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Background: Plexins are large transmembrane receptors for the semaphorin family of signalling proteins. Semaphorin-plexin signalling controls cellular interactions that are critical during development as well as in adult life stages. Nine plexin genes have been identified in humans, but despite the apparent importance of plexins in development, only biallelic PLXND1 and PLXNA1 variants have so far been associated with Mendelian genetic disease., Methods: Eight individuals from six families presented with a recessively inherited variable clinical condition, with core features of amelogenesis imperfecta (AI) and sensorineural hearing loss (SNHL), with variable intellectual disability. Probands were investigated by exome or genome sequencing. Common variants and those unlikely to affect function were excluded. Variants consistent with autosomal recessive inheritance were prioritised. Variant segregation analysis was performed by Sanger sequencing. RNA expression analysis was conducted in C57Bl6 mice., Results: Rare biallelic pathogenic variants in plexin B2 ( PLXNB2 ), a large transmembrane semaphorin receptor protein, were found to segregate with disease in all six families. The variants identified include missense, nonsense, splicing changes and a multiexon deletion. Plxnb2 expression was detected in differentiating ameloblasts., Conclusion: We identify rare biallelic pathogenic variants in PLXNB2 as a cause of a new autosomal recessive, phenotypically diverse syndrome with AI and SNHL as core features. Intellectual disability, ocular disease, ear developmental abnormalities and lymphoedema were also present in multiple cases. The variable syndromic human phenotype overlaps with that seen in Plxnb2 knockout mice, and, together with the rarity of human PLXNB2 variants, may explain why pathogenic variants in PLXNB2 have not been reported previously., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Background: Collagen XVII is most typically associated with human disease when biallelic COL17A1 variants (>230) cause junctional epidermolysis bullosa (JEB), a rare, genetically heterogeneous, mucocutaneous blistering disease with amelogenesis imperfecta (AI), a developmental enamel defect. Despite recognition that heterozygous carriers in JEB families can have AI, and that heterozygous COL17A1 variants also cause dominant corneal epithelial recurrent erosion dystrophy (ERED), the importance of heterozygous COL17A1 variants causing dominant non-syndromic AI is not widely recognised., Methods: Probands from an AI cohort were screened by single molecule molecular inversion probes or targeted hybridisation capture (both a custom panel and whole exome sequencing) for COL17A1 variants. Patient phenotypes were assessed by clinical examination and analyses of affected teeth., Results: Nineteen unrelated probands with isolated AI (no co-segregating features) had 17 heterozygous, potentially pathogenic COL17A1 variants, including missense, premature termination codons, frameshift and splice site variants in both the endo-domains and the ecto-domains of the protein. The AI phenotype was consistent with enamel of near normal thickness and variable focal hypoplasia with surface irregularities including pitting., Conclusion: These results indicate that COL17A1 variants are a frequent cause of dominantly inherited non-syndromic AI. Comparison of variants implicated in AI and JEB identifies similarities in type and distribution, with five identified in both conditions, one of which may also cause ERED. Increased availability of genetic testing means that more individuals will receive reports of heterozygous COL17A1 variants. We propose that patients with isolated AI or ERED, due to COL17A1 variants, should be considered as potential carriers for JEB and counselled accordingly, reflecting the importance of multidisciplinary care., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Background: Glioblastoma (GBM) brain tumors lacking IDH1 mutations (IDHwt) have the worst prognosis of all brain neoplasms. Patients receive surgery and chemoradiotherapy but tumors almost always fatally recur., Results: Using RNA sequencing data from 107 pairs of pre- and post-standard treatment locally recurrent IDHwt GBM tumors, we identify two responder subtypes based on longitudinal changes in gene expression. In two thirds of patients, a specific subset of genes is upregulated from primary to recurrence (Up responders), and in one third, the same genes are downregulated (Down responders), specifically in neoplastic cells. Characterization of the responder subtypes indicates subtype-specific adaptive treatment resistance mechanisms that are associated with distinct changes in the tumor microenvironment. In Up responders, recurrent tumors are enriched in quiescent proneural GBM stem cells and differentiated neoplastic cells, with increased interaction with the surrounding normal brain and neurotransmitter signaling, whereas Down responders commonly undergo mesenchymal transition. ChIP-sequencing data from longitudinal GBM tumors suggests that the observed transcriptional reprogramming could be driven by Polycomb-based chromatin remodeling rather than DNA methylation., Conclusions: We show that the responder subtype is cancer-cell intrinsic, recapitulated in in vitro GBM cell models, and influenced by the presence of the tumor microenvironment. Stratifying GBM tumors by responder subtype may lead to more effective treatment., (© 2024. The Author(s).)

Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAX Arg60Gln (Max ∗Arg60Gln ) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAX Arg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

D-type cyclins encode G1/S cell cycle checkpoint proteins, which play a crucial role in defining cell cycle exit and progression. Precise control of cell cycle exit is vital during embryonic development, with defects in the pathways regulating intracellular D-type cyclins resulting in abnormal initiation of stem cell differentiation in a variety of different organ systems. Furthermore, stabilisation of D-type cyclins is observed in a wide range of disorders characterized by cellular over-proliferation, including cancers and overgrowth disorders. In this review, we will summarize and compare the roles played by each D-type cyclin during development and provide examples of how their intracellular dysregulation can be an underlying cause of disease.

Purpose: Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) regulates cell growth in response to nutritional status. Central to the mTORC1 function is the Rag-GTPase heterodimer. One component of the Rag heterodimer is RagC (Ras-related GTP-binding protein C), which is encoded by the RRAGC gene., Methods: Genetic testing via trio exome sequencing was applied to identify the underlying disease cause in 3 infants with dilated cardiomyopathy, hepatopathy, and brain abnormalities, including pachygyria, polymicrogyria, and septo-optic dysplasia. Studies in patient-derived skin fibroblasts and in a HEK293 cell model were performed to investigate the cellular consequences., Results: We identified 3 de novo missense variants in RRAGC (NM_022157.4: c.269C>A, p.(Thr90Asn), c.353C>T, p.(Pro118Leu), and c.343T>C, p.(Trp115Arg)), which were previously reported as occurring somatically in follicular lymphoma. Studies of patient-derived fibroblasts carrying the p.(Thr90Asn) variant revealed increased cell size, as well as dysregulation of mTOR-related p70S6K (ribosomal protein S6 kinase 1) and transcription factor EB signaling. Moreover, subcellular localization of mTOR was decoupled from metabolic state. We confirmed the key findings for all RRAGC variants described in this study in a HEK293 cell model., Conclusion: The above results are in line with a constitutive overactivation of the mTORC1 pathway. Our study establishes de novo missense variants in RRAGC as cause of an early-onset mTORopathy with unfavorable prognosis., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The widespread adoption of exome sequencing has greatly increased the rate of genetic diagnosis for inherited conditions. However, the detection and validation of large deletions remains challenging. While numerous bioinformatics approaches have been developed to detect deletions from whole - exome sequencing and targeted panels, further work is typically required to define the physical breakpoints or integration sites. Accurate characterisation requires either expensive follow - up whole - genome sequencing or the time - consuming, laborious process of PCR walking, both of which are challenging when dealing with the repeat sequences which frequently intersect deletion breakpoints. The aim of this study was to develop a cost-effective, long-range sequencing method to characterise deletions., Methods: Genomic DNA was amplified with primers spanning the deletion using long-range PCR and the products purified. Sequencing was performed on MinION flongle flowcells. The resulting fast5 files were basecalled using Guppy, trimmed using Porechop and aligned using Minimap2. Filtering was performed using NanoFilt. Nanopore sequencing results were verified by Sanger sequencing., Results: Four cases with deletions detected following comparative read-depth analysis of targeted short-read sequencing were analysed. Nanopore sequencing defined breakpoints at the molecular level in all cases including homozygous breakpoints in EYS, CNGA1 and CNGB1 and a heterozygous deletion in PRPF31. All breakpoints were verified by Sanger sequencing., Conclusions: In this study, a quick, accurate and cost - effective method is described to characterise deletions identified from exome, and similar data, using nanopore sequencing., (© 2023 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing., Competing Interests: PS is an employee of Molecular Loop Biosciences Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Panneman, Hitti-Malin, Holtes, de Bruijn, Reurink, Boonen, Khan, Ali, Andréasson, De Baere, Banfi, Bauwens, Ben-Yosef, Bocquet, De Bruyne, Cerda, Coppieters, Farinelli, Guignard, Inglehearn, Karali, Kjellström, Koenekoop, de Koning, Leroy, McKibbin, Meunier, Nikopoulos, Nishiguchi, Poulter, Rivolta, Rodríguez de la Rúa, Saunders, Simonelli, Tatour, Testa, Thiadens, Toomes, Tracewska, Tran, Ushida, Vaclavik, Verhoeven, van de Vorst, Gilissen, Hoischen, Cremers and Roosing.)

