Phenotype variability and natural history of X-linked myopathy with excessive autophagy.

OBJECTIVE: X-linked myopathy with excessive autophagy (XMEA) linked to the VMA21 gene leads to autophagy failure with progressive vacuolation and atrophy of skeletal muscles. Current knowledge of this rare disease is limited. Our objective was to define the clinical, radiological, and natural history of XMEA. METHODS: We conducted a retrospective study collecting clinical, genetic, muscle imaging, and biopsy data of XMEA patients followed in France and reviewed the literature for additional cases. RESULTS: Eighteen males had genetically confirmed XMEA in France, carrying four different VMA21 variants. Mean age at disease onset was 9.4 ± 9.9 (range 1-40) years. In 14/18 patients (77.8%), onset occurred during childhood (< 15 years); however in four patients, the disease started in adulthood. Patients had anterior and medial compartment thigh muscle weakness, distal contractures (56.3%), elevated CK levels (1287.9 ± 757.8 U/l) and autophagic vacuoles with sarcolemmal features on muscle histopathology. Muscle MRI (n = 10) showed a characteristic pattern of lower limb muscle involvement. In 11 patients, outcome measures were available for an average follow-up period of 10.6 ± 9.8 years and six of them show disease progression. Mean change of functional outcomes was 0.5 ± 1.2 points for Brooke and 2.2 ± 2.5 points for Vignos score, 7/16 patients (43.8%) needed a walking aid and 3/16 (18.8%) were wheelchair-bound (median age of 40 years old, range 39-48). The variant c.164-7 T > G was associated with a later onset of symptoms. Respiratory insufficiency was common (57.1%) but cardiac involvement rare (12.5%). INTERPRETATION: XMEA has variable age of onset, but a characteristic clinical, histopathological, and muscle imaging presentation, guiding the diagnosis. Although slowly, motor disability progresses with time, and relevant genotype-phenotype correlations will help design future clinical trials.

Cerebral dural arteriovenous fistulas in patients with PTEN-related hamartoma tumor syndrome.

Central nervous system (CNS) dural arteriovenous fistulas (DAVF) have been reported in PTEN-related hamartoma tumor syndrome (PHTS). However, PHTS-associated DAVF remain an underexplored field of the PHTS clinical landscape. Here, we studied cases with a PTEN pathogenic variant identified between 2007 and 2020 in our laboratory (n = 58), and for whom brain imaging was available. Two patients had DAVF (2/58, 3.4%), both presenting at advanced stages: a 34-year-old man with a left lateral sinus DAVF at immediate risk of hemorrhage, and a 21-year-old woman with acute intracranial hypertension due to a torcular DAVF. Interestingly, not all patients had 3D TOF/MRA, the optimal sequences to detect DAVF. Early diagnosis of DAVF can be lifesaving, and is easier to treat compared to developed, proliferative, or complex lesions. As a result, one should consider brain MRI with 3D TOF/MRA in PHTS patients at genetic diagnosis, with subsequent surveillance on a case-by-case basis.

MYH7 p.(Arg1712Gln) is pathogenic founder variant causing hypertrophic cardiomyopathy with overall relatively delayed onset.

INTRODUCTION: The MYH7 c.5135G > A p.(Arg1712Gln) variant has been identified in several patients worldwide and is classified as pathogenic in the ClinVar database. We aimed to delineate its associated phenotype and evaluate a potential founder effect. METHODS: We retrospectively collected clinical and genetic data of 22 probands and 74 family members from an international cohort. RESULTS: In total, 53 individuals carried the MYH7 p.(Arg1712Gln) variant, of whom 38 (72%) were diagnosed with hypertrophic cardiomyopathy (HCM). Mean age at HCM diagnosis was 48.8 years (standard deviation: 18.1; range: 8-74). The clinical presentation ranged from asymptomatic HCM to arrhythmias (atrial fibrillation and malignant ventricular arrhythmias). Aborted sudden cardiac death (SCD) leading to the diagnosis of HCM occurred in one proband at the age of 68 years, and a family history of SCD was reported by 39% (5/13) probands. Neither heart failure deaths nor heart transplants were reported. Women had a generally later-onset disease, with 14% of female carriers diagnosed with HCM at age 50 years compared with 54% of male carriers. In both sexes, the disease was fully penetrant by age 75 years. Haplotypes were reconstructed for 35 patients and showed a founder effect in a subset of patients. CONCLUSION: MYH7 p.(Arg1712Gln) is a pathogenic founder variant with a consistent HCM phenotype that may present with delayed penetrance. This suggested that clinical follow-up should be pursued after the seventh decade in healthy carriers and that longer intervals between screening may be justified in healthy women < 30 years.

