Μοριακή γενετική διερεύνηση ασθενών με πρώιμη επιληπτική και πρώιμη μιτοχονδριακή εγκεφαλοπάθεια

Εισαγωγή: Οι αναπτυξιακές και επιληπτικές εγκεφαλοπάθειες (ΑΕΕ) αποτελούν μία υποκατηγορία επιληπτικών συνδρόμων με την γενετική βάση να αφορά κυρίως σε γονίδια που κωδικοποιούν για διαύλους, πρωτεΐνες απαραίτητες για την ορθή λειτουργία των συνάψεων, μεταγραφικούς παράγοντες καθώς και για άλλες ρυθμιστικές πρωτεΐνες με ρόλο σε βασικές κυτταρικές διεργασίες. Μέχρι σήμερα, καταγράφονται περισσότερες από 100 διαφορετικές ΑΕΕ ενώ σε καθημερινή βάση ανακαλύπτονται και νέα γονίδια που σχετίζονται με αυτήν την κλινική εικόνα. Η γενετική διάγνωση των ΑΕΕ αφορά την ανίχνευση παθογόνων ή πιθανώς παθογόνων παραλλαγών σε ένα (ή περισσότερα) γονίδια και συνιστά μια τεράστια πρόκληση. Η μεγάλη φαινοτυπική ετερογένεια, η αλληλοεπικάλυψη κλινικών χαρακτηριστικών ανάμεσα στα διαφορετικά επιληπτικά σύνδρομα καθώς και ο μεγάλος αριθμός γονιδίων που εμπλέκονται στην επιληπτογένεση δυσχεραίνουν τη διαφορική διάγνωση και καθιστούν την αποκάλυψη της γενετικής βλάβης και του υποκείμενου μηχανισμού απαραίτητα για την τελική διάγνωση. Σκοπός: Η παρούσα μελέτη έχει ως σκοπό την μελέτη των υποκείμενων γενετικών μηχανισμών επιληπτογένεσης με στοχευμένη μελέτη παραλλαγών των γονιδίων MECP2, CDKL5, FOXG1, ARX, SCN1A ή POLG, σε ασθενείς με ΑΕΕ ή μέσω αλληλούχησης επόμενης γενιάς (Next generation sequencing, ΝGS) ασθενών με σπασμούς με ή χωρίς συνοδή ψυχοκινητική καθυστέρηση. Στην ευρύτερη προσέγγιση περιλαμβάνεται και η μελέτη του προτύπου απενεργοποίησης του χρωμοσώματος Χ σε περιπτώσεις ανίχνευσης παραλλαγών σε φυλοσύνδετα γονίδια σε θήλεα άτομα, η καταγραφή φαινοτυπικής ετερογένειας και η καταχώρηση νέων παραλλαγών σε βάσεις δεδομένων σύμφωνα με την παθογονικότητα τους, η αναζήτηση νέων συσχετίσεων γονοτύπου- φαινοτύπου και η σύγκριση της διαγνωστικής απόδοσης των διαφορετικών τεχνικών. Υλικά και μέθοδοι: Μελετήθηκαν 48 ασθενείς με στοχευμένο έλεγχο των γονιδίων CDKL5 και FOXG1, 42 στο γονίδιο ARX, 6 στο γονίδιο POLG, 122 στο γονίδιο MECP2 και 270 στο γονίδιο SCN1A. Με αλληλούχηση επόμενης γενιάς μελετήθηκαν 107 ασθενείς. Αποτελέσματα: Μέσω του NGS ανιχνεύθηκαν παθογόνες ή πιθανώς παραλλαγές σε 64 ασθενείς αναδεικνύοντας ποσοστό συνολικής διαγνωστικής απόδοσης 59,8%, ενώ με τους στοχευμένους ελέγχους διαγνώσθηκε το 9,88% των ασθενών. Στις παραλλαγές περιλαμβάνονται αλλοιώσεις σε νέα γονίδια ή και σε γονίδια που δεν ήταν σαφές ότι εμπλέκονται στην επιληπτογένεση και τα αποτελέσματα συνέβαλαν στον χαρακτηρισμό νέων και την κατανόηση ήδη υπαρχόντων κλινικών οντοτήτων. Συμπεράσματα: Η σύγχρονη διαγνωστική προσπέλαση της γενετικής βάσης των επιληπτικών συνδρόμων είναι σαφώς αποτελεσματικότερη με τη χρήση WES σε σχέση με τη μελέτη μεμονομένων ή και ομαδοποιημένων (panel) γονιδίων όπως καταγράφεται και σε διεθνείς κατευθυντήριες οδηγίες. Η ευελιξία της επανανάλυσης των δεδομένων του NGS συμβάλει στην τελική διάγνωση των ασθενών λόγω της διαρκούς αποκάλυψης νέων συσχετίσεων γονοτύπου-φαινοτύπου, ενώ η αποκάλυψη μοριακής βλάβης διαφορετικής από