Skeletal Anomalies Research Articles

Exome and genome sequencing to unravel the precise breakpoints of partial trisomy 6q and partial Monosomy 2q

BackgroundPatients with complex phenotypes and a chromosomal translocation are particularly challenging, since several potentially pathogenic mechanisms need to be investigated.Case presentationHere, we combined exome and genome sequencing techniques to identify the precise breakpoints of heterozygous microduplications in the 6q25.3-q27 region and microdeletions in the 2q37.1-q37.3 region in a proband. The 5-year-old girl exhibited a severe form of congenital cranial dysinnervation disorder (CCDD) in addition to skeletal dysmorphism anomalies and severe intellectual disability. This is the second case affecting chromosomes 2q and 6q. The individual’s karyotype showed an unbalanced translocation 46,XX,del(2)t(2;6)(q37.1;q25.3), which was inherited from her unaffected father [46,XY,t(2;6)(q37.1;q25.3)]. We also obtained the precise breakpoints of a de novo heterozygous copy number deletion [del(2)(q37.1q37.3)chr2:g.232963568_24305260del] and a copy number duplication [dup(6)(q25.3q27)chr6:g.158730978_170930050dup]. The parental origin of the observed balanced translocation was not clear because the parents declined genetic testing.ConclusionPatients with a 2q37 deletion and 6q25.3 duplication may exhibit severe significant neurological and skeletal dysmorphisms, and the utilization of exome and genome sequencing techniques has the potential to unveil the entire translocation of the CNV and the precise breakpoint.

Associations across 22 dental and craniovertebral anomalies or variations, sagittal skeletal relationships, and vertical growth patterns: a comprehensive epidemiological study of 43 dentoskeletal traits

IntroductionDespite researchers' recent interest in identifying links between some dental and craniovertebral abnormalities, there are many important, unassessed gaps in our knowledge of this matter. In addition, previous samples were small. This large study aimed to examine, for the first time, the occurrence/severity of numerous dental and skeletal anomalies or variations and their correlations with each other and with growth patterns.MethodsThis epidemiological study was conducted on pretreatment radiographs of 1194 patients from 3 cities (815 females). Skeletal sagittal skeletal relationships and vertical growth patterns were determined. The occurrence/severity were assessed for: cervical vertebral fusion (CVF), atlas posterior arch deficiency (APAD), ponticulus posticus (PP), sella turcica bridging (STB), hypodontia, oligodontia, hyperdontia, missing of maxillary laterals, microdontia, macrodontia, root dilaceration, odontoma, taurodontism, dental fusion, dental gemination, enamel pearl, permanent molar ankylosis, primary molar ankylosis, dens in dente, dens invaginatus, dental impaction, ectopic eruption, and dental transposition. Incidental findings were recorded as well. Concurrent anomalies, sex dimorphism, and correlations across variables were examined statistically, adjusting for the false discovery rate (α = 0.05).ResultsPrevalence was calculated for 43 dentoskeletal traits/anomalies (22 abnormalities/variations [plus their severities/types] as well as 21 incidentally found traits/anomalies). Dental impaction may be more common in hyperdivergent and severer cases of sella bridging; also, primary molar ankylosis was associated with missing teeth. Dental impaction was associated only with STB and not with PP, APAD, or vertebral fusion. The only association observed among the four skeletal anomalies was seen between APAD and CVF. Merely the variables ‘sagittal skeletal relationships, vertical growth patterns, PP, and APAD’ showed sexual dimorphism; of these, only vertical growth pattern and APAD remained sexually dimorphic after adjusting for the FDR; still, the other two remained marginally significant and worth further evaluations. Sex dimorphism did not exist in concurrent abnormalities. The skeletal Class III was associated with the concurrent occurrence of craniovertebral, dental, and dentoskeletal abnormalities. Skeletal Class I was associated with fewer occurrences of concurrent dental anomalies. Vertical growth patterns were not associated with concurrent dental or dentoskeletal anomalies. However, the hyperdivergent pattern was associated with fewer cases of concurrent craniovertebral abnormalities.ConclusionsSTB and hyperdivergent pattern were associated with dental impaction. However, APAD, CVF, or PP were not associated with dental impaction. APAD was associated with CVF. Sexual dimorphism existed conclusively in the case of vertical growth patterns and APAD. Concurrent abnormalities (dental, skeletal, and dentoskeletal) were associated with skeletal Class III.

