Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development.

Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a Tbx18:Cre (Tbx18Cre/+) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 (Smad4f/f ) in the limbs of mice. We found that the Smad4-deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan, in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 (Runx2), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4-deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia.

Tbx20 acts upstream of Wnt signaling to regulate endocardial cushion formation and valve remodeling during mouse cardiogenesis.

Cardiac valves are essential to direct forward blood flow through the cardiac chambers efficiently. Congenital valvular defects are prevalent among newborns and can cause an immediate threat to survival as well as long-term morbidity. Valve leaflet formation is a rigorously programmed process consisting of endocardial epithelial-mesenchymal transformation (EMT), mesenchymal cell proliferation, valve elongation and remodeling. Currently, little is known about the coordination of the diverse signals that regulate endocardial cushion development and valve elongation. Here, we report that the T-box transcription factor Tbx20 is expressed in the developing endocardial cushions and valves throughout heart development. Ablation of Tbx20 in endocardial cells causes severe valve elongation defects and impaired cardiac function in mice. Our study reveals that endocardial Tbx20 is crucial for valve endocardial cell proliferation and extracellular matrix development, but is not required for initiation of EMT. Elimination of Tbx20 also causes aberrant Wnt/ß-catenin signaling in the endocardial cushions. In addition, Tbx20 regulates Lef1, a key transcriptional mediator for Wnt/ß-catenin signaling, in this developmental process. Our study suggests a model in which Tbx20 regulates the Wnt pathway to direct endocardial cushion maturation and valve elongation, and provides new insights into the etiology of valve defects in humans.

Tbx18 is essential for normal development of vasculature network and glomerular mesangium in the mammalian kidney.

Tbx18 has been shown to be essential for ureteral development. However, it remains unclear whether it plays a direct role in kidney development. Here we addressed this by focusing on examining the pattern and contribution of Tbx18+ cells in the kidney and its role in kidney vascular development. Expression studies and genetic lineage tracing revealed that Tbx18 is expressed in renal capsule, vascular smooth muscle cells and pericytes and glomerular mesangial cells in the kidney and that Tbx18-expressing progenitors contribute to these cell types. Examination of Tbx18(-/-) kidneys revealed large reduction in vasculature density and dilation of glomerular capillary loops. While SMA+ cells were reduced in the mutant, PDGFRß+ cells were seen in early capillary loop renal corpuscles in the mutant, but fewer than in the controls, and further development of the mesangium failed. Analysis of kidney explants cultured from E12.5 excluded the possibility that the defects observed in the mutant were caused by ureter obstruction. Reduced proliferation in glomerular tuft and increased apoptosis in perivascular mesenchyme were observed in Tbx18(-/-) kidneys. Thus, our analyses have identified a novel role of Tbx18 in kidney vasculature development.

Mesodermal Nkx2.5 is necessary and sufficient for early second heart field development.

The vertebrate heart develops from mesoderm and requires inductive signals secreted from early endoderm. During embryogenesis, Nkx2.5 acts as a key transcription factor and plays essential roles for heart formation from Drosophila to human. In mice, Nkx2.5 is expressed in the early first heart field, second heart field pharyngeal mesoderm, as well as pharyngeal endodermal cells underlying the second heart field. Currently, the specific requirements for Nkx2.5 in the endoderm versus mesoderm with regard to early heart formation are incompletely understood. Here, we performed tissue-specific deletion in mice to dissect the roles of Nkx2.5 in the pharyngeal endoderm and mesoderm. We found that heart development appeared normal after endodermal deletion of Nkx2.5 whereas mesodermal deletion engendered cardiac defects almost identical to those observed on Nkx2.5 null embryos (Nkx2.5(-/-)). Furthermore, re-expression of Nkx2.5 in the mesoderm rescued Nkx2.5(-/-) heart defects. Our findings reveal that Nkx2.5 in the mesoderm is essential while endodermal expression is dispensable for early heart formation in mammals.

Speciation analysis of mercury in water samples by dispersive liquid-liquid microextraction coupled to capillary electrophoresis.

