Short Tandem Repeats in the era of next-generation sequencing: from historical loci to population databases.

In this study, we explore the landscape of short tandem repeats (STRs) within the human genome through the lens of evolving technologies to detect genomic variations. STRs, which encompass approximately 3% of our genomic DNA, are crucial for understanding human genetic diversity, disease mechanisms, and evolutionary biology. The advent of high-throughput sequencing methods has revolutionized our ability to accurately map and analyze STRs, highlighting their significance in genetic disorders, forensic science, and population genetics. We review the current available methodologies for STR analysis, the challenges in interpreting STR variations across different populations, and the implications of STRs in medical genetics. Our findings underscore the urgent need for comprehensive STR databases that reflect the genetic diversity of global populations, facilitating the interpretation of STR data in clinical diagnostics, genetic research, and forensic applications. This work sets the stage for future studies aimed at harnessing STR variations to elucidate complex genetic traits and diseases, reinforcing the importance of integrating STRs into genetic research and clinical practice.

Unraveling the role of non-coding rare variants in epilepsy.

The discovery of new variants has leveled off in recent years in epilepsy studies, despite the use of very large cohorts. Consequently, most of the heritability is still unexplained. Rare non-coding variants have been largely ignored in studies on epilepsy, although non-coding single nucleotide variants can have a significant impact on gene expression. We had access to whole genome sequencing (WGS) from 247 epilepsy patients and 377 controls. To assess the functional impact of non-coding variants, ExPecto, a deep learning algorithm was used to predict expression change in brain tissues. We compared the burden of rare non-coding deleterious variants between cases and controls. Rare non-coding highly deleterious variants were significantly enriched in Genetic Generalized Epilepsy (GGE), but not in Non-Acquired Focal Epilepsy (NAFE) or all epilepsy cases when compared with controls. In this study we showed that rare non-coding deleterious variants are associated with epilepsy, specifically with GGE. Larger WGS epilepsy cohort will be needed to investigate those effects at a greater resolution. Nevertheless, we demonstrated the importance of studying non-coding regions in epilepsy, a disease where new discoveries are scarce.

Syngap1 Disruption Induced by Recombination between Inverted loxP Sites Is Associated with Hippocampal Interneuron Dysfunction.

SYNGAP1 haploinsufficiency in humans causes intellectual disability (ID). SYNGAP1 is highly expressed in cortical excitatory neurons and, reducing its expression in mice accelerates the maturation of excitatory synapses during sensitive developmental periods, restricts the critical period window for plasticity, and impairs cognition. However, its specific role in interneurons remains largely undetermined. In this study, we investigated the effects of conditional Syngap1 disruption in medial ganglionic eminence (MGE)-derived interneurons on hippocampal interneuron firing properties and excitatory synaptic inputs, as well as on pyramidal cell synaptic inhibition and synaptic integration. We show that conditional Syngap1 disruption in MGE-derived interneurons results in cell-specific impairment of firing properties of hippocampal Nkx2.1 fast-spiking interneurons, with enhancement of their AMPA receptor (AMPAR)-mediated excitatory synaptic inputs but compromised short-term plasticity. In contrast, regular-spiking Nkx2.1 interneurons are largely unaffected. These changes are associated with impaired pyramidal cell synaptic inhibition and enhanced summation of excitatory responses. Unexpectedly, we found that the Syngap1flox allele used in this study contains inverted loxP sites and that its targeted recombination in MGE-derived interneurons induces some cell loss during embryonic development and the reversible inversion of the sequence flanked by the loxP sites in postmitotic cells. Together, these results suggest that Syngap1 plays a role in cell-specific regulation of hippocampal interneuron function and inhibition of pyramidal cells in mice. However, because of our finding that the Syngap1flox allele used in this study contains inverted loxP sites, it will be important to further investigate interneuron function using a different Syngap1 conditional allele.

Bi-allelic variants in WNT7B disrupt the development of multiple organs in humans.

