Affect Protein Function Research Articles

CircBCL11B acts as a ceRNA to facilitate 1,2-dichloroethane-induced astrocyte swelling via miR-29b-3p/AQP4 axis in SVG p12 cells

1,2-Dichloroethane (1,2-DCE) is a pervasive environmental pollutant found in ambient and residential air, as well as ground and drinking water. Brain edema is the primary pathological consequence of 1,2-DCE overexposure. We found that microRNA (miRNA)-29b dysregulation after 1,2-DCE exposure can aggravate brain edema by suppressing aquaporin 4 (AQP4). Moreover, circular RNAs (circRNAs) can regulate the expression of downstream target genes through miRNA, and affect protein function. However, circRNAs’ role in 1,2-DCE-induced brain edema via miR-29b-3p/AQP4 axis remains unclear. To address the mechanism’s bottleneck, we explored the circRNA-miRNA-mRNA network underlying 1,2-DCE-driven astrocyte swelling in SVG p12 cells by circRNA sequencing, electron microscopy and isotope 3H labeling combined with the 3-O-methylglucose uptake method. The results showed that 25 and 50 mM 1,2-DCE motivated astrocyte swelling, characterized by increased water content, enlarged cell vacuoles, and mitochondrial swelling. This was accompanied by miR-29b-3p downregulation and AQP4 upregulation. We verified that AQP4 were negatively regulated by miR-29b-3p in 1,2-DCE-induced astrocyte swelling. Also, circRNA sequencing highlighted that circBCL11B was upregulated by 1,2-DCE. This was manifested as circBCL11B overexpression playing an endogenous competitive role via upregulating AQP4 by binding to miR-29b-3p, thus leading to astrocyte swelling. Conversely, circBCL11B knockdown reversed the 1,2-DCE-motivated AQP4 upregulation and alleviated the cell swelling. Finally, we demonstrated that the circBCL11B was targeted to miR-29b-3p by fluorescence in situ hybridization and dual-luciferase reporter assay. In conclusion, our findings indicate that circBCL11B acts as a competing endogenous RNA to facilitate 1,2-DCE-caused astrocyte swelling via miR-29b-3p/AQP4 axis. These observations provide new insight into the epigenetic mechanisms underlying 1,2-DCE-induced brain edema.

Whole-genome sequences from wild-type and laboratory-evolved strains define the alleleome and establish its hallmarks

The genomic diversity across strains of a species forms the genetic basis for differences in their behavior. A large-scale assessment of sequence variation has been made possible by the growing availability of strain-specific whole-genome sequences (WGS) and with the advent of large-scale databases of laboratory-acquired mutations. We define the Escherichia coli "alleleome" through a genome-scale assessment of amino acid (AA) sequence diversity in open reading frames across 2,661 WGS from wild-type strains. We observe a highly conserved alleleome enriched in mutations unlikely to affect protein function. In contrast, 33,000 mutations acquired in laboratory evolution experiments result in more severe AA substitutions that are rarely achieved by natural selection. Large-scale assessment of the alleleome establishes a method for the quantification of bacterial allelic diversity, reveals opportunities for synthetic biology to explore novel sequence space, and offers insights into the constraints governing evolution.

PRDM1 DNA-binding zinc finger domain is required for normal limb development and is disrupted in split hand/foot malformation.

Split hand/foot malformation (SHFM) is a rare limb abnormality with clefting of the fingers and/or toes. For many individuals, the genetic etiology is unknown. Through whole-exome and targeted sequencing, we detected three novel variants in a gene encoding a transcription factor, PRDM1, that arose de novo in families with SHFM or segregated with the phenotype. PRDM1 is required for limb development; however, its role is not well understood and it is unclear how the PRDM1 variants affect protein function. Using transient and stable overexpression rescue experiments in zebrafish, we show that the variants disrupt the proline/serine-rich and DNA-binding zinc finger domains, resulting in a dominant-negative effect. Through gene expression assays, RNA sequencing, and CUT&RUN in isolated pectoral fin cells, we demonstrate that Prdm1a directly binds to and regulates genes required for fin induction, outgrowth and anterior/posterior patterning, such as fgfr1a, dlx5a, dlx6a and smo. Taken together, these results improve our understanding of the role of PRDM1 in the limb gene regulatory network and identified novel PRDM1 variants that link to SHFM in humans.