Somatic mutations in UBA1 cause vacuoles, E1 ubiquitin-activating enzyme, X-linked, autoinflammatory somatic (VEXAS) syndrome, an adult-onset inflammatory disease with an overlap of hematologic manifestations. VEXAS syndrome is characterized by a high mortality rate and significant clinical heterogeneity. We sought to determine independent predictors of survival in VEXAS and to understand the mechanistic basis for these factors. We analyzed 83 patients with somatic pathogenic variants in UBA1 at p.Met41 (p.Met41Leu/Thr/Val), the start codon for translation of the cytoplasmic isoform of UBA1 (UBA1b). Patients with the p.Met41Val genotype were most likely to have an undifferentiated inflammatory syndrome. Multivariate analysis showed ear chondritis was associated with increased survival, whereas transfusion dependence and the p.Met41Val variant were independently associated with decreased survival. Using in vitro models and patient-derived cells, we demonstrate that p.Met41Val variant supports less UBA1b translation than either p.Met41Leu or p.Met41Thr, providing a molecular rationale for decreased survival. In addition, we show that these 3 canonical VEXAS variants produce more UBA1b than any of the 6 other possible single-nucleotide variants within this codon. Finally, we report a patient, clinically diagnosed with VEXAS syndrome, with 2 novel mutations in UBA1 occurring in cis on the same allele. One mutation (c.121 A>T; p.Met41Leu) caused severely reduced translation of UBA1b in a reporter assay, but coexpression with the second mutation (c.119 G>C; p.Gly40Ala) rescued UBA1b levels to those of canonical mutations. We conclude that regulation of residual UBA1b translation is fundamental to the pathogenesis of VEXAS syndrome and contributes to disease prognosis., (© 2022 by The American Society of Hematology.)

Background: Primary ciliopathies represent a group of inherited disorders due to defects in the primary cilium, the 'cell's antenna'. The 100,000 Genomes Project was launched in 2012 by Genomics England (GEL), recruiting National Health Service (NHS) patients with eligible rare diseases and cancer. Sequence data were linked to Human Phenotype Ontology (HPO) terms entered by recruiting clinicians., Methods: Eighty-three prescreened probands were recruited to the 100,000 Genomes Project suspected to have congenital malformations caused by ciliopathies in the following disease categories: Bardet-Biedl syndrome (n=45), Joubert syndrome (n=14) and 'Rare Multisystem Ciliopathy Disorders' (n=24). We implemented a bespoke variant filtering and analysis strategy to improve molecular diagnostic rates for these participants., Results: We determined a research molecular diagnosis for n=43/83 (51.8%) probands. This is 19.3% higher than previously reported by GEL (n=27/83 (32.5%)). A high proportion of diagnoses are due to variants in non-ciliopathy disease genes (n=19/43, 44.2%) which may reflect difficulties in clinical recognition of ciliopathies. n=11/83 probands (13.3%) had at least one causative variant outside the tiers 1 and 2 variant prioritisation categories (GEL's automated triaging procedure), which would not be reviewed in standard 100,000 Genomes Project diagnostic strategies. These include four structural variants and three predicted to cause non-canonical splicing defects. Two unrelated participants have biallelic likely pathogenic variants in LRRC45 , a putative novel ciliopathy disease gene., Conclusion: These data illustrate the power of linking large-scale genome sequence to phenotype information. They demonstrate the value of research collaborations in order to maximise interpretation of genomic data., Competing Interests: Competing interests: Disclosure: HB, RPJB and AS are employed by Genomics England, UK. GW is employed by Illumina. The other authors declare no conflict of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Leukodystrophies are a heterogenous group of genetic disorders, characterised by abnormal development of cerebral white matter. Pelizaeus-Merzbacher disease is caused by mutations in PLP1, encoding major myelin-resident protein required for myelin sheath assembly. We report a missense variant p.(Ala109Asp) in MAL as causative for a rare, hypomyelinating leukodystrophy similar to Pelizaeus-Merzbacher disease. MAL encodes a membrane proteolipid that directly interacts with PLP1, ensuring correct distribution during myelin assembly. In contrast to wild-type MAL, mutant MAL was retained in the endoplasmic reticulum but was released following treatment with 4-phenylbutyrate. Proximity-dependent identification of wild-type MAL interactants implicated post-Golgi vesicle-mediated protein transport and protein localisation to membranes, whereas mutant MAL interactants suggested unfolded protein responses. Our results suggest that mislocalisation of MAL affects PLP1 distribution, consistent with known pathomechanisms for hypomyelinating leukodystrophies., (© 2022. The Author(s).)