BACKGROUND: Pulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia and Cardiac defects delineate the PDAC syndrome. We aim to identify the cause of PDAC syndrome in patients who do not carry pathogenic variants in RARB and STRA6, which have been previously associated with this disorder. METHODS: We sequenced the exome of patients with unexplained PDAC syndrome and performed functional validation of candidate variants. RESULTS: We identified bi-allelic variants in WNT7B in fetuses with PDAC syndrome from two unrelated families. In one family, the fetus was homozygous for the c.292C>T (p.(Arg98*)) variant whereas the fetuses from the other family were compound heterozygous for the variants c.225C>G (p.(Tyr75*)) and c.562G>A (p.(Gly188Ser)). Finally, a molecular autopsy by proxy in a consanguineous couple that lost two babies due to lung hypoplasia revealed that both parents carry the p.(Arg98*) variant. Using a WNT signalling canonical luciferase assay, we demonstrated that the identified variants are deleterious. In addition, we found that wnt7bb mutant zebrafish display a defect of the swimbladder, an air-filled organ that is a structural homolog of the mammalian lung, suggesting that the function of WNT7B has been conserved during evolution for the development of these structures. CONCLUSION: Our findings indicate that defective WNT7B function underlies a form of lung hypoplasia that is associated with the PDAC syndrome, and provide evidence for involvement of the WNT-ß-catenin pathway in human lung, tracheal, ocular, cardiac, and renal development.

Assessment of burden and segregation profiles of CNVs in patients with epilepsy.

OBJECTIVE: Microdeletions are associated with different forms of epilepsy but show incomplete penetrance, which is not well understood. We aimed to assess whether unmasked variants or double CNVs could explain incomplete penetrance. METHODS: We analyzed copy number variants (CNVs) in 603 patients with four different subgroups of epilepsy and 945 controls. CNVs were called from genotypes and validated on whole-genome (WGS) or whole-exome sequences (WES). CNV burden difference between patients and controls was obtained by fitting a logistic regression. CNV burden was assessed for small and large (>1 Mb) deletions and duplications and for deletions overlapping different gene sets. RESULTS: Large deletions were enriched in genetic generalized epilepsies (GGE) compared to controls. We also found enrichment of deletions in epilepsy genes and hotspots for GGE. We did not find truncating or functional variants that could have been unmasked by the deletions. We observed a double CNV hit in two patients. One patient also carried a de novo deletion in the 22q11.2 hotspot. INTERPRETATION: We could corroborate previous findings of an enrichment of large microdeletions and deletions in epilepsy genes in GGE. We could also replicate that microdeletions show incomplete penetrance. However, we could not validate the hypothesis of unmasked variants nor the hypothesis of double CNVs to explain the incomplete penetrance. We found a de novo CNV on 22q11.2 that could be of interest. We also observed GGE families carrying a deletion on 15q13.3 hotspot that could be investigated in the Quebec founder population.

The role of common genetic variation in presumed monogenic epilepsies.

BACKGROUND: The developmental and epileptic encephalopathies (DEEs) are the most severe group of epilepsies which co-present with developmental delay and intellectual disability (ID). DEEs usually occur in people without a family history of epilepsy and have emerged as primarily monogenic, with damaging rare mutations found in 50% of patients. Little is known about the genetic architecture of patients with DEEs in whom no pathogenic variant is identified. Polygenic risk scoring (PRS) is a method that measures a person's common genetic burden for a trait or condition. Here, we used PRS to test whether genetic burden for epilepsy is relevant in individuals with DEEs, and other forms of epilepsy with ID. METHODS: Genetic data on 2,759 cases with DEEs, or epilepsy with ID presumed to have a monogenic basis, and 447,760 population-matched controls were analysed. We compared PRS for 'all epilepsy', 'focal epilepsy', and 'genetic generalised epilepsy' (GGE) between cases and controls. We performed pairwise comparisons between cases stratified for identifiable rare deleterious genetic variants and controls. FINDINGS: Cases of presumed monogenic severe epilepsy had an increased PRS for 'all epilepsy' (p<0.0001), 'focal epilepsy' (p<0.0001), and 'GGE' (p=0.0002) relative to controls, which explain between 0.08% and 3.3% of phenotypic variance. PRS was increased in cases both with and without an identified deleterious variant of major effect, and there was no significant difference in PRS between the two groups. INTERPRETATION: We provide evidence that common genetic variation contributes to the aetiology of DEEs and other forms of epilepsy with ID, even when there is a known pathogenic variant of major effect. These results provide insight into the genetic underpinnings of the severe epilepsies and warrant a shift in our understanding of the aetiology of the DEEs as complex, rather than monogenic, disorders. FUNDING: Science foundation Ireland, Human Genome Research Institute; National Heart, Lung, and Blood Institute; German Research Foundation.

Sensory processing dysregulations as reliable translational biomarkers in SYNGAP1 haploinsufficiency.

