Prospective Investigation of Optical Genome Mapping for Prenatal Genetic Diagnosis.

BACKGROUND: Optical genome mapping (OGM) is a novel assay for detecting structural variants (SVs) and has been retrospectively evaluated for its performance. However, its prospective evaluation in prenatal diagnosis remains unreported. This study aimed to prospectively assess the technical concordance of OGM with standard of care (SOC) testing in prenatal diagnosis. METHODS: A prospective cohort of 204 pregnant women was enrolled in this study. Amniotic fluid samples from these women were subjected to OGM and SOC testing, which included chromosomal microarray analysis (CMA) and karyotyping (KT) in parallel. The diagnostic yield of OGM was evaluated, and the technical concordance between OGM and SOC testing was assessed. RESULTS: OGM successfully analyzed 204 cultured amniocyte samples, even with a cell count as low as 0.24 million. In total, 60 reportable SVs were identified through combined OGM and SOC testing, with 22 SVs detected by all 3 techniques. The diagnostic yield for OGM, CMA, and KT was 25% (51/204), 22.06% (45/204), and 18.14% (37/204), respectively. The highest diagnostic yield (29.41%, 60/204) was achieved when OGM and KT were used together. OGM demonstrated a concordance of 95.56% with CMA and 75.68% with KT in this cohort study. CONCLUSIONS: Our findings suggest that OGM can be effectively applied in prenatal diagnosis using cultured amniocytes and exhibits high concordance with SOC testing. The combined use of OGM and KT appears to yield the most promising diagnostic outcomes.

Nitrogen atom insertion into arenols to access benzazepines.

Advances in site-selective molecular editing have enabled structural modification on complex molecules. However, thus far, their applications have been restricted to C-H functionalization chemistry. The modification of the underlying molecular skeleton remains limited. Here, we describe a skeletal editing approach that provides access to benzazepine structures through direct nitrogen atom insertion into arenols. Using widely available arenols as benzazepine precursors, this alternative approach allowed the streamlined assembly of benzazepines with broad functional group tolerance. Experimental mechanistic studies support a reaction pathway involving dearomatizative azidation and then aryl migration. This study further highlights the potential for carbon-nitrogen transmutation sequences through combinations with oxidative carbon atom deletion, providing an alternative for the development of N-heteroarenes and demonstrating significant potential in materials chemistry.

Rapid syntheses of N-fused heterocycles via acyl-transfer in heteroaryl ketones.

The wide-ranging potencies of bioactive N-fused heterocycles inspire the development of synthetic transformations that simplify preparation of their complex, diverse structural motifs. Heteroaryl ketones are ubiquitous, readily available, and inexpensive molecular scaffolds, and are thus synthetically appealing as precursors in preparing N-fused heterocycles via intramolecular acyl-transfer. To best of our knowledge, acyl-transfer of unstrained heteroaryl ketones remains to be demonstrated. Here, we show an acyl transfer-annulation to convert heteroaryl ketones to N-fused heterocycles. Driven via aromatisation, the acyl of a heteroaryl ketone can be transferred from the carbon to the nitrogen of the corresponding heterocycle. The reaction commences with the spiroannulation of a heteroaryl ketone and an alkyl bromide, with the resulting spirocyclic intermediate undergoing aromatisation-driven intramolecular acyl transfer. The reaction conditions are optimised, with the reaction exhibiting a broad substrate scope in terms of the ketone and alkyl bromide. The utility of this protocol is further demonstrated via application to complex natural products and drug derivatives to yield heavily functionalised N-fused heterocycles.

Anti-Obesity and Gut Microbiota Modulation Effect of Secoiridoid-Enriched Extract from Fraxinus mandshurica Seeds on High-Fat Diet-Fed Mice.

