Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Genetic complexity of diagnostically unresolved Ehlers-Danlos syndrome.

BACKGROUND: The Ehlers-Danlos syndromes (EDS) are heritable disorders of connective tissue (HDCT), reclassified in the 2017 nosology into 13 subtypes. The genetic basis for hypermobile Ehlers-Danlos syndrome (hEDS) remains unknown. METHODS: Whole exome sequencing (WES) was undertaken on 174 EDS patients recruited from a national diagnostic service for complex EDS and a specialist clinic for hEDS. Patients had already undergone expert phenotyping, laboratory investigation and gene sequencing, but were without a genetic diagnosis. Filtered WES data were reviewed for genes underlying Mendelian disorders and loci reported in EDS linkage, transcriptome and genome-wide association studies (GWAS). A genetic burden analysis (Minor Allele Frequency (MAF) <0.05) incorporating 248 Avon Longitudinal Study of Parents and Children (ALSPAC) controls sequenced as part of the UK10K study was undertaken using TASER methodology. RESULTS: Heterozygous pathogenic (P) or likely pathogenic (LP) variants were identified in known EDS and Loeys-Dietz (LDS) genes. Multiple variants of uncertain significance where segregation and functional analysis may enable reclassification were found in genes associated with EDS, LDS, heritable thoracic aortic disease (HTAD), Mendelian disorders with EDS symptomatology and syndromes with EDS-like features. Genetic burden analysis revealed a number of novel loci, although none reached the threshold for genome-wide significance. Variants with biological plausibility were found in genes and pathways not currently associated with EDS or HTAD. CONCLUSIONS: We demonstrate the clinical utility of large panel-based sequencing and WES for patients with complex EDS in distinguishing rare EDS subtypes, LDS and related syndromes. Although many of the P and LP variants reported in this cohort would be identified with current panel testing, they were not at the time of this study, highlighting the use of extended panels and WES as a clinical tool for complex EDS. Our results are consistent with the complex genetic architecture of EDS and suggest a number of novel hEDS and HTAD candidate genes and pathways.

Cingulin and paracingulin tether myosins-2 to junctions to mechanoregulate the plasma membrane.

The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.

Potential implications of the glycosylation patterns in collagen α1(I) and α2(I) chains for fibril assembly and growth.

O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.

50 Years of the steric-blocking mechanism in vertebrate skeletal muscle: a retrospective.

Fifty years have now passed since Parry and Squire proposed a detailed structural model that explained how tropomyosin, mediated by troponin, played a steric-blocking role in the regulation of vertebrate skeletal muscle. In this Special Issue dedicated to the memory of John Squire it is an opportune time to look back on this research and to appreciate John's key contributions. A review is also presented of a selection of the developments and insights into muscle regulation that have occurred in the years since this proposal was formulated.

An experimental model of contusion injury in humans.

INTRODUCTION: Contusion injuries are common in sport, but our knowledge of the responses to injury primarily come from animal studies and research using eccentric exercise. Therefore, the aim of this study was to develop a model of contusion injury in human participants and, additionally, investigate and compare physiological responses to four impact loads. METHODS: Thirty-two males were exposed to a single impact of either 4.2, 5.2, 6.2 or 7.2kg, dropped from 67 cm, on to the vastus lateralis of one leg. Maximum voluntary and electrically induced quadriceps force, and pressure pain threshold were measured, and blood sampling carried out, prior to and 30min, 24, 48 and 72h post-impact. Magnetic resonance imaging was carried out 24h post-impact to quantify oedema. RESULTS: Despite impact force with 7.2kg (1681.4 ± 235.6 N) not being different to 6.2kg (1690.7 ± 117.6 N), 7.2kg resulted in greater volume of oedema, voluntary force loss, pain and elevations in creatine kinase than the other loads. Although electrically induced force changed over time, post-hoc analysis failed to identify any changes. Interleukin-6 and prostaglandin-E2 did not change over time for any of the loads. Significant correlations were found between oedema volume, pressure pain threshold and maximum voluntary contraction force. CONCLUSIONS: This is the first experimental study to investigate traumatic loading of skeletal muscle and the subsequent physiological responses associated with contusion injuries in humans. The absence of immediate elevations in creatine kinase and changes in electrically induced force suggest impact, with forces similar to those experienced in contact sport, does not cause significant, direct damage to skeletal muscle. However, the relationship between oedema volume, changes in pressure pain threshold and maximum voluntary contraction force suggests central inhibition plays a role in contusion-related muscle dysfunction.