Mutational investigation of 17 causative genes in a cohort of 113 families with nonsyndromic early-onset high myopia in northwestern China

High myopia (HM) is a leading cause of visual impairment in the world. To expand the genotypic and phenotypic spectra of HM in the Chinese population, we investigated genetic variations in a cohort of 113 families with nonsyndromic early-onset high myopia from northwestern China by whole-exome sequencing, with focus on 17 known genes. Sixteen potentially pathogenic variants predicted to affect protein function in eight of seventeen causative genes for HM in fifteen (13.3%) families were revealed, including seven novel variants, c.767 + 1G > A in ARR3, c.3214C > A/p.H1072N, and c.2195C > T/p.A732V in ZNF644, c.1270G > T/p.V424L in CPSF1, c.1918G > C/p.G640R and c.2786T > G/p.V929G in XYLT1, c.601G > C/p.E201Q in P4HA2; six rare variants, c.799G > A/p.E267K in NDUFAF7, c.1144C > T/p.R382W in TNFRSF21, c.1100C > T/p.P367L in ZNF644, c.3980C > T/p.S1327L in CPSF1, c.145G > A/p.E49K and c.325G > T/p.G109W in SLC39A5; and three known variants, c.2014A > G/p.S672G and c.3261A > C/p.E1087D in ZNF644, c.605C > T/p.P202L in TNFRSF21. Ten of them were co-segregated with HM. The mean (± SD) examination age of these 15 probands was 14.7 (± 11.61) years. The median spherical equivalent was -9.50 D (IQ -8.75 ~ -12.00) for the right eye and -11.25 D (IQ -9.25 ~ -14.13) for the left eye. The median axial length was 26.67mm (IQ 25.83 ~ 27.13) for the right eye and 26.25mm (IQ 25.97 ~ 27.32) for the left eye. These newly identified genetic variations not only broaden the genetic and clinical spectra, but also offer convincing evidence that the genes ARR3, NDUFAF7, TNFRSF21, and ZNF644 contribute to hereditable HM. This work improves further understanding of molecular mechanism of HM.

Adaptive protein evolution through length variation of short tandem repeats in Arabidopsis.

Intrinsically disordered protein regions are of high importance for biotic and abiotic stress responses in plants. Tracts of identical amino acids accumulate in these regions and can vary in length over generations because of expansions and retractions of short tandem repeats at the genomic level. However, little attention has been paid to what extent length variation is shaped by natural selection. By environmental association analysis on 2514 length variable tracts in 770 whole-genome sequenced Arabidopsis thaliana, we show that length variation in glutamine and asparagine amino acid homopolymers, as well as in interaction hotspots, correlate with local bioclimatic habitat. We determined experimentally that the promoter activity of a light-stress gene depended on polyglutamine length variants in a disordered transcription factor. Our results show that length variations affect protein function and are likely adaptive. Length variants modulating protein function at a global genomic scale has implications for understanding protein evolution and eco-evolutionary biology.

Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila.

Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.

Deciphering local adaptation of native Indian cattle (Bos indicus) breeds using landscape genomics and in-silico prediction of deleterious SNP effects on protein structure and function.

The online version contains supplementary material available at 10.1007/s13205-023-03493-3.

Loss of LGR4/GPR48 causes severe neonatal salt wasting due to disrupted WNT signaling altering adrenal zonation.

Disorders of isolated mineralocorticoid deficiency, which cause potentially life-threatening salt-wasting crisis early in life, have been associated with gene variants of aldosterone biosynthesis or resistance; however, in some patients no such variants are found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone-producing adrenal zona glomerulosa (zG). Herein, we describe a highly consanguineous family with multiple perinatal deaths and infants presenting at birth with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Whole exome sequencing revealed a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C) regulating WNT signaling. The resulting transcripts affected protein function and stability and resulted in loss of Wnt/β-catenin signaling in vitro. The impact of LGR4 inactivation was analyzed by adrenal cortex-specific ablation of Lgr4, using Lgr4fl/fl mice mated with Sf1:Cre mice. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation with deficient zG, and reduced aldosterone production. Thus, human LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling, which results in abnormal zG differentiation and endocrine function. Therefore, variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism.