Previously we conducted a phytochemical study on the seeds of Fraxinus excelsior and isolated nine secoiridoid compounds with adipocyte differentiation inhibitory activity and peroxisome proliferator activated receptor alpha (PPARα) activation effects. However, the bioactive constituents and functions of Fraxinus mandshurica seeds have not been studied. In the present study, we investigated the secoiridoid compounds in F. mandshurica seed extract (FM) using column chromatography, 1H-NMR, 13C-NMR and HPLC-DAD methods. The pancreatic lipase inhibitory activities of isolated compounds were evaluated in vitro. Additionally, the anti-obesity and gut microbiota modulation effect of FM on high-fat diet-induced obesity in C57BL/6 mice were also studied in vivo. The results showed that 19 secoiridoids were isolated from FM and identified. The total content of secoiridoids in FM reached 181.35 mg/g and the highest content was nuzhenide (88.21 mg/g). All these secoiridoid compounds exhibited good pancreatic lipase inhibitory activity with inhibition rate ranged from 33.77% to 70.25% at the concentration of 100 µM. After obese mice were administrated with FM at 400 mg/kg.bw for 8 weeks, body weight was decreased by 15.81%. Moreover, FM could attenuate the lipid accumulation in serum and liver, relieve the damage in liver and kidney, and extenuate oxidative stress injury and inflammation caused by obesity in mice. FM could also modulate the structural alteration of gut microbiota in obese mice, increasing the proportion of anti-obesity gut microbiota (Bacteroidetes, Bacteroidia, S24-7 and Allobaculum), and reducing the proportion of obesogenic gut microbiota (Firmicutes and Dorea). This study suggests that F. mandshurica seeds or their secoiridoids may have potential for use as a dietary supplement for obesity management.

Rare Copy Number Variations Might Not be Involved in the Molecular Pathogenesis of PA-IVS in an Unselected Chinese Cohort.

Congenital heart defect (CHD) is one of the most common birth defects in China, while pulmonary atresia with intact ventricular septum (PA-IVS) is the life-threatening form of CHD. Numerous previous studies revealed that rare copy number variants (CNVs) play important roles in CHD, but little is known about the prevalence and role of rare CNVs in PA-IVS. In this study, we conducted a genome-wide scanning of rare CNVs in an unselected cohort consisted of 54 Chinese patients with PA-IVS and 20 patients with pulmonary atresia with ventricular septal defect (PA-VSD). CNVs were identified in 6/20 PA-VSD patients (30%), and three of these CNVs (15%) were considered potentially pathogenic. Two pathogenic CNVs occurred at a known CHD locus (22q11.2) and one likely pathogenic deletion located at 13q12.12. However, no rare CNVs were detected in patients with PA-IVS. Potentially pathogenic CNVs were more enriched in PA-VSD patients than in PA-IVS patients (p = 0.018). No rare CNVs were detected in patients with PA-IVS in our study. PA/IVS might be different from PA/VSD in terms of genetics as well as anatomy.

Controlled Ni-catalyzed mono- and double-decarbonylations of α-ketothioesters.

A method for Ni-catalyzed controlled decarbonylation of α-ketothioesters is described. Mono- and double-decarbonylations, which gave thioesters and thioethers, respectively, were selectively achieved by changing the ligand. A fundamental study of Ni-catalyzed decarbonylation of α-ketothioesters is presented.

Suzuki-Miyaura coupling of unstrained ketones via chelation-assisted C-C bond cleavage.

Herein, we report that unstrained ketones can be efficiently employed as electrophiles in Suzuki-Miyaura reactions via catalytic activation of unstrained C-C bonds assist by an N-containing directing group. A wide range of aromatic ketones directly coupled with boronic ester with excellent functional group tolerance. This strategy provides an alternative and versatile approach to constructing biaryls from unstrained ketones.

Synthesis of Acyl Azides from 1,3-Diketones via Oxidative Cleavage of Two C-C Bonds.

A metal-free PhI(OAc)2-mediated method for the synthesis of acyl azides through oxidative cleavage of 1,3-diketones is described. This method is shown to have a broad substrate scope, providing a useful tool for multiproduct synthesis in a single procedure. A possible reaction pathway is proposed based on mechanistic studies.

Directed Decarbonylation of Unstrained Aryl Ketones via Nickel-Catalyzed C-C Bond Cleavage.

The nickel-catalyzed decarbonylation of unstrained diaryl ketones has been developed. The reaction is catalyzed by a combination of Ni(cod)2 and an electron-rich N-heterocyclic carbene ligand. High functional group tolerance and excellent yields (up to 98%) are observed. This strategy provides an alternative and versatile approach to construct biaryls using an inexpensive nickel catalyst.

Three novel missense mutations in the filamin B gene are associated with isolated congenital talipes equinovarus.