Fatal thrombotic microangiopathy case following adeno-associated viral SMN gene therapy.

Adeno-associated virus (AAV) gene therapies are highly promising, such as the onasemnogene abeparvovec (Zolgensma) in spinal muscle atrophy (SMA). We report the first case of fatal systemic thrombotic microangiopathy (TMA) following onasemnogene abeparvovec in a 6-month-old child with SMA type 1, carrying a potential genetic predisposition in the complement factor I gene. Other cases of TMA have recently been reported after onasemnogene abeparvovec and after AAV9 minidystrophin therapy in Duchenne muscular dystrophy. The risk-benefit ratio of this therapy must therefore be assessed. Early recognition of TMA and targeted immunotherapy are fundamental to ensure the safety of patients treated with AAV gene therapies.

A recurrent RYR1 mutation associated with early-onset hypotonia and benign disease course.

The ryanodine receptor RyR1 is the main sarcoplasmic reticulum Ca2+ channel in skeletal muscle and acts as a connecting link between electrical stimulation and Ca2+-dependent muscle contraction. Abnormal RyR1 activity compromises normal muscle function and results in various human disorders including malignant hyperthermia, central core disease, and centronuclear myopathy. However, RYR1 is one of the largest genes of the human genome and accumulates numerous missense variants of uncertain significance (VUS), precluding an efficient molecular diagnosis for many patients and families. Here we describe a recurrent RYR1 mutation previously classified as VUS, and we provide clinical, histological, and genetic data supporting its pathogenicity. The heterozygous c.12083C>T (p.Ser4028Leu) mutation was found in thirteen patients from nine unrelated congenital myopathy families with consistent clinical presentation, and either segregated with the disease in the dominant families or occurred de novo. The affected individuals essentially manifested neonatal or infancy-onset hypotonia, delayed motor milestones, and a benign disease course differing from classical RYR1-related muscle disorders. Muscle biopsies showed unspecific histological and ultrastructural findings, while RYR1-typical cores and internal nuclei were seen only in single patients. In conclusion, our data evidence the causality of the RYR1 c.12083C>T (p.Ser4028Leu) mutation in the development of an atypical congenital myopathy with gradually improving motor function over the first decades of life, and may direct molecular diagnosis for patients with comparable clinical presentation and unspecific histopathological features on the muscle biopsy.

Structural analysis of the impact of a novel androgen receptor gene mutation in two adult patients with mild androgen insensitivity syndrome.

Androgen receptor gene (AR) mutations are responsible for androgen insensitivity syndrome (AIS) presenting with a clinical phenotype that ranges from gynaecomastia and/ or infertility in mild AIS (MAIS) to complete testicular feminisation in complete AIS. We report a novel AR gene mutation in two unrelated adult patients with MAIS and we studied its functional impact using 3D modelling. Patient 1, referred for infertility, presented with gynaecomastia, mild hypospadias and bilateral testicular hypotrophy contrasting with high testosterone levels, an elevated FSH, an elevated androgen sensitivity index (ASI) and oligoasthenoteratospermia. In vitro fertilisation and intracytoplasmic sperm injection resulted in a successful twin pregnancy. Patient 2 referred for a decrease in athletic performance had surgically treated gynaecomastia, oligoasthenospermia, high testosterone levels and an elevated ASI. Despite his impaired spermogram, he fathered two children without assisted reproductive technology. AR gene sequencing in the two patients revealed a common novel missense mutation, Ala699Thr, in exon 4 within the ligand-binding domain. 3D modelling studies showed that this mutation may impact dimer stability upon ligand binding or may affect allosteric changes upon dimerisation. This study illustrates the value of structural analysis for the functional study of mutations and expands the database of AR gene mutations.

