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IntroductionNeuromuscular disorders (NMDs) have a heterogeneous etiology. A genetic diagnosis is key to personalized healthcare and access to targeted treatment for the affected individuals.MethodsIn this study, 861 patients with NMDs were analyzed with genome sequencing and comprehensive variant calling including single nucleotide variants, small insertions/deletions (SNVs/INDELs), and structural variants (SVs) in a panel of 895 NMD genes, as well as short tandem repeat expansions (STRs) at 28 loci. In addition, for unsolved cases with an unspecific clinical presentation, the analysis of a panel with OMIM disease genes was added.ResultsIn the cohort, 27% (232/861) of the patients harbored pathogenic variants, of which STRs and SVs accounted for one-third of the patients (71/232). The variants were found in 107 different NMD genes. Furthermore, 18 pediatric patients harbored pathogenic variants in non-NMD genes.DiscussionOur results highlight that for children with unspecific hypotonia, a genome-wide analysis rather than a disease-based gene panel should be considered as a diagnostic approach. More importantly, our results clearly show that it is crucial to include STR- and SV-analyses in the diagnostics of patients with neuromuscular disorders.

Purpose. Dominant variants in the Retinoic Acid Receptor Beta (RARB) gene underlie a syndromic form of microphthalmia, known as MCOPS12, which is associated with other birth anomalies and global developmental delay with spasticity and/or dystonia. Here, we report 25 affected individuals with 17 novel pathogenic or likely pathogenic variants in RARB. This study aims to characterize the functional impact of these variants and describe the clinical spectrum of MCOPS12. Methods. We used in vitro transcriptional assays and in silico structural analysis to assess the functional relevance of RARB variants in affecting the normal response to retinoids. Results. We found that all RARB variants tested in our assays exhibited either a gain-of-function or a loss-of-function activity. Loss-of-function variants disrupted RARB function through a dominant-negative effect, possibly by disrupting ligand binding and/or co-activators’ recruitment. By reviewing clinical data from 52 affected individuals, we found that disruption of RARB is associated with a more variable phenotype than initially suspected, with the absence in some individuals of cardinal features of MCOPS12, such as developmental eye anomaly or motor impairment. Conclusion. Our study indicates that pathogenic variants in RARB are functionally heterogeneous and are associated with extensive clinical heterogeneity.

This paper focuses on genetic counselling in Phelan-McDermid syndrome (PMS), a rare neurodevelopmental disorder caused by a deletion 22q13.3 or a pathogenic variant in SHANK3. It is one of a series of papers written by the European PMS consortium as a consensus guideline. We reviewed the available literature based on pre-set questions to formulate recommendations on counselling, diagnostic work-up and surveillance for tumours related to ring chromosome 22. All recommendations were approved by the consortium, which consists of professionals and patient representatives, using a voting procedure. PMS can only rarely be diagnosed based solely on clinical features and requires confirmation via genetic testing. In most cases, the family will be referred to a clinical geneticist for counselling after the genetic diagnosis has been made. Family members will be investigated and, if indicated, the chance of recurrence discussed with them. Most individuals with PMS have a de novo deletion or a pathogenic variant of SHANK3. The 22q13.3 deletion can be a simple deletion, a ring chromosome 22, or the result of a parental balanced chromosomal anomaly, influencing the risk of recurrence. Individuals with a ring chromosome 22 have an increased risk of NF2-related schwannomatosis (formerly neurofibromatosis type 2) and atypical teratoid rhabdoid tumours, which are associated with the tumour-suppressor genes NF2 and SMARCB1, respectively, and both genes are located on chromosome 22. The prevalence of PMS due to a ring chromosome 22 is estimated to be 10–20%. The risk of developing a tumour in an individual with a ring chromosome 22 can be calculated as 2–4%. However, those individuals who do develop tumours often have multiple. We recommend referring all individuals with PMS and their parents to a clinical geneticist or a comparably experienced medical specialist for genetic counselling, further genetic testing, follow-up and discussion of prenatal diagnostic testing in subsequent pregnancies. We also recommend karyotyping to diagnose or exclude a ring chromosome 22 in individuals with a deletion 22q13.3 detected by molecular tests. If a ring chromosome 22 is found, we recommend discussing personalised follow-up for NF2-related tumours and specifically cerebral imaging between the age of 14 and 16 years.

Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.