Skeletal Morphogenesis and Anomalies in Gilthead Seabream: A Comprehensive Review.

The gilthead seabream, one of the most important species in Mediterranean aquaculture, with an increasing status of exploitation in terms of production volume and aquafarming technologies, has become an important research topic over the years. The accumulation of knowledge from several studies conducted during recent decades on their functional and biological characteristics has significantly improved their aquacultural aspects, namely their reproductive success, survival, and growth. Despite the remarkable progress in the aquaculture industry, hatchery conditions are still far from ideal, resulting in frequent abnormalities at the beginning of intensive culture, entailing significant economic losses. Those deformities are induced during the embryonic and post-embryonic periods of life, and their development is still poorly understood. In the present review, we created a comprehensive synthesis that covers the various aspects of skeletal morphogenesis and anomalies in the gilthead seabream, highlighting the genetic, environmental, and nutritional factors contributing to bone deformities and emphasized the potential of the gilthead seabream as a model organism for understanding bone morphogenesis in both aquaculture and translational biological research. This review article addresses the existing lack in the literature regarding gilthead seabream bone deformities, as there are currently no comprehensive reviews on this subject.

Specific heterozygous variants in MGP lead to endoplasmic reticulum stress and cause spondyloepiphyseal dysplasia

Matrix Gla protein (MGP) is a vitamin K-dependent post-translationally modified protein, highly expressed in vascular and cartilaginous tissues. It is a potent inhibitor of extracellular matrix mineralization. Biallelic loss-of-function variants in the MGP gene cause Keutel syndrome, an autosomal recessive disorder characterized by widespread calcification of various cartilaginous tissues and skeletal and vascular anomalies. In this study, we report four individuals from two unrelated families with two heterozygous variants in MGP, both altering the cysteine 19 residue to phenylalanine or tyrosine. These individuals present with a spondyloepiphyseal skeletal dysplasia characterized by short stature with a short trunk, diffuse platyspondyly, midface retrusion, progressive epiphyseal anomalies and brachytelephalangism. We investigated the cellular and molecular effects of one of the heterozygous deleterious variants (C19F) using both cell and genetically modified mouse models. Heterozygous ‘knock-in’ mice expressing C19F MGP recapitulate most of the skeletal anomalies observed in the affected individuals. Our results suggest that the main underlying mechanism leading to the observed skeletal dysplasia is endoplasmic reticulum stress-induced apoptosis of the growth plate chondrocytes. Overall, our findings support that heterozygous variants in MGP altering the Cys19 residue cause autosomal dominant spondyloepiphyseal dysplasia, a condition distinct from Keutel syndrome both clinically and molecularly.

Determination of Bone Developments of Rat Anterior and Posterior Extremity Bones in Prenatal and Postnatal Period by Double Staining Method

Aim: In our study, we aimed to determine the morphological development of the bones of the anterior and posterior extremity by staining the rat fetus and offspring skeletons with the double staining method. Method: In the current study, seven groups three prenatal (16th, 18th, and 20th days) and four postnatal (0th, 3th, 7th and 12th days) were formed from the foetuses and offsprings obtained from 13 pregnant rats. Then, it was stained with double staining method. Anterior and posterior extremity images of the fetuses and offsprings were examined under a stereo microscope, and ossification findings were determined. Total bone and ossification lengths as well as ossification areas were measured using the ImageJ software. Results: The first cartilage destruction in fetuses occurred on the 16th day of pregnancy in the clavicle, scapula, humerus, radius and ulna; It was seen in the femur, tibia and fibula on the 18th day of pregnancy. The first ossification centres were in the clavicle, scapula and humerus on the 18th day of pregnancy; It was seen in the radius, ulna, femur, tibia, fibula and 2-5 metatarsal bones on the 20th day of pregnancy. The secondary ossification centre was seen on the 0th day (birthday) in the scapula and humerus, on the 7th day after birth in the ulna and radius, and on the 12th day after birth in the femur and tibia. In the study, while the ossification rate in 20-day-old foetuses was 48.9% in the humerus, 53.2% in the radius, 55.7% in the ulna, 33.6% in the femur, 43.2% in the tibia, 44.3% in the fibula, it was determined that it reached 69.7% in the humerus, 78.4% in the radius, 73.3% in the ulna, 63.5% in the femur, 75.5% in the tibia, and 69.2% in the fibula on the 12th day after birth. Conclusion: In this study, we revealed the morphological changes of the anterior and posterior extremity bones of fetuses and offsprings in the normal developmental course. We think that these results will shed light on the studies to be conducted on the detection of skeletal anomalies in teratological studies and contribute to a more comprehensive evaluation of the findings to be obtained from the studies to be conducted.