Background: poor prognosis primary breast cancers are typically treated with cytotoxic chemotherapy. However, recurrences remain relatively common even after this aggressive therapy. Comparison of matched tumours pre- and post-chemotherapy can allow identification of molecular characteristics of therapy resistance and thereby potentially aid discovery of novel predictive markers or targets for chemosensitisation. Through this comparison, we aimed to identify microRNAs associated with chemoresistance, define microRNA target genes, and assess targets as predictors of chemotherapy response., Methods: cancer cells were laser microdissected from matched breast cancer tissues pre- and post-chemotherapy from estrogen receptor positive/HER2 negative breast cancers showing partial responses to epirubicin/cyclophosphamide chemotherapy ( n = 5). MicroRNA expression was profiled using qPCR arrays. MicroRNA/mRNA expression was manipulated in estrogen receptor positive/HER2 negative breast cancer cell lines (MCF7 and MDA-MB-175 cells) with mimics, inhibitors or siRNAs, and chemoresponse was assessed using MTT and colony forming survival assays. MicroRNA targets were identified by RNA-sequencing of microRNA mimic pull-downs, and comparison of these with mRNAs containing predicted microRNA binding sites. Survival correlations were tested using the METABRIC expression dataset ( n = 1979)., Results: miR-195 and miR-26b were consistently up-regulated after therapy, and changes in their expression in cell lines caused significant differences in chemotherapy sensitivity, in accordance with up-regulation driving resistance. SEMA6D was defined and confirmed as a target of the microRNAs. Reduced SEMA6D expression was significantly associated with chemoresistance, in accordance with SEMA6D being a down-stream effector of the microRNAs. Finally, low SEMA6D expression in breast cancers was significantly associated with poor survival after chemotherapy, but not after other therapies., Conclusions: microRNAs and their targets influence chemoresponse, allowing the identification of SEMA6D as a predictive marker for chemotherapy response that could be used to direct therapy or as a target in chemosensitisation strategies.

Background: The HERC2 gene encodes a 527 kDa E3 ubiquitin protein ligase that has key roles in cell cycle regulation, spindle formation during mitosis, mitochondrial functions and DNA damage responses. It has essential roles during embryonic development, particularly for neuronal and muscular functions. To date, missense mutations in HERC2 have been associated with an autosomal recessive neurodevelopmental disorder with some phenotypical similarities to Angelman syndrome, and a homozygous deletion spanning HERC2 and OCA2 causing a more severe neurodevelopmental phenotype., Methods and Results: We ascertained a consanguineous family with a presumed autosomal recessive severe neurodevelopmental disorder that leads to paediatric lethality. In affected individuals, we identified a homozygous HERC2 frameshift variant that results in a premature stop codon and complete loss of HERC2 protein. Functional characterisation of this variant in fibroblasts, from one living affected individual, revealed impaired mitochondrial network and function as well as disrupted levels of known interacting proteins such as XPA., Conclusion: This study extends the genotype-phenotype correlation for HERC2 variants to include a distinct lethal neurodevelopmental disorder, highlighting the importance of further characterisation for HERC2 -related disorders., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

We report a consanguineous family in which schizophrenia segregates in a manner consistent with recessive inheritance of a rare, partial-penetrance susceptibility allele. From 4 marriages between 2 sets of siblings who are half first cousins, 6 offspring have diagnoses of psychotic disorder. Homozygosity mapping revealed a 6.1-Mb homozygous region on chromosome 13q22.2-31.1 shared by all affected individuals, containing 13 protein-coding genes. Microsatellite analysis confirmed homozygosity for the affected haplotype in 12 further apparently unaffected members of the family. Psychiatric reports suggested an endophenotype of milder psychiatric illness in 4 of these individuals. Exome and genome sequencing revealed no potentially pathogenic coding or structural variants within the risk haplotype. Filtering for noncoding variants with a minor allele frequency of <0.05 identified 17 variants predicted to have significant effects, the 2 most significant being within or adjacent to the SCEL gene. RNA sequencing of blood from an affected homozygote showed the upregulation of transcription from NDFIP2 and SCEL. NDFIP2 is highly expressed in brain, unlike SCEL, and is involved in determining T helper (Th) cell type 1 and Th2 phenotypes, which have previously been implicated with schizophrenia., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center.)

