ABSTRACT
The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important pathogenic DNA mutation signature in genomic disorders and cancer genomes, its architecture remains unresolved. Here, we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the DNA of 24 patients identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted structural variant (SV) haplotypes. Using a combination of short-read genome sequencing (GS), long-read GS, optical genome mapping, and single-cell DNA template strand sequencing (strand-seq), the haplotype structure was resolved in 18 samples. The point of template switching in 4 samples was shown to be a segment of â¼2.2-5.5 kb of 100% nucleotide similarity within inverted repeat pairs. These data provide experimental evidence that inverted low-copy repeats act as recombinant substrates. This type of CGR can result in multiple conformers generating diverse SV haplotypes in susceptible dosage-sensitive loci.
Subject(s)
Haplotypes , Humans , Haplotypes/genetics , Comparative Genomic Hybridization , Genomic Structural Variation/genetics , Genome, Human/genetics , Gene Duplication/geneticsABSTRACT
Both long-read genome sequencing (lrGS) and the recently published Telomere to Telomere (T2T) reference genome provide increased coverage and resolution across repetitive regions promising heightened structural variant detection and improved mapping. Inversions (INV), intrachromosomal segments which are rotated 180° and inserted back into the same chromosome, are a class of structural variants particularly challenging to detect due to their copy-number neutral state and association with repetitive regions. Inversions represent about 1/20 of all balanced structural chromosome aberrations and can lead to disease by gene disruption or altering regulatory regions of dosage sensitive genes in cis . Here we remapped the genome data from six individuals carrying unsolved cytogenetically detected inversions. An INV6 and INV10 were resolved using GRCh38 and T2T-CHM13. Finally, an INV9 required optical genome mapping, de novo assembly of lrGS data and T2T-CHM13. This inversion disrupted intron 25 of EHMT1, confirming a diagnosis of Kleefstra syndrome 1 (MIM#610253). These three inversions, only mappable in specific references, prompted us to investigate the presence and population frequencies of differential reference regions (DRRs) between T2T-CHM13, GRCh37, GRCh38, the chimpanzee and bonobo, and hundreds of megabases of DRRs were identified. Our results emphasize the significance of the chosen reference genome and the added benefits of lrGS and optical genome mapping in solving rearrangements in challenging regions of the genome. This is particularly important for inversions and may impact clinical diagnostics.
ABSTRACT
The detection, validation, and subsequent interpretation of potentially mosaic single-nucleotide variants (SNV) within next-generation sequencing data remains a challenge in both research and clinical laboratory settings. The ability to identify mosaic variants in high genome coverage sequencing data at levels of ≤1% underscores the necessity for developing guidelines and best practices to verify these variants orthogonally. Droplet digital PCR (ddPCR) has proven to be a powerful and precise method that allows for the determination of low-level variant fractions within a given sample. Herein we describe two precise ddPCR methods using either a fluorescent TaqMan hydrolysis probe approach or an EvaGreen fluorescent dye protocol. The TaqMan approach relies on two different fluorescent probes (FAM and HEX/VIC), each designed to amplify selectively only in the presence of a single nucleotide change denoting the variant or reference position. The fractional abundance is then calculated to determine the relative quantities of both alleles in the final sample. The EvaGreen protocol relies on two independent reactions with oligonucleotide primers designed with the single nucleotide change denoting the variant at the penultimate position of the primer. The relative amplification efficiency of both primer sets (reference and variant) can be compared to determine the mosaic level of a given variant. As the cost of high-coverage sequencing continues to decrease, the identification of potentially mosaic variants will also increase. The approaches outlined will allow clinicians and researchers a more precise determination of the true mosaic level of a given variant allowing them to better assess not only its potential pathogenicity but also its possible recurrence risk when offering genetic counseling to families. © 2024 Wiley Periodicals LLC. Basic Protocol: Droplet digital PCR (ddPCR) with TaqMan hydrolysis probes Alternate Protocol: EvaGreen oligonucleotide-specific ddPCR.
