Whole-Genome Sequencing Identified New Structural Variations in the DMD Gene That Cause Duchenne Muscular Dystrophy in Two Girls.

Dystrophinopathies are the most common muscle diseases, especially in men. In women, on the other hand, a manifestation of Duchenne muscular dystrophy is rare due to X-chromosomal inheritance. We present two young girls with severe muscle weakness, muscular dystrophies, and creatine kinase (CK) levels exceeding 10,000 U/L. In the skeletal muscle tissues, dystrophin staining reaction showed mosaicism. The almost entirely skewed X-inactivation in both cases supported the possibility of a dystrophinopathy. Despite standard molecular diagnostics (including multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) gene panel sequencing), the genetic cause of the girls' conditions remained unknown. However, whole-genome sequencing revealed two reciprocal translocations between their X chromosomes and chromosome 5 and chromosome 19, respectively. In both cases, the breakpoints on the X chromosomes were located directly within the DMD gene (in introns 54 and 7, respectively) and were responsible for the patients' phenotypes. Additional techniques such as Sanger sequencing, conventional karyotyping and fluorescence in situ hybridization (FISH) confirmed the disruption of DMD gene in both patients through translocations. These findings underscore the importance of accurate clinical data combined with histopathological analysis in pinpointing the suspected underlying genetic disorder. Moreover, our study illustrates the viability of whole-genome sequencing as a time-saving and highly effective method for identifying genetic factors responsible for complex genetic constellations in Duchenne muscular dystrophy (DMD).

Closing the Gap - Detection of 5q-Spinal Muscular Atrophy by Short-Read Next-Generation Sequencing and Unexpected Results in a Diagnostic Patient Cohort.

BACKGROUND: The importance of early diagnosis of 5q-Spinal muscular atrophy (5q-SMA) has heightened as early intervention can significantly improve clinical outcomes. In 96% of cases, 5q-SMA is caused by a homozygous deletion of SMN1. Around 4 % of patients carry a SMN1 deletion and a single-nucleotide variant (SNV) on the other allele. Traditionally, diagnosis is based on multiplex ligation probe amplification (MLPA) to detect homozygous or heterozygous exon 7 deletions in SMN1. Due to high homologies within the SMN1/SMN2 locus, sequence analysis to identify SNVs of the SMN1 gene is unreliable by standard Sanger or short-read next-generation sequencing (srNGS) methods. OBJECTIVE: The objective was to overcome the limitations in high-throughput srNGS with the aim of providing SMA patients with a fast and reliable diagnosis to enable their timely therapy. METHODS: A bioinformatics workflow to detect homozygous SMN1 deletions and SMN1 SNVs on srNGS analysis was applied to diagnostic whole exome and panel testing for suggested neuromuscular disorders (1684 patients) and to fetal samples in prenatal diagnostics (260 patients). SNVs were detected by aligning sequencing reads from SMN1 and SMN2 to an SMN1 reference sequence. Homozygous SMN1 deletions were identified by filtering sequence reads for the ,, gene-determining variant" (GDV). RESULTS: 10 patients were diagnosed with 5q-SMA based on (i) SMN1 deletion and hemizygous SNV (2 patients), (ii) homozygous SMN1 deletion (6 patients), and (iii) compound heterozygous SNVs in SMN1 (2 patients). CONCLUSIONS: Applying our workflow in srNGS-based panel and whole exome sequencing (WES) is crucial in a clinical laboratory, as otherwise patients with an atypical clinical presentation initially not suspected to suffer from SMA remain undiagnosed.

Homozygous Inversion on Chromosome 13 Involving SGCG Detected by Short Read Whole Genome Sequencing in a Patient Suffering from Limb-Girdle Muscular Dystrophy.

New techniques in molecular genetic diagnostics now allow for accurate diagnosis in a large proportion of patients with muscular diseases. Nevertheless, many patients remain unsolved, although the clinical history and/or the muscle biopsy give a clear indication of the involved genes. In many cases, there is a strong suspicion that the cause must lie in unexplored gene areas, such as deep-intronic or other non-coding regions. In order to find these changes, next-generation sequencing (NGS) methods are constantly evolving, making it possible to sequence entire genomes to reveal these previously uninvestigated regions. Here, we present a young woman who was strongly suspected of having a so far genetically unsolved sarcoglycanopathy based on her clinical history and muscle biopsy. Using short read whole genome sequencing (WGS), a homozygous inversion on chromosome 13 involving SGCG and LINC00621 was detected. The breakpoint in intron 2 of SGCG led to the absence of γ-sarcoglycan, resulting in the manifestation of autosomal recessive limb-girdle muscular dystrophy 5 (LGMDR5) in the young woman.