An unexpected presentation of very severe hypertriglyceridemia in a boy with Coffin-Lowry syndrome: a case report

BackgroundCoffin-Lowry syndrome (CLS) is a rare X-linked condition with intellectual disability, growth retardation, characteristic facies and skeletal anomalies. To date, hypertriglyceridemia has not been reported in literature to be associated with CLS.Case PresentationHerein, we report a case of very severe hypertriglyceridemia 32 mmol/L (2834 mg/dL) detected incidentally at three months old in an otherwise well boy born late preterm with intrauterine growth restriction, when he presented with lipaemic plasma. He was later diagnosed with CLS. No pathogenic mutations were found for hypertriglyceridemia, and no secondary causes could explain his very severe hypertriglyceridemia.ConclusionsThe very severe hypertriglyceridemia in this case may appear to be a serious presentation of an unrecognised clinical feature of CLS, further expanding its phenotype.

Case Report: A rare case of Fanconi anaemia

Fanconi anaemia (FA) is autosomal recessive but can also be an autosomal dominant or X-linked recessive disease. In India, Fanconi anaemia is a very rarely seen disorder. Clinical findings in FA include pancytopenia, hyperpigmentation or hypopigmentation, skeletal anomalies, small stature or growth retardation, endocrine abnormalities, anal atresia, deafness, malignancy of head and neck, and it has a tendency to run in families. Diagnosis of FA can be made on clinical findings and laboratory examination. Recommended prenatal testing like chorionic villus sampling and amniocentesis. Androgen therapy and hematopoietic stem cell transplantation (HSCT) are treatment modalities recommended for FA. Follow-up of all diagnosed FA patients is essential throughout their lifetime. Having a healthcare team with diverse expertise is vital in preventing anemia and malignancy. A meticulous plan and unwavering support are also crucial. Outcomes of FA patients is desolate due to complications associated with it. Our aim of the study is to increase awareness among physicians about the presence of such rare diseases. Well-timed diagnosis with appropriate treatment is needed to prevent complication.

Identification of coral disease within the high-latitude reef, Lord Howe Island Marine Park

Coral disease prevalence has significantly increased under a changing climate, impacting coral community structure and functionality. The impacts and ecology of coral diseases are unclear in most high-latitude reefs (coral reefs above 28° north and below 28° south). High-latitude locations are vulnerable to climate change; therefore, identifying diseases and developing region-specific baselines are important for local management. We report the first coral disease findings at the UNESCO World Heritage listed Lord Howe Island Marine Park (31.5°S, 159°E), the southernmost coral reef system. This study assessed coral disease prevalence during November 2018, March 2019 and October 2019. Surveys from three lagoonal reefs identified four coral diseases: white syndrome, skeletal eroding band, growth anomalies and endolithic hypermycosis impacting six coral taxa (Acropora, Isopora, Montipora, Pocillopora, Porites and Seriatopora). Overall, disease prevalence was 5 ± 1% and significantly differed between time and site. Disease prevalence was highest in November 2018 (10 ± 1%) and significantly lower during March 2019 (5 ± 1%), coinciding with a bleaching event. White syndrome was the most prevalent disease (4 ± 1%) with 83 colonies of six taxa affected, predominately Isopora. Acroporids recorded the highest disease susceptibility, with three of the four diseases observed. Documenting baseline coral disease prevalence and monitoring throughout a bleaching event assists our understanding of disease ecology dynamics under current climate change impacts at high-latitude reefs.