Subject(s)
Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Polymorphism, Single Nucleotide/genetics , Humans , Polymerase Chain Reaction/methods , Mosaicism , Fluorescent Dyes/chemistry , High-Throughput Nucleotide Sequencing/methodsABSTRACT
BACKGROUND: NODAL signaling plays a critical role in embryonic patterning and heart development in vertebrates. Genetic variants resulting in perturbations of the TGF-ß/NODAL signaling pathway have reproducibly been shown to cause laterality defects in humans. To further explore this association and improve genetic diagnosis, the study aims to identify and characterize a broader range of NODAL variants in a large number of individuals with laterality defects. METHODS: We re-analyzed a cohort of 321 proband-only exomes of individuals with clinically diagnosed laterality congenital heart disease (CHD) using family-based, rare variant genomic analyses. To this cohort we added 12 affected subjects with known NODAL variants and CHD from institutional research and clinical cohorts to investigate an allelic series. For those with candidate contributory variants, variant allele confirmation and segregation analysis were studied by Sanger sequencing in available family members. Array comparative genomic hybridization and droplet digital PCR were utilized for copy number variants (CNV) validation and characterization. We performed Human Phenotype Ontology (HPO)-based quantitative phenotypic analyses to dissect allele-specific phenotypic differences. RESULTS: Missense, nonsense, splice site, indels, and/or structural variants of NODAL were identified as potential causes of heterotaxy and other laterality defects in 33 CHD cases. We describe a recurrent complex indel variant for which the nucleic acid secondary structure predictions implicate secondary structure mutagenesis as a possible mechanism for formation. We identified two CNV deletion alleles spanning NODAL in two unrelated CHD cases. Furthermore, 17 CHD individuals were found (16/17 with known Hispanic ancestry) to have the c.778G > A:p.G260R NODAL missense variant which we propose reclassification from variant of uncertain significance (VUS) to likely pathogenic. Quantitative HPO-based analyses of the observed clinical phenotype for all cases with p.G260R variation, including heterozygous, homozygous, and compound heterozygous cases, reveal clustering of individuals with biallelic variation. This finding provides evidence for a genotypic-phenotypic correlation and an allele-specific gene dosage model. CONCLUSION: Our data further support a role for rare deleterious variants in NODAL as a cause for sporadic human laterality defects, expand the repertoire of observed anatomical complexity of potential cardiovascular anomalies, and implicate an allele specific gene dosage model.
Subject(s)
Heart Defects, Congenital , Heterotaxy Syndrome , Transposition of Great Vessels , Animals , Humans , Arteries , Comparative Genomic Hybridization , Heart Defects, Congenital/genetics , Heterotaxy Syndrome/genetics , PhenotypeABSTRACT
Repeated sequences spread throughout the genome play important roles in shaping the structure of chromosomes and facilitating the generation of new genomic variation. Through a variety of mechanisms, repeats are involved in generating structural rearrangements such as deletions, duplications, inversions, and translocations, which can have the potential to impact human health. Despite their significance, repetitive regions including tandem repeats, transposable elements, segmental duplications, and low-copy repeats remain a challenge to characterize due to technological limitations inherent to many sequencing methodologies. We performed genome-wide analyses and comparisons of direct and inverted repeated sequences in the latest available human genome reference assemblies including GRCh37 and GRCh38 and the most recent telomere-to-telomere alternate assembly (T2T-CHM13). Overall, the composition and distribution of direct and inverted repeats identified remains similar among the three assemblies but we observed an increase in the number of repeated sequences detected in the T2T-CHM13 assembly versus the reference assemblies. As expected, there is an enrichment of repetitive regions in the short arms of acrocentric chromosomes, which had been previously unresolved in the human genome reference assemblies. We cross-referenced the identified repeats with protein-coding genes across the genome to identify those at risk for being involved in genomic disorders. We observed that certain gene categories, such as olfactory receptors and immune response genes, are enriched among those impacted by repeated sequences likely contributing to human diversity and adaptation. Through this analysis, we have produced a catalogue of direct and inversely oriented repeated sequences across the currently three most widely used human genome assemblies. Bioinformatic analyses of these repeats and their contribution to genome architecture can reveal regions that are most susceptible to genomic instability. Understanding how the architectural genomic features of repeat pairs such as their homology, size and distance can lead to complex genomic rearrangement formation can provide further insights into the molecular mechanisms leading to genomic disorders and genome evolution. Author summary: This study focused on the characterization of intrachromosomal repeated sequences in the human genome that can play important roles in shaping chromosome structure and generating new genomic variation in three human genome assemblies. We observed an increase in the number of repeated sequence pairs detected in the most recent telomere-to-telomere alternate assembly (T2T-CHM13) compared to the reference assemblies (GRCh37 and GRCh38). We observed an enrichment of repeats in the T2T-CHM13 acrocentric chromosomes, which had been previously unresolved. Importantly, our study provides a catalogue of direct and inverted repeated sequences across three commonly used human genome assemblies, which can aid in the understanding of genomic architecture instability, evolution, and disorders. Our analyses provide insights into repetitive regions in the human genome that may contribute to complex genomic rearrangements.