Detection of pericentric inversion with breakpoint in DMD by whole genome sequencing.

BACKGROUND: Dystrophinopathies caused by variants in the DMD gene are a well-studied muscle disease. The most common type of variant in DMD are large deletions. Very rarely reported forms of variants are chromosomal translocations, inversions and deep intronic variants (DIVs) because they are not detectable by standard diagnostic techniques (sequencing of coding sequence, copy number variant detection). This might be the reason that some clinically and histologically proven dystrophinopathy cases remain unsolved. METHODS: We used whole genome sequencing (WGS) to screen the entire DMD gene for variants in one of two brothers suffering from typical muscular dystrophy with strongly elevated creatine kinase levels. RESULTS: Although a pathogenic DIV could not be detected, we were able to identify a pericentric inversion with breakpoints in DMD intron 44 and Xq13.3, which could be confirmed by Sanger sequencing in the index as well as in his brother and mother. As this variation affects a major part of DMD it is most likely disease causing. CONCLUSION: Our findings elucidate that WGS is capable of detecting large structural rearrangements and might be suitable for the genetic diagnostics of dystrophinopathies in the future. In particular, inversions might be a more frequent cause for dystrophinopathies as anticipated and should be considered in genetically unsolved dystrophinopathy cases.

X-chromosomal inactivation patterns in women with Fabry disease.

BACKGROUND: Although Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA), women may develop severe symptoms. We investigated X-chromosomal inactivation patterns (XCI) as a potential determinant of symptom severity in FD women. PATIENTS AND METHODS: We included 95 women with mutations in GLA (n = 18 with variants of unknown pathogenicity) and 50 related men, and collected mouth epithelial cells, venous blood, and skin fibroblasts for XCI analysis using the methylation status of the androgen receptor gene. The mutated X-chromosome was identified by comparison of samples from relatives. Patients underwent genotype categorization and deep clinical phenotyping of symptom severity. RESULTS: 43/95 (45%) women carried mutations categorized as classic. The XCI pattern was skewed (i.e., ≥75:25% distribution) in 6/87 (7%) mouth epithelial cell samples, 31/88 (35%) blood samples, and 9/27 (33%) skin fibroblast samples. Clinical phenotype, α-galactosidase A (GAL) activity, and lyso-Gb3 levels did not show intergroup differences when stratified for X-chromosomal skewing and activity status of the mutated X-chromosome. CONCLUSIONS: X-inactivation patterns alone do not reliably reflect the clinical phenotype of women with FD when investigated in biomaterial not directly affected by FD. However, while XCI patterns may vary between tissues, blood frequently shows skewing of XCI patterns.

CRISPR/Cas9-edited PKP2 knock-out (JMUi001-A-2) and DSG2 knock-out (JMUi001-A-3) iPSC lines as an isogenic human model system for arrhythmogenic cardiomyopathy (ACM).

Arrhythmogenic cardiomyopathy (ACM) is characterized by fibro-fatty replacement of the myocardium, heart failure and life-threatening ventricular arrhythmias. Causal mutations were identified in genes encoding for proteins of the desmosomes, predominantly plakophilin-2 (PKP2) and desmoglein-2 (DSG2). We generated gene-edited knock-out iPSC lines for PKP2 (JMUi001-A-2) and DSG2 (JMUi001-A-3) using the CRISPR/Cas9 system in a healthy control iPSC background (JMUi001-A). Stem cell-like morphology, robust expression of pluripotency markers, embryoid body formation and normal karyotypes confirmed the generation of high quality iPSCs to provide a novel isogenic human in vitro model system mimicking ACM when differentiated into cardiomyocytes.

New Insights on Genetic Diagnostics in Cardiomyopathy and Arrhythmia Patients Gained by Stepwise Exome Data Analysis.