Pathogenic variants in MYH3 cause distal arthrogryposis type 2A and type 2B3 as well as contractures, pterygia and spondylocarpotarsal fusion syndromes types 1A and 1B. These disorders are ultra-rare and their natural course and phenotypic variability are not well described. In this study, we summarize the clinical features and genetic findings of 17 patients from 10 unrelated families with vertebral malformations caused by dominant or recessive pathogenic variants in MYH3. Twelve novel pathogenic variants in MYH3 (NM_002470.4) were identified: three of them were de novo or inherited in autosomal dominant way and nine were inherited in autosomal recessive way. The patients had vertebral segmentation anomalies accompanied with variable joint contractures, short stature and dysmorphic facial features. There was a significant phenotypic overlap between dominant and recessive MYH3-associated conditions regarding the degree of short stature as well as the number of vertebral fusions. All monoallelic variants caused significantly decreased SMAD3 phosphorylation, which is consistent with the previously proposed pathogenic mechanism of impaired canonical TGF-beta signaling. Most of the biallelic variants were predicted to be protein-truncating, while one missense variant c.4244T>G,p.(Leu1415Arg), which was inherited in an autosomal recessive way, was found to alter the phosphorylation level of p38, suggesting an inhibition of the non-canonical pathway of TGF-beta signaling. In conclusion, the identification of 12 novel pathogenic variants and overlapping phenotypes in 17 affected individuals from 10 unrelated families expands the mutation and phenotype spectrum of MYH3-associated skeletal disorders. We show that disturbances of canonical or non-canonical TGF-beta signaling pathways are involved in pathogenesis of MYH3-associated skeletal fusion (MASF) syndrome. Funding Agencies|National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [81930068, 81772299, 81822030, 82072391, 81972132, 81672123, 81972037, 81902178]; Beijing Natural Science FoundationBeijing Natural Science Foundation [JQ20032, 7191007]; CAMS Innovation Fund for Medical Sciences (CIFMS) [2021-I2M-1-051, 2021-I2M-1-052]; Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences [2019PT320025]; Tsinghua University-Peking Union Medical College Hospital Initiative Scientific Research Program; PUMC Youth Fund & the Fundamental Research Funds for the Central Universities [3332019021]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [K2015-54X-22 736-01-4, 2015-02227, 2018-03046]; Swedish Governmental Agency for Innovation Systems (Vinnova)Vinnova [2014-01438]; Marianne and Marcus Wallenberg Foundation; IngaBritt och Arne Lundbergs forskningsstiftelse; Byggmastare Olle Engkvist Stiftelse; Promobilia; Nyckelfonden; Stiftelsen Frimurare Barnhuset i Stockholm; Region Stockholm; Karolinska Institutet, Stockholm, Sweden; orebro University, orebro, Sweden; Sallskapet Barnavard; Karolinska InstitutetKarolinska Institutet; Stiftelsen Sallsyntafonden; Stiftelsen Samariten; Stiftelsen Promobilia; Region Stockholm [20180131, 20200500]; US National Institutes of Health (NIH), National Institute of Neurological Disorders and Stroke [NINDS R35 NS105078]; National Human Genome Research Institute/National Heart, Lung, and Blood Institute [NHGRI/NHLBI UM1 HG006542]; US NIH National Human Genome Research Institute [NHGRI K08 HG008986]

Mowat-Wilson syndrome (MWS) is a complex developmental syndrome caused by heterozygous mutations in the Zinc finger E-box-binding homeobox 2 gene (ZEB2). We generated the first human iPSC lines from primary fibroblasts of two siblings with MWS carrying a heterozygous ZEB2 stop mutation (c.1027C > T; p.Arg343*) using the Sendai virus reprogramming system. Both iPSC lines were free from reprogramming vector genes, expressed pluripotency markers and showed potential to differentiate into the three germ layers. Genetic analysis confirmed normal karyotypes and a preserved stop mutation. These iPSC lines will provide a useful resource to study altered neural lineage fate and neuropathophysiology in MWS.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities.

Rubinstein–Taybi syndrome (RTS) is a rare autosomal dominant congenital disorder characterized by distinctive facial features, broad thumbs and halluces, growth retardation, and a variable degree of cognitive impairment. CREBBP is the major causative gene and mutations in EP300 are the cause of RTS in a minority of patients. In this study, 17 patients with a clinical diagnosis of RTS were investigated with direct sequencing, MLPA, and array‐CGH in search for mutations in these two genes. Eleven patients (64.7%) had disease‐causing point mutations or a deletion in CREBBP and in one patient (5.9%) a causal de novo frameshift mutation in EP300 was identified. This patient had broad thumbs, mild intellectual disability, and autism. In addition, an inherited missense mutation of uncertain clinical significance was identified in EP300 in one patient and his healthy father, and three patients had intronic nucleotide changes of uncertain clinical significance in CREBBP. Snoring and sleep apnea were common in both groups and four of the patients' mothers had preeclampsia during pregnancy. Importantly, difficulties associated with anesthesia were frequently reported and included delayed or complicated emergency in 53.3% of patients.

International audience; KAT6B sequence variants have been identified previously in both patients with the Say-Barber-Biesecker type of blepharophimosis mental retardation syndromes (SBBS) and in the more severe genitopatellar syndrome (GPS). We report on the findings in a previously unreported group of 57 individuals with suggestive features of SBBS or GPS. Likely causative variants have been identified in 34/57 patients and were commonly located in the terminal exons of KAT6B. Of those where parental samples could be tested, all occurred de novo. Thirty out of thirty-four had truncating variants, one had a missense variant and the remaining three had the same synonymous change predicted to affect splicing. Variants in GPS tended to occur more proximally to those in SBBS patients, and genotype/phenotype analysis demonstrated significant clinical overlap between SBBS and GPS. The de novo synonymous change seen in three patients with features of SBBS occurred more proximally in exon 16. Statistical analysis of clinical features demonstrated that KAT6B variant-positive patients were more likely to display hypotonia, feeding difficulties, long thumbs/great toes and dental, thyroid and patella abnormalities than KAT6B variant-negative patients. The few reported patients with KAT6B haploinsufficiency had a much milder phenotype, though with some features overlapping those of SBBS. We report the findings in a previously unreported patient with a deletion of the KAT6B gene to further delineate the haploinsufficiency phenotype. The molecular mechanisms giving rise to the SBBS and GPS phenotypes are discussed.