Dystrophin (DMD) Missense Variant in Cats with Becker-Type Muscular Dystrophy

Muscular dystrophy due to dystrophin deficiency in humans is phenotypically divided into a severe Duchenne and milder Becker type. Dystrophin deficiency has also been described in a few animal species, and few DMD gene variants have been identified in animals. Here, we characterize the clinical, histopathological, and molecular genetic aspects of a family of Maine Coon crossbred cats with clinically mild and slowly progressive muscular dystrophy. Two young adult male littermate cats exhibited abnormal gait and muscular hypertrophy with macroglossia. Serum creatine kinase activities were highly increased. Histopathologically, dystrophic skeletal muscle exhibited marked structural changes including atrophic, hypertrophic, and necrotic muscle fibers. Immunohistochemistry showed irregularly reduced expression of dystrophin but the staining of other muscle proteins such as β- and γ-sarcoglycans as well as desmin was also diminished. Whole genome sequencing of one affected cat and genotyping of the littermate found both to be hemizygous mutant at a single DMD missense variant (c.4186C>T). No other protein-changing variants in candidate genes for muscular dystrophy were detected. In addition, one clinically healthy male littermate was hemizygous wildtype, while the queen and one female littermate were clinically healthy, but heterozygous. The predicted amino acid exchange (p.His1396Tyr) resides in a conserved central rod spectrin domain of dystrophin. Various protein modeling programs did not predict major disruption of the dystrophin protein by this substitution, but the altered charge of the region may still affect protein function. This study represents the first genotype-to-phenotype correlation of Becker-type dystrophin deficiency in companion animals.

Packing and elasticity in membrane mechanical properties.

Here we address how membrane properties involving head group size, acyl chain unsaturation, and lipid packing affect their structure and dynamics. Strong out-of-plane proteolipid couplings considered by the flexible surface model (FSM) affect protein function through elastic forces described by the spontaneous curvature and the bending rigidity. We hypothesized that the mechanical properties and the molecular packing resulting from different lipid types are captured by introducing a perdeuterated POPC-d31 probe lipid. Di-monounsaturated phosphatidylcholine and phosphatidylethanolamine (DOPC, DOPE), and different ratios of DOPC/DOPE mixtures were studied using MD simulations combined with experimental solid-state 2H NMR spectroscopy. Residual quadrupolar couplings obtained from de-Paked powder-type spectra yielded order parameter (SCD) profiles for the individual acyl segments giving insights into average membrane structural properties such as area per lipid and bilayer thickness [1]. The corresponding spin-lattice relaxation rates (R1Z) for the resolved peaks followed a theoretical square-law dependence on SCD providing the bending rigidity [2]. Furthermore, MD simulations for the lipid systems were carried out and order parameters were calculated and compared against the NMR-observed SCD values. Utilization of the probe lipid POPC-d31 (1−10 mol%)affected the lamellar to hexagonal phase transition temperature of DOPE lipid systems but did not affect the quadrupolar splittings of DOPE and DOPC systems. Higher bending rigidity was observed for DOPE membranes versus DOPC supporting the idea that the membrane bending rigidity is primarily governed by increased lipid packing and is related to the area-compressibility modulus. Use of a probe lipid to capture membrane mechanical properties of complex lipid systems allow investigation of the relationship of membrane mechanics in vital biological functions.

Lipid composition of Xenopus oocytes used for heterologous membrane protein expression.

Biological membranes are characterized by a dynamic, heterogeneous lipid composition that can vary by cell type and species origin. Lipids are estimated to comprise 50% of the plasma membrane by weight. A growing body of evidence supports the view that membrane proteins and their adjacent lipids interact in a bidirectional manner. For example, lipid-protein interactions can affect protein function and contribute to membrane organization, such as the formation of domains. The Xenopus oocyte is a well-established classical heterologous expression system for the functional analysis of ion channels and transporters, especially those prevalent in neural tissue. This pilot research was undertaken with the goal of identifying lipids present in unfertilized Xenopus oocytes, specifically those known for their role in plasma membrane composition. Unfertilized Xenopus oocytes were purchased from a commercial vendor, frozen in liquid nitrogen, and shipped to the West Coast Metabolomics Center (WCMC) for lipid extraction. Six samples (20-30 oocytes each) were processed with an Agilent 6530B Q-TOF. During spectral analysis of the output LC/MS, over 1200 distinct lipids were identified. The MS-DIAL program (Version 4.48) assigned nomenclature and external database identifiers (InChI key; LIPIDMAPS) to 380 lipids that were grouped into 12 lipid subclasses, including canonical membrane lipids (phosphatidylcholine; phosphatidylethanolamine; sphingomyelin; ceramide; cholesterol). Estimates of relative abundance suggest that membrane lipids and triacylglycerols comprise 10% and 50% of the Xenopus oocyte lipidome, respectively. Preliminary findings may have implications for heterologous functional analysis of ion channels and other membrane transporters from the nervous system in Xenopus oocytes. Outcomes from this research are being incorporated into computational models of the membrane environment and lipid-protein interactions.

