signs in 10 patients, mostly boys, suggested nonspecific cerebral dysfunctions, e.g., developmental delay, nonspecific epilepsy, headaches, or benign macrocephaly. Spaces were sometimes round, subsequently mimicking microcystic malacic lesions. In two patients, abnormal magnetic resonance imaging signals were evident in white matter contiguous to widened perivascular spaces, suggesting a broader disorder of fluid exchanges. Four cases occurred in two sibships. In two families, other patients exhibited early developmental difficulties. Long-term clinical and magnetic resonance imaging surveillance will clarify which cases of primary Virchow-Robin space dilatation imply a benign prognosis. Performance of magnetic resonance imaging on any relative exhibiting minor neuropsychologic handicaps would permit estimations of real genetic incidence.

Exhaustive analysis of BH4 and dopamine biosynthesis genes in patients with Dopa-responsive dystonia.

Dopa-responsive dystonia is a childhood-onset dystonic disorder, characterized by a dramatic response to low dose of L-Dopa. Dopa-responsive dystonia is mostly caused by autosomal dominant mutations in the GCH1 gene (GTP cyclohydrolase1) and more rarely by autosomal recessive mutations in the TH (tyrosine hydroxylase) or SPR (sepiapterin reductase) genes. In addition, mutations in the PARK2 gene (parkin) which causes autosomal recessive juvenile parkinsonism may present as Dopa-responsive dystonia. In order to evaluate the relative frequency of the mutations in these genes, but also in the genes involved in the biosynthesis and recycling of BH4, and to evaluate the associated clinical spectrum, we have studied a large series of index patients (n = 64) with Dopa-responsive dystonia, in whom dystonia improved by at least 50% after L-Dopa treatment. Fifty seven of these patients were classified as pure Dopa-responsive dystonia and seven as Dopa-responsive dystonia-plus syndromes. All patients were screened for point mutations and large rearrangements in the GCH1 gene, followed by sequencing of the TH and SPR genes, then PTS (pyruvoyl tetrahydropterin synthase), PCBD (pterin-4a-carbinolamine dehydratase), QDPR (dihydropteridin reductase) and PARK2 (parkin) genes. We identified 34 different heterozygous point mutations in 40 patients, and six different large deletions in seven patients in the GCH1 gene. Except for one patient with mental retardation and a large deletion of 2.3 Mb encompassing 10 genes, all patients had stereotyped clinical features, characterized by pure Dopa-responsive dystonia with onset in the lower limbs and an excellent response to low doses of L-Dopa. Dystonia started in the first decade of life in 40 patients (85%) and before the age of 1 year in one patient (2.2%). Three of the 17 negative GCH1 patients had mutations in the TH gene, two in the SPR gene and one in the PARK2 gene. No mutations in the three genes involved in the biosynthesis and recycling of BH4 were identified. The clinical presentations of patients with mutations in TH and SPR genes were strikingly more complex, characterized by mental retardation, oculogyric crises and parkinsonism and they were all classified as Dopa-responsive dystonia-plus syndromes. Patient with mutation in the PARK2 gene had Dopa-responsive dystonia with a good improvement with L-Dopa, similar to Dopa-responsive dystonia secondary to GCH1 mutations. Although the yield of mutations exceeds 80% in pure Dopa-responsive dystonia and Dopa-responsive dystonia-plus syndromes groups, the genes involved are clearly different: GCH1 in the former and TH and SPR in the later.

Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as regulator of the innate immune response.

Aicardi-Goutières syndrome is a mendelian mimic of congenital infection and also shows overlap with systemic lupus erythematosus at both a clinical and biochemical level. The recent identification of mutations in TREX1 and genes encoding the RNASEH2 complex and studies of the function of TREX1 in DNA metabolism have defined a previously unknown mechanism for the initiation of autoimmunity by interferon-stimulatory nucleic acid. Here we describe mutations in SAMHD1 as the cause of AGS at the AGS5 locus and present data to show that SAMHD1 may act as a negative regulator of the cell-intrinsic antiviral response.

Rapid screening for nuclear genes mutations in isolated respiratory chain complex I defects.

Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype-genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.