Amongst the numerous genes associated with intellectual disability, SYNGAP1 stands out for its frequency and penetrance of loss-of-function variants found in patients, as well as the wide range of co-morbid disorders associated with its mutation. Most studies exploring the pathophysiological alterations caused by Syngap1 haploinsufficiency in mouse models have focused on cognitive problems and epilepsy; however, whether and to what extent sensory perception and processing are altered by Syngap1 haploinsufficiency is less clear. By performing EEG recordings in awake mice, we identified specific alterations in multiple aspects of auditory and visual processing, including increased baseline gamma oscillation power, increased theta/gamma phase amplitude coupling following stimulus presentation and abnormal neural entrainment in response to different sensory modality-specific frequencies. We also report lack of habituation to repetitive auditory stimuli and abnormal deviant sound detection. Interestingly, we found that most of these alterations are present in human patients as well, thus making them strong candidates as translational biomarkers of sensory-processing alterations associated with SYNGAP1/Syngap1 haploinsufficiency.

De novo coding variants in the AGO1 gene cause a neurodevelopmental disorder with intellectual disability.

BACKGROUND: High-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD). METHODS: This study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28). RESULTS: A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations. CONCLUSION: Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2-related NDD.

Variant-specific effects define the phenotypic spectrum of HNRNPH2-associated neurodevelopmental disorders in males.

Bain type of X-linked syndromic intellectual developmental disorder, caused by pathogenic missense variants in HRNRPH2, was initially described in six female individuals affected by moderate-to-severe neurodevelopmental delay. Although it was initially postulated that the condition would not be compatible with life in males, several affected male individuals harboring pathogenic variants in HNRNPH2 have since been documented. However, functional in-vitro analyses of identified variants have not been performed and, therefore, possible genotype-phenotype correlations remain elusive. Here, we present eight male individuals, including a pair of monozygotic twins, harboring pathogenic or likely pathogenic HNRNPH2 variants. Notably, we present the first individuals harboring nonsense or frameshift variants who, similarly to an individual harboring a de novo p.(Arg29Cys) variant within the first quasi-RNA-recognition motif (qRRM), displayed mild developmental delay, and developed mostly autistic features and/or psychiatric co-morbidities. Additionally, we present two individuals harboring a recurrent de novo p.(Arg114Trp), within the second qRRM, who had a severe neurodevelopmental delay with seizures. Functional characterization of the three most common HNRNPH2 missense variants revealed dysfunctional nucleocytoplasmic shuttling of proteins harboring the p.(Arg206Gln) and p.(Pro209Leu) variants, located within the nuclear localization signal, whereas proteins with p.(Arg114Trp) showed reduced interaction with members of the large assembly of splicing regulators (LASR). Moreover, RNA-sequencing of primary fibroblasts of the individual harboring the p.(Arg114Trp) revealed substantial alterations in the regulation of alternative splicing along with global transcriptome changes. Thus, we further expand the clinical and variant spectrum in HNRNPH2-associated disease in males and provide novel molecular insights suggesting the disorder to be a spliceopathy on the molecular level.

Ectopic expression of Irx3 and Irx5 in the paraventricular nucleus of the hypothalamus contributes to defects in Sim1 haploinsufficiency.

The paraventricular nucleus of the hypothalamus (PVH) contains a heterogeneous cluster of Sim1-expressing neurons critical for feeding regulation. Sim1 haploinsufficiency results in hyperphagic obesity with disruption of PVH neurons, yet the molecular profiles of PVH neurons and the mechanism underlying the defects of Sim1 haploinsufficiency are not well understood. By single-cell RNA sequencing, we identified two major populations of Sim1+ PVH neurons, which are differentially affected by Sim1 haploinsufficiency. The Iroquois homeobox genes Irx3 and Irx5 have been implicated in the hypothalamic control of energy homeostasis. We found that Irx3 and Irx5 are ectopically expressed in the Sim1+ PVH cells of Sim1+/− mice. By reducing their dosage and PVH-specific deletion of Irx3, we demonstrate that misexpression of Irx3 and Irx5 contributes to the defects of Sim1+/− mice. Our results illustrate abnormal hypothalamic activities of Irx3 and Irx5 as a central mechanism disrupting PVH development and feeding regulation in Sim1 haploinsufficiency.

Differential auditory brain response abnormalities in two intellectual disability conditions: SYNGAP1 mutations and Down syndrome.

