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Non-syndromic hearing loss (NSHL) remains the most common sensory disorder, affecting individuals across the lifespan, from approximately one in every 500 newborns to more than two-thirds of octogenarians. (1,2) Because the condition presents without additional clinical features, gene discovery and molecular diagnosis are essential for counselling families, guiding rehabilitation strategies, paving the way for precision therapeutics, and informing public health policies. With more than 120 genes already implicated and new loci reported each year (3,4), the field now faces two intertwined challenges: (a) resolving the extraordinary genetic heterogeneity of NSHL across global populations, and (b) translating that knowledge into equitable, high-yield diagnostic workflows.The four articles gathered in this Research Topic take complementary approaches to those challenges and, taken together, expand the catalog of pathogenic variants and outline an up-todate roadmap for the next phase of NSHL genetics.Studying a socio-economically and ethnically diverse Brazilian cohort, Antunes et al. (2024) examined 90 probands with presumed autosomal-recessive NSHL. Using two complementary NGS strategies (a 99-gene hearing-loss panel and whole-exome sequencing) they identified pathogenic variants in 32 of 90 probands (36.7 %), uncovering 39 alleles across 24 genes, including 10 previously unreported variants. Notably, causative variants in SIX1 and P2RX2 revealed autosomal-dominant inheritance in two families that pedigree analysis had suggested were recessive, and several probands carried variants in genes classically linked to syndromic deafness (e.g., USH2A), underscoring the need to differentiate isolated from syndromic presentations because the latter may mandate extra-auditory surveillance. As one of the largest NGS surveys of hearing loss in Latin America, the study confirms the extreme locus heterogeneity of NSHL and demonstrates that multi-gene NGS testing is a highly effective firsttier diagnostic strategy. 2024) applied whole-exome sequencing to 11 Emirati families, of which 45.5 % were consanguineous. A molecular diagnosis was achieved in 6 probands (54.5 %), identifying pathogenic or likely pathogenic variants in MYO15A, SLC26A4, and GJB2; one nonsense variant in MYO15A (p.Tyr1962Ter) was previously unreported. In the remaining five families, the authors detected six variants of uncertain significance in CDH23, COL11A1, ADGRV1, NLRP3, and GDF6 and, through an extensive in-silico pipeline, classified them as potentially deleterious. The study demonstrates that robust genetic diagnosis is feasible in populations with high consanguinity and underscores the role of molecular results in guiding prognosis, therapy, and reproductive counselling, including pre-conception carrier testing and embryo selection.Studying a cohort of 152 Chinese families with heterogeneous clinical presentations, Zeng et al. (2022) implemented a four-tier stepwise strategy that combined multiplex PCR + highthroughput sequencing, Sanger sequencing, MLPA of the STRC gene, and reflex exome sequencing. This pipeline yielded a high diagnostic rate of 73 % (111/152). Notably, exome sequencing contributed an additional 18.4 % of solved cases and uncovered 21 novel variants in 15 deafness genes. It also re-classified 11 probands as having syndromic hearing loss, a distinction that triggers surveillance of extra-auditory organs and systems. The authors emphasize the value of tailoring test menus to local allele spectra, laboratory infrastructure, and cost constraints. They further observe that although precision therapies remain scarce, advances in inner-ear gene delivery suggest that gene therapy may become a viable option for selected molecular subtypes.The traditional focus on autosomes has left potential sex-linked contributions underexplored. Naderi et al. (2023) investigated X-chromosome variants associated with age-related hearing loss using data from the UK Biobank, encompassing over 460,000 individuals of White European ancestry. To date, the genetic basis of this kind of hearing loss remains incompletely understood, and X-linked contributions have received limited attention. Through genome-wide association analysis, the authors identified significant associations between age-related hearing loss and variants near ZNF185, MAP7D2, and LOC101928437 on the X chromosome. They concluded that X-linked variants account for only a small proportion of the genetic variance in this kind of hearing loss, suggesting a limited role for the X chromosome in this condition. The authors emphasize that replication of these findings in populations with different genetic backgrounds will be essential to validate the observed associations and to determine their broader relevance.In conclusion, the studies in this Topic not only deepen our understanding of the genes and variants that shape the auditory landscape but also chart practical paths toward precision medicine for all individuals living with NSHL, including (a) awareness of their molecular diagnosis, which has significant psychological, familial, and social implications; (b) individualized surveillance and management, particularly for those with syndromic forms of hearing loss that require specialized, multisystem care; (c) informed family planning; and (d) access to assistedreproduction options such as pre-implantation genetic testing. Nevertheless, a substantial proportion of cases remains genetically unresolved, underscoring the need for novel approaches to close the diagnostic gap.The study of oligogenic and multifactorial variation, X-linked modifiers, and non-coding regulatory regions of the genome are among the approaches needed to move beyond a strictly Mendelian script. Multi-omics strategies, such as long-read sequencing to resolve structural variants and single-cell epigenomics to map cochlear regulatory landscapes, are poised to uncover additional unsolved forms of...