New splicing pathogenic variant in EBP causing extreme familial variability of Conradi-Hünermann-Happle Syndrome.

X-linked dominant chondrodysplasia punctata (CDPX2 or Conradi-Hünermann-Happle syndrome, MIM #302960) is caused by mutations in the EBP gene. Affected female patients present with Blaschkolinear ichthyosis, coarse hair or alopecia, short stature, and normal psychomotor development. The disease is usually lethal in boys. Nevertheless, few male patients have been reported; they carry a somatic mosaicism in EBP or present with Klinefelter syndrome. Here, we report CDPX2 patients belonging to a three-generation family, carrying the splice variant c.301 + 5 G > C in intron 2 of EBP. The grandfather carries the variant as mosaic state and presents with short stature and mild ichthyosis. The mother also presents with short stature and mild ichthyosis and the female fetus with severe limb and vertebrae abnormalities and no skin lesions, with random X inactivation in both. This further characterizes the phenotypical spectrum of CDPX2, as well as intrafamilial variability, and raises the question of differential EBP mRNA splicing between the different target tissues.

De Novo Missense Mutations in DHX30 Impair Global Translation and Cause a Neurodevelopmental Disorder.

DHX30 is a member of the family of DExH-box helicases, which use ATP hydrolysis to unwind RNA secondary structures. Here we identified six different de novo missense mutations in DHX30 in twelve unrelated individuals affected by global developmental delay (GDD), intellectual disability (ID), severe speech impairment and gait abnormalities. While four mutations are recurrent, two are unique with one affecting the codon of one recurrent mutation. All amino acid changes are located within highly conserved helicase motifs and were found to either impair ATPase activity or RNA recognition in different in vitro assays. Moreover, protein variants exhibit an increased propensity to trigger stress granule (SG) formation resulting in global translation inhibition. Thus, our findings highlight the prominent role of translation control in development and function of the central nervous system and also provide molecular insight into how DHX30 dysfunction might cause a neurodevelopmental disorder.

Progressive Atrial Conduction Defects Associated With Bone Malformation Caused by a Connexin-45 Mutation.

BACKGROUND: Inherited cardiac conduction disease is a rare bradyarrhythmia associated with mutations in various genes that affect action potential propagation. It is often characterized by isolated conduction disturbance of the His-Purkinje system, but it is rarely described as a syndromic form. OBJECTIVES: The authors sought to identify the genetic defect in families with a novel bradyarrhythmia syndrome associated with bone malformation. METHODS: The authors genetically screened 15 European cases with genotype-negative de novo atrioventricular (AV) block and their parents by trio whole-exome sequencing, plus 31 Japanese cases with genotype-negative familial AV block or sick sinus syndrome by targeted exon sequencing of 457 susceptibility genes. Functional consequences of the mutation were evaluated using an in vitro cell expression system and in vivo knockout mice. RESULTS: The authors identified a connexin-45 (Cx45) mutation (p.R75H) in 2 unrelated families (a de novo French case and a 3-generation Japanese family) who presented with progressive AV block, which resulted in atrial standstill without ventricular conduction abnormalities. Affected individuals shared a common extracardiac phenotype: a brachyfacial pattern, finger deformity, and dental dysplasia. Mutant Cx45 expressed in Neuro-2a cells showed normal hemichannel assembly and plaque formation. However, Lucifer yellow dye transfer and gap junction conductance between cell pairs were severely impaired, which suggested that mutant Cx45 impedes gap junction communication in a dominant-negative manner. Tamoxifen-induced, cardiac-specific Cx45 knockout mice showed sinus node dysfunction and atrial arrhythmia, recapitulating the intra-atrial disturbance. CONCLUSIONS: Altogether, the authors showed that Cx45 mutant p.R75H is responsible for a novel disease entity of progressive atrial conduction system defects associated with craniofacial and dentodigital malformation.