Laws: Local alignment of water sites-Describing allosteric water networks in enzymes.

Water is an essential component of biological systems; it affects the structure and stability of biological machinery. Highly conserved water networks have been shown to play a crucial role in the motion of protein domains and affect protein function by mediating allosteric communication. Perturbations to these networks have also been implicated in human diseases. Data from x-ray crystallography provide the locations of crystallographic water sites (CWS) coordinated by the protein which can be potentially used to study the conserved water networks. Here, we propose a computational method called local alignment for water sites (LAWS) to identify conserved water networks in molecular dynamics (MD) simulations. This method is based on multilateration — an algorithm widely used in GPS tracking. LAWS considers the contacts formed by CWS and protein atoms in the crystal structure and uses these interaction distances to track ordered waters in a simulation. We apply our method to simulations of an enzyme, fluoroacetate dehalogenase, and analyze the occupancy, residence times and correlated motions of individual water molecules coupled to the protein. Our results help to determine a highly conserved water network involved in allosteric regulation.

A nanobody toolbox to investigate localisation and dynamics of Drosophila titins and other key sarcomeric proteins.

Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understanding protein function. However, to date, this is challenging, as the accessibility of large antibodies to dense tissues is often limited, and fluorescent proteins inserted close to a domain of interest may affect protein function. These complications apply in particular to muscle sarcomeres, arguably one of the most protein-dense assemblies in nature, which complicates studying sarcomere morphogenesis at molecular resolution. Here, we introduce a toolbox of nanobodies recognising various domains of the two Drosophila titin homologs, Sallimus and Projectin, as well as the key sarcomeric proteins Obscurin, α-Actinin, and Zasp52. We verified the superior labelling qualities of our nanobodies in muscle tissue as compared to antibodies. By applying our toolbox to larval muscles, we found a gigantic Sallimus isoform stretching more than 2 µm to bridge the sarcomeric I-band, while Projectin covers almost the entire myosin filaments in a polar orientation. Transgenic expression of tagged nanobodies confirmed their high affinity-binding without affecting target protein function. Finally, adding a degradation signal to anti-Sallimus nanobodies suggested that it is difficult to fully degrade Sallimus in mature sarcomeres; however, expression of these nanobodies caused developmental lethality. These results may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.

Evolutionary dissection of monkeypox virus: Positive Darwinian selection drives the adaptation of virus-host interaction proteins.

The emerging and ongoing outbreak of human monkeypox (hMPX) in 2022 is a serious global threat. An understanding of the evolution of the monkeypox virus (MPXV) at the single-gene level may provide clues for exploring the unique aspects of the current outbreak: rapidly expanding and sustained human-to-human transmission. For the current investigation, alleles of 156 MPXV coding genes (which account for >95% of the genomic sequence) have been gathered from roughly 1,500 isolates, including those responsible for the previous outbreaks. Using a range of molecular evolution approaches, we demonstrated that intra-species homologous recombination has a negligible effect on MPXV evolution. Despite the fact that the majority of the MPXV genes (64.10%) were subjected to negative selection at the whole gene level, 10 MPXV coding genes (MPXVgp004, 010, 012, 014, 044, 098, 138, 178, 188, and 191) were found to have a total of 15 codons or amino acid sites that are known to evolve under positive Darwinian selection. Except for MPXVgp138, almost all of these genes encode proteins that interact with the host. Of these, five ankyrin proteins (MPXVgp004, 010, 012, 178, and 188) and one Bcl-2-like protein (MPXVgp014) are involved in poxviruses' host range determination. We discovered that the majority (80%) of positive amino acid substitutions emerged several decades ago, indicating that these sites have been under constant selection pressure and that more adaptable alleles have been circulating in the natural reservoir. This finding was also supported by the minimum spanning networks of the gene alleles. The three positive amino acid substitutions (T/A426V in MPXVgp010, A423D in MPXVgp012, and S105L in MPXVgp191) appeared in 2019 or 2022, indicating that they would be crucial for the virus' eventual adaptation to humans. Protein modeling suggests that positive amino acid substitutions may affect protein functions in a variety of ways. Further study should focus on revealing the biological effects of positive amino acid substitutions in the genes for viral adaptation to humans, virulence, transmission, and so on. Our study advances knowledge of MPXV's adaptive mechanism and provides insights for exploring factors that are responsible for the unique aspects of the current outbreak.