ABSTRACT
Calling structural variations (SVs) is technically challenging, but using long reads remains the most accurate way to identify complex genomic alterations. Here we present Sniffles2, which improves over current methods by implementing a repeat aware clustering coupled with a fast consensus sequence and coverage-adaptive filtering. Sniffles2 is 11.8 times faster and 29% more accurate than state-of-the-art SV callers across different coverages (5-50×), sequencing technologies (ONT and HiFi) and SV types. Furthermore, Sniffles2 solves the problem of family-level to population-level SV calling to produce fully genotyped VCF files. Across 11 probands, we accurately identified causative SVs around MECP2, including highly complex alleles with three overlapping SVs. Sniffles2 also enables the detection of mosaic SVs in bulk long-read data. As a result, we identified multiple mosaic SVs in brain tissue from a patient with multiple system atrophy. The identified SV showed a remarkable diversity within the cingulate cortex, impacting both genes involved in neuron function and repetitive elements.
ABSTRACT
PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.
Subject(s)
Intellectual Disability , Neurodevelopmental Disorders , Animals , Humans , Mice , Bicarbonates/metabolism , Chloride-Bicarbonate Antiporters/metabolism , Intellectual Disability/genetics , Membrane Transport Proteins , Mice, Knockout , Neurodevelopmental Disorders/genetics , Sodium/metabolism , Sodium Bicarbonate/metabolism , Sodium-Bicarbonate Symporters/geneticsABSTRACT
Homozygous duplications contribute to genetic disease by altering gene dosage or disrupting gene regulation and can be more deleterious to organismal biology than heterozygous duplications. Intragenic exonic duplications can result in loss-of-function (LoF) or gain-of-function (GoF) alleles that when homozygosed, i.e. brought to homozygous state at a locus by identity by descent or state, could potentially result in autosomal recessive (AR) rare disease traits. However, the detection and functional interpretation of homozygous duplications from exome sequencing data remains a challenge. We developed a framework algorithm, HMZDupFinder, that is designed to detect exonic homozygous duplications from exome sequencing (ES) data. The HMZDupFinder algorithm can efficiently process large datasets and accurately identifies small intragenic duplications, including those associated with rare disease traits. HMZDupFinder called 965 homozygous duplications with three or less exons from 8,707 ES with a recall rate of 70.9% and a precision of 16.1%. We experimentally confirmed 8/10 rare homozygous duplications. Pathogenicity assessment of these copy number variant alleles allowed clinical genomics contextualization for three homozygous duplications alleles, including two affecting known OMIM disease genes EDAR (MIM# 224900), TNNT1(MIM# 605355), and one variant in a novel candidate disease gene: PAAF1.
Subject(s)
DNA Copy Number Variations , Exome Sequencing , Software , Humans , Adaptor Proteins, Signal Transducing , Homozygote , Rare Diseases/geneticsABSTRACT
Background: The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a type of complex genomic rearrangement (CGR) hypothesized to result from replicative repair of DNA due to replication fork collapse. It is often mediated by a pair of inverted low-copy repeats (LCR) followed by iterative template switches resulting in at least two breakpoint junctions in cis . Although it has been identified as an important mutation signature of pathogenicity for genomic disorders and cancer genomes, its architecture remains unresolved and is predicted to display at least four structural variation (SV) haplotypes. Results: Here we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the genomic DNA of 24 patients with neurodevelopmental disorders identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted SV haplotypes. Using a combination of short-read genome sequencing (GS), long- read GS, optical genome mapping and StrandSeq the haplotype structure was resolved in 18 samples. This approach refined the point of template switching between inverted LCRs in 4 samples revealing a DNA segment of â¼2.2-5.5 kb of 100% nucleotide similarity. A prediction model was developed to infer the LCR used to mediate the non-allelic homology repair. Conclusions: These data provide experimental evidence supporting the hypothesis that inverted LCRs act as a recombinant substrate in replication-based repair mechanisms. Such inverted repeats are particularly relevant for formation of copy-number associated inversions, including the DUP-TRP/INV-DUP structures. Moreover, this type of CGR can result in multiple conformers which contributes to generate diverse SV haplotypes in susceptible loci .