την αναμενόμενη υπογραμμίζει την διαρκώς εξελισσόμενη γνώση του γενετικού υποβάθρου που οδηγεί σε επιληπτογένεση.Introduction: Developmental and epileptic encephalopathies (DEEs) are a specific category of epileptic syndromes and are caused due to defects in neuron-expressed genes encoding for either channels, or proteins that have crucial role in the correct function of synapses, or transcription factors (as well as other proteins) with regulatory action in various cellular processes. To date over 100 different DEEs have been described and almost daily new genes are discovered and associated with this clinical entity. Genetic diagnosis of DEEs involves the detection of pathogenic or likely pathogenic variants in a potentially large number of genes, and this constitutes a large challenge. The broad phenotypic heterogeneity, the overlap of clinical features between different epileptic syndromes and the large number of genes involved in epileptogenesis make differential diagnosis difficult, and thus the identification of the specific genetic defect(s) and the underlying mechanism(s) are necessary for a final diagnosis in most patients. Aim: The present work aims to study the underlying genetic mechanisms that lead to epileptogenesis in patients with DEE through the targeted screening for variants in previously associated genes (MECP2, CDKL5, FOXG1, ARX, SCN1A or POLG) and, in epilepsy patients with or without accompanying global developmental delay via Next generation sequencing (ΝGS). The overall study will also include the pattern of X-inactivation in females with variants in X-linked genes, the recording of phenotypic heterogeneity, the submission of novel variants in databases according to their classification, the search for new genotype-phenotype associations and the comparison of the diagnostic yield using different molecular techniques. Materials and Methods: 48 patients were studied for variants in CDKL5 and FOXG1 genes, 42 in ARX, 6 in POLG, 122 in MECP2 and 270 in SCN1A. With NGS 107 patients were studied. Results: NGS detected pathogenic or likely pathogenic variants in 64 patients leading to a diagnostic yield of 59,8%, in comparison to targeted gene studies that resulted in a diagnostic yield of 9,88%. Amongst the variants observed, there are molecular defects in new genes or in genes not yet clearly involved in epileptogenesis, whereby such results potentially contribute to the elucidation of new, or clearer characterization of existing clinical entities. Conclusions: The current diagnostic approach is clearly more efficient with the use of NGS than the targeted screening of a single gene or the use of epilepsy gene panels as recorded by international guidelines when testing for epilepsy syndromes. The ability to reanalyze NGS data contributes to the final diagnosis of the patients due to the continuous discovery of new genotype-phenotype associations; while the discovery of unexpected genetic defects reflects the ever-evolving knowledge of the genetic background underlying epileptogenesis