In this study, a method of pretreatment and speciation analysis of mercury by dispersive liquid-liquid microextraction along with CE was developed. The method was based on the fact that mercury species including methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg), and Hg(II) were complexed with 1-(2-pyridylazo)-2-naphthol to form hydrophobic chelates and l-cysteine could displace 1-(2-pyridylazo)-2-naphthol to form hydrophilic chelates with the four mercury species. Factors affecting complex formation and extraction efficiency, such as pH value, type, and volume of extractive solvent and disperser solvent, concentration of the chelating agent, ultrasonic time, and buffer solution were investigated. Under the optimal conditions, the enrichment factors were 102, 118, 547, and 46, and the LODs were 1.79, 1.62, 0.23, and 1.50 µg/L for MeHg, EtHg, PhHg, and Hg(II), respectively. Method precisions (RSD, n = 5) were in the range of 0.29-0.54% for migration time, and 3.08-7.80% for peak area. Satisfactory recoveries ranging from 82.38 to 98.76% were obtained with seawater, lake, and tap water samples spiked at three concentration levels, respectively, with RSD (n = 5) of 1.98-7.18%. This method was demonstrated to be simple, convenient, rapid, cost-effective, and environmentally benign, and could be used as an ideal alternative to existing methods for analyzing trace residues of mercury species in water samples.

A method of percutaneous vertebroplasty under the guidance of two C-arm fluoroscopes.

OBJECTIVE: To compare the clinical application in the percutaneous vertebroplasty under the guidance of one or two C-arm fluoroscopes. METHODS: One hundred forty three elderly patients with Osteoporotic vertebral compression fractures (OVCFs) underwent percutaneous vertebroplasty under the guidance of one or two C-arm fluoroscopes. The number of pulsed imagings, the time of operation and the incidence of cement leakage were recorded. RESULTS: The average number of pulsed imagings was 16.00±1.58 vs 13.07±2.00 per patient under the guidance of one vs two C-arm fluoroscopes. The average time of operation was 48.42±5.00 minutes vs 39.70±7.42 minutes per patient under the guidance of one vs two C-arm fluoroscopes. The incidence of cement leakage was 20% vs 15.7% of the patients under the guidance of one vs two C-arm fluoroscopes. The differences in the number of pulsed imagings and the time of operation were statistically significant. The difference in incidence of cement leakage was not statistically significant. CONCLUSION: The two-fluoroscopic technique reduce the labor cost, the radiation, the time of operation and the operation risk.

Coupling High Hardness and Zn Affinity in Amorphous-Crystalline Diamond for Stable Zn Metal Anodes.

The highly reversible plating/stripping of Zn is plagued by dendrite growth and side reactions on metallic Zn anodes, retarding the commercial application of aqueous Zn-ion batteries. Herein, a distinctive nano dual-phase diamond (NDPD) comprised of an amorphous-crystalline heterostructure is developed to regulate Zn deposition and mechanically block dendrite growth. The rich amorphous-crystalline heterointerfaces in the NDPD endow modified Zn anodes with enhanced Zn affinity and result in homogeneous nucleation. In addition, the unparalleled hardness of the NDPD effectively overcomes the high growth stress of dendrites and mechanically impedes their proliferation. Moreover, the hydrophobic surfaces of the NDPD facilitate the desolvation of hydrate Zn2+ and prevent water-mediated side reactions. Consequently, the Zn@NDPD presents an ultrastable lifespan exceeding 3200 h at 5 mA cm-2 and 1 mAh cm-2. The practical application potential of Zn@NDPD is further demonstrated in full cells. This work exhibits the great significance of a chemical-mechanical synergistic anode modification strategy in constructing high-performance aqueous Zn-ion batteries.

Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies.

TET2 is mutated/deleted with high frequencies in multiple forms of myeloid malignancies including MDS, CMML, MPN, and AML. However, little is known regarding the biological function of TET2 and its role in the pathogenesis of myeloid malignancies. To study the function of TET2 in vivo, we generated a Tet2 knock out mouse model. Deletion of Tet2 in mice led to dramatic reduction in the 5-hydroxymethylcytosine levels and concomitant increase in the 5-methylcytosine levels in the genomic DNA of BM cells. The Tet2(-/-) mice contained an increased Lin(-)Sca-1(+)c-Kit(+) (LSK) cell pool before the development of myeloid malignancies. A competitive reconstitution assay revealed that Tet2(-/-) LSK cells had an increased hematopoietic repopulating capacity with an altered cell differentiation skewing toward monocytic/granulocytic lineages. Approximately 1/3 of Tet2(-/-) and 8% of Tet2(+/-) mice died within 1 year of age because of the development of myeloid malignancies resembling characteristics of CMML, MPD-like myeloid leukemia, and MDS. Furthermore, transplantation of Tet2(-/-), but not wild-type (WT) or Tet2(+/-) BM cells, led to increased WBC counts, monocytosis, and splenomegaly in WT recipient mice. These data indicate that Tet2-deficient mice recapitulate patients with myeloid malignancies, implying that Tet2 functions as a tumor suppressor to maintain hematopoietic cell homeostasis.