Characterization of degradation signals at protein C-termini.

Protein termini are critical for protein functions. They are often more accessible than internal regions and thus are frequently subjected to various modifications that affect protein function. Protein termini also contribute to regulating protein lifespan. Recent studies have revealed a series of degradation signals located at protein C-termini, termed C-degrons or C-end degrons. C-degrons have been implicated as underlying a protein quality surveillance system that eliminates truncated, cleaved and mislocalized proteins. Despite the importance of C-degrons, our knowledge of them remains sparse. Here, we describe an established framework for the characterization of C-degrons by Global Protein Stability (GPS) profiling assay, a fluorescence-based reporter system for measuring protein stability in cellulo. Furthermore, we apply an approach that couples GPS with random peptide libraries for unbiased and context-independent characterization of C-degron motifs. Our methodology provides a robust and efficient platform for analyzing the degron potencies of C-terminal peptides, which can significantly accelerate our understanding of C-degrons.

UNC45A-related osteo-oto-hepato-enteric syndrome in a Chinese neonate

Unexplained diarrhea and cholestasis are common clinical phenotypes in newborns, indicating there is only a little common genetic basis for these conditions. However, it has been reported that defects in the UNC45A gene can lead to osteo-oto-hepato-enteric syndrome. However, to date, only 10 patients with this syndrome have been reported in 2 studies; therefore, there is still a lack of analysis regarding the correlation between disease phenotype and genotype. Trio-whole exome sequencing was conducted using DNA samples from a newborn with congenital diarrhea and cholestasis from a Chinese Han family. The UNC45A variants were verified using Sanger sequencing. In addition, we applied a crystal structure model to analyze the potential hazards associated with the variants. The plasmids were constructed in vitro and transfected into human 293T cells for Western blot (WB) analysis. After the mutant protein was fused with the Green Fluorescent Protein label, intracellular localization was observed using laser confocal microscopy. The gene detection results showed that the UNC45A gene of the newborn examined in the present study harbored the compound heterozygous variants p.Arg819Ter, and p.Leu237Pro; this was confirmed via Sanger sequencing. Analysis of the Leu237Pro crystal structure model suggested that this variant may decrease local structural stability and affect protein function. The Western blot and laser confocal microscopy observation results suggested that the Leu237Pro mutation leads to reduced protein expression, while the Arg819Ter mutation completely inhibits the expression of the protein. The compound heterozygous variants of UNC45A (p.Arg819Ter and p.Leu237Pro) may be pathogenic factors of congenital diarrhea and cholestasis in this neonatal patient. Therefore, UNC45A deficiency should be considered when intractable diarrhea and cholestasis occur in newborns.

Store-Operated Calcium Entry in Breast Cancer Cells Is Insensitive to Orai1 and STIM1 N-Linked Glycosylation

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

Genome-Wide Association Screening Determines Peripheral Players in Male Fertility Maintenance

Deciphering the functional relationships of genes resulting from genome-wide screens for polymorphisms that are associated with phenotypic variations can be challenging. However, given the common association with certain phenotypes, a functional link should exist. We have tested this prediction in newly sequenced exomes of altogether 100 men representing different states of fertility. Fertile subjects presented with normal semen parameters and had naturally fathered offspring. In contrast, infertile probands were involuntarily childless and had reduced sperm quantity and quality. Genome-wide association study (GWAS) linked twelve non-synonymous single-nucleotide polymorphisms (SNPs) to fertility variation between both cohorts. The SNPs localized to nine genes for which previous evidence is in line with a role in male fertility maintenance: ANAPC1, CES1, FAM131C, HLA-DRB1, KMT2C, NOMO1, SAA1, SRGAP2, and SUSD2. Most of the SNPs residing in these genes imply amino acid exchanges that should only moderately affect protein functionality. In addition, proteins encoded by genes from present GWAS occupied peripheral positions in a protein-protein interaction network, the backbone of which consisted of genes listed in the Online Mendelian Inheritance in Man (OMIM) database for their implication in male infertility. Suggestive of an indirect impact on male fertility, the genes focused were indeed linked to each other, albeit mediated by other interactants. Thus, the chances of identifying a central player in male infertility by GWAS could be limited in general. Furthermore, the SNPs determined and the genes containing these might prove to have potential as biomarkers in the diagnosis of male fertility.

A Novel De Novo Frameshift Pathogenic Variant in the FAM111B Resulting in Progressive Osseous Heteroplasia Phenotype.

