Biallelic variation in the choline and ethanolamine transporter FLVCR1 underlies a pleiotropic disease spectrum from adult neurodegeneration to severe developmental disorders.

FLVCR1 encodes Feline leukemia virus subgroup C receptor 1 (FLVCR1), a solute carrier (SLC) transporter within the Major Facilitator Superfamily. FLVCR1 is a widely expressed transmembrane protein with plasma membrane and mitochondrial isoforms implicated in heme, choline, and ethanolamine transport. While Flvcr1 knockout mice die in utero with skeletal malformations and defective erythropoiesis reminiscent of Diamond-Blackfan anemia, rare biallelic pathogenic FLVCR1 variants are linked to childhood or adult-onset neurodegeneration of the retina, spinal cord, and peripheral nervous system. We ascertained from research and clinical exome sequencing 27 individuals from 20 unrelated families with biallelic ultra-rare missense and predicted loss-of-function (pLoF) FLVCR1 variant alleles. We characterize an expansive FLVCR1 phenotypic spectrum ranging from adult-onset retinitis pigmentosa to severe developmental disorders with microcephaly, reduced brain volume, epilepsy, spasticity, and premature death. The most severely affected individuals, including three individuals with homozygous pLoF variants, share traits with Flvcr1 knockout mice and Diamond-Blackfan anemia including macrocytic anemia and congenital skeletal malformations. Pathogenic FLVCR1 missense variants primarily lie within transmembrane domains and reduce choline and ethanolamine transport activity compared with wild-type FLVCR1 with minimal impact on FLVCR1 stability or subcellular localization. Several variants disrupt splicing in a mini-gene assay which may contribute to genotype-phenotype correlations. Taken together, these data support an allele-specific gene dosage model in which phenotypic severity reflects residual FLVCR1 activity. This study expands our understanding of Mendelian disorders of choline and ethanolamine transport and demonstrates the importance of choline and ethanolamine in neurodevelopment and neuronal homeostasis.

Unraveling the genetic landscape of undiagnosed cerebellar ataxia in Brazilian patients.

INTRODUCTION: Hereditary ataxias (HAs) encompass a diverse and genetically intricate group of rare neurodegenerative disorders, presenting diagnostic challenges. Whole-exome sequencing (WES) has significantly improved diagnostic success. This study aimed to elucidate genetic causes of cerebellar ataxia within a diverse Brazilian cohort. METHODS: Biological samples were collected from individuals with sporadic or familial cerebellar ataxia, spanning various ages and phenotypes, excluding common SCAs and Friedreich ataxia. RFC1 biallelic AAGGG repeat expansion was screened in all patients. For AAGGG-negative cases, WES targeting 441 ataxia-related genes was performed, followed by ExpansionHunter analysis for repeat expansions, including the recently described GGC-ZFHX3. Variant classification adhered to ClinGen guidelines, yielding definitive or probable diagnoses. RESULTS: The study involved 76 diverse Brazilian families. 16 % received definitive diagnoses, and another 16 % received probable ones. RFC1-related ataxia was predominant, with two definitive cases, followed by KIF1A (one definitive and one probable) and SYNE-1 (two probable). Early-onset cases exhibited higher diagnostic rates. ExpansionHunter improved diagnosis by 4 %.We did not detected GGC-ZFHX3 repeat expansion in this cohort. CONCLUSION: This study highlights diagnostic complexities in cerebellar ataxia, even with advanced genetic methods. RFC1, KIF1A, and SYNE1 emerged as prevalent mutations. ZFHX3 repeat expansion seem to be rare in Brazilian population. Early-onset cases showed higher diagnostic success. WES coupled with ExpansionHunter holds promise as a primary diagnostic tool, emphasizing the need for broader NGS accessibility in Brazil.

Frequency of GAA-FGF14 Ataxia in a Large Cohort of Brazilian Patients With Unsolved Adult-Onset Cerebellar Ataxia.

Objectives: Intronic FGF14 GAA repeat expansions have recently been found to be a common cause of hereditary ataxia (GAA-FGF14 ataxia; SCA27B). The global epidemiology and regional prevalence of this newly reported disorder remain to be established. In this study, we investigated the frequency of GAA-FGF14 ataxia in a large cohort of Brazilian patients with unsolved adult-onset ataxia. Methods: We recruited 93 index patients with genetically unsolved adult-onset ataxia despite extensive genetic investigation and genotyped the FGF14 repeat locus. Patients were recruited across 4 different regions of Brazil. Results: Of the 93 index patients, 8 (9%) carried an FGF14 (GAA)≥250 expansion. The expansion was also identified in 1 affected relative. Seven patients were of European descent, 1 was of African descent, and 1was of admixed American ancestry. One patient carrying a (GAA)376 expansion developed ataxia at age 28 years, confirming that GAA-FGF14 ataxia can occur before the age of 30 years. One patient displayed episodic symptoms, while none had downbeat nystagmus. Cerebellar atrophy was observed on brain MRI in 7 of 8 patients (87%). Discussion: Our results suggest that GAA-FGF14 ataxia is a common cause of adult-onset ataxia in the Brazilian population, although larger studies are needed to fully define its epidemiology.