Lessons learned from additional research analyses of unsolved clinical exome cases.

BACKGROUND: Given the rarity of most single-gene Mendelian disorders, concerted efforts of data exchange between clinical and scientific communities are critical to optimize molecular diagnosis and novel disease gene discovery. METHODS: We designed and implemented protocols for the study of cases for which a plausible molecular diagnosis was not achieved in a clinical genomics diagnostic laboratory (i.e. unsolved clinical exomes). Such cases were recruited to a research laboratory for further analyses, in order to potentially: (1) accelerate novel disease gene discovery; (2) increase the molecular diagnostic yield of whole exome sequencing (WES); and (3) gain insight into the genetic mechanisms of disease. Pilot project data included 74 families, consisting mostly of parent-offspring trios. Analyses performed on a research basis employed both WES from additional family members and complementary bioinformatics approaches and protocols. RESULTS: Analysis of all possible modes of Mendelian inheritance, focusing on both single nucleotide variants (SNV) and copy number variant (CNV) alleles, yielded a likely contributory variant in 36% (27/74) of cases. If one includes candidate genes with variants identified within a single family, a potential contributory variant was identified in a total of ~51% (38/74) of cases enrolled in this pilot study. The molecular diagnosis was achieved in 30/63 trios (47.6%). Besides this, the analysis workflow yielded evidence for pathogenic variants in disease-associated genes in 4/6 singleton cases (66.6%), 1/1 multiplex family involving three affected siblings, and 3/4 (75%) quartet families. Both the analytical pipeline and the collaborative efforts between the diagnostic and research laboratories provided insights that allowed recent disease gene discoveries (PURA, TANGO2, EMC1, GNB5, ATAD3A, and MIPEP) and increased the number of novel genes, defined in this study as genes identified in more than one family (DHX30 and EBF3). CONCLUSION: An efficient genomics pipeline in which clinical sequencing in a diagnostic laboratory is followed by the detailed reanalysis of unsolved cases in a research environment, supplemented with WES data from additional family members, and subject to adjuvant bioinformatics analyses including relaxed variant filtering parameters in informatics pipelines, can enhance the molecular diagnostic yield and provide mechanistic insights into Mendelian disorders. Implementing these approaches requires collaborative clinical molecular diagnostic and research efforts.

Germline De Novo Mutations in GNB1 Cause Severe Neurodevelopmental Disability, Hypotonia, and Seizures.

Whole-exome sequencing of 13 individuals with developmental delay commonly accompanied by abnormal muscle tone and seizures identified de novo missense mutations enriched within a sub-region of GNB1, a gene encoding the guanine nucleotide-binding protein subunit beta-1, Gß. These 13 individuals were identified among a base of 5,855 individuals recruited for various undiagnosed genetic disorders. The probability of observing 13 or more de novo mutations by chance among 5,855 individuals is very low (p = 7.1 × 10(-21)), implicating GNB1 as a genome-wide-significant disease-associated gene. The majority of these 13 mutations affect known Gß binding sites, which suggests that a likely disease mechanism is through the disruption of the protein interface required for Gα-Gßγ interaction (resulting in a constitutively active Gßγ) or through the disruption of residues relevant for interaction between Gßγ and certain downstream effectors (resulting in reduced interaction with the effectors). Strikingly, 8 of the 13 individuals recruited here for a neurodevelopmental disorder have a germline de novo GNB1 mutation that overlaps a set of five recurrent somatic tumor mutations for which recent functional studies demonstrated a gain-of-function effect due to constitutive activation of G protein downstream signaling cascades for some of the affected residues.

Expanding the clinical spectrum of hereditary fibrosing poikiloderma with tendon contractures, myopathy and pulmonary fibrosis due to FAM111B mutations.