Follicular regulatory T cells eliminate HIV-1-infected follicular helper T cells in an IL-2 concentration dependent manner.

Follicular helper CD4+ T cells (TFH) are highly permissive to HIV and major foci of virus expression in both untreated and treated infection. Follicular regulatory CD4+ T cells (TFR) limit TFH numbers and function in vitro and in vivo. We evaluated the hypothesis that TFR suppress HIV replication in TFH using a well-established model of ex vivo HIV infection that employs tonsil cells from HIV uninfected individuals spinoculated with CXCR4- and CCR5-tropic HIV-GFP reporter viruses. Both CXCR4 and CCR5-tropic HIV replication were reduced in TFH cultured with TFR as compared to controls. Blocking antibodies to CD39, CTLA-4, IL-10, and TGF-beta failed to reverse suppression of HIV replication by TFR, and there were no sex differences in TFR suppressive activity. TFR reduced viability of TFH and even more so reduced HIV infected TFH as assessed by total and integrated HIV DNA. Exogenous IL-2 enhanced TFH viability and particularly numbers of GFP+ TFH in a concentration dependent manner. TFR reduced productively infected TFH at low and moderate IL-2 concentrations, and this was associated with decreases in extracellular IL-2. Both IL-2 expressing cells and larger numbers of FoxP3+CD4+ cells were detected in follicles and germinal centers of lymph nodes of people living with HIV. TFR may deplete TFH in vivo through restriction of IL-2 and thereby contribute to decay of HIV expressing cells in B cell follicles during HIV infection.

Novel SIX6 mutations cause recessively inherited congenital cataract, microcornea, and corneal opacification with or without coloboma and microphthalmia.

Purpose: To investigate the molecular basis of recessively inherited congenital cataract, microcornea, and corneal opacification with or without coloboma and microphthalmia in two consanguineous families. Methods: Conventional autozygosity mapping was performed using single nucleotide polymorphism (SNP) microarrays. Whole-exome sequencing was completed on genomic DNA from one affected member of each family. Exome sequence data were also used for homozygosity mapping and copy number variation analysis. PCR and Sanger sequencing were used to confirm the identification of mutations and to screen further patients. Evolutionary conservation of protein sequences was assessed using CLUSTALW, and protein structures were modeled using PyMol. Results: In family MEP68, a novel homozygous nucleotide substitution in SIX6 was found, c.547G>C, that converts the evolutionarily conserved aspartic acid residue at the 183rd amino acid in the protein to a histidine, p.(Asp183His). This residue mapped to the third helix of the DNA-binding homeobox domain in SIX6, which interacts with the major groove of double-stranded DNA. This interaction is likely to be disrupted by the mutation. In family F1332, a novel homozygous 1034 bp deletion that encompasses the first exon of SIX6 was identified, chr14:g.60975890_60976923del. Both mutations segregated with the disease phenotype as expected for a recessive condition and were absent from publicly available variant databases. Conclusions: Our findings expand the mutation spectrum in this form of inherited eye disease and confirm that homozygous human SIX6 mutations cause a developmental spectrum of ocular phenotypes that includes not only the previously described features of microphthalmia, coloboma, and congenital cataract but also corneal abnormalities.

Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.

Loss of Integrin-Linked Kinase Sensitizes Breast Cancer to SRC Inhibitors.

SRC is a nonreceptor tyrosine kinase with key roles in breast cancer development and progression. Despite this, SRC tyrosine kinase inhibitors have so far failed to live up to their promise in clinical trials, with poor overall response rates. We aimed to identify possible synergistic gene-drug interactions to discover new rational combination therapies for SRC inhibitors. An unbiased genome-wide CRISPR-Cas9 knockout screen in a model of triple-negative breast cancer revealed that loss of integrin-linked kinase (ILK) and its binding partners α-Parvin and PINCH-1 sensitizes cells to bosutinib, a clinically approved SRC/ABL kinase inhibitor. Sensitivity to bosutinib did not correlate with ABL dependency; instead, bosutinib likely induces these effects by acting as a SRC tyrosine kinase inhibitor. Furthermore, in vitro and in vivo models showed that loss of ILK enhanced sensitivity to eCF506, a novel and highly selective inhibitor of SRC with a unique mode of action. Whole-genome RNA sequencing following bosutinib treatment in ILK knockout cells identified broad changes in the expression of genes regulating cell adhesion and cell-extracellular matrix. Increased sensitivity to SRC inhibition in ILK knockout cells was associated with defective adhesion, resulting in reduced cell number as well as increased G1 arrest and apoptosis. These findings support the potential of ILK loss as an exploitable therapeutic vulnerability in breast cancer, enhancing the effectiveness of clinical SRC inhibitors. SIGNIFICANCE: A CRISPR-Cas9 screen reveals that loss of integrin-linked kinase synergizes with SRC inhibition, providing a new opportunity for enhancing the clinical effectiveness of SRC inhibitors in breast cancer.