Alternative Genetic Diagnoses in Axenfeld-Rieger Syndrome Spectrum.

Axenfeld-Rieger anomaly (ARA) is a specific ocular disorder that is frequently associated with other systemic abnormalities. PITX2 and FOXC1 variants explain the majority of individuals with Axenfeld-Rieger syndrome (ARS) but leave ~30% unsolved. Here, we present pathogenic/likely pathogenic variants in nine families with ARA/ARS or similar phenotypes affecting five different genes/regions. USP9X and JAG1 explained three families each. USP9X was recently linked with syndromic cognitive impairment that includes hearing loss, dental defects, ventriculomegaly, Dandy-Walker malformation, skeletal anomalies (hip dysplasia), and other features showing a significant overlap with FOXC1-ARS. Anterior segment anomalies are not currently associated with USP9X, yet our cases demonstrate ARA, congenital glaucoma, corneal neovascularization, and cataracts. The identification of JAG1 variants, linked with Alagille syndrome, in three separate families with a clinical diagnosis of ARA/ARS highlights the overlapping features and high variability of these two phenotypes. Finally, intragenic variants in CDK13, BCOR, and an X chromosome deletion encompassing HCCS and AMELX (linked with ocular and dental anomalies, correspondingly) were identified in three additional cases with ARS. Accurate diagnosis has important implications for clinical management. We suggest that broad testing such as exome sequencing be applied as a second-tier test for individuals with ARS with normal results for PITX2/FOXC1 sequencing and copy number analysis, with attention to the described genes/regions.

Phenotype, genotype, and clinical outcome of Taiwanese with congenital nephrotic syndrome

Congenital nephrotic syndrome (CNS) is one of the important causes of end-stage kidney disease in children. Studies on the genotype, phenotype, and clinical outcome in infants with CNS caused by genetic mutations are scarce. We analyzed the genetic background, clinical manifestations, treatment response, and prognosis of pediatric patients with CNS in Taiwan. Fifteen infants with CNS were enrolled, and 11 patients of median age 21 (interquartile range 3∼44) days caused by genetic mutations from 10 unrelated families were included in the study. Of the eleven patients, 9 had extra-renal manifestations including microcephaly, facial dysmorphism, and skeletal anomalies. More than two-thirds of the patients had disease onset before 1 month of age. Diffuse meningeal sclerosis was the most common histological characteristic. Whole exome sequencing followed by direct Sanger sequence revealed mutations in OSGEP (R247Q), WT1 (R366H and R467Q), LAMB2 (Q1209∗ and c. 5432-5451 19 bp deletion), NUP93 (D302V), and LAGE3 (c.188+1G>A). Three of the variants were novel. Corticosteroids and/or immunosuppressants were administered in 2 patients, but both were refractory to treatment. During the mean 3.5 years of follow-up, all but two died of uremia and sepsis. The two survivors reached end-stage kidney disease and required peritoneal dialysis, and one of them underwent uneventful renal transplantation. The majority of patients with CNS in Taiwan were caused by OSGEP followed by WT1 mutation. R247Q is the hotspot mutation of OSGEP in Taiwan. CNS patients in Taiwan suffer from significant morbidity and mortality.

