Whole-exome sequencing in children with dyslexia implicates rare variants in CLDN3 and ion channel genes.

We sequenced the genomes of 200 individuals from 41 families multiply affected with bipolar disorder (BD) to identify contributions of rare variants to genetic risk. We initially focused on 3,087 candidate genes with known synaptic functions or prior evidence from genome-wide association studies. BD pedigrees had an increased burden of rare variants in genes encoding neuronal ion channels, including subunits of GABAA receptors and voltage-gated calcium channels. Four uncommon coding and regulatory variants also showed significant association, including a missense variant in GABRA6. Targeted sequencing of 26 of these candidate genes in an additional 3,014 cases and 1,717 controls confirmed rare variant associations in ANK3, CACNA1B, CACNA1C, CACNA1D, CACNG2, CAMK2A, and NGF. Variants in promoters and 5' and 3' UTRs contributed more strongly than coding variants to risk for BD, both in pedigrees and in the case-control cohort. The genes and pathways identified in this study regulate diverse aspects of neuronal excitability. We conclude that rare variants in neuronal excitability genes contribute to risk for BD.

Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease affecting the central nervous system with many known genetic risk factors. Although genome-wide association studies (GWAS) have identified common genetic variants with small effects associated with MS, the role of rare variants with large effects in MS aetiology remains underexplored. We hypothesized that rare variants in MS-associated genes from GWAS studies (GWAS-associated genes) are more likely to contribute to familial MS (FMS) risk than to sporadic MS (SMS). Therefore, we aimed to assess the burden of rare, predicted pathogenic (RPP) variants in GWAS-associated genes in FMS and SMS patients compared to controls. Rare genetic variants in 111 GWAS-associated genes were assessed in 87 FMS, 89 SMS and 3866 control cases. We demonstrate that RPP variants were significantly overrepresented in the FMS cohort whereas their frequency was not increased in the SMS cohort compared to controls (p-values 5.27 × 10− 74 and 1.00, respectively). Six genes (ALPK2, ANKRD55, INTS8, IQCB1, JADE2, and MALT1) significantly contributed to the burden of RPP in the FMS group. We conclude that rare variants in genes identified by GWAS might contribute to the genetic predisposition of familial MS patients.

Genetic counseling in direct-to-consumer exome sequencing: a case report.

Chiari Malformation Type 1 (CM-1) is characterized by herniation of the cerebellar tonsils below the foramen magnum and the presence of headaches and other neurologic symptoms. Cranial bone constriction is suspected to be the most common biologic mechanism leading to CM-1. However, other mechanisms may also contribute, particularly in the presence of connective tissue disorders (CTDs), such as Ehlers Danlos Syndrome (EDS). Accumulating data suggest CM-1 with connective tissue disorders (CTD+) may have a different patho-mechanism and different genetic risk factors than CM-1 without CTDs (CTD-). To identify CM-1 genetic risk variants, we performed whole exome sequencing on a single large, multiplex family from Spain and targeted sequencing on a cohort of 186 unrelated adult, Caucasian females with CM-1. Targeted sequencing captured the coding regions of 21 CM-1 and EDS candidate genes, including two genes identified in the Spanish family. Using gene burden analysis, we compared the frequency of rare, functional variants detected in CM-1 cases versus publically available ethnically-matched controls from gnomAD. A secondary analysis compared the presence of rare variants in these genes between CTD+ and CTD- CM-1 cases. In the Spanish family, rare variants co-segregated with CM-1 in COL6A5, ADGRB3 and DST. A variant in COL7A1 was present in affected and unaffected family members. In the targeted sequencing analysis, rare variants in six genes (COL7A1, COL5A2, COL6A5, COL1A2, VEGFB, FLT1) were significantly more frequent in CM-1 cases compared to public controls. In total, 47% of CM-1 cases presented with rare variants in at least one of the four significant collagen genes and 10% of cases harbored variants in multiple significant collagen genes. Moreover, 26% of CM-1 cases presented with rare variants in the COL6A5 gene. We also identified two genes (COL7A1, COL3A1) for which the burden of rare variants differed significantly between CTD+ and CTD- CM-1 cases. A higher percentage of CTD+ patients had variants in COL7A1 compared to CTD+ patients, while CTD+ patients had fewer rare variants in COL3A1 than did CTD- patients. In summary, rare variants in several collagen genes are particularly frequent in CM-1 cases and those in COL6A5 co-segregated with CM-1 in a Spanish multiplex family. COL6A5 has been previously associated with musculoskeletal phenotypes, but this is the first association with CM-1. Our findings underscore the contribution of rare genetic variants in collagen genes to CM-1, and suggest that CM-1 in the presence and absence of CTD symptoms is driven by different genes.