Congenital talipes equinovarus (CTEV) is one of the most common musculoskeletal disorders. Genetic factors have been suggested to be an important contributor to its pathogenesis. Some genes, including PITX1, TBX4, and RBM10, have been associated with CTEV. We aimed to determine the disease-causing mutations in Chinese patients with isolated CTEV. Genomic DNA was extracted from peripheral blood samples of a three-generation pedigree and 53 sporadic patients with CTEV. Whole-exome sequencing and Sanger sequencing were used to identify and validate disease-causing mutations, respectively. A putative pathogenic mutation c.4717G>T (p.D1573Y) in the filamin B (FLNB) gene, which co-segregated with CETV, was identified in the pedigree. Two additional novel missense mutations in the same gene [c.1897A>G (p.M633V) and c.2195A>G (p.Y732C)] were identified from the 53 sporadic patients. Plasmids expressing wild-type or mutant constructs were transfected into HEK293T cells to determine whether these amino acid substitutions affect protein activity. All three (M633V, Y732C, and D1573Y) affected FLNB protein expression and led to cytoplasmic focal accumulation. Our results provide evidence for the involvement of FLNB in the pathogenesis of isolated CTEV and have expanded the clinical spectrum of FLNB mutations.

A novel missense mutation of TNNI2 in a Chinese family cause distal arthrogryposis type 1.

The distal arthrogryposis (DA) syndromes are a group of disorders characterized by congenital contractures of limbs. According to phenotypical characteristics, DA syndromes have been clinically classified into 10 types. Currently, at least nine disease causing genes have been identified for different types of DA. Here, we report a 3-generation Chinese pedigree with three DA affected members. We performed whole exome sequencing on two affected and one unaffected individuals of this family and successfully identified a novel missense mutation in TNNI2 as the pathogenic mutation. The TNNI2 gene encodes a subunit of the troponin complex, a contractile machinery of the muscle. The mutation p.F178C that could change the H-bond formation of a neighboring residue occurs at a highly conserved position, suggesting that this variation probably affects the TNNI2 protein function. Our study also demonstrates the power of whole exome sequencing in causal mutation identification for phenotypically variable and genetically heterogeneous disorders.

A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal.

Growth and differentiation factors (GDFs) are secreted signaling molecules within the BMP family that have critical roles in joint morphogenesis during skeletal development in mice and humans. Using genetic data obtained from a six-generation Chinese family, we identified a missense variant in GDF6 (NP_001001557.1; p.Y444N) that fully segregates with a novel autosomal dominant synostoses (SYNS) phenotype, which we designate as SYNS4. Affected individuals display bilateral wrist and ankle deformities at birth and progressive conductive deafness after age 40 years. We find that the Y444N variant affects a highly conserved residue of GDF6 in a region critical for binding of GDF6 to its receptor(s) and to the BMP antagonist NOG, and show that this mutant GDF6 is a more potent stimulator of the canonical BMP signaling pathway compared with wild-type GDF6. Further, we determine that the enhanced BMP activity exhibited by mutant GDF6 is attributable to resistance to NOG-mediated antagonism. Collectively, our findings indicate that increased BMP signaling owing to a GDF6 gain-of-function mutation is responsible for loss of joint formation and profound functional impairment in patients with SYNS4. More broadly, our study highlights the delicate balance of BMP signaling required for proper joint morphogenesis and reinforces the critical role of BMP signaling in skeletal development.

Chromosome microarray testing for patients with congenital heart defects reveals novel disease causing loci and high diagnostic yield.

BACKGROUND: Congenital heart defects (CHD), as the most common congenital anomaly, have been reported to be frequently associated with pathogenic copy number variants (CNVs). Currently, patients with CHD are routinely offered chromosomal microarray (CMA) testing, but the diagnostic yield of CMA on CHD patients has not been extensively evaluated based on a large patient cohort. In this study, we retrospectively assessed the detected CNVs in a total of 514 CHD cases (a 422-case clinical cohort from Boston Children's Hospital (BCH) and a 92-case research cohort from Shanghai Children's Medical Center (SCMC)) and conducted a genotype-phenotype analysis. Furthermore, genes encompassed in pathogenic/likely pathogenic CNVs were prioritized by integrating several tools and public data sources for novel CHD candidate gene identification. RESULTS: Based on the BCH cohort, the overall diagnostic yield of CMA testing for CHD patients was 12.8(pathogenic CNVs)-18.5% (pathogenic and likely pathogenic CNVs). The diagnostic yield of CMA for syndromic CHD was 14.1-20.6% (excluding aneuploidy cases), whereas the diagnostic yield for isolated CHD was 4.3-9.3%. Four recurrent genomic loci (4q terminal region, 15q11.2, 16p12.2 and Yp11.2) were more significantly enriched in cases than in controls. These regions are considered as novel CHD loci. We further identified 20 genes as the most likely novel CHD candidate genes through gene prioritization analysis. CONCLUSION: The high clinical diagnostic yield of CMA in this study provides supportive evidence for CMA as the first-line genetic diagnostic tool for CHD patients. The CNVs detected in our study suggest a number of CHD candidate genes that warrant further investigation.