Noninvasive Evaluation of Fetal Zygosity in Twin Pregnancies Involving a Binary Analysis of Single-Nucleotide Polymorphisms.

Twin pregnancy constitutes significant risks for maternal and fetal health, which is usually detected by ultrasound examination at early gestation. However, the imaging-based approach may not accurately identify all twins confounded by practical or clinical variables. The analysis of fetal cell-free DNA in noninvasive prenatal screening assays can completement the ultrasound method for twin detection, which differentiates fraternal or identical twins based on their distinct genotypes. Here, a new noninvasive prenatal screening employing high-coverage next-generation sequencing for targeted nucleotide polymorphisms was developed for detection of zygosity and determination of fetal fraction in twin pregnancies. This method utilizes a binary analysis of both the number and allelic fraction of fetus-specific single-nucleotide polymorphisms to infer the zygosity. In 323 samples collected from 215 singleton, 90 dizygotic, and 18 monozygotic twin pregnancies, all 90 dizygotic twins were correctly detected, with a 100% sensitivity and a 100% specificity. In addition, this method can detect complex pregnancies, such as egg donors, contamination, and twins with complete hydatidiform mole. The fetus-specific fetal fraction change was monitored in nine dizygotic twin pregnancies, which demonstrated highly variable dynamics of fetal cell-free DNA turnover up to 7 weeks after twin reduction. Overall, this study provides a new noninvasive prenatal screening strategy for the accurate identification of twin zygosity and quantification of fetal fraction, which has important clinical implications for the management of twin pregnancies.

Myocardial Tbx20 regulates early atrioventricular canal formation and endocardial epithelial-mesenchymal transition via Bmp2.

During early embryogenesis, the formation of the cardiac atrioventricular canal (AVC) facilitates the transition of the heart from a linear tube into a chambered organ. However, the genetic pathways underlying this developmental process are poorly understood. The T-box transcription factor Tbx20 is expressed predominantly in the AVC of early heart tube. It was shown that Tbx20 activates Nmyc1 and suppresses Tbx2 expression to promote proliferation and specification of the atrial and ventricular chambers, yet it is not known if Tbx20 is involved in early AVC development. Here, we report that mice lacking Tbx20 in the AVC myocardium fail to form the AVC constriction, and the endocardial epithelial-mesenchymal transition (EMT) is severely perturbed. Tbx20 maintains expression of a variety of genes, including Bmp2, Tbx3 and Hand1 in the AVC myocardium. Intriguingly, we found Bmp2 downstream genes involved in the EMT initiation are also downregulated. In addition, re-expression of Bmp2 in the AVC myocardium substantially rescues the EMT defects resulting from the lack of Tbx20, suggesting Bmp2 is one of the key downstream targets of Tbx20 in AVC development. Our data support a complex signaling network with Tbx20 suppressing Tbx2 in the AVC myocardium but also indirectly promoting Tbx2 expression through Bmp2. The spatiotemporal expression of Tbx2 in the AVC appears to be balanced between these two opposing signals. Overall, our study provides genetic evidence that Tbx20 has essential roles in regulating AVC development that coordinate early cardiac chamber formation.

Bipolar hemiarthroplasty compared with internal fixation for unstable intertrochanteric fractures in elderly patients.

BACKGROUND: The optimal treatment for unstable intertrochanteric fractures in elderly patients remains controversial. We aimed to compare internal fixation and bipolar hemiarthroplasty for the treatment of unstable intertrochanteric fractures in elderly patients. METHODS: 124 patients aged over 70 years were enrolled into this study (64 internal fixations, 60 bipolar hemiarthroplasties). Patients were followed for two years, and had a clinical, radiological, and functional review at three, six, and twelve months as well as two years. RESULTS: In the internal fixation group, the fracture reduction and internal fixation were regarded as satisfactory in 44 cases and unsatisfactory in 20 cases. Five patients in the internal fixation group (two with satisfactory results and three with unsatisfactory results) and three patients in the arthroplasty group died before the final two-year follow-up. Five patients in the internal fixation group who had unsatisfactory results suffered complications. At 24 months post-operation, patients who were treated satisfactorily with internal fixation had higher Harris scores, less pain, and better walking ability than those treated with hemiarthroplasty and unsatisfactory internal fixation. CONCLUSIONS: Internal fixation with good reduction and fixation quality should be the preferred therapeutic method for elderly unstable intertrochanteric fractures, even when severe osteoporosis is present.

