Mariana Santos

Common forms of migraine are complex disorders characterized by significant clinical diversity. Their genetic basis has been extensively studied but remains unclear. This study represents the first pilot genome-wide association study (GWAS) integrating a polygenic risk score (PRS) in the Portuguese population, designed to identify migraine susceptibility loci through a case–control study and unravel population-specific variants. Genotyping data was acquired with Applied Biosystems Axiom™ PMDA array, producing 12,035,248 single-nucleotide polymorphisms (SNPs) post-imputation, providing a comprehensive scope for GWAS analysis. PRS models were created and tested using a k-folds cross-validation framework and the optimal significance threshold was assessed. We detected 12 potential risk loci corresponding to 12 lead SNPs (RP11-204N11.2, CTA-481E9.4/CTA-481E9.3, RAP1A, TIGD4, CADPS2, RP11-46E17.6, RP4-569D19.5, RP11-398K14.1, PCBP1-AS1, TCF15, IL6R and UNC13A). The top three variants (RP11-204N11.2, CTA-481E9.4/CTA-481E9.3 and RAP1A) were also supported by the PRS model. We highlight that several variants present putative biological relevance to migraine pathophysiology, reinforcing the importance of neurotransmitter release, synaptic transmission and the involvement of vascular components in migraine pathophysiology.

Tau tubulin kinase 2 (TTBK2) is a ubiquitous serine-threonine protein kinase implicated in diverse cellular processes, including microtubule regulation, ciliogenesis, synaptic signaling, and the phosphorylation of key proteins like TDP-43. Despite its relevance, many aspects of TTBK2 function in both physiological and pathological conditions remain poorly understood. Truncating variants in TTBK2 gene cause s pinocerebellar ataxia type 11 (SCA11), a rare form of autosomal dominant cerebellar ataxia. However, the functional consequences and pathogenic potential of missense variants have yet to be elucidated. In this study, we developed a CRISPR/Cas9 knock-in cell model harboring a missense variant in TTBK2 kinase domain (NM_173500.4:c.625 C > T; p.Leu209Phe) to evaluate its impact on TTBK2 expression, associated protein levels, and phosphoproteomic profiles. TTBK2 missense variant (TTBK2-L209F) was associated with reduced TTBK2 protein levels, altered levels of cytoskeleton-related proteins, and impaired kinase activity, namely toward TDP-43. Phosphoproteomic analyses identified dysregulation in pathways linked to gene regulation, protein degradation, cytoskeletal organization, and TGF-β signaling. These findings provide valuable insights into the biological roles of TTBK2 in cellular signaling. Moreover, this study underscores the importance of functional studies to better understand the consequences of TTBK2 missense variants, particularly those affecting the kinase domain, and their potential contribution to disease.

Hereditary cerebellar ataxia (HCA) and hereditary spastic paraplegia (HSP) are rare neurologic disorders that often represent opposite ends of a shared clinical spectrum. Spastic ataxia, defined by the co-occurrence of cerebellar syndrome and overt spasticity, remains comparatively underexplored and is associated with relatively few genetic causes. The aim of this study was to characterize the clinical and genetic features of spastic ataxia in a large HCA cohort and compare them with those of nonspastic HCA. A prospective HCA cohort was initiated in 2017, incorporating annual assessments following a structured and standardized protocol. Spastic ataxia was defined as ataxia occurring in conjunction with spasticity grade ≥2 on the Modified Ashworth Scale. Patients meeting this criterion were identified; their clinical and genetic data were analyzed and compared with those of patients with nonspastic forms of HCA. The Movement Disorder Society's nomenclature for genetic disorders was adopted, using dual-prefix notation for combined phenotypes (e.g., HSP/ATX for spastic ataxia), except for entities such as MJD/SCA3 or ARSACS, which are more readily recognized by their original designations. Of 249 patients assessed (164 families), 56 (22.5%; from 46 families) exhibited a spastic ataxia phenotype. Compared with nonspastic HCA, these patients had earlier onset and longer disease duration. Spastic ataxia was significantly associated with autosomal recessive inheritance and conventional (nonrepeat expansion) variants. Thirty-eight probands (80.8%) had a definite genetic diagnosis, involving 22 causal genes. The most frequent diagnoses were ARSACS (17.4%), ATX-SYNE1 (6.5%), ATX-ANO10, HSP/ATX-KIF1C, HSP/ATX-PGN, HSP-ZFYVE26, MxMD-ATP13A2, and ATX/HSP-KCNA2 (4.3% each). A noncerebellar presentation was observed in 30 patients with spastic ataxia (53.6%) while 26 (46.6%) had cerebellar onset. After adjustment for disease duration, patients with spastic ataxia had significantly higher baseline scores on the Scale for the Assessment and Rating of Ataxia, reflecting a greater disease burden. In addition, falls were more frequent in this group. Spastic ataxia represented a clinically and genetically distinct subgroup within HCA, marked by recessive inheritance, large genetic heterogeneity, and more severe motor impairment. Greater awareness of its heterogeneous presentations and progressive disability over time is crucial for timely diagnosis, genetic counseling, and development of tailored management strategies for these patients.