Context: Rare copy number variants (CNVs) have been associated with the development of severe obesity. However, the potential disease-causing contribution of individual genes within the region of CNVs is often not known. Objective: Screening of rare variants in genes involved in CNVs in Finnish patients with severe early-onset obesity to find candidate genes linked to severe obesity. Methods: Custom-made targeted exome sequencing panel to search for rare (minor allele frequency <0.1%) variants in genes affected by previously identified CNVs in 92 subjects (median age 14 years) with early-onset severe obesity (median body mass index (BMI) Z-score + 4.0). Results: We identified thirteen rare heterozygous variants of unknown significance in eleven subjects in twelve of the CNV genes. Two rare missense variants (p.Pro405Arg and p.Tyr232Cys) were found in SORCS1, a gene highly expressed in the brain and previously linked to diabetes risk. Four rare variants were in genes in the proximal 16p11.2 region (a frameshift variant in TAOK2 and missense variants in SEZ6L2, ALDOA and KIF22) and three rare missense variants were in genes in the 22q11.21 region (AIFM3, ARVCF and KLHL22). Conclusion: We report several rare variants in CNV genes in subjects with childhood obesity. However, the role of the individual genes in the previously identified rare CNVs to development of obesity remains uncertain. More studies are needed to understand the potential role of the specific genes within obesity associated CNVs.

Current genetic screening for breast cancer predisposition is limited to the analysis of coding regions (exons) and intron/exon boundaries of BRCA1/2 genes. There is limited data on the prevalence and clinical significance of variants in the non-coding regions of these genes. Consequently, the majority of variants identified in these regions remain unclassified, and approximately 80% of germline BRCA1/2 tests are not considered in the daily management of patients with triple-negative breast cancer (TNBC). Emerging evidence suggests that non-coding variants can impact cancer risk and response to treatment. This study aimed to investigate the prevalence of variants in the non-coding regulatory regions of BRCA1/2 and other breast cancer predisposition genes in TNBC patients selected based on age at cancer diagnosis and/or family history of cancer. Additionally, we sought to explore the functional role of identified variants of uncertain significance (VUS) through ongoing analyses. We enrolled 144 TNBC patients who had previously tested negative for germline variants in the coding regions of BRCA1/2 and other cancer predisposition genes. Next-generation sequencing (NGS) analysis identified 635 rare variants in the non-coding regions of 28 selected genes involved in breast/ovarian cancer predisposition. In our TNBC cohort, we observed a higher prevalence of rare variants in the genes CDH1 (1.3%), STK11 (11.2%), ATM (10.7%), PTEN (7.40%), and PMS2 (5.04%). Germline variants in BRCA2 were statistically significantly associated with worse overall survival (p-value=0.017). CDH1 rare variants were associated with the highest percentage of non-pathologic complete response after neoadjuvant chemotherapy (p=0.0273). MLH1 and PALB2 rare variants were both associated with bilateral breast cancer (p=0.015 and p=0.0005, respectively). Rare variants of the ATM gene were associated with a positive family history (p=0.041). Preliminary single nucleotide variant (SNV) data analysis showed that the most significant functional score for alterations were detected in the promoter of MSH6, potentially associated with chromatin effects. Further analyses are ongoing to elucidate the functional impact of these variants. Due to the small sample size, these analyses should be considered exploratory, and larger studies are needed to confirm these findings and establish the clinical utility of screening for non-coding variants in TNBC patients. Citation Format: Michela Palleschi, Alessandra Virga, Emanuela Scarpi, Eugenio Fonzi, Filippo Merloni, Samanta Sarti, Rita Danesi, Mila Ravegnani, Chiara Casadei, Marianna Sirico, Caterina Gianni, Roberta Maltoni, Sara Bravaccini, Daniele Calistri, Valentina Arcangeli, Valentina Zampiga, Ilaria Cangini, Erika Bandini, Francesca Mannozzi, Fabio Falcini, Ugo De Giorgi, Paola Ulivi, Gianluca Tedaldi. Investigating the Potential Role of Rare Germline Non-Coding Variants in Cancer Predisposition Genes in Patients with Triple-Negative Breast [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-05-10.