Assessing the toxicity of aegerolysin-based bioinsecticidal complexes using the sea urchin Paracentrotus lividus as model organism

The use of alternative solutions for pest management to replace pesticides in agriculture is of great interest. Proteinaceous complexes deriving from edible oyster mushrooms were recently proposed as environmentally friendly bioinsecticides. Such complexes, composed of ostreolysin A6 (OlyA6) and pleurotolysin B (PlyB), target invertebrate-specific membrane sphingolipids in insect's midgut, causing death through the formation of transmembrane pores. In this work, the potential impact of OlyA6/PlyB complexes was tested in the Mediterranean sea urchin Paracentrotus lividus, as an indicator of environmental quality. The ability of the fluorescently tagged OlyA6 to bind sea urchin gametes (sperm, eggs), the lipidome of sea urchin gametes, and the potential toxic effects and developmental anomalies caused by OlyA6/PlyB complexes on P. lividus early development (embryo, larvae) were investigated. The binding of the fluorescently tagged OlyA6 could be observed only in sea urchin eggs, which harbor OlyA6 sphingolipid membrane receptors, conversely to sperm. High protein concentrations affected sea urchin fertilization (>750 µg/L) and early development (> 375 µg/L in embryos; >100 µg/L in larvae), by causing toxicity and morphological anomalies in embryos and larvae. The main anomalies consisted in delayed embryos and incorrect migration of the primary mesenchyme cells that caused larval skeletal anomalies. The classification of these anomalies indicated a slight environmental impact of OlyA6/PlyB complexes at concentrations higher than 750 µg/L. Such impact should not persist in the marine environment, due to the reversible anomalies observed in sea urchin embryos and larvae that may promote defense strategies. However, before promoting the use of OlyA6/PlyB complexes as bio-pesticides at low concentrations, further studies on other marine coastal species are needed.

SBDSR126T rescues survival of sbds -/- zebrafish in a dose-dependent manner independently of Tp53.

Defects in ribosomal biogenesis profoundly affect organismal development and cellular function, and these ribosomopathies produce a variety of phenotypes. One ribosomopathy, Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, pancreatic exocrine insufficiency, and skeletal anomalies. SDS results from biallelic mutations in SBDS, which encodes a ribosome assembly factor. Some individuals express a missense mutation, SBDS R126T , along with the common K62X mutation. We reported that the sbds-null zebrafish phenocopies much of SDS. We further showed activation of Tp53-dependent pathways before the fish died during the larval stage. Here, we expressed SBDS R126T as a transgene in the sbds -/- background. We showed that one copy of the SBDS R126T transgene permitted the establishment of maternal zygotic sbds-null fish which produced defective embryos with cdkn1a up-regulation, a Tp53 target involved in cell cycle arrest. None survived beyond 3 dpf. However, two copies of the transgene resulted in normal development and lifespan. Surprisingly, neutropenia persisted. The surviving fish displayed suppression of female sex differentiation, a stress response in zebrafish. To evaluate the role of Tp53 in the pathogenesis of sbds -/- fish phenotype, we bred the fish with a DNA binding deficient allele, tp53 M214K Expression of the loss-of-function tp53 M214K did not rescue neutropenia or survival in sbds-null zebrafish. Increased expression of cdkn1a was abrogated in the tp53 M214K/M214K ;sbds -/- fish. We conclude that the amount of SBDSR126T protein is important for development, inactivation of Tp53 fails to rescue neutropenia or survival in the sbds-null background, and cdkn1a up-regulation was dependent on WT tp53 We hypothesize that additional pathways are involved in the pathophysiology of SDS.

Novel inherited CDX2 variant segregating in a family with diverse congenital malformations of the genitourinary system.

Anorectal malformations (ARMs) constitute a group of congenital defects of the gastrointestinal and urogenital systems. They affect males and females, with an estimated worldwide prevalence of 1 in 5000 live births. These malformations are clinically heterogeneous and can be part of a syndromic presentation (syndromic ARM) or as a nonsyndromic entity (nonsyndromic ARM). Despite the well-recognized heritability of nonsyndromic ARM, the genetic etiology in most patients is unknown. In this study, we describe three siblings with diverse congenital anomalies of the genitourinary system, anemia, delayed milestones, and skeletal anomalies. Genome sequencing identified a novel, paternally inherited heterozygous Caudal type Homeobox 2 (CDX2) variant (c.722A > G (p.Glu241Gly)), that was present in all three affected siblings. The variant identified in this family is absent from population databases and predicted to be damaging by most in silico pathogenicity tools. So far, only two other reports implicate variants in CDX2 with ARMs. Remarkably, the individuals described in these studies had similar clinical phenotypes and genetic alterations in CDX2 CDX2 encodes a transcription factor and is considered the master regulator of gastrointestinal development. This variant maps to the homeobox domain of the encoded protein, which is critical for interaction with DNA targets. Our finding provides a potential molecular diagnosis for this family's condition and supports the role of CDX2 in anorectal anomalies. It also highlights the clinical heterogeneity and variable penetrance of ARM predisposition variants, another well-documented phenomenon. Finally, it underscores the diagnostic utility of genomic profiling of ARMs to identify the genetic etiology of these defects.