Cerebral palsy (CP) is a neurodevelopmental disorder of motor function, with genetic etiologies, particularly de novo variants, identified in approximately one-third of cases. The contribution of consanguinity – long-recognized as a CP risk factor – has remained undefined. Here, we report findings from 188 primarily consanguineous Middle Eastern families with CP and identified putative causative genes in nearly three-quarters. The majority demonstrated recessive inheritance, although multi-level consanguinity and multilocus pathogenic variants complicated Mendelian assortment analyses. We identified 110 known CP-associated genes – five with phenotypic expansions and three others exhibiting new recessive inheritance patterns – and 24 novel candidates. We characterized ten candidates as high-confidence based on independent replication and protein modeling. We experimentally validated a role for SUCO variants in CP and newly identified a role for mid-gestational migrating excitatory neurons in the disorder. These findings highlight new genes, pathways, and phenotypes that reveal striking genomic diversity in CP.

Migraine is a complex neurological disorder, and the most common migraine categories are migraine with aura and without aura. The higher prevalence of migraine in related individuals compared to the general population indicates a potential genetic predisposition; however, gene expression, which is influenced by both genetic and environmental factors, can also be a major factor in the migraine susceptibility. Given the high number of Portuguese migraine patients whose diagnosis and treatment have not yet been well established, we decided to carry out a whole transcriptome analysis within a migraine Portuguese cohort. This study aims to identify potential biomarkers that could contribute to improved migraine therapy. We performed total RNA sequencing on whole blood samples from 15 migraine patients and 12 age-matched controls. Differential expression analysis and gene set enrichment analysis were performed in different migraine subgroups. Finally, we performed the protein-protein interaction networks of differentially expressed genes. Gene set enrichment analysis comparing migraine patients with controls highlighted upregulated pathways linked to metabolism, and downregulated immuno-inflammatory pathways. Moreover, the groups of female migraine patients and female migraine without aura patients emphasized significant upregulated pathways, including G protein-coupled receptors signalling pathways, when compared with female controls. Interestingly, we found two important differentially expressed genes related to parathyroid hormone: PTH1R and PTH2. PTH1R was upregulated in female migraine without aura versus female controls, while PTH2 was both upregulated between female migraine patients and female controls, as well as between female migraine without aura and controls. Here, we show, for the first time, the involvement of parathyroid hormone receptors and their associated gene expression patterns in female migraine patients. These molecules stand out as sturdy and promising biomarkers for innovative therapeutic in female migraine patients.

Background: Hereditary cerebellar ataxia (HCA) represents a complex group of disorders, with a wide spectrum of neurological symptoms. Among these, non‐ataxia movement disorders (MD) have been increasingly acknowledged, with variable frequency across different forms. Objectives: To characterize the type and frequency of MD in patients with HCA. To identify factors associated with MD and analyze their impact on disability. Methods: We conducted a prospective study starting in 2017, with annual visits according to a structured protocol. Patients were selected from the study database and their clinical and genetic features analyzed. Results: The cohort comprised 193 symptomatic patients. Machado‐Joseph disease (MJD, also SCA3 or ATX‐ ATXN3 ) and cerebellar ataxia, neuropathy and vestibular areflexia syndrome (ATX‐ RFC1 ) were the most common autosomal dominant (AD) or recessive forms, with a frequency of 14.0% and 15.0%, respectively. MD were present in 95 (54.4%), with dystonia being the most common (49.2%). Tremor was identified in 10.9%, Parkinsonism in 4.1% and chorea in 3.6% patients. Myoclonus and tics were rare (2.6% and 0.5%). The presence of MD was associated with AD inheritance and ATXN3 . MD, regardless of type, correlated with higher SARA score at baseline, increased fall frequency, confinement to wheelchair, and earlier occurrence of falls and of permanent use of walking aid. Conclusions: Movement disorders, particularly dystonia, were common in our cohort. This highlights the possible role of the cerebellum in MD, but also extra‐cerebellar involvement in some HCA. Presence of MD significantly worsened motor disability, highlighting the need for strategies of early identification and tailored management.