ABSTRACT
Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by aplasia of the female reproductive tract; the syndrome can include renal anomalies, absence or dysgenesis, and skeletal anomalies. While functional models have elucidated several candidate genes, only WNT4 (MIM: 603490) variants have been definitively associated with a subtype of MRKH with hyperandrogenism (MIM: 158330). DNA from 148 clinically diagnosed MRKH probands across 144 unrelated families and available family members from North America, Europe, and South America were exome sequenced (ES) and by family-based genomics analyzed for rare likely deleterious variants. A replication cohort consisting of 442 Han Chinese individuals with MRKH was used to further reproduce GREB1L findings in diverse genetic backgrounds. Proband and OMIM phenotypes annotated using the Human Phenotype Ontology were analyzed to quantitatively delineate the phenotypic spectrum associated with GREB1L variant alleles found in our MRKH cohort and those previously published. This study reports 18 novel GREB1L variant alleles, 16 within a multiethnic MRKH cohort and two within a congenital scoliosis cohort. Cohort-wide analyses for a burden of rare variants within a single gene identified likely damaging variants in GREB1L (MIM: 617782), a known disease gene for renal hypoplasia and uterine abnormalities (MIM: 617805), in 16 of 590 MRKH probands. GREB1L variant alleles, including a CNV null allele, were found in 8 MRKH type 1 probands and 8 MRKH type II probands. This study used quantitative phenotypic analyses in a worldwide multiethnic cohort to identify and strengthen the association of GREB1L to isolated uterine agenesis (MRKH type I) and syndromic MRKH type II.
Subject(s)
46, XX Disorders of Sex Development , Urogenital Abnormalities , Female , Humans , 46, XX Disorders of Sex Development/genetics , Uterus/abnormalitiesABSTRACT
Protein phosphatase 1 regulatory subunit 35 (PPP1R35) encodes a centrosomal protein required for recruiting microtubule-binding elongation machinery. Several proteins in this centriole biogenesis pathway correspond to established primary microcephaly (MCPH) genes, and multiple model organism studies hypothesize PPP1R35 as a candidate MCPH gene. Here, using exome sequencing (ES) and family-based rare variant analyses, we report a homozygous, frameshifting indel deleting the canonical stop codon in the last exon of PPP1R35 [Chr7: c.753_*3delGGAAGCGTAGACCinsCG (p.Trp251Cysfs*22)]; the variant allele maps in a 3.7 Mb block of absence of heterozygosity (AOH) in a proband with severe MCPH (-4.3 SD at birth, -6.1 SD by 42 months), pachygyria, and global developmental delay from a consanguineous Turkish kindred. Droplet digital PCR (ddPCR) confirmed mutant mRNA expression in fibroblasts. In silico prediction of the translation of mutant PPP1R35 is expected to be elongated by 18 amino acids before encountering a downstream stop codon. This complex indel allele is absent in public databases (ClinVar, gnomAD, ARIC, 1000 genomes) and our in-house database of 14,000+ exomes including 1800+ Turkish exomes supporting predicted pathogenicity. Comprehensive literature searches for PPP1R35 variants yielded two probands affected with severe microcephaly (-15 SD and -12 SD) with the same homozygous indel from a single, consanguineous, Iranian family from a cohort of 404 predominantly Iranian families. The lack of heterozygous cases in two large cohorts representative of the genetic background of these two families decreased our suspicion of a founder allele and supports the contention of a recurrent mutation. We propose two potential secondary structure mutagenesis models for the origin of this variant allele mediated by hairpin formation between complementary GC rich segments flanking the stop codon via secondary structure mutagenesis.
