Högskolan i Skövde

Limb-girdle muscular dystrophies (LGMDs) are disorders with an important clinical heterogeneity, usually involving proximal limb muscles. One subtype, LGMD R16 (LGMD 2P), is an autosomal recessive condition caused by pathogenic variants in DAG1, with clinical presentations ranging from mild to extremely severe forms. DAG1 is responsible for producing dystroglycan, an essential complex in the muscular protein network. Following translation, dystroglycan is cleaved into alpha-dystroglycan, which undergoes glycosylation and acts as a sarcolemmal receptor for extracellular proteins, and beta-dystroglycan, which connects to dystrophin. In recent years, heterozygous nonsense or frameshift DAG1 variants have been linked with asymptomatic hyperCKemia (increased serum creatine kinase levels) or mild muscular phenotypes characterized by fatigability and myalgia. Here, we describe a Romanian family comprising four affected individuals (one father and three sons) carrying the heterozygous missense DAG1 variant NM_004393.6:c.887G>A, NP_004384.5:p.(Gly296Asp) and showing a mild–moderate muscular phenotype similar to previous cases of DAG1 haploinsufficiency. Three of the affected individuals exhibit myopathic changes in muscle biopsies (increased fiber size variability, internalized nuclei, and regenerating fibers), while two demonstrate reduced alpha-dystroglycan glycosylation in muscle tissue. Atomic force microscopy findings in myoblasts from one patient showed a significantly lower stiffness compared to controls. These findings align with prior reports and further support the pathogenicity of this variant.

The dystonin gene (DST) encodes three major isoforms, DST-a, DST-b and DST-e. Biallelic pathogenic variants in DST have previously been associated with two allelic monogenic disorders: hereditary sensory and autonomic neuropathy type VI (caused by a loss of DST-a) and epidermolysis bullosa simplex 3 (caused by a loss of DST-e). We investigated patients diagnosed with congenital myopathy using exome or genome sequencing. In 19 affected individuals from 14 unrelated families, we identified nine different variants in biallelic state located in exons 40-41, specific to DST-b. Affected individuals presented with severe neonatal myopathy characterized by arthrogryposis, hypotonia and dilated cardiomyopathy. Postnatal CPAP ventilation was required in nine patients, and seven died within the first three years of life. Survivors showed an improvement of symptoms, with the oldest three patients, now over 25 years old, exhibiting normal cognition and being ambulatory. RNA analyses demonstrated that transcripts encoding DST-b are predominantly expressed in skeletal muscle, heart tissue and cultured fibroblasts, but not in brain, matching the phenotypic spectrum. Patient-derived fibroblasts exhibited reduced DST mRNA expression. Proteomic analysis confirmed a reduction of DST protein levels due to an absence of the DST-b isoform. Muscle biopsies from four patients aged 1 month to 3 years revealed mild, non-specific myopathic changes. Ultrastructural analysis in three individuals showed mild and focal myofibrillar disruption and non-specific undulating nuclear membranes, with these changes observed in two cases each. Additionally, we identified two homozygous variants affecting both DST-a and DST-b isoforms in four patients from two unrelated families; all presented with severe arthrogryposis and died intrauterine or shortly after birth. Genotype-phenotype correlation in these patients and previously published cases with respective variants resulted in the definition of a DST-associated lethal congenital contracture syndrome.

Background: FDXR-related disorders (FRDs) are rare mitochondrial conditions typically presenting with progressive optic atrophy and neuropathy. We report identical twins presenting with acute-onset flaccid quadriparesis following a respiratory infection, expanding the known phenotypic spectrum of FDXR variants.

Case Presentation: 22-year-old monozygotic twins from a consanguineous Iranian family developed progressive weakness after an upper respiratory infection, initially diagnosed as Guillain-Barré syndrome. Despite IVIG and plasmapheresis, both progressed to flaccid quadriplegia. Twin A. had additional manifestations including blindness, dysarthria, and incontinence, that were not present in Twin B. Extensive workup excluded inflammatory, infectious, and some metabolic etiologies. Whole exome sequencing (WES) revealed a homozygous FDXR missense variant (c.463C>T, p.Arg155Trp) in Twin A.

Conclusions: This represents the first report of the mentioned FDXR variant in monozygotic twins. The twins' phenotypic discordance despite genetic identity suggests environmental or epigenetic modifiers. Our findings underscore:

1. FDXR variants should be considered in acute neuropathies unresponsive to immunotherapy.

2. The c.463C>T variant may cause both acute and chronic presentations.

Neurodevelopmental disorders (NDDs) are a highly diverse group of conditions that manifest through motor, cognitive, and behavioral impairments, representing the most common chronic condition encountered in children. As these often have a genetic cause or component, extensive genetic testing, particularly whole exome sequencing (WES), plays a critical role in their diagnosis, management, and prevention. Here, we present detailed clinical and genetic data on 54 Romanian patients included in the NeuroMyoDredger project, and provide an overview of the current landscape of genetic testing availability for NDDs in Romania, commenting on current barriers and the importance of integrating advanced genomic technologies into national healthcare strategies. A total of 54 undiagnosed Romanian patients with an initial clinical suspicion of unspecific NDD have benefited from singleton WES (including CNV and mitochondrial DNA analysis), with a diagnostic yield of 50%. Furthermore, a substantial proportion of cases (eight patients, 14.81%) yielded nondefinitive results involving variants of uncertain significance, with potential pathogenic relevance. Our findings align with the existing literature data, supporting the integration of singleton WES with CNV and mitochondrial variants detection as a first-line investigation in the diagnostic workflow for NDDs.