Biallelic MED27 variants lead to variable ponto-cerebello-lental degeneration with movement disorders.

MED27 is a subunit of the Mediator multiprotein complex, which is involved in transcriptional regulation. Biallelic MED27 variants have recently been suggested to be responsible for an autosomal recessive neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. We further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families with biallelic MED27 variants. Using exome sequencing and extensive international genetic data sharing, 39 unpublished affected individuals from 18 independent families with biallelic missense variants in MED27 have been identified (29 females, mean age at last follow-up 17 ± 12.4 years, range 0.1-45). Follow-up and hitherto unreported clinical features were obtained from the published 12 families. Brain MRI scans from 34 cases were reviewed. MED27-related disease manifests as a broad phenotypic continuum ranging from developmental and epileptic-dyskinetic encephalopathy to variable neurodevelopmental disorder with movement abnormalities. It is characterized by mild to profound global developmental delay/intellectual disability (100%), bilateral cataracts (89%), infantile hypotonia (74%), microcephaly (62%), gait ataxia (63%), dystonia (61%), variably combined with epilepsy (50%), limb spasticity (51%), facial dysmorphism (38%) and death before reaching adulthood (16%). Brain MRI revealed cerebellar atrophy (100%), white matter volume loss (76.4%), pontine hypoplasia (47.2%) and basal ganglia atrophy with signal alterations (44.4%). Previously unreported 39 affected individuals had seven homozygous pathogenic missense MED27 variants, five of which were recurrent. An emerging genotype-phenotype correlation was observed. This study provides a comprehensive clinical-radiological description of MED27-related disease, establishes genotype-phenotype and clinical-radiological correlations and suggests a differential diagnosis with syndromes of cerebello-lental neurodegeneration and other subtypes of 'neuro-MEDopathies'.

The clinical and genetic spectrum of autosomal-recessive TOR1A-related disorders.

In the field of rare diseases, progress in molecular diagnostics led to the recognition that variants linked to autosomal-dominant neurodegenerative diseases of later onset can, in the context of biallelic inheritance, cause devastating neurodevelopmental disorders and infantile or childhood-onset neurodegeneration. TOR1A-associated arthrogryposis multiplex congenita 5 (AMC5) is a rare neurodevelopmental disorder arising from biallelic variants in TOR1A, a gene that in the heterozygous state is associated with torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with AMC5-TOR1A have been reported (less than 10 in detail), a systematic investigation of the full disease-associated spectrum has not been conducted. Here, we assess the clinical, radiological and molecular characteristics of 57 individuals from 40 families with biallelic variants in TOR1A. Median age at last follow-up was 3 years (0-24 years). Most individuals presented with severe congenital flexion contractures (95%) and variable developmental delay (79%). Motor symptoms were reported in 79% and included lower limb spasticity and pyramidal signs, as well as gait disturbances. Facial dysmorphism was an integral part of the phenotype, with key features being a broad/full nasal tip, narrowing of the forehead and full cheeks. Analysis of disease-associated manifestations delineated a phenotypic spectrum ranging from normal cognition and mild gait disturbance to congenital arthrogryposis, global developmental delay, intellectual disability, absent speech and inability to walk. In a subset, the presentation was consistent with foetal akinesia deformation sequence with severe intrauterine abnormalities. Survival was 71%, with higher mortality in males. Death occurred at a median age of 1.2 months (1 week-9 years), due to respiratory failure, cardiac arrest or sepsis. Analysis of brain MRI studies identified non-specific neuroimaging features, including a hypoplastic corpus callosum (72%), foci of signal abnormality in the subcortical and periventricular white matter (55%), diffuse white matter volume loss (45%), mega cisterna magna (36%) and arachnoid cysts (27%). The molecular spectrum included 22 distinct variants, defining a mutational hotspot in the C-terminal domain of the Torsin-1A protein. Genotype-phenotype analysis revealed an association of missense variants in the 3-helix bundle domain to an attenuated phenotype, while missense variants near the Walker A/B motif as well as biallelic truncating variants were linked to early death. In summary, this systematic cross-sectional analysis of a large cohort of individuals with biallelic TOR1A variants across a wide age-range delineates the clinical and genetic spectrum of TOR1A-related autosomal-recessive disease and highlights potential predictors for disease severity and survival.

Biallelic PRMT7 pathogenic variants are associated with a recognizable syndromic neurodevelopmental disorder with short stature, obesity, and craniofacial and digital abnormalities.