ABSTRACTBackground:Polycystic ovary syndrome (PCOS) is a complex genetic trait, the pathogenesis of which is governed by an interplay of genetic and epigenetic factors. However, the aetiology of PCOS is not fully understood.Aims:The objective of this study was to investigate the genetic causes of PCOS by identifying rare variants in genes implicated in its pathophysiology.Settings and Design:This was a hospital-based observational study.Materials and Methods:We used whole-exome sequencing for 52 PCOS women to identify the rare variants in genes related to PCOS pathogenesis. Subsequently, we analysed these variants using in silico prediction software to determine their functional effects. We then assessed the relationship between these variants and the clinical outcomes of the patients.Statistical Analysis Used:Student’s t-test and Fisher’s exact test were used to compare clinical parameters and frequency differences amongst PCOS patients with and without variants.Results:A total of four rare exonic variants in obesity- and hyperinsulinaemia-related genes including UCP1 (p.Thr227Ile), UCP2 (p.Arg88Cys), IRS1 (p.Ser892Gly) and GHRL (p.Leu72Met) were identified in eight patients. Significant differences were observed between the patients carrying variants and those without variants. PCOS patients with identified variants exhibited significantly higher average body mass index and fasting insulin levels of PCOS subjects with identified variants compared to those without variants (P < 0.05). Additionally, there were significant differences in the variant frequencies of four variants when compared to the population database (P < 0.05).Conclusion:This study shows a prevalence of rare variants in obesity and hyperinsulinaemia-related genes in a cohort of PCOS women, thereby underscoring the impact of the identified rare variants on the development of obesity and associated metabolic derangements in PCOS women.

Acquired long QT syndrome (aLQTS) is a serious unpredictable adverse drug reaction. Pharmacogenomic markers may predict risk. Among 153 aLQTS patients (mean age 58 years [range, 14-88], 98.7% White, 85.6% symptomatic), computational methods identified proteins interacting most significantly with 216 QT-prolonging drugs. All cases underwent sequencing of 31 candidate genes arising from this analysis or associating with congenital LQTS. Variants were filtered using a minor allele frequency <1% and classified for susceptibility for aLQTS. Gene-burden analyses were then performed comparing the primary cohort to control exomes (n=452) and an independent replication aLQTS exome sequencing cohort. In 25.5% of cases, at least one rare variant was identified: 22.2% of cases carried a rare variant in a gene associated with congenital LQTS, and in 4% of cases that variant was known to be pathogenic or likely pathogenic for congenital LQTS; 7.8% cases carried a cytochrome-P450 (CYP) gene variant. Of 12 identified CYP variants, 11 (92%) were in an enzyme known to metabolize at least one culprit drug to which the subject had been exposed. Drug-drug interactions that affected culprit drug metabolism were found in 19% of cases. More than one congenital LQTS variant, CYP gene variant, or drug interaction was present in 7.8% of cases. Gene-burden analyses of the primary cohort compared to control exomes (n=452), and an independent replication aLQTS exome sequencing cohort (n=67) and drug-tolerant controls (n=148) demonstrated an increased burden of rare (minor allele frequency<0.01) variants in CYP genes but not LQTS genes. Rare susceptibility variants in CYP genes are emerging as potentially important pharmacogenomic risk markers for aLQTS and could form part of personalized medicine approaches in the future.

Analysis of exome-sequenced UK Biobank subjects implicates genes affecting risk of hyperlipidaemia

BackgroundThe interleukin (IL)-1 pathway is primarily associated with innate immunological defense and plays a major role in the induction and regulation of inflammation. Both common and rare genetic variation in this pathway underlies various inflammation-mediated diseases, but the role of rare variants relative to common variants in immune response variability in healthy individuals remains unclear.MethodsWe performed molecular inversion probe sequencing on 48 IL-1 pathway-related genes in 463 healthy individuals from the Human Functional Genomics Project. We functionally grouped common and rare variants, over gene, subpathway, and inflammatory levels and performed the Sequence Kernel Association Test to test for association with in vitro stimulation-induced cytokine responses; specifically, IL-1β and IL-6 cytokine measurements upon stimulations that represent an array of microbial infections: lipopolysaccharide (LPS), phytohaemagglutinin (PHA), Candida albicans (C. albicans), and Staphylococcus aureus (S. aureus).ResultsWe identified a burden of NCF4 rare variants with PHA-induced IL-6 cytokine and showed that the respective carriers are in the 1% lowest IL-6 producers. Collapsing rare variants in IL-1 subpathway genes produces a bidirectional association with LPS-induced IL-1β cytokine levels, which is reflected by a significant Spearman correlation. On the inflammatory level, we identified a burden of rare variants in genes encoding for proteins with an anti-inflammatory function with S. aureus-induced IL-6 cytokine. In contrast to these rare variant findings which were based on different types of stimuli, common variant associations were exclusively identified with C. albicans-induced cytokine over various levels of grouping, from the gene, to subpathway, to inflammatory level.ConclusionsIn conclusion, this study shows that functionally grouping common and rare genetic variants enables the elucidation IL-1-mediated biological mechanisms, specifically, for IL-1β and IL-6 cytokine responses induced by various stimuli. The framework used in this study may allow for the analysis of rare and common genetic variants in a wider variety of (non-immune) complex phenotypes and therefore has the potential to contribute to better understanding of unresolved, complex traits and diseases.