Microtubule-actin cross-linking factor 1 (MACF1) is a large protein of the spectraplakin family, which is essential for brain development. MACF1 interacts with microtubules through the growth arrest-specific 2 (Gas2)-related (GAR) domain. Heterozygous MACF1 missense variants affecting the zinc-binding residues in this domain result in a distinctive cortical and brain stem malformation. Evidence for other MACF1-associated disorders is still limited. Here, we present a cohort of 45 individuals with heterozygous or bi-allelic MACF1 variants to explore the phenotypic spectrum and assess possible pathogenic relevance. We observe that de novo heterozygous missense variants in the EF-hand domains also result in distinctive brain malformation and provide experimental evidence that variants in the EF-hand/GAR module increase microtubule binding, suggestive of a toxic gain of function. Notably, no phenotype-genotype correlation was possible for the remaining heterozygous variants in other domains. A clinical review of eight families with bi-allelic variants reveals a possible complex neurodevelopmental syndrome of the central and peripheral nervous systems. In these individuals, bi-allelic variants mostly affect the Plakin domain. Furthermore, RNA sequencing and chromatin immunoprecipitation (ChIP) analyses of human fetal brain tissue reveal five MACF1 isoforms with region-specific expression, differing in their exon 1 transcription start sites but splicing to a common exon 2. This differential expression explains the frontal-predominant lissencephaly in an individual with a homozygous stop-gain in exon 1 (MACF1-204: c.70C>T [p.Arg24∗]), as this isoform is preferentially expressed in the frontal cortex. We conclude that MACF1-related disorders are strictly linked to domain function and the level of transcript expression, explaining the observed wide clinical heterogeneity.

Biallelic variants in NADH-ubiquinone oxidoreductase 1 alpha subcomplex 13 (NDUFA13) have been linked to mitochondrial complex I deficiency, nuclear type 28, based on three affected individuals from two families. With only two families reported, the clinical and molecular spectrum of NDUFA13-related diseases remains unclear. We report 10 additional affected individuals from nine independent families, identifying four missense variants (including recurrent c.170G>A) and three ultra-rare or novel predicted loss-of-function biallelic variants. Updated clinical-radiological data from previously reported families and a literature review compiling clinical features of all reported patients with isolated complex I deficiency caused by 43 genes encoding complex I subunits and assembly factors are also provided.

Our cohort (mean age 7.8 ± 5.4 years; range 2.5–18) predominantly presented a moderate-to-severe neurodevelopmental syndrome with oculomotor abnormalities (84%), spasticity/hypertonia (83%), hypotonia (69%), cerebellar ataxia (66%), movement disorders (58%), and epilepsy (46%). Neuroimaging revealed bilateral symmetric T2 hyperintense substantia nigra lesions (91.6%) and optic nerve atrophy (66.6%). Protein modeling suggests missense variants destabilize a critical junction between the hydrophilic and membrane arms of complex I. Fibroblasts from two patients showed reduced complex I activity and compensatory complex IV activity increase. This study characterizes NDUFA13-related disease in 13 individuals, highlighting genotype-phenotype correlations.

Background

DYNC1H1 is a critical gene implicated in neurodevelopmental and neuromuscular disorders with overlapping and variable phenotypes that challenge diagnosis.

Methods and Results

Whole exome sequencing in patients presenting with motor neuron disease symptoms and a predominant pattern of lower-limb muscle weakness revealed pathogenic DYNC1H1 variants. This expands the known phenotypic spectrum to include rare features such as scapular winging and camptocormia. Clinical evaluations of affected individuals revealed features consistent with SMA-LED, reinforcing the role of DYNC1H1 in neuromuscular disorders. A review of 208 published DYNC1H1 variants highlighted significant clustering in the tail domain, primarily associated with neuromuscular conditions like SMA-LED. Approximately 28% of variants exhibited overlapping neuromuscular and neurodevelopmental features, emphasizing the diagnostic challenges posed by phenotypic overlap. These findings underscore the necessity of comprehensive clinical and genetic evaluations to address the variability observed within families and improve genotype–phenotype correlations.