PURPOSE: Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyzes the methylation of arginine residues on several protein substrates. Biallelic pathogenic PRMT7 variants have previously been associated with a syndromic neurodevelopmental disorder characterized by short stature, brachydactyly, intellectual developmental disability, and seizures. To our knowledge, no comprehensive study describes the detailed clinical characteristics of this syndrome. Thus, we aim to delineate the phenotypic spectrum of PRMT7-related disorder. METHODS: We assembled a cohort of 51 affected individuals from 39 different families, gathering clinical information from 36 newly described affected individuals and reviewing data of 15 individuals from the literature. RESULTS: The main clinical characteristics of the PRMT7-related syndrome are short stature, mild to severe developmental delay/intellectual disability, hypotonia, brachydactyly, and distinct facial morphology, including bifrontal narrowing, prominent supraorbital ridges, sparse eyebrows, short nose with full/broad nasal tip, thin upper lip, full and everted lower lip, and a prominent or squared-off jaw. Additional variable findings include seizures, obesity, nonspecific magnetic resonance imaging abnormalities, eye abnormalities (i.e., strabismus or nystagmus), and hearing loss. CONCLUSION: This study further delineates and expands the molecular, phenotypic spectrum and natural history of PRMT7-related syndrome characterized by a neurodevelopmental disorder with skeletal, growth, and endocrine abnormalities.

A recurrent homozygous missense DPM3 variant leads to muscle and brain disease.

Biallelic pathogenic variants in the genes encoding the dolichol-phosphate mannose synthase subunits (DPM) which produce mannosyl donors for glycosylphosphatidylinositols, N-glycan and protein O- and C-mannosylation, are rare causes of congenital disorders of glycosylation. Pathogenic variants in DPM1 and DPM2 are associated with muscle-eye-brain (MEB) disease, whereas DPM3 variants have mostly been reported in patients with isolated muscle disease-dystroglycanopathy. Thus far, only one affected individual with compound heterozygous DPM3 variants presenting with myopathy, mild intellectual disability, seizures, and nonspecific white matter abnormalities (WMA) around the lateral ventricles has been described. Here we present five affected individuals from four unrelated families with global developmental delay/intellectual disability ranging from mild to severe, microcephaly, seizures, WMA, muscle weakness and variable cardiomyopathy. Exome sequencing of the probands revealed an ultra-rare homozygous pathogenic missense DPM3 variant NM_018973.4:c.221A>G, p.(Tyr74Cys) which segregated with the phenotype in all families. Haplotype analysis indicated that the variant arose independently in three families. Functional analysis did not reveal any alteration in the N-glycosylation pathway caused by the variant; however, this does not exclude its pathogenicity in the function of the DPM complex and related cellular pathways. This report provides supporting evidence that, besides DPM1 and DPM2, defects in DPM3 can also lead to a muscle and brain phenotype.

Bi-allelic loss-of-function variants in PPFIBP1 cause a neurodevelopmental disorder with microcephaly, epilepsy, and periventricular calcifications.

PPFIBP1 encodes for the liprin-ß1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

Molecular mechanisms of the anti-cancer drug, LY2874455, in overcoming the FGFR4 mutation-based resistance.

In recent years, many strategies have been used to overcome the fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors (TKIs) resistance caused by different mutations. LY2874455 (or 6LF) is a pan-FGFR inhibitor which is identified as the most efficient TKI for all resistant mutations in FGFRs. Here, we perform a comparative dynamics study of wild type (WT) and the FGFR4 V550L mutant for better understanding of the 6LF inhibition mechanism. Our results confirm that the pan-FGFR inhibitor 6LF can bind efficiently to both WT and V550L FGFR4. Moreover, the communication network analysis indicates that in apo-WT FGFR4, αD-αE loop behaves like a switch between open and close states of the substrate-binding pocket in searching of its ligand. In contrast, V550L mutation induces the active conformation of the FGFR4 substrate-binding pocket through disruption of αD-αE loop and αG helix anti-correlation. Interestingly, 6LF binding causes the rigidity of hinge and αD helix regions, which results in overcoming V550L induced resistance. Collectively, the results of this study would be informative for designing more efficient TKIs for more effective targeting of the FGFR signaling pathway.

A novel carcinogenic PI3Kα mutation suggesting the role of helical domain in transmitting nSH2 regulatory signals to kinase domain.

AIMS: Mutations in PIK3CA, which encodes p110α subunit of PI3K class IA enzymes, are highly frequent in breast cancer. Here, we aimed to probe mutations in exon 9 of PIK3CA and computationally simulate their function. MATERIALS AND METHODS: PCR/HRM and PCR/sequencing were used for mutation detection in 40 breast cancer specimens. The identified mutations were queried via in silico algorithms to check the pathogenicity. The molecular dynamics (MD) simulations were utilized to assess the function of mutant proteins. KEY FINDINGS: Three samples were found to harbor at least one of the E542K, E545K and L551Q mutations of which L551Q has not been reported previously. All mutations were confirmed to be pathogenic and MD simulations revealed their impact on protein function and regulation. The novel L551Q mutant dynamics was similar to that of previously found carcinogenic mutants, E542K and E545K. A functional role for the helical domain was also suggested by which the inhibitory signal of p85α is conducted to kinase domain via helical domain. Helical domain mutations lead to impairment of kinase domain allosteric regulation. Interestingly, our results show that p110α substrate binding pocket of kinase domain in mutants may have differential affinity for enzyme substrates, including anit-p110α drugs. SIGNIFICANCE: The novel p110α L551Q mutation could have carcinogenic feature similar to previously known helical domain mutations.