OBJECTIVE: Altered sensory processing is common in intellectual disability (ID). Here, we study electroencephalographic responses to auditory stimulation in human subjects presenting a rare condition (mutations in SYNGAP1) which causes ID, epilepsy and autism. METHODS: Auditory evoked potentials, time-frequency and inter-trial coherence analyses were used to compare subjects with SYNGAP1 mutations with Down syndrome (DS) and neurotypical (NT) participants (N = 61 ranging from three to 19 years of age). RESULTS: Altered synchronization in the brain responses to sound were found in both ID groups. The SYNGAP1 mutations group showed less phase-locking in early time windows and lower frequency bands compared to NT, and in later time windows compared to NT and DS. Time-frequency analysis showed more power in beta-gamma in the SYNGAP1 group compared to NT participants. CONCLUSIONS: This study indicated reduced synchronization as well as more high frequencies power in SYNGAP1 mutations, while maintained synchronization was found in the DS group. These results might reflect dysfunctional sensory information processing caused by excitation/inhibition imbalance, or an imperfect compensatory mechanism in SYNGAP1 mutations individuals. SIGNIFICANCE: Our study is the first to reveal brain response abnormalities in auditory sensory processing in SYNGAP1 mutations individuals, that are distinct from DS, another ID condition.

FBXO28 causes developmental and epileptic encephalopathy with profound intellectual disability.

Chromosome 1q41-q42 deletion syndrome is a rare cause of intellectual disability, seizures, dysmorphology, and multiple anomalies. Two genes in the 1q41-q42 microdeletion, WDR26 and FBXO28, have been implicated in monogenic disease. Patients with WDR26 encephalopathy overlap clinically with those with 1q41-q42 deletion syndrome, whereas only one patient with FBXO28 encephalopathy has been described. Seizures are a prominent feature of 1q41-q42 deletion syndrome; therefore, we hypothesized that pathogenic FBXO28 variants cause developmental and epileptic encephalopathies (DEEs). We describe nine new patients with FBXO28 pathogenic variants (four missense, including one recurrent, three nonsense, and one frameshift) and analyze all 10 known cases to delineate the phenotypic spectrum. All patients had epilepsy and 9 of 10 had DEE, including infantile spasms (3) and a progressive myoclonic epilepsy (1). Median age at seizure onset was 22.5 months (range 8 months to 5 years). Nine of 10 patients had intellectual disability, which was profound in six of nine and severe in three of nine. Movement disorders occurred in eight of 10 patients, six of 10 had hypotonia, four of 10 had acquired microcephaly, and five of 10 had dysmorphic features, albeit different to those typically seen in 1q41-q42 deletion syndrome and WDR26 encephalopathy. We distinguish FBXO28 encephalopathy from both of these disorders with more severe intellectual impairment, drug-resistant epilepsy, and hyperkinetic movement disorders.

A framework for an evidence-based gene list relevant to autism spectrum disorder.

Autism spectrum disorder (ASD) is often grouped with other brain-related phenotypes into a broader category of neurodevelopmental disorders (NDDs). In clinical practice, providers need to decide which genes to test in individuals with ASD phenotypes, which requires an understanding of the level of evidence for individual NDD genes that supports an association with ASD. Consensus is currently lacking about which NDD genes have sufficient evidence to support a relationship to ASD. Estimates of the number of genes relevant to ASD differ greatly among research groups and clinical sequencing panels, varying from a few to several hundred. This Roadmap discusses important considerations necessary to provide an evidence-based framework for the curation of NDD genes based on the level of information supporting a clinically relevant relationship between a given gene and ASD.

A variant of neonatal progeroid syndrome, or Wiedemann-Rautenstrauch syndrome, is associated with a nonsense variant in POLR3GL.

Neonatal progeroid syndrome, also known as Wiedemann-Rautenstrauch syndrome, is a rare condition characterized by severe growth retardation, apparent macrocephaly with prominent scalp veins, and lipodystrophy. It is caused by biallelic variants in POLR3A, a gene encoding for a subunit of RNA polymerase III. All variants reported in the literature lead to at least a partial loss-of-function (when considering both alleles together). Here, we describe an individual with several clinical features of neonatal progeroid syndrome in whom exome sequencing revealed a homozygous nonsense variant in POLR3GL (NM_032305.2:c.358C>T; p.(Arg120Ter)). POLR3GL also encodes a subunit of RNA polymerase III and has recently been associated with endosteal hyperostosis and oligodontia in three patients with a phenotype distinct from the patient described here. Given the important role of POLR3GL in the same complex as the protein implicated in neonatal progeroid syndrome, the nature of the variant identified, our RNA studies suggesting nonsense-mediated decay, and the clinical overlap, we propose POLR3GL as a gene causing a variant of neonatal progeroid syndrome and therefore expand the phenotype associated with POLR3GL variants.