Inherited cardiomyopathies are characterized by clinical and genetic heterogeneity that challenge genetic diagnostics. In this study, we examined the diagnostic benefit of exome data compared to targeted gene panel analyses, and we propose new candidate genes. We performed exome sequencing in a cohort of 61 consecutive patients with a diagnosis of cardiomyopathy or primary arrhythmia, and we analyzed the data following a stepwise approach. Overall, in 64% of patients, a variant of interest (VOI) was detected. The detection rate in the main sub-cohort consisting of patients with dilated cardiomyopathy (DCM) was much higher than previously reported (25/36; 69%). The majority of VOIs were found in disease-specific panels, while a further analysis of an extended panel and exome data led to an additional diagnostic yield of 13% and 5%, respectively. Exome data analysis also detected variants in candidate genes whose functional profile suggested a probable pathogenetic role, the strongest candidate being a truncating variant in STK38. In conclusion, although the diagnostic yield of gene panels is acceptable for routine diagnostics, the genetic heterogeneity of cardiomyopathies and the presence of still-unknown causes favor exome sequencing, which enables the detection of interesting phenotype-genotype correlations, as well as the identification of novel candidate genes.

Generation of two patient-derived iPSC lines from siblings (LIBUCi001-A and LIBUCi002-A) and a genetically modified iPSC line (JMUi001-A-1) to mimic dilated cardiomyopathy with ataxia (DCMA) caused by a homozygous DNAJC19 mutation.

Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from mutations in DNAJC19. Two patient-derived dermal fibroblast cell lines of siblings with the same homozygous splice acceptor site mutation in DNAJC19 (NM_145261.4):c.130-1G>C were reprogrammed into induced pluripotent stem cell (iPSC) lines (LIBUCi001-A and LIBUCi002-A) using non-integrative Sendai virus. Additionally, a third DNAJC19tv (truncation variant) iPSC line (JMUi001-A-1) was generated by CRISPR/Cas9 in healthy control iPSCs (JMUi001-A). All three DCMA iPSC lines present normal karyotypes, high expression of pluripotency markers and the capacity to differentiate into cells of all three germ layers.

Targeted Gene Expression Profile Reveals CDK4 as Therapeutic Target for Selected Patients With Adrenocortical Carcinoma.

Adrenocortical carcinomas (ACC) are aggressive tumors with a heterogeneous prognosis and limited therapeutic options for advanced stages. This study aims to identify novel drug targets for a personalized treatment in ACC. RNA was isolated from 40 formalin-fixed paraffin-embedded ACC samples. We evaluated gene expression of 84 known cancer drug targets by reverse transcriptase quantitative real time-PCR and calculated fold change using 5 normal adrenal glands as reference (overexpression by fold change >2.0). The most promising candidate cyclin-dependent kinase 4 (CDK4) was investigated at protein level in 104 ACC samples and tested by in vitro experiments in two ACC cell lines (NCI-H295R and MUC1). The most frequently overexpressed genes were TOP2A (100% of cases, median fold change = 16.5), IGF2 (95%, fold change = 52.9), CDK1 (80%, fold change = 6.7), CDK4 (62%, fold change = 2.6), PLK4 (60%, fold change = 2.8), and PLK1 (52%, fold change = 2.3). CDK4 was chosen for functional validation, as it is actionable by approved CDK4/6-inhibitors (e.g., palbociclib). Nuclear immunostaining of CDK4 significantly correlated with mRNA expression (R = 0.52, P < 0.005). We exposed both NCI-H295R and MUC1 cell lines to palbociclib and found a concentration- and time-dependent reduction of cell viability, which was more pronounced in the NCI-H295R cells in line with higher CDK4 expression. Furthermore, we tested palbociclib in combination with insulin-like growth factor 1/insulin receptor inhibitor linsitinib showing an additive effect. In conclusion, we demonstrate that RNA profiling is useful to discover potential drug targets and that CDK4/6 inhibitors are promising candidates for treatment of selected patients with ACC.

Exon-4 Mutations in KRAS Affect MEK/ERK and PI3K/AKT Signaling in Human Multiple Myeloma Cell Lines.