Subject(s)
Microcephaly , Infant, Newborn , Humans , Microcephaly/genetics , Codon, Terminator , Iran , Microtubule-Associated Proteins/genetics , Frameshift Mutation/genetics , PedigreeABSTRACT
BACKGROUND: The multiple de novo copy number variant (MdnCNV) phenotype is described by having four or more constitutional de novo CNVs (dnCNVs) arising independently throughout the human genome within one generation. It is a rare peri-zygotic mutational event, previously reported to be seen once in every 12,000 individuals referred for genome-wide chromosomal microarray analysis due to congenital abnormalities. These rare families provide a unique opportunity to understand the genetic factors of peri-zygotic genome instability and the impact of dnCNV on human diseases. METHODS: Chromosomal microarray analysis (CMA), array-based comparative genomic hybridization, short- and long-read genome sequencing (GS) were performed on the newly identified MdnCNV family to identify de novo mutations including dnCNVs, de novo single-nucleotide variants (dnSNVs), and indels. Short-read GS was performed on four previously published MdnCNV families for dnSNV analysis. Trio-based rare variant analysis was performed on the newly identified individual and four previously published MdnCNV families to identify potential genetic etiologies contributing to the peri-zygotic genomic instability. Lin semantic similarity scores informed quantitative human phenotype ontology analysis on three MdnCNV families to identify gene(s) driving or contributing to the clinical phenotype. RESULTS: In the newly identified MdnCNV case, we revealed eight de novo tandem duplications, each ~ 1 Mb, with microhomology at 6/8 breakpoint junctions. Enrichment of de novo single-nucleotide variants (SNV; 6/79) and de novo indels (1/12) was found within 4 Mb of the dnCNV genomic regions. An elevated post-zygotic SNV mutation rate was observed in MdnCNV families. Maternal rare variant analyses identified three genes in distinct families that may contribute to the MdnCNV phenomenon. Phenotype analysis suggests that gene(s) within dnCNV regions contribute to the observed proband phenotype in 3/3 cases. CNVs in two cases, a contiguous gene duplication encompassing PMP22 and RAI1 and another duplication affecting NSD1 and SMARCC2, contribute to the clinically observed phenotypic manifestations. CONCLUSIONS: Characteristic features of dnCNVs reported here are consistent with a microhomology-mediated break-induced replication (MMBIR)-driven mechanism during the peri-zygotic period. Maternal genetic variants in DNA repair genes potentially contribute to peri-zygotic genomic instability. Variable phenotypic features were observed across a cohort of three MdnCNV probands, and computational quantitative phenotyping revealed that two out of three had evidence for the contribution of more than one genetic locus to the proband's phenotype supporting the hypothesis of de novo multilocus pathogenic variation (MPV) in those families.
Subject(s)
DNA Copy Number Variations , Genomic Instability , Humans , Comparative Genomic Hybridization , Mutation , DNA , Nucleotides , DNA-Binding Proteins/genetics , Transcription Factors/geneticsABSTRACT
Xia-Gibbs syndrome (XGS; MIM# 615829) is a rare mendelian disorder characterized by Development Delay (DD), intellectual disability (ID), and hypotonia. Individuals with XGS typically harbor de novo protein-truncating mutations in the AT-Hook DNA binding motif containing 1 (AHDC1) gene, although some missense mutations can also cause XGS. Large de novo heterozygous deletions that encompass the AHDC1 gene have also been ascribed as diagnostic for the disorder, without substantial evidence to support their pathogenicity. We analyzed 19 individuals with large contiguous deletions involving AHDC1, along with other genes. One individual bore the smallest known contiguous AHDC1 deletion (â¼350 Kb), encompassing eight other genes within chr1p36.11 (Feline Gardner-Rasheed, IFI6, FAM76A, STX12, PPP1R8, THEMIS2, RPA2, SMPDL3B) and terminating within the first intron of AHDC1. The breakpoint junctions and phase of the deletion were identified using both short and long read sequencing (Oxford Nanopore). Quantification of RNA expression patterns in whole blood revealed that AHDC1 exhibited a mono-allelic expression pattern with no deficiency in overall AHDC1 expression levels, in contrast to the other deleted genes, which exhibited a 50% reduction in mRNA expression. These results suggest that AHDC1 expression in this individual is compensated by a novel regulatory mechanism and advances understanding of mutational and regulatory mechanisms in neurodevelopmental disorders.