Parkinson's disease (PD) is a neurodegenerative movement disorder which can be either familial or sporadic. While it is well known that monogenic mutations are not a very common cause of PD, GWAS studies have shown that an additional fraction of the PD heritability could be explained by rare or common variants. To identify the rare variants that could influence the risk of PD in the Moroccan population, a cohort of 94 sporadic PD patients negative for the LRRK2 G2019S mutation was subjected to NGS gene panel sequencing, and gene dosage using the MLPA method. Mean age of onset at enrollment was 51.7 ± 11.51years, and 60% of patients were men. We identified 70 rare variants under 0.5% of frequency in 16 of the 20 genes analyzed, of which 7 were novel. Biallelic disease-causing variants in genes with recessive inheritance were found in 5 PD cases (5.31%), whereas 13 patients (13.8%) carried likely pathogenic variants in genes with dominant inheritance. Moreover, 8 patients (8.5%) carried a single variant in MAPT or POLG, whereas co-occurrence of rare variants involving more than one gene was observed in 28 patients (30%). PD patients with variants in recessive genes had a younger mean age at onset than patients with dominant ones (33.40 (12.77) vs. 53.15 (6.63), p < 0.001), while their clinical features were similar. However, patients with rare variants in the risk factor genes or in more than one gene tended to have less resting tremor (p < 0.04), but more dystonia (p < 0.006) and dementia (p < 0.002) than those without any rare variants in known PD-associated genes. Our results showed a significant enrichment of rare variants particularly in LRRK2, VPS13C, POLG, and MAPT and underline their impact on the risk of sporadic form of the disease.

Brain injuries are associated with oxidative stress and a need to restore neuronal homeostasis. Mutations in ion channel genes, in particular CACNA1A, have been implicated in familial hemiplegic migraine (FHM) and in the development of concussion-related symptoms in response to trivial head trauma. The aim of this study was to explore the potential role of variants in other ion channel genes in the development of such responses. We conducted whole exome sequencing (WES) on16 individuals who developed a range of neurological and concussion-related symptoms following minor or trivial head injuries. All individuals were initially tested and shown to be negative for mutations in known FHM genes.Variants identified from the WES results were filtered to identify rare variants (minor allele frequency [MAF] <0.01) in genes related to neural processes as well as genes highly expressed in the brain using a combination of in silico prediction tools (SIFT, PolyPhen, PredictSNP, Mutation Taster, and Mutation Assessor). Rare (MAF <0.001) or novel heterozygous variants in 7 ion channel genes were identified in 37.5% (6/16) of the cases (CACNA1I, CACNA1C, ATP10A, ATP7B, KCNAB1, KCNJ10, and SLC26A4), rare variants in neurotransmitter genes were found in 2 cases (GABRG1 and GRIK1), and rare variants in 3 ubiquitin-related genes identified in 4 cases (SQSTM1, TRIM2, and HECTD1).In this study, the largest proportion of potentially pathogenic variants in individuals with severe responses to minor head trauma were identified in genes previously implicated in migraine and seizure-related autosomal recessive neurological disorders. Together with results implicating variants in the hemiplegic migraine genes, CACNA1A and ATP1A2, in severe head trauma response, our results support a role for heterozygous deleterious mutations in genes implicated in neurological dysfunction and potentially increasing the risk of poor response to trivial head trauma.