Toll-like receptor 6 V327M polymorphism is associated with an increased risk of Klebsiella pneumoniae infection.

BACKGROUND: Klebsiella pneumoniae is a common cause of nosocomial pneumonia, especially in children. Toll-like receptors plays an important role in defense against this pathogen. The impact of human TLR6 polymorphisms on susceptibility to K. pneumoniae infection is poorly understood. The aim of the present work was to determine whether single nucleotide polymorphisms in TLR6 are associated with altered immune responses to K. pneumoniae. METHODS: The TLR6 coding region was sequenced in 126 K. pneumoniae culture-positive patients and 142 hospitalized K. pneumoniae culture-negative controls. RESULTS: The frequency of V327M polymorphism was found to be significantly higher in patients than that in controls (16.7% vs. 7.7%). In vitro studies showed that V327M polymorphism did not impair TLR6 expression in transfected HEK 293T cells. Further studies demonstrated that V327M polymorphism was associated with increased IL-8 mRNA expression in transfected HEK 293T cells when stimulated with K. pneumoniae and the specific ligand for TLR2/TLR6 heterodimers known as Pam2CSK4. The present data showed V327M polymorphism to be associated with increased apoptosis of HEK 293T cells when challenged with K. pneumoniae. CONCLUSIONS: Taken together, these data indicated that TLR6 V327M may be involved in mediating deleterious inflammatory responses and modulating host susceptibility to K. pneumoniae. These results provide new insight into the pathophysiologic role of TLR6 V327M in the innate immune response to bacterial infection in human.

Elevated NKT cell levels in adults with severe chronic immune thrombocytopenia.

The aim of this study was to investigate the frequency of circulating natural killer T (NKT) cells and regulatory T cells (Tregs), as well as serum cytokine profiles, in adult chronic primary immune thrombocytopenia (ITP). The frequency of circulating T cell receptor (TCR) Vα24+Vß11+CD3+ NKT cells and CD4+CD25+CD127-/low Tregs was measured using multi-color flow cytometry. The serum concentrations of 11 cytokines were determined with a cytometric bead assay. The frequency of circulating NKT cells in patients with ITP was 0.13±0.03%, whereas the frequency in healthy controls was 0.07±0.01% of CD3+ (P>0.05). However, the frequency of NKT cells in patients with ITP with platelet counts ≤20×109/l (0.22±0.05%) was significantly higher than that in patients with platelet counts >20×109/l (0.05±0.01%; P<0.05) and that in healthy controls (0.07±0.01%; P<0.05). The frequency of peripheral Tregs was comparable between patients with ITP (3.97±0.44% of CD4+) and healthy controls (3.69±0.31%; P>0.05). No significant differences were observed in the serum concentrations of 11 cytokines between patients with ITP and healthy controls, despite the fact that the serum levels of interleukin (IL)-12p70, IL-8, IL-4, interferon (IFN)-γ and tumor necrosis factor (TNF)-α in patients with ITP were higher than those in the healthy controls. The platelet count was negatively correlated with the frequency of circulating NKT cells in chronic ITP. These results indicate that NKT cells may be involved in ITP with severe thrombocytopenia, and NKT and Tregs may be important in cytokine deregulation in chronic ITP.

Molecular defects identified by whole exome sequencing in a child with Fanconi anemia.