A Case of Prune Belly Syndrome: Radiological Aspects of a Rare Disease

Prune Belly Syndrome (PBS) is a rare but morbid entity usually found in male neonates. It is classically defined by a triad of cardinal features that includes deficient abdominal wall musculature, urinary tract malformation and in males, cryptorchidism. Most of the patients have associated pulmonary, cardiac, skeletal and gastrointestinal tract anomalies. Children often require numerous surgical interventions including bilateral orchidopexy as well as individually tailored urinary tract and abdominal wall reconstruction. The prognosis of infants with PBS may be improved by quality antenatal follow-up, to enable the early diagnosis and preparation for prompt surgical intervention. We describe a case of PBS and emphasize the role of imaging, in the process of diagnosis and briefly review the subject.

A novel epidermal nevus syndrome associated with congenital pulmonary airway malformation

Epidermal nevus syndromes involve a group of disorders characterized by the type of epidermal nevus and are associated with an extracutaneous abnormality. Most epidermal nevus syndromes involve skin lesions associated with central nervous system, ocular or skeletal anomalies. We report a previously undescribed type of epidermal nevus syndrome in a preterm infant. The face and mouth were remarkable for large exophytic verrucous lesions and extensive pedunculated tumors involving the scalp. The infant was additionally affected by type 3 Congenital Pulmonary Airway Malformation (CPAM). Microscopic examination of the skin lesions revealed multiple layers of immature epithelium and the complete absence of adnexal structures. Chromosomal microarray analysis demonstrated no abnormalities. Family history was significant for numerous maternal relatives with multiple first and second trimester pregnancy losses. The distribution of lesions in this patient was characteristic of postzygotic mosaicism. We believe this case may represent a new histological subtype of epidermal nevi associated with a CPAM lesion. Future molecular research may indicate whether this disorder may be categorized as a distinct entity.

Maternal-fetal safety evaluation of an aqueous extract of Casearia sylvestris leaves in rats.

This study evaluated the maternal, embryotoxic, and teratogenic effects of the aqueous extract of Casearia sylvestris (AECS), a species listed in the Unique Health System of Brazil, and widely used for treating several conditions, such as diarrhea, wounds, pain, and ulcers. Pregnant rats were daily treated orally with 0, 175, 350, or 700 mg/kg/body weight of AECS, from gestational day (GD) 6 to 15 (organogenesis period). On GD 20, the pregnant rats were euthanized, and the litters submitted to an assessment of fetal development. No clinical signs of toxicity were observed in the dams during the treatment. In the embryo-fetal development study, a significant increase in the basal zone height of the placenta was observed in the intermediate dose group. Furthermore, there was a significant increase in the relative anogenital distance measurement of female fetuses in the lowest and intermediate dose groups. Although no visceral abnormalities were observed in the treated-fetuses, skeletal anomalies evidenced by changes in the ossification of the sternum and the presence of supernumerary ribs were found in the intermediate and high dose groups. In conclusion, the treatment with AECS during organogenesis at this dose level had detrimental effects on the normal development of fetuses.