The prevalence of neurodegenerative disease has increased significantly in recent years, and with a rapidly aging global population, this trend is expected to continue. These diseases are characterised by a progressive neuronal loss in the brain or peripheral nervous system, and generally involve protein aggregation, as well as metabolic abnormalities and immune dysregulation. Although the vast majority of neurodegeneration is idiopathic, there are many known genetic and environmental triggers. In the past decade, research exploring low-grade systemic inflammation and its impact on the development and progression of neurodegenerative disease has increased. A particular research focus has been whether systemic inflammation arises only as a secondary effect of disease or is also a cause of pathology. The inflammasomes, and more specifically the NLRP3 inflammasome, a crucial component of the innate immune system, is usually activated in response to infection or tissue damage. Dysregulation of the NLRP3 inflammasome has been implicated in the progression of several neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases. This review aims to summarise current literature on the role of the NLRP3 inflammasome in the pathogenesis of neurodegenerative diseases, and recent work investigating NLRP3 inflammasome inhibition as a potential future therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Holbrook, Jarosz-Griffiths, Caseley, Lara-Reyna, Poulter, Williams-Gray, Peckham and McDermott.)

Purpose: Determining the role of DYNC2H1 variants in nonsyndromic inherited retinal disease (IRD)., Methods: Genome and exome sequencing were performed for five unrelated cases of IRD with no identified variant. In vitro assays were developed to validate the variants identified (fibroblast assay, induced pluripotent stem cell [iPSC] derived retinal organoids, and a dynein motility assay)., Results: Four novel DYNC2H1 variants (V1, g.103327020&#95;103327021dup; V2, g.103055779A>T; V3, g.103112272C>G; V4, g.103070104A>C) and one previously reported variant (V5, g.103339363T>G) were identified. In proband 1 (V1/V2), V1 was predicted to introduce a premature termination codon (PTC), whereas V2 disrupted the exon 41 splice donor site causing incomplete skipping of exon 41. V1 and V2 impaired dynein-2 motility in vitro and perturbed IFT88 distribution within cilia. V3, homozygous in probands 2-4, is predicted to cause a PTC in a retina-predominant transcript. Analysis of retinal organoids showed that this new transcript expression increased with organoid differentiation. V4, a novel missense variant, was in trans with V5, previously associated with Jeune asphyxiating thoracic dystrophy (JATD)., Conclusion: The DYNC2H1 variants discussed herein were either hypomorphic or affecting a retina-predominant transcript and caused nonsyndromic IRD. Dynein variants, specifically DYNC2H1 variants are reported as a cause of non syndromic IRD.

Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system., (© 2020 by The American Society of Hematology.)

Amelogenesis is the process of enamel formation. For amelogenesis to proceed, the cells of the inner enamel epithelium (IEE) must first proliferate and then differentiate into the enamel-producing ameloblasts. Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective or absent tooth enamel. We identified a 2 bp variant c.817&#95;818GC>AA in SP6, the gene encoding the SP6 transcription factor, in a Caucasian family with autosomal dominant hypoplastic AI. The resulting missense protein change, p.(Ala273Lys), is predicted to alter a DNA-binding residue in the first of three zinc fingers. SP6 has been shown to be crucial to both proliferation of the IEE and to its differentiation into ameloblasts. SP6 has also been implicated as an AI candidate gene through its study in rodent models. We investigated the effect of the missense variant in SP6 (p.(Ala273Lys)) using surface plasmon resonance protein-DNA binding studies. We identified a potential SP6 binding motif in the AMBN proximal promoter sequence and showed that wild-type (WT) SP6 binds more strongly to it than the mutant protein. We hypothesize that SP6 variants may be a very rare cause of AI due to the critical roles of SP6 in development and that the relatively mild effect of the missense variant identified in this study is sufficient to affect amelogenesis causing AI, but not so severe as to be incompatible with life. We suggest that current AI cohorts, both with autosomal recessive and dominant disease, be screened for SP6 variants., (© The Author(s) 2020. Published by Oxford University Press.)

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility., (Copyright © 2018. Published by Elsevier Inc.)