Conclusion

This study reinforces the importance of DYNC1H1 in motor neuron function and its pivotal role in neurodevelopmental and neuromuscular disease mechanisms. The integration of exome sequencing in clinical practice is essential for identifying rare and novel variants, enhancing diagnostic accuracy. We recommend incorporating DYNC1H1 screening into diagnostic workflows to advance understanding and management of conditions with overlapping phenotypes.

Purpose: Biallelic INPP4A variants have recently been associated with severe neurodevelopmental disease in single-case reports. Here, we expand and elucidate the clinical-genetic spectrum and provide a pathomechanistic explanation for genotype-phenotype correlations.

Methods: Clinical and genomic investigations of 30 individuals were undertaken alongside molecular and in silico modelling and translation reinitiation studies.

Results: We characterize a clinically variable disorder with cardinal features, including global developmental delay, severe-profound intellectual disability, microcephaly, limb weakness, cerebellar signs, and short stature. A more severe presentation associated with biallelic INPP4A variants downstream of exon 4 has additional features of (ponto)cerebellar hypoplasia, reduced cerebral volume, peripheral spasticity, contractures, intractable seizures, and cortical visual impairment. Our studies identify the likely pathomechanism of this genotype-phenotype correlation entailing translational reinitiation in exon 4 resulting in an N-terminal truncated INPP4A protein retaining partial functionality, associated with less severe disease. We also identified identical reinitiation site conservation in Inpp4a−/− mouse models displaying similar genotype-phenotype correlation. Additionally, we show fibroblasts from a single affected individual exhibit disrupted endocytic trafficking pathways, indicating the potential biological basis of the condition.

Conclusion: Our studies comprehensively characterize INPP4A-related neurodevelopmental disorder and suggest genotype-specific clinical assessment guidelines. We propose that the potential mechanistic basis of observed genotype-phenotype correlations entails exon 4 translation reinitiation. 

To evaluate the diagnostic yield of prenatal exome sequencing (pES) in fetuses with structural anomalies detected by prenatal ultrasound in a consanguineous population. This was a prospective study of 244 anomalous fetuses from unrelated consanguineous Egyptian families. Detailed phenotyping was performed throughout pregnancy and postnatally, and pES data analysis was conducted. Genetic variants were prioritized based on the correlation of their corresponding human phenotype ontology terms with the ultrasound findings. Analyses were carried out to determine the diagnostic efficiency of pES and its correlation to the organ systems involved. The largest clinical category of fetuses referred for pES was those manifesting multisystem anomalies (104/244, 42.6%). pES provided a definitive diagnosis explaining the fetal anomalies in 47.1% (115/244) of the cases, with the identification of 122 pathogenic or likely pathogenic variants completely fitting with the phenotype. Variants of uncertain significance associated with the fetal phenotypes were detected in 84 fetuses (34%), while 18.44% (45/244) had negative results. Positive consanguinity is associated with a high diagnostic yield of ES. The novel variants and new fetal manifestations, described in our cohort, further expand the mutational and phenotypic spectrum of a wide variety of genetic disorders presenting with congenital malformations. 

OBJECTIVE: Autophagy is a fundamental biological pathway with vital roles in intracellular homeostasis. During autophagy, defective cargoes including mitochondria are targeted to lysosomes for clearance and recycling. Recessive truncating variants in the autophagy gene EPG5 have been associated with Vici syndrome, a severe early-onset neurodevelopmental disorder with extensive multisystem involvement. Here, we aimed to delineate the extended, age-dependent EPG5-related disease spectrum.

METHODS: We investigated clinical, radiological, and molecular features from the largest cohort of EPG5-related patients identified to date, complemented by experimental investigation of cellular and animal models of EPG5 defects.

RESULTS: Through worldwide collaboration, we identified 211 patients, 97 of them previously unpublished, with recessive EPG5 variants. The phenotypic spectrum ranged from antenatally lethal presentations to milder isolated neurodevelopmental disorders. A novel Epg5 knock-in mouse model of a recurrent EPG5 missense variant featured motor impairments and defective autophagy in brain areas particularly relevant for the neurological disorders in milder presentations. Novel age-dependent neurodegenerative manifestations in our cohort included adolescent-onset parkinsonism and dystonia with cognitive decline, and myoclonus. Radiological features suggested an emerging continuum with brain iron accumulation disorders. Patient fibroblasts showed defects in PINK1-Parkin-dependent mitophagic clearance and α-synuclein overexpression, indicating a cellular basis for the observed neurodegenerative phenotypes. In Caenorhabditis elegans, EPG5 knockdown caused motor impairments, defective mitophagic clearance, and changes in mitochondrial respiration comparable to observations in C. elegans knockdown of parkinsonism-related genes.

INTERPRETATION: Our findings illustrate a lifetime neurological disease continuum associated with pathogenic EPG5 variants, linking neurodevelopmental and neurodegenerative disorders through the common denominator of defective autophagy. ANN NEUROL 2025;98:932-950.