Approximately 20% of multiple myeloma (MM) cases harbor a point mutation in KRAS. However, there is still no final consent on whether KRAS-mutations are associated with disease outcome. Specifically, no data exist on whether KRAS-mutations have an impact on survival of MM patients at diagnosis in the era of novel agents. Direct blockade of KRAS for therapeutic purposes is mostly impossible, but recently a mutation-specific covalent inhibitor targeting KRASp.G12C entered into clinical trials. However, other KRAS hotspot-mutations exist in MM patients, including the less common exon-4 mutations. For the current study, the coding regions of KRAS were deep-sequenced in 80 newly diagnosed MM patients, uniformely treated with three cycles of bortezomib plus dexamethasone and cyclophosphamide (VCD)-induction, followed by high-dose chemotherapy and autologous stem cell transplantation. Moreover, the functional impact of KRASp.G12A and the exon-4 mutations p.A146T and p.A146V on different survival pathways was investigated. Specifically, KRASWT, KRASp.G12A, KRASp.A146T, and KRASp.A146V were overexpressed in HEK293 cells and the KRASWT MM cell lines JJN3 and OPM2 using lentiviral transduction and the Sleeping Beauty vector system. Even though KRAS-mutations were not correlated with survival, all KRAS-mutants were found capable of potentially activating MEK/ERK- and sustaining PI3K/AKT-signaling in MM cells.

Biallelic mutation in MYH7 and MYBPC3 leads to severe cardiomyopathy with left ventricular noncompaction phenotype.

Dominant mutations in the MYH7 and MYBPC3 genes are common causes of inherited cardiomyopathies, which often demonstrate variable phenotypic expression and incomplete penetrance across family members. Biallelic inheritance is rare but allows gaining insights into the genetic mode of action of single variants. Here, we present three cases carrying a loss-of-function (LoF) variant in a compound heterozygous state with a missense variant in either MYH7 or MYBPC3 leading to severe cardiomyopathy with left ventricular noncompaction. Most likely, MYH7 haploinsufficiency due to one LoF allele results in a clinical phenotype only in compound heterozygous form with a missense variant. In contrast, haploinsufficiency in MYBPC3 results in a severe early-onset ventricular noncompaction phenotype requiring heart transplantation when combined with a de novo missense variant on the second allele. In addition, the missense variant may lead to an unstable protein, as overall only 20% of the MYBPC3 protein remain detectable in affected cardiac tissue compared to control tissue. In conclusion, in patients with early disease onset and atypical clinical course, biallelic inheritance or more complex variants including copy number variations and de novo mutations should be considered. In addition, the pathogenic consequence of variants may differ in heterozygous versus compound heterozygous state.

The identification of patient-specific mutations reveals dual pathway activation in most patients with melanoma and activated receptor tyrosine kinases in BRAF/NRAS wild-type melanomas.

BACKGROUND: Increasing knowledge of cancer genomes has triggered the development of specific targeted inhibitors, thus providing a valuable therapeutic pool. METHODS: In this report, the authors analyze the presence of targetable alterations in 136 tumor samples from 92 patients with melanoma using a comprehensive approach based on targeted DNA sequencing and supported by RNA and protein analysis. Three topics of high clinical relevance are addressed: the identification of rare, activating alterations; the detection of patient-specific, co-occurring single nucleotide variants (SNVs) and copy number variations (CNVs) in parallel pathways; and the presence of cancer-relevant germline mutations. RESULTS: The analysis of patient-matched blood and tumor samples was done with a custom-designed gene panel that was enriched for genes from clinically targetable pathways. To detect alterations with high therapeutic relevance for patients with unknown driver mutations, genes that are untypical for melanoma also were included. Among all patients, CNVs were identified in one-third of samples and contained amplifications of druggable kinases, such as CDK4, ERBB2, and KIT. Considering SNVs and CNVs, 60% of patients with metastases exhibited co-occurring activations of at least 2 pathways, thus providing a rationale for individualized combination therapies. Unexpectedly, 9% of patients carry potentially protumorigenic germline mutations frequently affecting receptor tyrosine kinases. Remarkably two-thirds of BRAF/NRAS wild-type melanomas harbor activating mutations or CNVs in receptor tyrosine kinases. CONCLUSIONS: The results indicate that the integrated analysis of SNVs, CNVs, and germline mutations reveals new druggable targets for combination tumor therapy.

Targeted Molecular Analysis in Adrenocortical Carcinomas: A Strategy Toward Improved Personalized Prognostication.