To date, over 150 disease-associated variants in CRB1 have been described, resulting in a range of retinal disease phenotypes including Leber congenital amaurosis and retinitis pigmentosa. Despite this, no genotype-phenotype correlations are currently recognised. We performed a retrospective review of electronic patient records to identify patients with macular dystrophy due to bi-allelic variants in CRB1. In total, seven unrelated individuals were identified. The median age at presentation was 21 years, with a median acuity of 0.55 decimalised Snellen units (IQR = 0.43). The follow-up period ranged from 0 to 19 years (median = 2.0 years), with a median final decimalised Snellen acuity of 0.65 (IQR = 0.70). Fundoscopy revealed only a subtly altered foveal reflex, which evolved into a bull's-eye pattern of outer retinal atrophy. Optical coherence tomography identified structural changes-intraretinal cysts in the early stages of disease, and later outer retinal atrophy. Genetic testing revealed that one rare allele (c.498&#95;506del, p.(Ile167&#95;Gly169del)) was present in all patients, with one patient being homozygous for the variant and six being heterozygous. In trans with this, one variant recurred twice (p.(Cys896Ter)), while the four remaining alleles were each observed once (p.(Pro1381Thr), p.(Ser478ProfsTer24), p.(Cys195Phe) and p.(Arg764Cys)). These findings show that the rare CRB1 variant, c.498&#95;506del, is strongly associated with localised retinal dysfunction. The clinical findings are much milder than those observed with bi-allelic, loss-of-function variants in CRB1, suggesting this in-frame deletion acts as a hypomorphic allele. This is the most prevalent disease-causing CRB1 variant identified in the non-Asian population to date.

We identified two homozygous missense variants (c.428C>T, p.(T143M) and c.746C>T, p.(P249L)) in ACPT, the gene encoding acid phosphatase, testicular, which segregates with hypoplastic amelogenesis imperfecta in two unrelated families. ACPT is reported to play a role in odontoblast differentiation and mineralisation by supplying phosphate during dentine formation. Analysis by computerised tomography and scanning electron microscopy of a primary molar tooth from an individual homozygous for the c.746C>T variant revealed an enamel layer that was hypoplastic, but mineralised with prismatic architecture. These findings implicate variants in ACPT as a cause of early failure of amelogenesis during the secretory phase.

Amelogenesis imperfecta (AI) is the name given to a heterogeneous group of conditions characterized by inherited developmental enamel defects. AI enamel is abnormally thin, soft, fragile, pitted and/or badly discolored, with poor function and aesthetics, causing patients problems such as early tooth loss, severe embarrassment, eating difficulties, and pain. It was first described separately from diseases of dentine nearly 80 years ago, but the underlying genetic and mechanistic basis of the condition is only now coming to light. Mutations in the gene AMELX , encoding an extracellular matrix protein secreted by ameloblasts during enamel formation, were first identified as a cause of AI in 1991. Since then, mutations in at least eighteen genes have been shown to cause AI presenting in isolation of other health problems, with many more implicated in syndromic AI. Some of the encoded proteins have well documented roles in amelogenesis, acting as enamel matrix proteins or the proteases that degrade them, cell adhesion molecules or regulators of calcium homeostasis. However, for others, function is less clear and further research is needed to understand the pathways and processes essential for the development of healthy enamel. Here, we review the genes and mutations underlying AI presenting in isolation of other health problems, the proteins they encode and knowledge of their roles in amelogenesis, combining evidence from human phenotypes, inheritance patterns, mouse models, and in vitro studies. An LOVD resource (http://dna2.leeds.ac.uk/LOVD/) containing all published gene mutations for AI presenting in isolation of other health problems is described. We use this resource to identify trends in the genes and mutations reported to cause AI in the 270 families for which molecular diagnoses have been reported by 23rd May 2017. Finally we discuss the potential value of the translation of AI genetics to clinical care with improved patient pathways and speculate on the possibility of novel treatments and prevention strategies for AI.