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 to torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with TOR1A-AMC5 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 fetal 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

Molecular genetic investigation of patients with early epileptic and early mitochondrial encephalopathy

Introduction: Developmental and epileptic encephalopathies (DEEs) are a specific category of epileptic syndromes and are caused due to defects in neuron-expressed genes encoding for either channels, or proteins that have crucial role in the correct function of synapses, or transcription factors (as well as other proteins) with regulatory action in various cellular processes. To date over 100 different DEEs have been described and almost daily new genes are discovered and associated with this clinical entity. Genetic diagnosis of DEEs involves the detection of pathogenic or likely pathogenic variants in a potentially large number of genes, and this constitutes a large challenge. The broad phenotypic heterogeneity, the overlap of clinical features between different epileptic syndromes and the large number of genes involved in epileptogenesis make differential diagnosis difficult, and thus the identification of the specific genetic defect(s) and the underlying mechanism(s) are necessary for a final diagnosis in most patients. Aim: The present work aims to study the underlying genetic mechanisms that lead to epileptogenesis in patients with DEE through the targeted screening for variants in previously associated genes (MECP2, CDKL5, FOXG1, ARX, SCN1A or POLG) and, in epilepsy patients with or without accompanying global developmental delay via Next generation sequencing (ΝGS). The overall study will also include the pattern of X-inactivation in females with variants in X-linked genes, the recording of phenotypic heterogeneity, the submission of novel variants in databases according to their classification, the search for new genotype-phenotype associations and the comparison of the diagnostic yield using different molecular techniques. Materials and Methods: 48 patients were studied for variants in CDKL5 and FOXG1 genes, 42 in ARX, 6 in POLG, 122 in MECP2 and 270 in SCN1A. With NGS 107 patients were studied. Results: NGS detected pathogenic or likely pathogenic variants in 64 patients leading to a diagnostic yield of 59,8%, in comparison to targeted gene studies that resulted in a diagnostic yield of 9,88%. Amongst the variants observed, there are molecular defects in new genes or in genes not yet clearly involved in epileptogenesis, whereby such results potentially contribute to the elucidation of new, or clearer characterization of existing clinical entities. Conclusions: The current diagnostic approach is clearly more efficient with the use of NGS than the targeted screening of a single gene or the use of epilepsy gene panels as recorded by international guidelines when testing for epilepsy syndromes. The ability to reanalyze NGS data contributes to the final diagnosis of the patients due to the continuous discovery of new genotype-phenotype associations; while the discovery of unexpected genetic defects reflects the ever-evolving knowledge of the genetic background underlying epileptogenesis.