Over the last decade, whole-exome sequencing (WES) has become a standard diagnostic tool, significantly transforming the landscape of clinical genetics and playing a pivotal role in the diagnosis of neurogenetic diseases. This revolutionary shift has left a lasting impact on the field of neurology in Brazil. The current review article examines key developments and milestones achieved in Brazil through the application of WES in neurology and discusses forthcoming challenges and essential steps to advance molecular diagnosis. Several studies report the use of WES to diagnose genetic disorders with neurological manifestations in Brazil, underscoring the growing importance of molecular diagnosis in neurogenetics. These studies often provide detailed phenotypic analyses and clinical descriptions, offering valuable insights into the genetic underpinnings of several neurological conditions. Many reports highlight the use of WES in the investigation of complex neurological conditions in Brazil, such as neurodevelopmental disorders, hereditary spastic paraplegia, movement disorders, and ataxia. The discovery of new genes implicated in monogenic diseases with neurological manifestations through WES was a significant breakthrough. Despite these advances, the availability of large cohort studies on rare diseases in Brazil remains limited, hindering the ability to generalize findings and explore the full spectrum of genetic diversity. However, a few larger cohort studies have substantially contributed to our understanding of rare diseases and specific neurological disorders.While WES has limitations and may eventually be supplanted by more advanced diagnostic tools, it left a permanent mark on the neurology field in Brazil. The field of neurogenetics is set to become increasingly important in the future.

Purpose: Secondary findings (SF) are pathogenic or likely pathogenic variants in genes unrelated to the primary purpose of genetic testing. The American College of Medical Genetics (ACMG) provides guidelines on which SF should be reported, involving 81 genes linked to different conditions. With the increasing use of genome sequencing (GS), SF are more frequently detected, presenting challenges for healthcare systems. The Brazilian population is often underrepresented in genomic studies, which limits population-specific knowledge. Objective: This study aimed to outline the profile of SF in the Brazilian Rare Genomes Project (BRGP). Methods: We analyzed retrospectively SF (ACMG) data from GS of 5,402 BRGP individuals. Results: Of the 5,316 cases who consented to receive SF, 3.6% (191 cases) had at least one SF. The most common genes identified were TTR, TTN, and BRCA2 . SF were mainly related to cardiovascular conditions (40.2%) and cancer predisposition (37.6%). Some variants, such as TTR: c.424G>A; p. (Val142Ile) and TP53: c.1010G>A; p. (Arg337His), were recurrent, reflecting population-specific traits and founder effects. Novel variants were 10.6% of SF. Conclusion: SF rate varies across studies and populations. While SF can aid early diagnosis, their relevance is debated due to potential psychological and healthcare burdens. Effective genetic counseling and public health policies are essential.

Advances in DNA sequencing technologies, especially next-generation sequencing (NGS), which is the basis for whole-exome sequencing (WES) and whole-genome sequencing (WGS), have profoundly transformed immune-mediated rheumatic disease diagnosis. Recently, substantial cost reductions have facilitated access to these diagnostic tools, expanded the capacity of molecular diagnostics and enabled the pursuit of precision medicine in rheumatology. Understanding the fundamental principles of genetics and diversity in genetic variant classification is a crucial milestone in rheumatology. However, despite the growing availability of DNA sequencing platforms, a significant number of autoinflammatory diseases (AIDs), neuromuscular disorders, hereditary collagen diseases, and monogenic bone diseases remain unsolved, and variants of uncertain significance (VUS) pose a formidable challenge to addressing these unmet needs in the coming decades. This article aims to provide an overview of the clinical indications and interpretation of comprehensive genetic testing in the medical field, addressing the related complexities and implications.

Background: Lactose tolerant test (LTT) is the most broadly used diagnostic test for lactose intolerance in Brazil, is an indirect, minimally invasive and a low-cost test that is widely available in primary care and useful in clinical practice. The C/T-13910 polymorphism in lactase persistence has been well characterized in Caucasian populations, but there are no studies evaluating the concordance between C/T-13910 polymorphism genotyping results and LTT results in Brazil, where the population is highly mixed. Objective: We aimed to evaluate agreement between presence of C/T-13910 polymorphism genotyping and malabsorption in LTT results. Methods: This is a retrospective analysis of a Brazilian population whose data were collected from a single laboratory database present in several Brazilian states. Results of individuals who underwent both genetic testing for lactose intolerance (C/T-13910 polymorphism genotyping) and an LTT from April 2016 until February 2019 were analysed to evaluate agreement between tests. Groups were classified according to age (<10-year-old (yo), 10-17 yo, ≥18 yo groups) and state of residence (São Paulo or Rio Grande do Sul). Results: Among the 404 patients evaluated, there was agreement between the genotyping and LTT results in 325 (80.4%) patients and discordance in 79 (19.6%) patients (k=0.42 -moderate agreement). Regarding the genotype, 47 patients with genotype C/C (lactase nonpersistence) had normal LTT results, and 32 with genotype C/T or T/T (indicating lactase persistence) had abnormal LTT results. Neither age nor state of residence (Rio Grande do Sul or São Paulo) affected the agreement between test results. Conclusion: Considering the moderate agreement between C/T-13910 polymorphism genotyping and LTT results (κ=0.42) in the Brazilian population, we hypothesize that an analysis of other polymorphisms could be a strategy to improve the agreement between genotyping and established tests and suggest that additional studies should focus on exploring this approach.