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder characterized by the abnormal development of the retinal vasculature. The majority of mutations identified in FEVR are found within four genes that encode the receptor complex (FZD4, LRP5, and TSPAN12) and ligand (NDP) of a molecular pathway that controls angiogenesis, the Norrin-β-catenin signaling pathway. However, half of all FEVR-affected case subjects do not harbor mutations in these genes, indicating that further mutated genes remain to be identified. Here we report the identification of mutations in CTNNB1, the gene encoding β-catenin, as a cause of FEVR. We describe heterozygous mutations (c.2142&#95;2157dup [p.His720 ∗ ] and c.2128C>T [p.Arg710Cys]) in two dominant FEVR-affected families and a de novo mutation (c.1434&#95;1435insC [p.Glu479Argfs ∗ 18]) in a simplex case subject. Previous studies have reported heterozygous de novo CTNNB1 mutations as a cause of syndromic intellectual disability (ID) and autism spectrum disorder, and somatic mutations are linked to many cancers. However, in this study we show that Mendelian inherited CTNNB1 mutations can cause non-syndromic FEVR and that FEVR can be a part of the syndromic ID phenotype, further establishing the role that β-catenin signaling plays in the development of the retinal vasculature., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Purpose: Recessive mutations in CLN7/MFSD8 usually cause variant late-infantile onset neuronal ceroid lipofuscinosis (vLINCL), a poorly understood neurodegenerative condition, though mutations may also cause nonsyndromic maculopathy. A series of 12 patients with nonsyndromic retinopathy due to novel CLN7/MFSD8 mutation combinations were investigated in this study., Methods: Affected patients and their family members were recruited in ophthalmic clinics at each center where they were examined by retinal imaging and detailed electrophysiology. Whole exome or genome next generation sequencing was performed on genomic DNA from at least one affected family member. Immunofluorescence confocal microscopy of murine retina cross-sections were used to localize the protein., Results: Compound heterozygous alleles were identified in six cases, one of which was always p.Glu336Gln. Such combinations resulted in isolated macular disease. Six further cases were homozygous for the variant p.Met454Thr, identified as a founder mutation of South Asian origin. Those patients had widespread generalized retinal disease, characterized by electroretinography as a rod-cone dystrophy with severe macular involvement. In addition, the photopic single flash electroretinograms demonstrated a reduced b- to a-wave amplitude ratio, suggesting dysfunction occurring after phototransduction. Immunohistology identified MFSD8 in the outer plexiform layer of the retina, a site rich in photoreceptor synapses., Conclusions: This study highlights a hierarchy of MFSD8 variant severity, predicting three consequences of mutation: (1) nonsyndromic localized maculopathy, (2) nonsyndromic widespread retinopathy, or (3) syndromic neurological disease. The data also shed light on the underlying pathogenesis by implicating the photoreceptor synaptic terminals as the major site of retinal disease.

'Amelogenesis imperfecta' (AI) describes a group of inherited diseases of dental enamel that have major clinical impact. Here, we identify the aetiology driving AI in mice carrying a p.S55I mutation in enamelin; one of the most commonly mutated proteins underlying AI in humans. Our data indicate that the mutation inhibits the ameloblast secretory pathway leading to ER stress and an activated unfolded protein response (UPR). Initially, with the support of the UPR acting in pro-survival mode, Enamp.S55I heterozygous mice secreted structurally normal enamel. However, enamel secreted thereafter was structurally abnormal; presumably due to the UPR modulating ameloblast behaviour and function in an attempt to relieve ER stress. Homozygous mutant mice failed to produce enamel. We also identified a novel heterozygous ENAMp.L31R mutation causing AI in humans. We hypothesize that ER stress is the aetiological factor in this case of human AI as it shared the characteristic phenotype described above for the Enamp.S55I mouse. We previously demonstrated that AI in mice carrying the Amelxp.Y64H mutation is a proteinopathy. The current data indicate that AI in Enamp.S55I mice is also a proteinopathy, and based on comparative phenotypic analysis, we suggest that human AI resulting from the ENAMp.L31R mutation is another proteinopathic disease. Identifying a common aetiology for AI resulting from mutations in two different genes opens the way for developing pharmaceutical interventions designed to relieve ER stress or modulate the UPR during enamel development to ameliorate the clinical phenotype., (© The Author 2017. Published by Oxford University Press.)

Heimler syndrome (HS) consists of recessively inherited sensorineural hearing loss, amelogenesis imperfecta (AI) and nail abnormalities, with or without visual defects. Recently HS was shown to result from hypomorphic mutations in PEX1 or PEX6, both previously implicated in Zellweger Syndrome Spectrum Disorders (ZSSD). ZSSD are a group of conditions consisting of craniofacial and neurological abnormalities, sensory defects and multi-organ dysfunction. The finding of HS-causing mutations in PEX1 and PEX6 shows that HS represents the mild end of the ZSSD spectrum, though these conditions were previously thought to be distinct nosological entities. Here, we present six further HS families, five with PEX6 variants and one with PEX1 variants, and show the patterns of Pex1, Pex14 and Pex6 immunoreactivity in the mouse retina. While Ratbi et al. found more HS-causing mutations in PEX1 than in PEX6, as is the case for ZSSD, in this cohort PEX6 variants predominate, suggesting both genes play a significant role in HS. The PEX6 variant c.1802G>A, p.(R601Q), reported previously in compound heterozygous state in one HS and three ZSSD cases, was found in compound heterozygous state in three HS families. Haplotype analysis suggests a common founder variant. All families segregated at least one missense variant, consistent with the hypothesis that HS results from genotypes including milder hypomorphic alleles. The clinical overlap of HS with the more common Usher syndrome and lack of peroxisomal abnormalities on plasma screening suggest that HS may be under-diagnosed. Recognition of AI is key to the accurate diagnosis of HS., Competing Interests: The authors declare no conflict of interest.