FRI387 Reproductive Capacity Of Znhit3 Knockout Mouse Model

Abstract Disclosure: D. Kira: None. Z.A. Hawkins: None. L.P. Chorich: None. S. Brakta: None. L. Gan: None. L.C. Layman: None. Introduction: Zinc finger, HIT domain containing 3 (ZNHIT3), also known as thyroid hormone receptor-interacting protein 3 (TRIP-3), is a 155 amino acid protein with an N-terminus Zinc finger HIT domain (11-42a.a) that is involved in pre-ribosomal RNA processing. It also has a C-terminus short conserved LxxLL binding motif that interacts with the ligand binding domain of the thyroid receptor in the presence of thyroid hormone as a co-activator associated with hepatocyte nuclear factor-4α (HNF-4α). ZNHIT3 is localized to the cytoplasm and the nucleus. Biallelic ZNHIT3 pathogenic variants in the N-terminus have been identified in persons with Progressive encephalopathy with Edema, Hypsarrhythmia, and Optic atrophy (PEHO) syndrome, a severe autosomal recessive neurodevelopmental disorder characterized by extreme cerebellar atrophy due to almost total loss of granule neurons. Heterozygous pathogenic variants in ZNHIT3 affecting the C-terminus have been demonstrated in individuals with Mayer-Rokitansky-Küster-Hauser (MRKH). MRKH consists of the congenital absence of the uterus and vagina, either as isolated Mullerian aplasia (type 1) or associated with renal, skeletal, cardiac, and auditory anomalies (type 2). Previous studies of animal models utilizing morpholino injection-induced knockdown and CRISPR editing of znhit3 in zebrafish both showed cerebellar defects and microcephaly. We are not aware of a mouse model of Znhit3.Hypothesis: We hypothesize that the Znhit3 knockout (KO) mouse will demonstrate reproductive abnormalities and an MRKH-related phenotype. Methods: Heterozygous Znhit3 (Znhit3+/-) KO mice were generated by CRISPR. A 1 base deletion was introduced in exon 2, creating a frameshift and a stop codon in exon 3, which leads to a truncated protein. We then crossed male and female Znhit3+/- mice and Znhit3+/- and wild-type Znhit3+/+ (WT) mice. Pups' genotypes were confirmed with DNA sequencing of tail samples. Pups were weighed weekly from age 3 weeks to 16 weeks. Results: Mating 17 pairs of Znhit3+/- mice resulted in a total of 73 pups born, of which 50 were Znhit3+/- and, 23 were WT with a ratio of almost 2:1. No homozygous Znhit3 (Znhit3-/-) KO pups were born. There was no significant difference in weights between Znhit3+/- and WT pups. Conclusion: Heterozygous Znhit3+/- animals appeared to be phenotypically normal with similar weights to WT. However, the Hardy-Weinberg equilibrium (1 homozygous wild-type: 2 heterozygous: 1 homozygous variant) was disrupted, as no Znhit3−/− homozygotes were born, suggesting embryonic lethality. It is possible that central nervous system, cardiac, and/or renal defects could be responsible for this lethality, which will be studied in embryos at E18.5, 15.5, and 12.5. A detailed phenotypic characterization will be performed with regard to reproductive function and MRKH-related organ development. Presentation: Friday, June 16, 2023

Drug Loaded 3D-Printed Poly(ε-Caprolactone) Scaffolds for Local Antibacterial or Anti-Inflammatory Treatment in Bone Regeneration.

Annual bone grafting surgeries due to bone fractures, resections of affected bones, skeletal anomalies, osteoporosis, etc. exceed two million worldwide. In this regard, the creation of new materials for bone tissue repair is one of the urgent tasks of modern medicine. Additive manufacturing, or 3D printing, offers great opportunities for the development of materials with diverse properties and designs. In this study, the one-pot technique for the production of 3D scaffolds based on poly(ε-caprolactone) (PCL) loaded with an antibiotic or anti-inflammatory drug was proposed. In contrast to previously described methods to prepare drug-containing scaffolds, drug-loaded PCL scaffolds were prepared by direct 3D printing from a polymer/drug blend. An investigation of the mechanical properties of 3D-printed scaffolds containing 0.5-5 wt% ciprofloxacin (CIP) or dexamethasone (DEX) showed almost no effect of the drug (compression modulus ~70-90 MPa) compared to unfilled PCL (74 MPa). At the same time, introducing the drug and increasing its content in the PCL matrix contributed to a 1.8-6.8-fold decrease in the specific surface area of the scaffold, depending on composition. The release of CIP and DEX in phosphate buffer solution and in the same buffer containing lipase revealed a faster release in enzyme-containing medium within 45 days. Furthermore, drug release was more intensive from scaffolds with a low drug load. Analysis of the release profiles using a number of mathematical dissolution models led to the conclusion that diffusion dominates over other probable factors. In vitro biological evaluation of the scaffolds containing DEX showed moderate toxicity against osteoblast-like and leukemia monocytic cells. Being 3D-printed together with PCL both drugs retain their biological activity. PCL/CIP and PCL/DEX scaffolds demonstrated antibacterial properties against Pseudomonas aeruginosa (a total inhibition after 48 h) and anti-inflammatory activity in experiments on TNFα-activated monocyte cells (a 4-time reduction in CD-54 expression relative to control), respectively.