Εισαγωγή: Οι αναπτυξιακές και επιληπτικές εγκεφαλοπάθειες (ΑΕΕ) αποτελούν μία υποκατηγορία επιληπτικών συνδρόμων με την γενετική βάση να αφορά κυρίως σε γονίδια που κωδικοποιούν για διαύλους, πρωτεΐνες απαραίτητες για την ορθή λειτουργία των συνάψεων, μεταγραφικούς παράγοντες καθώς και για άλλες ρυθμιστικές πρωτεΐνες με ρόλο σε βασικές κυτταρικές διεργασίες. Μέχρι σήμερα, καταγράφονται περισσότερες από 100 διαφορετικές ΑΕΕ ενώ σε καθημερινή βάση ανακαλύπτονται και νέα γονίδια που σχετίζονται με αυτήν την κλινική εικόνα. Η γενετική διάγνωση των ΑΕΕ αφορά την ανίχνευση παθογόνων ή πιθανώς παθογόνων παραλλαγών σε ένα (ή περισσότερα) γονίδια και συνιστά μια τεράστια πρόκληση. Η μεγάλη φαινοτυπική ετερογένεια, η αλληλοεπικάλυψη κλινικών χαρακτηριστικών ανάμεσα στα διαφορετικά επιληπτικά σύνδρομα καθώς και ο μεγάλος αριθμός γονιδίων που εμπλέκονται στην επιληπτογένεση δυσχεραίνουν τη διαφορική διάγνωση και καθιστούν την αποκάλυψη της γενετικής βλάβης και του υποκείμενου μηχανισμού απαραίτητα για την τελική διάγνωση. Σκοπός: Η παρούσα μελέτη έχει ως σκοπό την μελέτη των υποκείμενων γενετικών μηχανισμών επιληπτογένεσης με στοχευμένη μελέτη παραλλαγών των γονιδίων MECP2, CDKL5, FOXG1, ARX, SCN1A ή POLG, σε ασθενείς με ΑΕΕ ή μέσω αλληλούχησης επόμενης γενιάς (Next generation sequencing, ΝGS) ασθενών με σπασμούς με ή χωρίς συνοδή ψυχοκινητική καθυστέρηση. Στην ευρύτερη προσέγγιση περιλαμβάνεται και η μελέτη του προτύπου απενεργοποίησης του χρωμοσώματος Χ σε περιπτώσεις ανίχνευσης παραλλαγών σε φυλοσύνδετα γονίδια σε θήλεα άτομα, η καταγραφή φαινοτυπικής ετερογένειας και η καταχώρηση νέων παραλλαγών σε βάσεις δεδομένων σύμφωνα με την παθογονικότητα τους, η αναζήτηση νέων συσχετίσεων γονοτύπου- φαινοτύπου και η σύγκριση της διαγνωστικής απόδοσης των διαφορετικών τεχνικών. Υλικά και μέθοδοι: Μελετήθηκαν 48 ασθενείς με στοχευμένο έλεγχο των γονιδίων CDKL5 και FOXG1, 42 στο γονίδιο ARX, 6 στο γονίδιο POLG, 122 στο γονίδιο MECP2 και 270 στο γονίδιο SCN1A. Με αλληλούχηση επόμενης γενιάς μελετήθηκαν 107 ασθενείς. Αποτελέσματα: Μέσω του NGS ανιχνεύθηκαν παθογόνες ή πιθανώς παραλλαγές σε 64 ασθενείς αναδεικνύοντας ποσοστό συνολικής διαγνωστικής απόδοσης 59,8%, ενώ με τους στοχευμένους ελέγχους διαγνώσθηκε το 9,88% των ασθενών. Στις παραλλαγές περιλαμβάνονται αλλοιώσεις σε νέα γονίδια ή και σε γονίδια που δεν ήταν σαφές ότι εμπλέκονται στην επιληπτογένεση και τα αποτελέσματα συνέβαλαν στον χαρακτηρισμό νέων και την κατανόηση ήδη υπαρχόντων κλινικών οντοτήτων. Συμπεράσματα: Η σύγχρονη διαγνωστική προσπέλαση της γενετικής βάσης των επιληπτικών συνδρόμων είναι σαφώς αποτελεσματικότερη με τη χρήση WES σε σχέση με τη μελέτη μεμονομένων ή και ομαδοποιημένων (panel) γονιδίων όπως καταγράφεται και σε διεθνείς κατευθυντήριες οδηγίες. Η ευελιξία της επανανάλυσης των δεδομένων του NGS συμβάλει στην τελική διάγνωση των ασθενών λόγω της διαρκούς αποκάλυψης νέων συσχετίσεων γονοτύπου-φαινοτύπου, ενώ η αποκάλυψη μοριακής βλάβης διαφορετικής από την αναμενόμενη υπογραμμίζει την διαρκώς εξελισσόμενη γνώση του γενετικού υποβάθρου που οδηγεί σε επιληπτογένεση