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective dental enamel formation. Amelotin (AMTN) is a secreted protein thought to act as a promoter of matrix mineralization in the final stage of enamel development, and is strongly expressed, almost exclusively, in maturation stage ameloblasts. Amtn overexpression and Amtn knockout mouse models have defective enamel with no other associated phenotypes, highlighting AMTN as an excellent candidate gene for human AI. However, no AMTN mutations have yet been associated with human AI. Using whole exome sequencing, we identified an 8,678 bp heterozygous genomic deletion encompassing exons 3-6 of AMTN in a Costa Rican family segregating dominant hypomineralised AI. The deletion corresponds to an in-frame deletion of 92 amino acids, shortening the protein from 209 to 117 residues. Exfoliated primary teeth from an affected family member had enamel that was of a lower mineral density compared to control enamel and exhibited structural defects at least some of which appeared to be associated with organic material as evidenced using elemental analysis. This study demonstrates for the first time that AMTN mutations cause non-syndromic human AI and explores the human phenotype, comparing it with that of mice with disrupted Amtn function., (© The Author 2016. Published by Oxford University Press.)

Biallelic FAM20A mutations cause two conditions where Amelogenesis Imperfecta (AI) is the presenting feature: Amelogenesis Imperfecta and Gingival Fibromatosis Syndrome; and Enamel Renal Syndrome. A distinctive oral phenotype is shared in both conditions. On Sanger sequencing of FAM20A in cases with that phenotype, we identified two probands with single, likely pathogenic heterozygous mutations. Given the recessive inheritance pattern seen in all previous FAM20A mutation-positive families and the potential for renal disease, further screening was carried out to look for a second pathogenic allele. Reverse transcriptase-PCR on cDNA was used to determine transcript levels. CNVseq was used to screen for genomic insertions and deletions. In one family, FAM20A cDNA screening revealed only a single mutated FAM20A allele with the wild-type allele not transcribed. In the second family, CNV detection by whole genome sequencing (CNVseq) revealed a heterozygous 54.7 kb duplication encompassing exons 1 to 4 of FAM20A. This study confirms the link between biallelic FAM20A mutations and the characteristic oral phenotype. It highlights for the first time examples of FAM20A mutations missed by the most commonly used mutation screening techniques. This information informed renal assessment and ongoing clinical care.

Retinal dystrophies are an overlapping group of genetically heterogeneous conditions resulting from mutations in more than 250 genes. Here we describe five families affected by an adult-onset retinal dystrophy with early macular involvement and associated central visual loss in the third or fourth decade of life. Affected individuals were found to harbor disease-causing variants in DRAM2 (DNA-damage regulated autophagy modulator protein 2). Homozygosity mapping and exome sequencing in a large, consanguineous British family of Pakistani origin revealed a homozygous frameshift variant (c.140delG [p.Gly47Valfs(∗)3]) in nine affected family members. Sanger sequencing of DRAM2 in 322 unrelated probands with retinal dystrophy revealed one European subject with compound heterozygous DRAM2 changes (c.494G>A [p.Trp165(∗)] and c.131G>A [p.Ser44Asn]). Inspection of previously generated exome sequencing data in unsolved retinal dystrophy cases identified a homozygous variant in an individual of Indian origin (c.64&#95;66del [p.Ala22del]). Independently, a gene-based case-control association study was conducted via an exome sequencing dataset of 18 phenotypically similar case subjects and 1,917 control subjects. Using a recessive model and a binomial test for rare, presumed biallelic, variants, we found DRAM2 to be the most statistically enriched gene; one subject was a homozygote (c.362A>T [p.His121Leu]) and another a compound heterozygote (c.79T>C [p.Tyr27His] and c.217&#95;225del [p.Val73&#95;Tyr75del]). DRAM2 encodes a transmembrane lysosomal protein thought to play a role in the initiation of autophagy. Immunohistochemical analysis showed DRAM2 localization to photoreceptor inner segments and to the apical surface of retinal pigment epithelial cells where it might be involved in the process of photoreceptor renewal and recycling to preserve visual function., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)