Keratin intermediate filament chains in the European common wall lizard (Podarcis muralis) and a potential keratin filament crosslinker.

On the basis of sequence homology with mammalian α-keratins, and on the criteria that the coiled-coil segments and central linker in the rod domain of these molecules must have conserved lengths if they are to assemble into viable intermediate filaments, a total of 28 Type I and Type II keratin intermediate filament chains (KIF) have been identified from the genome of the European common wall lizard (Podarcis muralis). Using the same criteria this number may be compared to 33 found here in the green anole lizard (Anole carolinensis) and 25 in the tuatara (Sphenodon punctatus). The Type I and Type II KIF genes in the wall lizard fall in clusters on chromosomes 13 and 2 respectively. Although some differences occur in the terminal domains in the KIF chains of the two lizards and tuatara, the similarities between key indicator residues - cysteine, glycine and proline - are significant. The terminal domains of the KIF chains in the wall lizard also contain sequence repeats commonly based on glycine and large apolar residues and would permit the fine tuning of physical properties when incorporated within the intermediate filaments. The H1 domain in the Type II chain is conserved across the lizards, tuatara and mammals, and has been related to its role in assembly at the 2-4 molecule level. A KIF-like chain (K80) with an extensive tail domain comprised of multiple tandem repeats has been identified as having a potential filament-crosslinking role.

Microcephalic osteodysplastic primordial dwarfism type II is associated with global vascular disease.

BACKGROUND: Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) is the most common form of primordial dwarfism, caused by bialleic mutations in the pericentrin gene (PCNT). Aside from its classic features, there are multiple associated medical complications, including a well-documented risk of neurovascular disease. Over the past several years, it has become apparent that additional vascular issues, as well as systemic hypertension and kidney disease may also be related to MOPDII. However, the frequency and extent of the vasculopathy was unclear. To help address this question, a vascular substudy was initiated within our Primordial Dwarfism Registry. RESULTS: Medical records from 47 individuals, living and deceased, ranging in age from 3 to 41 years of age were interrogated for this purpose. Of the total group, 64% were diagnosed with moyamoya, intracranial aneurysms, or both. In general, the age at diagnosis for moyamoya was younger than aneurysms, but the risk for neurovascular disease was throughout the shortened lifespan. In addition to neurovascular disease, renal, coronary and external carotid artery involvement are documented. 43% of the total group was diagnosed with hypertension, and 17% had myocardial infarctions. A total of 32% of the entire cohort had some form of chronic kidney disease, with 4% of the total group necessitating a kidney transplant. In addition, 38% had diabetes/insulin resistance. Ages of diagnoses, treatment modalities employed, and location of vasculopathies were notated as available and applicable, as well as frequencies of other comorbidities. CONCLUSIONS: It is now clear that vascular disease in MOPDII is global and screening of the cardiac and renal vessels is warranted along with close monitoring of blood pressure. We recommend a blood pressure of 110/70 mmHg as a starting point for an upper limit, especially if the individual has a history of neurovascular disease, chronic kidney disease and/or diabetes. Additionally, providers need to be at high alert for the possibility of myocardial infarctions in young adults with MOPDII, so that appropriate treatment can be initiated promptly in an acute situation.

Structures of the ß-Keratin Filaments and Keratin Intermediate Filaments in the Epidermal Appendages of Birds and Reptiles (Sauropsids).