[Correlation of disability degree and Tile type of pelvic fracture caused by traffic accidents].

OBJECTIVE: To analyze the relevance of Tile type of pelvic fracture and the degree of disability and explore how to understand the malunion and severe malunion of pelvic fracture for the injured in the traffic accidents. METHODS: Eighty-six cases of pelvic fractures caused by traffic accidents from August 2008 to August 2011 in Forensic Judical Appraisal Institute of Suzhou Municipal Hospital were collected. At first, the grade of disability for every case was evaluated by 3 senior forensic experts independently. Then, the Tile type of pelvic fractures for every case was determined by 3 radiologists independently. At last, the correlation of the types of the fracture with the grades of disabilities was analyzed. RESULTS: In all the cases there were 19 cases determined as A-type fracture and evaluated as non-grade disability. There were 43 cases determined as B-type fracture. And in these cases there were 41 cases determined as tenth grade of disability, one case as non-grade disability and one case as ninth grade disability. There were 24 cases determined as C-type fracture. And in these cases there were 14 cases evaluated as tenth grade disability and 10 cases evaluated as ninth grade disability. There was a correlation between the grade of disability and the type of the fracture (r = 0.760). CONCLUSION: The disability degree caused by pelvic fracture correlates significantly with the type of the fracture. The finding is potentially useful to understand the malunion and severe malunion of pelvic fracture in forensic practice.

Biomechanics of the unilateral posterosuperior, unipedicular, and bipedicular approaches for treatment by percutaneous vertebroplasty: a comparative study.

Percutaneous vertebroplasty (PVP) via the unilateral posterosuperior approach has achieved good clinical results for the treatment of osteoporotic vertebral compression fractures. This study compared the biomechanical performance of a single vertebral body after PVP by the unilateral posterosuperior, unipedicular, and bipedicular approaches. Twenty-one vertebral bodies from the osteoporotic spine segments (T11-L1) of seven older female cadavers were randomly assigned to the unipedicular (group A), bipedicular (group B), or unilateral posterosuperior approach group (group C). After constructing the fracture compression model, PVP was performed by the different approaches. CT scans showed symmetrical, evenly distributed bone cement in groups B and C and unilaterally distributed cement in group A. The recovery rates of the anterior vertebral body height in groups B and C were significantly higher than those in group A after PVP (P<0.05). The left curvature elastic moduli after PVP were significantly higher in group A than in groups B and C; however, the right curvature moduli in group A were lower than in the other groups (P<0.05). The flexion, extension, and vertical compression elastic moduli were lowest in group B (P<0.05). After PVP, failure strength and stiffness in groups B and C were comparable (P>0.05) and higher than those in group A (P<0.05). PVP through the unilateral posterosuperior approach was superior to the unipedicular approach and comparable to the bipedicular approach based on the biomechanical performance of a single vertebral body. Due to its safety, simplicity, and efficacy, the unilateral posterosuperior approach is recommended for clinical application.

Genetic deconvolution of fetal and maternal cell-free DNA in maternal plasma enables next-generation non-invasive prenatal screening.

Current non-invasive prenatal screening (NIPS) analyzes circulating fetal cell-free DNA (cfDNA) in maternal peripheral blood for selected aneuploidies or microdeletion/duplication syndromes. Many genetic disorders are refractory to NIPS largely because the maternal genetic material constitutes most of the total cfDNA present in the maternal plasma, which hinders the detection of fetus-specific genetic variants. Here, we developed an innovative sequencing method, termed coordinative allele-aware target enrichment sequencing (COATE-seq), followed by multidimensional genomic analyses of sequencing read depth, allelic fraction, and linked single nucleotide polymorphisms, to accurately separate the fetal genome from the maternal background. Analytical confounders including multiple gestations, maternal copy number variations, and absence of heterozygosity were successfully recognized and precluded for fetal variant analyses. In addition, fetus-specific genomic characteristics, including the cfDNA fragment length, meiotic error origins, meiotic recombination, and recombination breakpoints were identified which reinforced the fetal variant assessment. In 1129 qualified pregnancies tested, 54 fetal aneuploidies, 8 microdeletions/microduplications, and 8 monogenic variants were detected with 100% sensitivity and 99.3% specificity. Using the comprehensive cfDNA genomic analysis tools developed, we found that 60.3% of aneuploidy samples had aberrant meiotic recombination providing important insights into the mechanism underlying meiotic nondisjunctions. Altogether, we show that the genetic deconvolution of the fetal and maternal cfDNA enables thorough and accurate delineation of fetal genome which paves the way for the next-generation prenatal screening of essentially all types of human genetic disorders.