Context: Adrenocortical carcinoma (ACC) has a heterogeneous prognosis, and current medical therapies have limited efficacy in its advanced stages. Genome-wide multiomics studies identified molecular patterns associated with clinical outcome. Objective: Here, we aimed at identifying a molecular signature useful for both personalized prognostic stratification and druggable targets, using methods applicable in clinical routine. Design: In total, 117 tumor samples from 107 patients with ACC were analyzed. Targeted next-generation sequencing of 160 genes and pyrosequencing of 4 genes were applied to formalin-fixed, paraffin-embedded (FFPE) specimens to detect point mutations, copy number alterations, and promoter region methylation. Molecular results were combined with clinical/histopathological parameters (tumor stage, age, symptoms, resection status, and Ki-67) to predict progression-free survival (PFS). Results: In addition to known driver mutations, we detected recurrent alterations in genes not previously associated with ACC (e.g., NOTCH1, CIC, KDM6A, BRCA1, BRCA2). Best prediction of PFS was obtained integrating molecular results (more than one somatic mutation, alterations in Wnt/ß-catenin and p53 pathways, high methylation pattern) and clinical/histopathological parameters into a combined score (P < 0.0001, χ2 = 68.6). Accuracy of prediction for early disease progress was 83.3% (area under the receiver operating characteristic curve: 0.872, 95% confidence interval 0.80 to 0.94). Furthermore, 17 potentially targetable alterations were found in 64 patients (e.g., in CDK4, NOTCH1, NF1, MDM2, and EGFR and in DNA repair system). Conclusions: This study demonstrates that molecular profiling of FFPE tumor samples improves prognostication of ACC beyond clinical/histopathological parameters and identifies new potential drug targets. These findings pave the way to precision medicine in this rare disease.

A new case expanding the mutation and phenotype spectrum of TMEM5-related alpha-dystroglycanopathy.

Dystroglycanopathies are a diverse group of neuromuscular disorders caused by aberrant glycosylation of alpha-dystroglycan. TMEM5 is one of many glycosyltransferases recently described to be associated with alpha-dystroglycanopathies. We report the case of a 15-year-old boy suffering from a congenital muscular dystrophy with elevated serum creatine kinase levels and an almost complete absence of alpha-dystroglycan in muscle biopsy. The clinical course was milder than any previously reported case and did not include brain or eye defects. Standard next-generation sequencing analysis revealed a homozygous mutation in the donor splice site region of exon 5 in TMEM5 (c.914+6 T>G). Available in-silico prediction tools anticipated a reduced efficiency of the splice site. Subsequent cDNA sequencing confirmed the expression of a truncated transcript of TMEM5 lacking exon 5, hence leading to an in-frame deletion in the exostosin domain of the protein. This report expands the clinical and mutation spectrum of alpha-dystroglycanopathies.

Distribution and Structure of DM2 Repeat Tract Alleles in the German Population.

Autosomal dominant inherited Myotonic dystrophy type 1 and 2 (DM1 and DM2) are the most frequent muscle dystrophies in the European population and are caused by repeat expansion mutations. For Germany cumulative empiric evidence suggests an estimated prevalence of DM2 of roughly 9 in 100,000, therefore being as prevalent as DM1. In DM2, a (CCTG)n repeat tract located in the first intron of the CNBP gene is expanded. The CCTG repeat tract is part of a complex repeat structure comprising not only CCTG tetraplets but also repeated TG dinucleotides and TCTG tetraplet elements as well as NCTG interruptions. Here, we provide the distribution of normal sized alleles in the German population, which was found to be highly similar to the Slovak population. Sequencing of 34 unexpanded healthy range alleles in DM2 positive patients (heterozygous for a full expansion) revealed that the CCTG repeat tract is usually interrupted by at least three tetraplets which according to current opinion is supposed to render it stable against expansion. Interestingly, only the largest analyzed normal allele had 23 uninterrupted CCTGs and consequently could represent an instable early premutation allele. In our diagnostic history of DM2 cases, a total of 18 premutations were detected in 16 independent cases. Here, we describe two premutation families, one with an expansion from a premutation allele and the other with a contraction of a full expansion down to a premutation allele. Our diagnostic results support the general assumption that the premutation range of unstable CCTG stretches lies obviously between 25 and 75 CCTGs. However, the clinical significance of premutation alleles is still unclear. In the light of the two described families we suggest incomplete penetrance. Thus, as it was proposed for other repeat expansion diseases (e.g., Huntington's disease), a fluid transition of penetrance is more likely rather than a clear cut CCTG number threshold.

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation-negative early-onset and high-risk breast cancer patients.