The epidermal appendages of birds and reptiles (the sauropsids) include claws, scales, and feathers. Each has specialized physical properties that facilitate movement, thermal insulation, defence mechanisms, and/or the catching of prey. The mechanical attributes of each of these appendages originate from its fibril-matrix texture, where the two filamentous structures present, i.e., the corneous ß-proteins (CBP or ß-keratins) that form 3.4 nm diameter filaments and the α-fibrous molecules that form the 7-10 nm diameter keratin intermediate filaments (KIF), provide much of the required tensile properties. The matrix, which is composed of the terminal domains of the KIF molecules and the proteins of the epidermal differentiation complex (EDC) (and which include the terminal domains of the CBP), provides the appendages, with their ability to resist compression and torsion. Only by knowing the detailed structures of the individual components and the manner in which they interact with one another will a full understanding be gained of the physical properties of the tissues as a whole. Towards that end, newly-derived aspects of the detailed conformations of the two filamentous structures will be discussed and then placed in the context of former knowledge.

Keratin intermediate filament chains in tuatara (Sphenodon punctatus): A comparison of tuatara and human sequences.

Determination of the sequences of the keratin intermediate filament chains in tuatara has shown that these are closely akin to the α-keratins in human and other vertebrates, especially in the central, coiled-coil rod region. The domain lengths within the rod are preserved exactly, both Type I and Type II chains have been recognised, and sequence identity/homology exists between their respective chains. Nonetheless, there are characteristic differences in amino acid composition and sequence between their respective head (N-terminal) domains and their tail (C-terminal) domains, though some similarities are retained. Further, there is evidence of tandem repeats of a variety of lengths in the tuatara heads and tails indicative of sequence duplication events. These are not evident in human α-keratins and would therefore have implications for the physical attributes of the tissues in the two species. Multiple families of keratin-associated proteins that are ultra-high cysteine-rich or glycine + tyrosine-rich in human and other species do not have direct equivalents in the tuatara. Although high-sulphur proteins are present in tuatara the cysteine residue contents are significantly lower than in human. Further, no sequence homologies between the HS proteins in the two species have been found, thereby casting considerable doubt as to whether any matrix-forming high-sulphur proteins exist in tuatara. These observations may be correlated with the numerous cysteine-rich ß-keratins (corneous ß-proteins) that are present in tuatara, but which are conspicuously absent in mammals.

Heterozygous lamin B1 and lamin B2 variants cause primary microcephaly and define a novel laminopathy.

PURPOSE: Lamins are the major component of nuclear lamina, maintaining structural integrity of the nucleus. Lamin A/C variants are well established to cause a spectrum of disorders ranging from myopathies to progeria, termed laminopathies. Phenotypes resulting from variants in LMNB1 and LMNB2 have been much less clearly defined. METHODS: We investigated exome and genome sequencing from the Deciphering Developmental Disorders Study and the 100,000 Genomes Project to identify novel microcephaly genes. RESULTS: Starting from a cohort of patients with extreme microcephaly, 13 individuals with heterozygous variants in the two human B-type lamins were identified. Recurrent variants were established to be de novo in nine cases and shown to affect highly conserved residues within the lamin ɑ-helical rod domain, likely disrupting interactions required for higher-order assembly of lamin filaments. CONCLUSION: We identify dominant pathogenic variants in LMNB1 and LMNB2 as a genetic cause of primary microcephaly, implicating a major structural component of the nuclear envelope in its etiology and defining a new form of laminopathy. The distinct nature of this lamin B-associated phenotype highlights the strikingly different developmental requirements for lamin paralogs and suggests a novel mechanism for primary microcephaly warranting future investigation.

Mutations in Spliceosomal Genes PPIL1 and PRP17 Cause Neurodegenerative Pontocerebellar Hypoplasia with Microcephaly.

Autosomal-recessive cerebellar hypoplasia and ataxia constitute a group of heterogeneous brain disorders caused by disruption of several fundamental cellular processes. Here, we identified 10 families showing a neurodegenerative condition involving pontocerebellar hypoplasia with microcephaly (PCHM). Patients harbored biallelic mutations in genes encoding the spliceosome components Peptidyl-Prolyl Isomerase Like-1 (PPIL1) or Pre-RNA Processing-17 (PRP17). Mouse knockouts of either gene were lethal in early embryogenesis, whereas PPIL1 patient mutation knockin mice showed neuron-specific apoptosis. Loss of either protein affected splicing integrity, predominantly affecting short and high GC-content introns and genes involved in brain disorders. PPIL1 and PRP17 form an active isomerase-substrate interaction, but we found that isomerase activity is not critical for function. Thus, we establish disrupted splicing integrity and "major spliceosome-opathies" as a new mechanism underlying PCHM and neurodegeneration and uncover a non-enzymatic function of a spliceosomal proline isomerase.