Sorption to dissolved humic acid and its impacts on the toxicity of imidazolium based ionic liquids.

Two typical ionic liquids (ILs), 1-butyl-3-methylimidazolium chloride ([C4MIM]Cl) and 1-octyl-3-methylimidazolium chloride ([C8MIM]Cl), are demonstrated to associate strongly with dissolved organic matter (DOM) with distribution coefficients (KDOC) in the range of 10(4.2) to 10(4.6) for Aldrich humic acid (used as model DOM). With the increase of humic acid concentration to 11 µg/mL DOC (dissolved organic carbon), the free fraction (ratio of freely dissolved to total concentration) of [C4MIM]Cl and [C8MIM]Cl reduced to about 0.85 and 0.79, respectively. This reduction of freely dissolved concentration gave rise to remarkable reduction of bioavailability and toxicity of the two ILs. MTT assay with HepG2 cell lines showed that the EC50 values were 459 µmol/L for [C4MIM]Cl and 12 µmol/L for [C8MIM]Cl, respectively, and the cell viability increased about 50% in the presence of trace amount of humic acid (1 µg/mL DOC). The SOS/umu test indicated mutagenicity for [C4MIM]Cl at levels above 664 µmol/L, and the genotoxicity was diminished with the addition of trace humic acid (0.00000374-0.374 µg/mL DOC). The studied ILs showed acute toxicity toward model fish medaka with a 96 h median lethal concentration (LC50) of 2254 µmol/L for [C4MIM]Cl and 366 µmol/L for [C8MIM]Cl. The addition of humic acid (5.49 µg/mL DOC for [C8MIM]Cl, 1.37 µg/mL DOC for [C4MIM]Cl) to IL solutions reduced the death rate of medaka to a minimum value of ∼25% of that at zero DOC. Our results suggest that DOM may play an important role in determining the environmental fate and toxicity of imidazolium-based ILs, and its effects should be taken into account in assessing the environmental risk of ILs.

Low-level germline mosaicism of a novel SMARCA2 missense variant: Expanding the phenotypic spectrum and mode of genetic transmission.

BACKGROUND: Nicolaides-Baraitser syndrome (NCBRS) is a severe neurodevelopmental disorder with multiple abnormalities. To date, all pathogenic variants in SMARCA2 causing NCBRS are de novo and most are missense variants located in the ATPase domain of SMARCA2 protein. METHODS: In this study, a familial trio whole-exome sequencing was performed on the proband presenting with intellectual disability, early-onset epilepsy, and autistic features. A novel missense variant c.553C>G (p.Gln185Glu) in SMARCA2 was identified, which is located in the QLQ domain. The same variant was subsequently also found in the mother's ongoing pregnancy. Samples from accessible tissues such as saliva and sperm other than blood were collected from the parents, and the detection of the target variant was performed by amplicon-based deep sequencing. RESULTS: Low-level mosaicism of the target variant c.553C>G (p.Gln185Glu) was detected in the father's sperm with allele fraction of 2.8% by amplicon-based deep sequencing, which was not detected in either parents' blood or saliva specimens. Heterozygosity of this variant was confirmed in the proband. CONCLUSION: This is the first report of paternal germline mosaicism for a SMARCA2 disease-causing variant. In addition, the missense variant c.553C>G (p.Gln185Glu) in the QLQ domain causes mainly neurological and developmental phenotypes with unremarkable characteristic facial features and limb abnormalities. Our findings expand the phenotypic spectrum and mode of genetic transmission associated with the SMARCA2 variants.

Comprehensive non-invasive prenatal screening for pregnancies with elevated risks of genetic disorders: protocol for a prospective, multicentre study.