BACKGROUND: Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients. METHODS: Clinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected. RESULTS: Key features consist of: (i) early-onset poikiloderma, hypotrichosis and hypohidrosis; (ii) multiple contractures, in particular triceps surae muscle contractures; (iii) diffuse progressive muscular weakness; (iv) pulmonary fibrosis in adulthood and (v) other features including exocrine pancreatic insufficiency, liver impairment and growth retardation. Muscle magnetic resonance imaging was informative and showed muscle atrophy and fatty infiltration. Histological examination of skeletal muscle revealed extensive fibroadipose tissue infiltration. Microscopy of the skin showed a scleroderma-like aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two recurrent mutations were found respectively in three and four independent families). All the mutations were predicted to localize in the trypsin-like cysteine/serine peptidase domain of the protein. We suggest gain-of-function or dominant-negative mutations resulting in FAM111B enzymatic activity changes. CONCLUSIONS: HFP with tendon contractures, myopathy and pulmonary fibrosis, is a multisystemic disorder due to autosomal dominant FAM111B mutations. Future functional studies will help in understanding the specific pathological process of this fibrosing disorder.

Muscle magnetic resonance imaging abnormalities in X-linked myopathy with excessive autophagy.

INTRODUCTION: X-linked myopathy with excessive autophagy (XMEA) is an X-linked recessive myopathy due to recently reported mutations in the VMA21 gene. METHODS: Four men from 2 separate families were studied. The clinical presentation, genetic data, muscle biopsy, and muscle MRI were analyzed. RESULTS: A known VMA21 mutation, c.163+4A>G, and a new mutation, c.163+3A>G, respectively, were found in the 2 families. The clinical course was characterized by onset in childhood and progressive muscle weakness with a limb-girdle pattern. Muscle biopsy revealed a mild myopathy with an increased number of giant autophagic vacuoles. Whole-body muscle MRI showed that pelvic girdle and proximal thighs were the most and earliest affected territories, with sparing of rectus femoris muscles. Muscle changes essentially consisted of degenerative fatty replacement. CONCLUSIONS: This study highlights a distinctive MRI pattern of muscle involvement, which can be helpful for diagnosis of XMEA, even before muscle biopsy or genetic analysis.

Clinical and electrophysiological features in a French family presenting with seipinopathy.

Seipinopathies are a group of inherited diseases affecting upper and lower motor neurons due to mutations in the Berardinelli-Seip congenital lipodystrophy 2 gene (BSCL2). We report a French family carrying the N88S mutation in the BSCL2 gene. A 12-yr-old girl complained of bilateral asymmetrical pes cavus with right hand motor deficit and amyotrophy, asymmetrical leg amyotrophy and pyramidal signs. Electrophysiological examination showed axonal asymmetrical motor neuropathy with distal predominance. Her father complained of right hand rest tremor with bilateral hand weakness. Physical examination revealed left leg, hand and forearm amyotrophy, akinesia and right arm rigidity, brisk reflexes in the lower limbs and bilateral Babinski sign. Nerve conduction studies showed distal asymmetrical axonal neuropathy with slight sensitive impairment with moderate decrease of nerve conduction velocity in some nerves. DNA sequencing revealed the presence of the known N88S mutation in the BSCL2 gene (dideoxy-nucleotide method on a 3730 DNA Analyzer, Life Technologies). BSCL2 gene mutations are associated with a wide spectrum of clinical and electrophysiological phenotypes and should be suspected in cases of distal hereditary motor neuropathy with pyramidal signs or early hand involvement. There may also be associated mild demyelination which may vary in severity within the same family. Clinical diagnosis was more difficult in this particular case due to the association with Parkinson symptoms.

New insights into genotype-phenotype correlation for GLI3 mutations.

The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype-phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype-phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues.

Mutations in FAM111B cause hereditary fibrosing poikiloderma with tendon contracture, myopathy, and pulmonary fibrosis.