To evaluate the role of constitutive epigenetic changes in normal body cells of BRCA1/BRCA2-mutation negative patients, we have developed a deep bisulfite sequencing assay targeting the promoter regions of 8 tumor suppressor (TS) genes (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, RB1) and the estrogene receptor gene (ESR1), which plays a role in tumor progression. We analyzed blood samples of two breast cancer (BC) cohorts with early onset (EO) and high risk (HR) for a heterozygous mutation, respectively, along with age-matched controls. Methylation analysis of up to 50,000 individual DNA molecules per gene and sample allowed quantification of epimutations (alleles with >50% methylated CpGs), which are associated with epigenetic silencing. Compared to ESR1, which is representative for an average promoter, TS genes were characterized by a very low (< 1%) average methylation level and a very low mean epimutation rate (EMR; < 0.0001% to 0.1%). With exception of BRCA1, which showed an increased EMR in BC (0.31% vs. 0.06%), there was no significant difference between patients and controls. One of 36 HR BC patients exhibited a dramatically increased EMR (14.7%) in BRCA1, consistent with a disease-causing epimutation. Approximately one third (15 of 44) EO BC patients exhibited increased rates of single CpG methylation errors in multiple TS genes. Both EO and HR BC patients exhibited global underexpression of blood TS genes. We propose that epigenetic abnormalities in normal body cells are indicative of disturbed mechanisms for maintaining low methylation and appropriate expression levels and may be associated with an increased BC risk.

Panel Sequencing Shows Recurrent Genetic FAS Alterations in Primary Cutaneous Marginal Zone Lymphoma.

Primary cutaneous marginal zone lymphoma (PCMZL) represents an indolent subtype of non-Hodgkin lymphoma that is clinically characterized by slowly growing skin tumors with a very low propensity for systemic dissemination. The underlying genetic basis of PCMZL has not been comprehensively elucidated. To gain deeper insight into the molecular pathogenesis of PCMZL, we performed hybridization-based panel sequencing of 38 patients with well-characterized PCMZL. In 32 of the 38 patients, we identified genetic alterations within 39 selected target genes. The most frequently detected alterations (24/38 patients, 63.2%) affected the FAS gene, of which 22 patients harbored alterations, which affect the functionally relevant death domain of the apoptosis-regulating FAS/CD95 protein in a dominant-negative manner. In addition, we identified highly recurrent mutations in three other genes, namely SLAMF1, SPEN, and NCOR2. Our molecular data suggest that apoptosis defects provide the molecular basis of the observed clinical features of PCMZL, which commonly presents with only slowly growing skin tumors, reflecting its invariably indolent behavior. From a diagnostic point of view, highly recurrent FAS mutations in PCMZL presumably separate this indolent lymphoma entity from pseudolymphoma, and this adds adjunctive discriminatory features at a molecular level.

Early and lethal neurodegeneration with myasthenic and myopathic features: A new ALG14-CDG.

OBJECTIVE: To describe the presentation and identify the cause of a new clinical phenotype, characterized by early severe neurodegeneration with myopathic and myasthenic features. METHODS: This case study of 5 patients from 3 families includes clinical phenotype, serial MRI, electrophysiologic testing, muscle biopsy, and full autopsy. Genetic workup included whole exome sequencing and segregation analysis of the likely causal mutation. RESULTS: All 5 patients showed severe muscular hypotonia, progressive cerebral atrophy, and therapy-refractory epilepsy. Three patients had congenital contractures. All patients died during their first year of life. In 2 of our patients, electrophysiologic testing showed abnormal decrement, but treatment with pyridostigmine led only to temporary improvement. Causative mutations in ALG14 were identified in all patients. The mutation c.220 G>A (p.Asp74Asn) was homozygous in 2 patients and heterozygous in the other 3 patients. Additional heterozygous mutations were c.422T>G (p.Val141Gly) and c.326G>A (p.Arg109Gln). In all cases, parents were found to be heterozygous carriers. None of the identified variants has been described previously. CONCLUSIONS: We report a genetic syndrome combining myasthenic features and severe neurodegeneration with therapy-refractory epilepsy. The underlying cause is a glycosylation defect due to mutations in ALG14. These cases broaden the phenotypic spectrum associated with ALG14 congenital disorders of glycosylation as previously only isolated myasthenia has been described.