A <i>TMEM63A</i> Nonsense Heterozygous Variant Linked to Infantile Transient Hypomyelinating Leukodystrophy Type 19?

Infantile onset transient hypomyelination (IOTH) is a rare form of leukodystrophy that is associated with transient motor impairment and delayed central nervous system myelination. Here, we report a case of a new mutation in the transmembrane protein 63A (TMEM63A) gene identified using Whole-Exome Sequencing (WES) in an 8.5-year-old boy with clinical symptoms similar to IOTH. The patient exhibited a mild developmental delay, including hypotonia and delayed motor milestones, as well as some notable phenotypic characteristics, such as macrocephaly and macrosomia. Despite the absence of early neuroimaging, genetic testing revealed a paternally inherited variant in TMEM63A (NM_14698.3:c.220A>T;p:(Arg74*)), potentially linked to infantile transient hypomyelinating leukodystrophy type 19. Our findings in this study and the patient’s favorable clinical course underscore the potential for successful myelination even with delayed initiation and may contribute to a better understanding of the genotype–phenotype correlation in IOTH, emphasizing the importance of genetic analysis in unresolved developmental delay cases and providing critical insights for accurate diagnosis, prognosis and potential therapeutic strategies in rare leukodystrophies

Lethal Complications and Complex Genotypes in Shwachman Diamond Syndrome: Report of a Family with Recurrent Neonatal Deaths and a Case-Based Brief Review of the Literature

Shwachman Diamond Syndrome (SDS) is a multi-system disease characterized by exocrine pancreatic insufficiency with malabsorption, infantile neutropenia and aplastic anemia. Life-threatening complications include progression to acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), critical deep-tissue infections and asphyxiating thoracic dystrophy. In most patients, SDS results from biallelic pathogenic variants in the SBDS gene, different combinations of which contribute to heterogenous clinical presentations. Null variants are not well tolerated, supporting the theory that the loss of SBDS expression is likely lethal in both mice and humans. A novel complex genotype (SBDS:c.[242C>G;258+2T>C];[460-1G>A]/WFS1:c.[2327A>T];[1371G>T]) was detected in a family with recurrent neonatal deaths. A female neonate died three hours after birth with hemolytic anemia, and a male neonate with severe anemia, thrombocytopenia and neutropenia succumbed on day 40 after Staphylococcus epidermidis infection. A subsequent review of the literature focused on fatal complications, complex SBDS genotypes and/or unusual clinical presentations and disclosed rare cases, of which some had unexpected combinations of genetic and clinical findings. The impact of pathogenic variants and associated phenotypes is discussed in the context of data sharing towards expanding scientific expert networks, consolidating knowledge and advancing an understanding of novel underlying genotypes and complex phenotypes, facilitating informed clinical decisions and disease management

The Diverse Genomic Landscape of Diamond–Blackfan Anemia: Two Novel Variants and a Mini-Review

Diamond–Blackfan anemia (DBA) is a ribosomopathy characterized by bone marrow erythroid hypoplasia, which typically presents with severe anemia within the first months of life. DBA is typically attributed to a heterozygous mutation in a ribosomal protein (RP) gene along with a defect in the ribosomal RNA (rRNA) maturation or levels. Besides classic DBA, DBA-like disease has been described with variations in 16 genes (primarily in GATA1, followed by ADA2 alias CECR1, HEATR3, and TSR2). To date, more than a thousand variants have been reported in RP genes. Splice variants represent 6% of identifiable genetic defects in DBA, while their prevalence is 14.3% when focusing on pathogenic and likely pathogenic (P/LP) variants, thus highlighting the impact of such alterations in RP translation and, subsequently, in ribosome levels. We hereby present two cases with novel pathogenic splice variants in RPS17 and RPS26. Associations of DBA-related variants with specific phenotypic features and malignancies and the molecular consequences of pathogenic variations for each DBA-related gene are discussed. The determinants of the spontaneous remission, cancer development, variable expression of the same variants between families, and selectivity of RP defects towards the erythroid lineage remain to be elucidated

Ovarian insufficiency and secondary amenorrhea in a patient with a novel variant within GDF9 gene.