Fanconi anemia is a rare genetic disease characterized by bone marrow failure, multiple congenital malformations, and an increased susceptibility to malignancy. At least 15 genes have been identified that are involved in the pathogenesis of Fanconi anemia. However, it is still a challenge to assign the complementation group and to characterize the molecular defects in patients with Fanconi anemia. In the current study, whole exome sequencing was used to identify the affected gene(s) in a boy with Fanconi anemia. A recurring, non-synonymous mutation was found (c.3971C>T, p.P1324L) as well as a novel frameshift mutation (c.989_995del, p.H330LfsX2) in FANCA gene. Our results indicate that whole exome sequencing may be useful in clinical settings for rapid identification of disease-causing mutations in rare genetic disorders such as Fanconi anemia.

Screening for coding variants in FTO and SH2B1 genes in Chinese patients with obesity.

OBJECTIVE: To investigate potential functional variants in FTO and SH2B1 genes among Chinese children with obesity. METHODS: Sanger sequencing of PCR products of all FTO and SH2B1 exons and their flanking regions were performed in 338 Chinese Han children with obesity and 221 age- and sex-matched lean controls. RESULTS: A total of seven and five rare non-synonymous variants were identified in FTO and SH2B1, respectively. The overall frequencies of FTO and SH2B1 rare non-synonymous variants were similar in obese and lean children (2.37% and 0.90% vs. 1.81% and 1.36%, P>0.05). However, four out of the seven variants in FTO were novel and all were unique to obese children (p>0.05). None of the novel variants was consistently being predicted to be deleterious. Four out of five variants in SH2B1 were novel and one was unique to obese children (p>0.05). One variant (L293R) that was consistently being predicted as deleterious in SH2B1 gene was unique to lean control. While rare missense mutations were more frequently detected in girls from obesity as well as lean control than boys, the difference was not statistically significant. In addition, it's shown that the prevalence of rare missense mutations of FTO as well as SH2B1 was similar across different ethnic groups. CONCLUSION: The rare missense mutations of FTO and SH2B1 did not confer risks of obesity in Chinese Han children in our cohort.

Two novel isovaleryl-CoA dehydrogenase gene mutations in a Chinese infant.

Isovaleric acidemia (IVA) is a rare inherited metabolic disease caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD). Newborn screening with tandem mass spectrometry leads to early identification of individuals with risk of IVA. The family specific mutations are useful for prenatal diagnosis. Molecular genetic analysis helps to further confirm the clinical diagnosis of IVA. We describe here the clinical and metabolic features of a Chinese infant with early onset IVA. Sequence analysis of the IVD gene identifies compound heterozygous mutations in this patient, c.39G>A (p.W13X) nonsense mutation and c.597C>G (p.I199 M) missense mutation, both of which are previously unreported. Structural analyses suggest that the p.I199 M missense mutation may destabilize the IVD monomer structure and affect the interaction between IVD and flavin adenine dinucleotide. Both the clinical and genetic features of this patient help to further expand our knowledge of IVA.

A unique combination of 17pter trisomy and 21qter monosomy in a boy with developmental delay, severe intellectual disability, growth retardation and dysmorphisms.

BACKGROUND: Microduplication at 17p13.3 and microdeletion at 21q22 are both rare chromosomal aberrations. The presence of both genomic imbalances in one patient has not been previously reported in literature. In this study, we performed a molecular diagnostic testing with a whole genome microarray on a 3-year-old boy with developmental delay, mental retardation and multiple malformations. METHODS: A routine G-banding karyotype analysis was performed using peripheral lymphocytes. Chromosome microarray analysis (CMA) was done using Affymetrix CytoScan™ HD array. Genomic imbalances were further confirmed by multiple ligation-dependent probe amplification (MLPA). RESULTS: The result of karyotyping was normal but CMA detected a 9.8 Mb microduplication at 17p13.3-13.1 (chr17: 1-9,875,545) and a 2.8 Mb microdeletion involving 21q22.3-qter (chr21: 45,239,077-48,097,372). The imbalances were due to a balanced translocation present in patient's mother. The patient was characterized with short stature, profound developmental delay, non-verbal, intellectual disability as well as craniofacial dysmorphism, subtle brain structural anomaly and sparse scalp hair. CONCLUSIONS: This is the first patient reported with a combination of a microduplication at 17p13.3-13.1 and a microdeletion at 21q22.3-qter. Both genomic imbalances were undetected by conventional karyotyping but were delineated with CMA test. Synergistic effect from the two rare genomic imbalances is likely responsible for the severe clinical phenotypes observed in this patient.