SummaryRare genetic variants in genes previously described to be involved in bone monogenic disorders were identified in postmenopausal women split into two groups according to extreme bone mineral density (BMD) values and lumbar spine Z-scores. A pathogenic variant in COL1A2 gene found in a woman with low BMD highlights the overlap between osteogenesis imperfecta and osteoporosis, which may share their genetic etiology. Other variants were not clearly associated with the extreme BMD, suggesting that there is little contribution of rare variants to postmenopausal osteoporosis.PurposeWe aimed to evaluate whether extreme values of bone mineral density (BMD) in a population-based cohort of postmenopausal women (BARCOS) could be determined by rare genetic variants in genes related to monogenic bone disorders.MethodsA panel of 127 genes related to different skeletal phenotypes was designed. Massive sequencing by targeted capture of these genes was performed in 104 DNA samples from those women of the BARCOS cohort that exhibited the highest (HZ group) and lowest (LZ group) LS Z-scores, ranging from + 0.70 to + 3.80 and from − 2.35 to − 4.26, respectively. 5’UTR, 3’UTR, splice region, missense, nonsense, and short indel variants with MAF < 0.01 were annotated with CADD version 1.6 and considered in the analysis.ResultsAfter filtering those variants with CADD > 25 and present only in one of the groups (either LZ or HZ), six variants were detected, most of which (5/6) were in the LZ group in TCIRG1, COL1A2, SEC24D, LRP4, and ANO5 genes, while only one, in the LMNA gene, was in the HZ group. According to the ClinVar database, the COL1A2 variant, causative of a recessive form of osteogenesis imperfecta, is described as pathogenic, while the other variants are considered of uncertain significance (VUS).ConclusionThe variant identified in COL1A2 in a woman from the LZ group highlights the genetic overlap between monogenic diseases such as osteogenesis imperfecta and complex diseases like osteoporosis. However, the other variants were not clearly associated with the extreme BMD, suggesting that there is little contribution of rare variants to postmenopausal osteoporosis.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which rare and common gene variants contribute to pathogenesis. Severe sporadic disease in children is often explained by "de novo" variants that can be uncovered by trio sequencing. Whole-exome sequencing was performed in 50 Chinese trios with childhood-onset SLE (cSLE). Rare coding variants in SLE-associated genes and all de novo variants were investigated. Gene pathway and expression analysis and interferon-β (IFNβ) luciferase assays were used to predict contribution to disease. Each proband carried at least one rare variant in an SLE-associated gene, with a median of six per child. At least two probands had monogenic disease, and one-third of probands carried novel or rare variants in genes well accepted to cause monogenic SLE: ACP5, C3, C4A, C4B, DNASE1, IFIH1, NRAS, RNASEH2B, RNASEH2C, and SAMHD1. Probands carried a median of one de novo, rare, coding variant. Intriguingly, although only two de novo variants occurred in genes previously associated with SLE, 12 of the 50 genes were enriched in the top 20 SLE-related pathways and were highly expressed in age-associated B cells and plasma B cells. These genes represent promising candidate lupus genes. Two de novo variants occurring in genes not previously linked to SLE or autoimmunity, DHX8 and ACTR5, enhanced type I IFN signaling. This study highlights the abundance of lupus-relevant rare gene variants in cSLE, supports frequent contribution of de novo variants to disease, and identifies genes that may constitute novel therapeutic targets of relevance to Chinese patients.

Cerebrovascular disease is a common comorbidity in patients with Alzheimer’s disease (AD) and other dementias. Accumulating evidence suggests that dysfunction of the cerebral vasculature and AD neuropathology interact in multiple ways. Additionally, common variants in COL4A1 and rare variants in HTRA1, NOTCH3, COL4A1, and CST3 have been associated with AD pathogenesis. We aimed to search for rare genetic variants in genes associated with monogenic small vessel disease in a cohort of Portuguese early-onset AD patients. We performed whole-exome sequencing in 104 thoroughly studied patients with early-onset AD who lacked known pathogenic variants in the genes associated with AD or frontotemporal dementia. We searched for rare (minor allele frequency < 0.001) non-synonymous variants in genes associated with small vessel disease: NOTCH3, HTRA1, COL4A1, COL4A2, CSTA, GLA, and TREX1. We identified 12 rare variants in 18 patients (17.3% of the cohort). Three male AD patients carried a pathogenic GLA variant (p.Arg118Cys). One of these patients had a definite neuropathological study, confirming the diagnosis of AD and showing concomitant Fabry pathology in CA1-CA4 and the subiculum. We also found several rare variants in other genes associated with cSVD (NOTCH3, COL4A2 and HTRA1), corroborating previous studies and providing further support for the possibility that cSVD genes may play a role in AD pathogenesis. The presence of the same GLA variant in 3 early-onset AD patients, with no other genetic cause for the disease, together with the colocalization of Fabry disease pathology in areas relevant for AD pathogenesis, suggest GLA may have a role in its pathophysiology, possibly parallel to that of GBA in Parkinson’s disease, meriting further studies.