OBJECTIVE: Premature ovarian insufficiency is a heterogeneous condition that can be caused by several factors, such as genetic, environmental, etc. and represents one of the main causes of female infertility. One of the genes implicated is GDF9, which encodes a member of the transforming growth factor-beta superfamily that participates in the coordination of somatic cell activity, female fertility, including folliculogenesis, and oocyte maturation. Damaging variants in GDF9-encoded growth factors can cause the production of inhibin, perturb oocyte granulosa cell microenvironments, and obstruct follicle development. A novel GDF9 variant is herein reported to consolidate the role of GDF9 in ovarian function and female fertility. METHODS: A 38-year-old female was referred for the investigation of secondary amenorrhea. Eventually, she was referred for genetic evaluation whereby conventional karyotyping and Fragile-X molecular testing were normal. Whole Exome Sequencing was performed, followed by targeted Sanger sequencing in all family members for variant confirmation and evaluation. RESULTS: In this study we report a patient presenting with secondary amenorrhea due to premature ovarian failure and a pituitary lesion with radiological characteristics compatible with a Rathke cyst or a macroadenoma, residing between the adenohypophysis and neurohypophysis. Whole exome sequencing revealed a novel heterozygous stop-loss variant c.1364A&gt;C, p.(*455Serext*8) in the GDF9 gene. CONCLUSIONS: Should the predicted elongated GDF9 protein and differentially configurated GDF9 mature protein molecule form unstable dimers, rapid proteolytic degradation may take place and inhibit homo/heterodimer formation

Retrospective analysis of persistent HyperCKemia with or without muscle weakness in a case series from Greece highlights vast <i>DMD</i> variants’ heterogeneity

Persistent hyperCKemia results from muscle dysfunction often attributed to genetic alterations of muscle-related genes, such as the dystrophin gene (DMD). Retrospective assessment of findings from DMD analysis, in association with persistent HyperCKemia, was conducted. Evaluation of medical records from 1354 unrelated cases referred during the period 1996–2021. Assessment of data concerning the detection of DMD gene rearrangements and nucleotide variants. A total of 730 individuals (657 cases, 569 of Greek and 88 of Albanian origins) were identified, allowing an overall estimation of dystrophinopathy incidence at ~1:3800 live male births. The heterogeneous spectrum of 275 distinct DMD alterations comprised exon(s) deletions/duplications, nucleotide variants, and rare events, such as chromosome translocation {t(X;20)}, contiguous gene deletions, and a fused gene involving the DMD and the DOCK8 genes. Ethnic-specific findings include a common founder variant in exon 36 (‘Hellenic’ variant). Some 50% of hyperCKemia cases were characterized as dystrophinopathies, highlighting that DMD variants may be considered the most common cause of hyperCKemia in Greece. Delineation of the broad genetic and clinical heterogeneity is fundamental for actionable public health decisions and theragnosis, as well as the establishment of guidelines addressing ethical considerations, especially related to the mild asymptomatic patient subgroup.</p

Expanded phenotypic spectrum of neurodevelopmental and neurodegenerative disorder Bryant-Li-Bhoj syndrome with 38 additional individuals.

Bryant-Li-Bhoj syndrome (BLBS), which became OMIM-classified in 2022 (OMIM: 619720, 619721), is caused by germline variants in the two genes that encode histone H3.3 (H3-3A/H3F3A and H3-3B/H3F3B) [1-4]. This syndrome is characterized by developmental delay/intellectual disability, craniofacial anomalies, hyper/hypotonia, and abnormal neuroimaging [1, 5]. BLBS was initially categorized as a progressive neurodegenerative syndrome caused by de novo heterozygous variants in either H3-3A or H3-3B [1-4]. Here, we analyze the data of the 58 previously published individuals along 38 unpublished, unrelated individuals. In this larger cohort of 96 people, we identify causative missense, synonymous, and stop-loss variants. We also expand upon the phenotypic characterization by elaborating on the neurodevelopmental component of BLBS. Notably, phenotypic heterogeneity was present even amongst individuals harboring the same variant. To explore the complex phenotypic variation in this expanded cohort, the relationships between syndromic phenotypes with three variables of interest were interrogated: sex, gene containing the causative variant, and variant location in the H3.3 protein. While specific genotype-phenotype correlations have not been conclusively delineated, the results presented here suggest that the location of the variants within the H3.3 protein and the affected gene (H3-3A or H3-3B) contribute more to the severity of distinct phenotypes than sex. Since these variables do not account for all BLBS phenotypic variability, these findings suggest that additional factors may play a role in modifying the phenotypes of affected individuals. Histones are poised at the interface of genetics and epigenetics, highlighting the potential role for gene-environment interactions and the importance of future research