Hereditary spastic paraplegias (HSP) are a diverse group of neurodegenerative diseases characterized by lower limb spasticity and weakness. To date, over 80 genes have been associated with HSP, but many families remain without a molecular diagnosis.In this study, linkage analysis and whole‐exome sequencing (WES) were performed to identify the causal gene in a HSP family with autosomal recessive inheritance. Multipoint linkage analysis revealed a maximum significant multipoint LOD score of 4.6 on chromosome 4. WES analysis focused on this region led to the identification of a homozygous missense variant in AIMP1 (c.223G>A). Minigene assays showed that the presumed missense variant in AIMP1 caused loss of the exon 3 donor splice site. Ultimately, this led to the use of an alternative splice site within the intron and the insertion of a premature stop codon.The identification of a novel AIMP1 causal variant contributes to the growing list of HSP genes. Furthermore, it shows that, considering also previous reported cases, disruption of AIMP1 causes a spectrum of disorders ranging from intellectual disability to more complex neurodegenerative diseases.

Hereditary transthyretin-related amyloidosis (ATTRv amyloidosis) is a rare and progressively debilitating disease characterized by the deposition of transthyretin (TTR) amyloid fibrils in various organs and tissues, most commonly in the heart and peripheral nerves. This pathological deposition can lead to significant organ dysfunction and, ultimately, organ failure. ATTRv amyloidosis exhibits a broad range of clinical presentations, from purely neurological symptoms to purely cardiac manifestations, as well as mixed phenotypes which result from both neurological and cardiac implications. This wide phenotypical spectrum realistically challenges disease diagnosis and prognosis, especially in individuals without or with an unknown family history. Multiple factors are thought to contribute to this variability, including genetic, epigenetic, and even environmental influences. Understanding these factors is crucial, as they can significantly affect disease expression and progression. This review aims to summarize each of these contributing factors, to help elucidate the current knowledge on the phenotypical variability of ATTRv amyloidosis.

Dystonia is a hyperkinetic movement disorder characterized by sustained or intermittent involuntary muscle contractions, causing abnormal postures and/or repetitive movements. In this report, we identified a novel heterozygous splice-site variant in VPS16 (NM_022575.4:c.240+3G>C) in a patient with cervical and upper limb dystonia without other neurological or extra-neurological features. Analysis of patient’s blood mRNA showed disruption of exon 3/intron 3 donor splice-site, leading to exon 3 skipping, which predictably results in a frameshift [p.(Ala48Valfs*14)]. Despite the scarcity of splice-affecting variants described in VPS16-related dystonia, our report contributes with the first fully characterized variant at the mRNA level.

Spinocerebellar ataxia type 11 (SCA11) is a rare type of autosomal dominant cerebellar ataxia, mainly characterized by progressive cerebellar ataxia, abnormal eye signs and dysarthria. SCA11 is caused by variants in TTBK2, which encodes tau tubulin kinase 2 (TTBK2) protein. Only a few families with SCA11 were described to date, all harbouring small deletions or insertions that result in frameshifts and truncated TTBK2 proteins. In addition, TTBK2 missense variants were also reported but they were either benign or still needed functional validation to ascertain their pathogenic potential in SCA11. The mechanisms behind cerebellar neurodegeneration mediated by TTBK2 pathogenic alleles are not clearly established. There is only one neuropathological report and a few functional studies in cell or animal models published to date. Moreover, it is still unclear whether the disease is caused by TTBK2 haploinsufficiency of by a dominant negative effect of TTBK2 truncated forms on the normal allele. Some studies point to a lack of kinase activity and mislocalization of mutated TTBK2, while others reported a disruption of normal TTBK2 function caused by SCA11 alleles, particularly during ciliogenesis. Although TTBK2 has a proven function in cilia formation, the phenotype caused by heterozygous TTBK2 truncating variants are not clearly typical of ciliopathies. Thus, other cellular mechanisms may explain the phenotype seen in SCA11. Neurotoxicity caused by impaired TTBK2 kinase activity against known neuronal targets, such as tau, TDP-43, neurotransmitter receptors or transporters, may contribute to neurodegeneration in SCA11.