Association of rare non-coding SNVs in the lung-specific FOXF1 enhancer with a mitigation of the lethal ACDMPV phenotype.

Haploinsufficiency of FOXF1 causes alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a lethal neonatal lung developmental disorder. We describe two similar heterozygous CNV deletions involving the FOXF1 enhancer and re-analyze FOXF1 missense mutation, all associated with an unexpectedly mitigated disease phenotype. In one case, the deletion of the maternal allele of the FOXF1 enhancer caused pulmonary hypertension and histopathologically diagnosed MPV without the typical ACD features. In the second case, the deletion of the paternal enhancer resulted in ACDMPV rather than the expected neonatal lethality. In both cases, FOXF1 expression in lung tissue was higher than usually seen or expected in patients with similar deletions, suggesting an increased activity of the remaining allele of the enhancer. Sequencing of these alleles revealed two rare SNVs, rs150502618-A and rs79301423-T, mapping to the partially overlapping binding sites for TFAP2s and CTCF in the core region of the enhancer. Moreover, in a family with three histopathologically-diagnosed ACDMPV siblings whose missense FOXF1 mutation was inherited from the healthy non-mosaic carrier mother, we have identified a rare SNV rs28571077-A within 2-kb of the above-mentioned non-coding SNVs in the FOXF1 enhancer in the mother, that was absent in the affected newborns and 13 unrelated ACDMPV patients with CNV deletions of this genomic region. Based on the low population frequencies of these three variants, their absence in ACDMPV patients, the results of reporter assay, RNAi and EMSA experiments, and in silico predictions, we propose that the described SNVs might have acted on FOXF1 enhancer as hypermorphs.

Biallelic variants in the transcription factor PAX7 are a new genetic cause of myopathy.

PURPOSE: Skeletal muscle growth and regeneration rely on muscle stem cells, called satellite cells. Specific transcription factors, particularly PAX7, are key regulators of the function of these cells. Knockout of this factor in mice leads to poor postnatal survival; however, the consequences of a lack of PAX7 in humans have not been established. METHODS: Here, we study five individuals with myopathy of variable severity from four unrelated consanguineous couples. Exome sequencing identified pathogenic variants in the PAX7 gene. Clinical examination, laboratory tests, and muscle biopsies were performed to characterize the disease. RESULTS: The disease was characterized by hypotonia, ptosis, muscular atrophy, scoliosis, and mildly dysmorphic facial features. The disease spectrum ranged from mild to severe and appears to be progressive. Muscle biopsies showed the presence of atrophic fibers and fibroadipose tissue replacement, with the absence of myofiber necrosis. A lack of PAX7 expression was associated with satellite cell pool exhaustion; however, the presence of residual myoblasts together with regenerating myofibers suggest that a population of PAX7-independent myogenic cells partially contributes to muscle regeneration. CONCLUSION: These findings show that biallelic variants in the master transcription factor PAX7 cause a new type of myopathy that specifically affects satellite cell survival.

Correction: IQSEC2-related encephalopathy in males and females: a comparative study including 37 novel patients.

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IQSEC2-related encephalopathy in males and females: a comparative study including 37 novel patients.

PURPOSE: Variants in IQSEC2, escaping X inactivation, cause X-linked intellectual disability with frequent epilepsy in males and females. We aimed to investigate sex-specific differences. METHODS: We collected the data of 37 unpublished patients (18 males and 19 females) with IQSEC2 pathogenic variants and 5 individuals with variants of unknown significance and reviewed published variants. We compared variant types and phenotypes in males and females and performed an analysis of IQSEC2 isoforms. RESULTS: IQSEC2 pathogenic variants mainly led to premature truncation and were scattered throughout the longest brain-specific isoform, encoding the synaptic IQSEC2/BRAG1 protein. Variants occurred de novo in females but were either de novo (2/3) or inherited (1/3) in males, with missense variants being predominantly inherited. Developmental delay and intellectual disability were overall more severe in males than in females. Likewise, seizures were more frequently observed and intractable, and started earlier in males than in females. No correlation was observed between the age at seizure onset and severity of intellectual disability or resistance to antiepileptic treatments. CONCLUSION: This study provides a comprehensive overview of IQSEC2-related encephalopathy in males and females, and suggests that an accurate dosage of IQSEC2 at the synapse is crucial during normal brain development.