INTRODUCTION: Chromosomal abnormalities and monogenic disorders account for ~15%-25% of recognisable birth defects. With limited treatment options, preconception and prenatal screening were developed to reduce the incidence of such disorders. Currently, non-invasive prenatal screening (NIPS) for common aneuploidies is implemented worldwide with superiority over conventional serum or sonographic screening approaches. However, the clinical validity for the screening of frequent chromosome segmental copy number variations and monogenic disorders still awaits to be proved. METHODS AND ANALYSIS: This study is a multicentre, prospective study. The participants were recruited from three tertiary hospitals in China starting from 10 April 2021. The study is expected to conclude before 10 October 2022. Pregnant women with abnormal prenatal screening results indicated for invasive prenatal diagnosis or those who decide to terminate their pregnancies due to abnormal ultrasound findings will be evaluated for enrolment. Cell-free DNA extracted from the maternal plasma will be used for an analytically validated comprehensive NIPS test developed by Beijing BioBiggen Technology Co. (Beijing, China). The diagnostic results from prenatal or postnatal specimens as well as the pregnancy outcome data will be collected to examine the clinical sensitivity, specificity, positive and negative predictive values of the test. ETHICS AND DISSEMINATION: This study was approved by the Obstetrics and Gynecology Hospital of Fudan University (2020-178). Results of this study will be disseminated to public through scientific conferences and a peer-reviewed journal. Written informed consents will be obtained from participants. TRIAL REGISTRATION NUMBER: ChiCTR2100045739.

Lessons learned from expanded reproductive carrier screening in self-reported Ashkenazi, Sephardi, and Mizrahi Jewish patients.

BACKGROUND: Next-generation sequencing (NGS)-based panels have gained traction as a strategy for reproductive carrier screening. Their value for screening Ashkenazi Jewish (AJ) individuals, who have benefited greatly from population-wide targeted testing, as well as Sephardi/Mizrahi Jewish (SMJ) individuals (an underserved population), has not been fully explored. METHODS: The clinical utilization by 6,805 self-reported Jewish individuals of an expanded NGS panel, along with several ancillary assays, was assessed retrospectively. Data were extracted for a subset of 96 diseases that, during the panel design phase, were classified as being AJ-, SMJ-, or pan-Jewish/pan-ethnic-relevant. RESULTS: 64.6% of individuals were identified as carriers of one or more of these 96 diseases. Over 80% of the reported variants would have been missed by following recommended AJ screening guidelines. 10.7% of variants reported for AJs were in "SMJ-relevant genes," and 31.2% reported for SMJs were in "AJ-relevant genes." Roughly 2.5% of individuals carried a novel, likely pathogenic variant. One in 16 linked cohort couples was identified as a carrier couple for at least one of these 96 diseases. CONCLUSION: For maximal carrier identification, this study supports using expanded NGS panels for individuals of all Jewish backgrounds. This approach can better empower at-risk couples for reproductive decision making.

Clinical Genetic Testing for Fragile X Syndrome by Polymerase Chain Reaction Amplification and Southern Blot Analyses.

Fragile X syndrome (FXS) is characterized by mental retardation and in the vast majority of cases it is caused by expansion of CGG trinucleotide repeats in the 5' untranslated region (or UTR) in the FMR1 gene on the X chromosome. The size and methylation status of CGG repeats are correlated with the clinical phenotype of FMR1-related disorders. The methods used for clinical genetic testing of FXS include polymerase chain reaction (PCR) amplification and Southern blot analyses, which effectively detect alleles with repeats in the normal, intermediate, premutation, and full mutation size ranges, as well as the methylation status of FMR1 promoter region.

Limited Regeneration Potential with Minimal Epicardial Progenitor Conversions in the Neonatal Mouse Heart after Injury.

The regeneration capacity of neonatal mouse heart is controversial. In addition, whether epicardial cells provide a progenitor pool for de novo heart regeneration is incompletely defined. Following apical resection of the neonatal mouse heart, we observed limited regeneration potential. Fate-mapping of Tbx18MerCreMer mice revealed that newly formed coronary vessels and a limited number of cardiomyocytes were derived from the T-box transcription factor 18 (Tbx18) lineage. However, further lineage tracing with SM-MHCCreERT2 and Nfactc1Cre mice revealed that the new smooth muscle and endothelial cells are in fact derivatives of pre-existing coronary vessels. Our data show that neonatal mouse heart can regenerate but that its potential is limited. Moreover, although epicardial cells are multipotent during embryogenesis, their contribution to heart repair through "stem" or "progenitor" cell conversion is minimal after birth. These observations suggest that early embryonic heart development and postnatal heart regeneration are distinct biological processes. Multipotency of epicardial cells is significantly decreased after birth.