Subject(s)
Abnormalities, Multiple , Intellectual Disability , Musculoskeletal Abnormalities , Neurodevelopmental Disorders , Humans , Abnormalities, Multiple/genetics , DNA-Binding Proteins/genetics , Endoribonucleases , Intellectual Disability/genetics , Neurodevelopmental Disorders/genetics , Phosphoprotein Phosphatases , Qa-SNARE Proteins , RNA-Binding Proteins , Sphingomyelin PhosphodiesteraseABSTRACT
Genetic heterogeneity, reduced penetrance, and variable expressivity, the latter including asymmetric body axis plane presentations, have all been described in families with congenital limb malformations (CLMs). Interfamilial and intrafamilial heterogeneity highlight the complexity of the underlying genetic pathogenesis of these developmental anomalies. Family-based genomics by exome sequencing (ES) and rare variant analyses combined with whole-genome array-based comparative genomic hybridization were implemented to investigate 18 families with limb birth defects. Eleven of 18 (61%) families revealed explanatory variants, including 7 single-nucleotide variant alleles and 3 copy number variants (CNVs), at previously reported "disease trait associated loci": BHLHA9, GLI3, HOXD cluster, HOXD13, NPR2, and WNT10B. Breakpoint junction analyses for all three CNV alleles revealed mutational signatures consistent with microhomology-mediated break-induced replication, a mechanism facilitated by Alu/Alu-mediated rearrangement. Homozygous duplication of BHLHA9 was observed in one Turkish kindred and represents a novel contributory genetic mechanism to Gollop-Wolfgang Complex (MIM: 228250), where triplication of the locus has been reported in one family from Japan (i.e., 4n = 2n + 2n versus 4n = 3n + 1n allelic configurations). Genes acting on limb patterning are sensitive to a gene dosage effect and are often associated with an allelic series. We extend an allele-specific gene dosage model to potentially assist, in an adjuvant way, interpretations of interconnections among an allelic series, clinical severity, and reduced penetrance of the BHLHA9-related CLM spectrum.
ABSTRACT
The leucine-rich glioma-inactivated (LGI) family consists of four highly conserved paralogous genes, LGI1-4, that are highly expressed in mammalian central and/or peripheral nervous systems. LGI1 antibodies are detected in subjects with autoimmune limbic encephalitis and peripheral nerve hyperexcitability syndromes (PNHSs) such as Isaacs and Morvan syndromes. Pathogenic variations of LGI1 and LGI4 are associated with neurological disorders as disease traits including familial temporal lobe epilepsy and neurogenic arthrogryposis multiplex congenita 1 with myelin defects, respectively. No human disease has been reported associated with either LGI2 or LGI3. We implemented exome sequencing and family-based genomics to identify individuals with deleterious variants in LGI3 and utilized GeneMatcher to connect practitioners and researchers worldwide to investigate the clinical and electrophysiological phenotype in affected subjects. We also generated Lgi3-null mice and performed peripheral nerve dissection and immunohistochemistry to examine the juxtaparanode LGI3 microarchitecture. As a result, we identified 16 individuals from eight unrelated families with loss-of-function (LoF) bi-allelic variants in LGI3. Deep phenotypic characterization showed LGI3 LoF causes a potentially clinically recognizable PNHS trait characterized by global developmental delay, intellectual disability, distal deformities with diminished reflexes, visible facial myokymia, and distinctive electromyographic features suggestive of motor nerve instability. Lgi3-null mice showed reduced and mis-localized Kv1 channel complexes in myelinated peripheral axons. Our data demonstrate bi-allelic LoF variants in LGI3 cause a clinically distinguishable disease trait of PNHS, most likely caused by disturbed Kv1 channel distribution in the absence of LGI3.