The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish models

Skeletal disorders are problematic aspects for the aquaculture industry as skeletal deformities, which affect most species of farmed fish, increase production costs and affect fish welfare. Following recent findings that show the presence of osteoactive compounds in marine organisms, we evaluated the osteogenic and mineralogenic potential of commercially available microalgae strains Skeletonema costatum and Tetraselmis striata CTP4 in several fish systems. Ethanolic extracts increased extracellular matrix mineralization in gilthead seabream (Sparus aurata) bone-derived cell cultures and promoted osteoblastic differentiation in zebrafish (Danio rerio) larvae. Long-term dietary exposure to both extracts increased bone mineralization in zebrafish and upregulated the expression of genes involved in bone formation (sp7, col1a1a, oc1, and oc2), bone remodeling (acp5a), and antioxidant defenses (cat, sod1). Extracts also improved the skeletal status of zebrafish juveniles by reducing the incidence of skeletal anomalies. Our results indicate that both strains of microalgae contain osteogenic and mineralogenic compounds, and that ethanolic extracts have the potential for an application in the aquaculture sector as dietary supplements to support fish bone health. Future studies should also identify osteoactive compounds and establish whether they can be used in human health to broaden the therapeutic options for bone erosive disorders such as osteoporosis.

Clinical observation of genetically confirmed Kabuki syndrome type 1, with an undetermined type of inheritance, in a 7-year-old child

Kabuki syndrome is a rare hereditary disease that has a specific clinical picture: facial features, growth retardation, muscular hypotonia, skeletal abnormalities, dermatoglyphic abnormalities, mild to moderate mental retardation, and postnatal growth deficiency. Kabuki syndrome (Kabuki mask syndrome / Kabuki makeup syndrome) got its name for the specific features of the appearance of patients who have a facial expression similar to a special makeup that highlights the eyes and eyebrows of the characters of the Japanese Kabuki theater. The main reasons for the development of Kabuki syndrome in 75% are mutations in the KMT2D gene (KS type 1) and in 5% are mutations in the KDM6A gene (Kabuki syndrome type 2), the etiology of about 20% of cases remains unknown. Most mutations in type 1 Kabuki syndrome are formed de novo, and cases of autosomal dominant inheritance have also been described. Type 2 Kabuki syndrome has sex-linked (X-linked) inheritance. Because Kabuki syndrome is a low-prevalence, multisystem disease, patients require a variety of diagnostic and screening tests with multidisciplinary evaluation of outcomes at different stages of their lives. Early diagnosis of the disease is crucial for timely treatment and rehabilitation measures and improving the prognosis and quality of life of the child. The interest of the presented clinical observation of Kabuki syndrome lies in the importance of early recognition of clinical manifestations, timely surgical treatment, hearing rehabilitation and improving the prognosis of the patients life. The article describes a clinical observation, genetically confirmed type 1 Kabuki syndrome, in a child born in 2015 (type of inheritance not established). The interest of the presented clinical observation of Kabuki syndrome, manifested by characteristic facial features, skeletal anomalies, postnatal growth retardation, dermatoglyphic anomalies, intellectual retardation, lies in the need for a multidisciplinary approach to the diagnosis and treatment of the patient.