Progressive osseous heteroplasia (POH) is a rare, debilitating disorder characterized by heterotopic ossification in the skin and muscles, resulting in contractures of the joints and progressive loss of function. While 60-70% of the POH patients have paternally inherited, inactivating pathogenic variants in GNAS, the remaining 30-40% have no known etiology. FAM111B pathogenic variants, located on chromosome 11q12.1, cause POIKTMP (hereditary fibrosing poikiloderma with tendon contractures, myopathy, and pulmonary fibrosis), a very rare, autosomal-dominant disorder with high frequency of de novo missense pathogenic variants, which affects multiple tissues and organs, causing extensive fibrosis and muscle adiposis, though the exact mechanism is unknown. To our knowledge, there are no reports of FAM111B associated with POH. We describe the first case of POH phenotype associated with a novel de novo frameshift pathogenic variant in the FAM111B and present an analysis of the protein structure and function caused by this genomic disruption. A 15-year-old African-American male presented with generalized calcific nodules, progressive contractures, and muscle weakness leading to immobility, beginning at 6years of age. Cutaneous examination showed generalized hard nodules varying from small to plaque-like ulcerated erupted skin lesions. Biochemical evaluation revealed 25(OH) vitamin D insufficiency (20ng/mL), and normal levels of parathyroid hormone, FGF-23, alkaline phosphatase, calcium, and phosphorus. Skeletal survey radiographs and computed tomography (CT) of the chest, abdomen, and pelvis showed extensive soft tissue and muscle heterotopic ossifications involving shoulders, axillae, trunk, abdomen, pelvis, upper and lower extremities, in a clumped, conglomerate distribution within muscle, subcutaneous fat, and in some areas extending to the skin. There was no pulmonary fibrosis on the chest CT. The clinical and radiographic findings were most consistent with POH. A trio-clinical exome sequencing revealed a de novo heterozygous likely pathogenic variant in the FAM111B (OMIM # 615584) (c.1462delT [p.Cys488Valfs*21]). The resulted frameshift change in exon 4 replaced C-terminal region with 21 alternative amino acids. Multiple, previously reported disease-associated variants appear to localize within the trypsin-like cysteine/serine peptidase domain in which this variant occurs, supporting the functional significance of this region, though none have been previously reported to be associated with POH phenotype. Our 3D protein modeling showed obliteration of predicted protein folding and structure, and elimination of the zinc-binding domain, likely severely affecting protein function. This is the first case of POH phenotype associated with a novel de novo pathogenic frameshift variant in FAM111B. Whether the frameshift change in FAM111B predicts POH remains unclear. Further evaluations are necessary to fully elucidate this finding and the potential role and mechanism by which the FAM111B variants contributes to POH phenotype.

Genetic diversity in the metronidazole metabolism genes nitroreductases and pyruvate ferredoxin oxidoreductases in susceptible and refractory clinical samples of Giardia lamblia

The effectiveness of metronidazole against the tetraploid intestinal parasite Giardia lamblia is dependent on its activation/inactivation within the cytoplasm. There are several activating enzymes, including pyruvate ferredoxin reductase (PFOR) and nitroreductase (NR) 1 which metabolize metronidazole into toxic forms, while NR2 on the other hand inactivates it. Metronidazole treatment failures have been increasing rapidly over the last decade, indicating genetic resistance mechanisms. Analyzing genetic variation in the PFOR and NR genes in susceptible and refractory Giardia isolates may help identify potential markers of resistance.Full length PFOR1, PFOR2, NR1 and NR2 genes from clinical culturable isolates and non-cultured clinical Giardia assemblage B samples were cloned, sequenced and single nucleotide variants (SNVs) were analyzed to assess genetic diversity and alleles.A similar ratio of amino acid changing SNVs per gene length was found for the NRs; 4.2% for NR1 and 6.4% for NR2, while the PFOR1 and PFOR2 genes had less variability with a ratio of 1.1% and 1.6%, respectively. One of the samples from a refractory case had a nonsense mutation which caused a truncated NR1 gene in one out of six alleles. Further, we found three NR2 alleles with frameshift mutations, possibly causing a truncated protein in two susceptible isolates. One of these isolates was homozygous for the affected NR2 allele. Three nsSNVs with potential for affecting protein function were found in the ferredoxin domain of the PFOR2 gene. The considerable variation and discovery of mutations possibly causing dysfunctional NR proteins in clinical Giardia assemblage B isolates, reveal a potential for genetic link to metronidazole susceptibility and resistance.