Congenital poikiloderma is characterized by a combination of mottled pigmentation, telangiectasia, and epidermal atrophy in the first few months of life. We have previously described a South African European-descent family affected by a rare autosomal-dominant form of hereditary fibrosing poikiloderma accompanied by tendon contracture, myopathy, and pulmonary fibrosis. Here, we report the identification of causative mutations in FAM111B by whole-exome sequencing. In total, three FAM111B missense mutations were identified in five kindreds of different ethnic backgrounds. The mutation segregated with the disease in one large pedigree, and mutations were de novo in two other pedigrees. All three mutations were absent from public databases and were not observed on Sanger sequencing of 388 ethnically matched control subjects. The three single-nucleotide mutations code for amino acid changes that are clustered within a putative trypsin-like cysteine/serine peptidase domain of FAM111B. These findings provide evidence of the involvement of FAM111B in congenital poikiloderma and multisystem fibrosis.

Palmo-Plantar hyperkeratosis, intellectual disability, and spastic paraplegia in two maternal half brothers: further evidence for an X-linked inheritance.

In 1983, Fitzsimmons et al. reported four brothers with an unrecognized disorder characterized by intellectual disability, spastic paraplegia, and palmo-plantar hyperkeratosis (OMIM 309500). In this report, we describe a family in which two males, maternal half-brothers, had learning disabilities. Both patients also showed spasticity in the lower limbs and palmo-plantar hyperkeratosis. The mother of the affected boys had learning difficulties but did not show any dermatological symptoms. This report confirms that the association of features reported by Fitzsimmons et al. is a distinct entity and further suggests an X-linked mode of inheritance.

Genetic and clinical specificity of 26 symptomatic carriers for dystrophinopathies at pediatric age.

The molecular basis underlying the clinical variability in symptomatic Duchenne muscular dystrophy (DMD) carriers are still to be precised. We report 26 cases of early symptomatic DMD carriers followed in the French neuromuscular network. Clinical presentation, muscular histological analysis and type of gene mutation, as well as X-chromosome inactivation (XCI) patterns using DNA extracted from peripheral blood or muscle are detailed. The initial symptoms were significant weakness (88%) or exercise intolerance (27%). Clinical severity varied from a Duchenne-like progression to a very mild Becker-like phenotype. Cardiac dysfunction was present in 19% of the cases. Cognitive impairment was worthy of notice, as 27% of the carriers are concerned. The muscular analysis was always contributive, revealing muscular dystrophy (83%), mosaic in immunostaining (81%) and dystrophin abnormalities in western blot analysis (84%). In all, 73% had exonic deletions or duplications and 27% had point mutations. XCI pattern was biased in 62% of the cases. In conclusion, we report the largest series of manifesting DMD carriers at pediatric age and show that exercise intolerance and cognitive impairment may reveal symptomatic DMD carriers. The complete histological and immunohistological study of the muscle is the key of the diagnosis leading to the dystrophin gene analysis. Our study shows also that cognitive impairment in symptomatic DMD carriers is associated with mutations in the distal part of the DMD gene. XCI study does not fully explain the mechanisms as well as the wide spectrum of clinical phenotype, though a clear correlation between the severity of the phenotype and inactivation bias was observed.

Dissection of the MYCN locus in Feingold syndrome and isolated oesophageal atresia.

Feingold syndrome (FS) is a syndromic microcephaly entity for which MYCN is the major disease-causing gene. We studied the expression pattern of MYCN at different stages of human embryonic development and collected a series of 17 FS and 12 isolated oesophageal atresia (IOA) cases. An MYCN gene deletion/mutation was identified in 47% of FS cases exclusively. We hypothesized that mutations or deletions of highly conserved non-coding elements (HCNEs) at the MYCN locus could lead to its misregulation and thereby to FS and/or IOA. We subsequently sequenced five HCNEs at the MYCN locus and designed a high-density tiling path comparative genomic hybridization array of 3.3 Mb at the MYCN locus. We found no mutations or deletions in this region, supporting the hypothesis of genetic heterogeneity in FS.