Background: Next-generation sequencing has had a significant impact on genetic disease diagnosis, but the interpretation of the vast amount of genomic data it generates can be challenging. To address this, the American College of Medical Genetics and Genomics and the Association for Molecular Pathology have established guidelines for standardized variant interpretation. In this manuscript, we present the updated Hospital Israelita Albert Einstein Standards for Constitutional Sequence Variants Classification, incorporating modifications from leading genetics societies and the ClinGen initiative. Results: First, we standardized the scientific publications, documents, and other reliable sources for this document to ensure an evidence-based approach. Next, we defined the databases that would provide variant information for the classification process, established the terminology for molecular findings, set standards for disease-gene associations, and determined the nomenclature for classification criteria. Subsequently, we defined the general rules for variant classification and the Bayesian statistical reasoning principles to enhance this process. We also defined bioinformatics standards for automated classification. Our workgroup adhered to gene-specific rules and workflows curated by the ClinGen Variant Curation Expert Panels whenever available. Additionally, a distinct set of specifications for criteria modulation was created for cancer genes, recognizing their unique characteristics. Conclusions: The development of an internal consensus and standards for constitutional sequence variant classification, specifically adapted to the Brazilian population, further contributes to the continuous refinement of variant classification practices. The aim of these efforts from the workgroup is to enhance the reliability and uniformity of variant classification.

Hearing loss (HL) is a common sensory deficit in humans and represents an important clinical and social burden. We studied whole-genome sequencing data of a cohort of 2,097 individuals from the Brazilian Rare Genomes Project who were unaffected by hearing loss to investigate pathogenic and likely pathogenic variants associated with nonsyndromic hearing loss (NSHL). We found relevant frequencies of individuals harboring these alterations: 222 heterozygotes (10.59%) for sequence variants, 54 heterozygotes (2.58%) for copy-number variants (CNV), and four homozygotes (0.19%) for sequence variants. The top five most frequent genes and their corresponding combined allelic frequencies (AF) were GJB2 (AF = 1.57%), STRC (AF = 1%), OTOA (AF = 0.69%), TMPRSS3 (AF = 0.41%), and OTOF (AF = 0.29%). The most frequent sequence variant was GJB2:c.35del (AF = 0.72%), followed by OTOA:p. (Glu787Ter) (AF = 0.61%), while the most recurrent CNV was a microdeletion of 57.9 kb involving the STRC gene (AF = 0.91%). An important fraction of these individuals (n = 104; 4.96%) presented variants associated with autosomal dominant forms of NSHL, which may imply the development of some hearing impairment in the future. Using data from the heterozygous individuals for recessive forms and the Hardy–Weinberg equation, we estimated the population frequency of affected individuals with autosomal recessive NSHL to be 1:2,222. Considering that the overall prevalence of HL in adults ranges from 4–15% worldwide, our data indicate that an important fraction of this condition may be associated with a monogenic origin and dominant inheritance.

De novo deleterious and heritable biallelic mutations in the DNA binding domain (DBD) of the transcription factor deformed epidermal autoregulatory factor 1 (DEAF1) result in a phenotypic spectrum of disorders termed DEAF1-associated neurodevelopmental disorders (DAND). RNA-sequencing using hippocampal RNA from mice with conditional deletion of Deaf1 in the central nervous system indicate that loss of Deaf1 activity results in the altered expression of genes involved in neuronal function, dendritic spine maintenance, development, and activity, with reduced dendritic spines in hippocampal regions. Since DEAF1 is not a dosage-sensitive gene, we assessed the dominant negative activity of previously identified de novo variants and a heritable recessive DEAF1 variant on selected DEAF1-regulated genes in 2 different cell models. While no altered gene expression was observed in cells over-expressing the recessive heritable variant, the gene expression profiles of cells over-expressing de novo variants resulted in similar gene expression changes as observed in CRISPR-Cas9-mediated DEAF1-deleted cells. Altered expression of DEAF1-regulated genes was rescued by exogenous expression of WT-DEAF1 but not by de novo variants in cells lacking endogenous DEAF1. De novo heterozygous variants within the DBD of DEAF1 were identified in 10 individuals with a phenotypic spectrum including autism spectrum disorder, developmental delays, sleep disturbance, high pain tolerance, and mild dysmorphic features. Functional assays demonstrate these variants alter DEAF1 transcriptional activity. Taken together, this study expands the clinical phenotypic spectrum of individuals with DAND, furthers our understanding of potential roles of DEAF1 on neuronal function, and demonstrates dominant negative activity of identified de novo variants.