Subject(s)
Myokymia , Nerve Tissue Proteins , Animals , Autoantibodies , Axons , Genomics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mammals/genetics , Mice , Nerve Tissue Proteins/genetics , Phenotype , Reverse GeneticsABSTRACT
PURPOSE: We aimed to identify the underlying genetic cause for a novel form of distal arthrogryposis. METHODS: Rare variant family-based genomics, exome sequencing, and disease-specific panel sequencing were used to detect ADAMTS15 variants in affected individuals. Adamts15 expression was analyzed at the single-cell level during murine embryogenesis. Expression patterns were characterized using in situ hybridization and RNAscope. RESULTS: We identified homozygous rare variant alleles of ADAMTS15 in 5 affected individuals from 4 unrelated consanguineous families presenting with congenital flexion contractures of the interphalangeal joints and hypoplastic or absent palmar creases. Radiographic investigations showed physiological interphalangeal joint morphology. Additional features included knee, Achilles tendon, and toe contractures, spinal stiffness, scoliosis, and orthodontic abnormalities. Analysis of mouse whole-embryo single-cell sequencing data revealed a tightly regulated Adamts15 expression in the limb mesenchyme between embryonic stages E11.5 and E15.0. A perimuscular and peritendinous expression was evident in in situ hybridization in the developing mouse limb. In accordance, RNAscope analysis detected a significant coexpression with Osr1, but not with markers for skeletal muscle or joint formation. CONCLUSION: In aggregate, our findings provide evidence that rare biallelic recessive trait variants in ADAMTS15 cause a novel autosomal recessive connective tissue disorder, resulting in a distal arthrogryposis syndrome.
Subject(s)
Arthrogryposis , Contracture , ADAMTS Proteins , Animals , Arthrogryposis/genetics , Consanguinity , Contracture/genetics , Homozygote , Humans , Mice , Mutation , Pedigree , PhenotypeABSTRACT
Complex genomic rearrangements (CGRs) are known contributors to disease but are often missed during routine genetic screening. Identifying CGRs requires (i) identifying copy number variants (CNVs) concurrently with inversions, (ii) phasing multiple breakpoint junctions incis, as well as (iii) detecting and resolving structural variants (SVs) within repeats. We demonstrate how combining cytogenetics and new sequencing methodologies is being successfully applied to gain insights into the genomic architecture of CGRs. In addition, we review CGR patterns and molecular features revealed by studying constitutional genomic disorders. These data offer invaluable lessons to individuals interested in investigating CGRs, evaluating their clinical relevance and frequency, as well as assessing their impact(s) on rare genetic diseases.
Subject(s)
Genome, Human , Rare Diseases , DNA Copy Number Variations/genetics , Gene Rearrangement/genetics , Genome, Human/genetics , Genomics/methods , Humans , Rare Diseases/geneticsABSTRACT
Joubert syndrome (JS), a well-established ciliopathy, is characterized by the distinctive molar tooth sign on brain MRI, ataxia, and neurodevelopmental features. Other manifestations can include polydactyly, accessory frenula, renal, or liver disease. Here, we report individuals meeting criteria for JS with de novo heterozygous variants in SLC30A7 (Chr1p21.2). The first individual is a female with history of unilateral postaxial polydactyly, classic molar tooth sign on MRI, macrocephaly, ataxia, ocular motor apraxia, neurodevelopmental delay, and precocious puberty. Exome sequencing detected a de novo heterozygous missense variant in SLC30A7: NM_133496.5: c.407 T > C, (p.Val136Ala). The second individual had bilateral postaxial polydactyly, molar tooth sign, macrocephaly, developmental delay, and an extra oral frenulum. A de novo deletion-insertion variant in SLC30A7, c.490_491delinsAG (p.His164Ser) was found. Both de novo variants affect highly conserved residues. Variants were not identified in known Joubert genes for either case. SLC30A7 has not yet been associated with a human phenotype. The SLC30 family of zinc transporters, like SLC30A7, permit cellular efflux of zinc, and although it is expressed in the brain its functions remain unknown. Published data from proteomic studies support SLC30A7 interaction with TCTN3, another protein associated with JS. The potential involvement of such genes in primary cilia suggest a role in Sonic Hedgehog signaling. SLC30A7 is a candidate JS-associated gene. Future work could be directed toward further characterization of SLC30A7 variants and understanding its function.