scholarly journals Severe Biallelic Loss-of-function Mutations in Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2) in Two Fetuses with Fetal Akinesia Deformation Sequence

2019 ◽  
Author(s):  
Marshall Lukacs ◽  
Jonathan Gilley ◽  
Yi Zhu ◽  
Giuseppe Orsomando ◽  
Carlo Angeletti ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Wallerian Degeneration ◽  
Molecular Evidence ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Nicotinamide Mononucleotide ◽  
Nicotinamide Mononucleotide Adenylyltransferase ◽  
Protein Variants ◽  
Axon Survival

AbstractThe three nicotinamide mononucleotide adenylyltransferase (NMNAT) family members synthesize the electron carrier nicotinamide adenine dinucleotide (NAD+) and are essential for cellular metabolism. In mammalian axons, NMNAT activity appears to be required for axon survival and is predominantly provided by NMNAT2. NMNAT2 has recently been shown to also function as a chaperone to aid in the refolding of misfolded proteins. Nmnat2 deficiency in mice, or in its ortholog dNmnat in Drosophila, results in axon outgrowth and survival defects. Peripheral nerve axons in NMNAT2-deficient mice fail to extend and innervate targets, and skeletal muscle is severely underdeveloped. In addition, removing NMNAT2 from established axons initiates axon death by Wallerian degeneration. We report here on two stillborn siblings with fetal akinesia deformation sequence (FADS), severely reduced skeletal muscle mass and hydrops fetalis. Clinical exome sequencing identified compound heterozygous NMNAT2 variant alleles in both cases. Both protein variants are incapable of supporting axon survival in mouse primary neuron cultures when overexpressed. In vitro assays demonstrate altered protein stability and/or defects in NAD+ synthesis and chaperone functions. Thus, both patient NMNAT2 alleles are null or severely hypo-morphic. These data indicate a previously unknown role for NMNAT2 in human neurological development and provide the first direct molecular evidence to support the involvement of Wallerian degeneration in a human axonal disorder.

scholarly journals Novel WEE2 compound heterozygous mutations identified in patients with fertilization failure or poor fertilization

2021 ◽  
Author(s):  
Jiamin Jin ◽  
Xiaomei Tong ◽  
Yin-Li Zhang ◽  
Weijie Yang ◽  
Yerong Ma ◽  
...  
Keyword(s):  
Hela Cells ◽  
Fertilization Rate ◽  
Chinese Patients ◽  
Compound Heterozygous ◽  
Fertilization Failure ◽  
Loss Of Function ◽  
Protein Levels ◽  
Compound Heterozygous Mutations ◽  
Poor Fertilization

Abstract Purpose To study associations between novel WEE2 mutations and patients with fertilization failure or poor fertilization. Methods Thirty-one Chinese patients who underwent treatment with assisted reproductive technology and suffered from repeated (at least two times) total fertilization failure (TFF) or a low fertilization rate were enrolled. Genomic DNA was extracted from patients for whole-exome sequencing. Suspicious mutations were validated by Sanger sequencing. WEE2 protein levels in oocytes from affected patients were examined by immunofluorescence. Disruptive effects of mutations on WEE2 protein stability, subcellular localization, and kinase function were analyzed through western blotting, immunofluorescence, and flow cytometry in HeLa cells. Results Three of thirty-one (9.6%) enrolled patients had six compound heterozygous mutations of the WEE2 gene, and three of them were reported here for the first time (c.115_116insT, c.756_758delTGA, and c.C1459T). Oocytes from affected patients showed decreased WEE2 immunofluorescence signals. In vitro experiments showed that the mutant WEE2 gene caused reduced WEE2 protein levels or cellular compartment translocation in HeLa cells, leading to decreased levels of the phosphorylated Cdc2 protein. Compared with the wild-type WEE2 protein, the mutant WEE2 proteins were also found to have different effects on the cell cycle. Conclusion Three novel compound heterozygous WEE2 variants were found in patients with pronucleus formation failure. This study provides new evidence that WEE2 mutations result in loss of function, which could result in fertilization failure.

scholarly journals Nicotinamide mononucleotide adenylyltransferase uses its NAD+ substrate-binding site to chaperone phosphorylated Tau

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xiaojuan Ma ◽  
Yi Zhu ◽  
Jinxia Lu ◽  
Jingfei Xie ◽  
Chong Li ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Single Molecule ◽  
Molecular Chaperones ◽  
Phosphorylated Tau ◽  
Amyloid Aggregation ◽  
Computational Approaches ◽  
Nad Metabolism ◽  
Nicotinamide Mononucleotide ◽  
Nicotinamide Mononucleotide Adenylyltransferase

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer’s disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD+ synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD+ metabolism and Tau homeostasis in aging and neurodegeneration.

scholarly journals Biallelic ERBB3 loss-of-function variants are associated with a novel multisystem syndrome without congenital contracture

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Niu Li ◽  
Yufei Xu ◽  
Yi Zhang ◽  
Guoqiang Li ◽  
Tingting Yu ◽  
...  
Keyword(s):  
Postnatal Growth ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Atrioventricular Canal ◽  
Multiple Systems ◽  
Feeding Difficulties ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Compound Heterozygous Variants

Abstract Background Gain-of-function pathogenic variants of the Erb-B2 receptor tyrosine kinase 3 (ERBB3) gene contribute to the occurrence and development of a variety of human carcinomas through activation of phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK) signaling. ERBB3 gene homozygous germline variants, whose loss of function may cause autosomal recessive congenital contractural syndrome, were recently identified. This study aims to identify the disease-causing gene in a Chinese pedigree with variable phenotypes involving multiple systems, including developmental delay, postnatal growth retardation, transient lower limb asymmetry, facial malformations, atrioventricular canal malformation, bilateral nystagmus and amblyopia, feeding difficulties, immunodeficiency, anemia, and liver damage, but without congenital contracture. Methods Trio-whole exome sequencing (WES) was performed to identify the disease-causing gene in a 24-month-old Chinese female patient. The pathogenicity of the identified variants was evaluated using in silico tools and in vitro functional studies. Results Trio-WES revealed compound heterozygous variants of c.1253 T > C (p.I418T) and c.3182dupA (p.N1061Kfs*16) in the ERBB3 gene. Functional studies showed that p.I418T resulted in normal expression of ERBB3, which was capable of interacting with ERBB2. However, the variant impaired ERBB3 phosphorylation, consequently blocking ERBB2 phosphorylation and AKT and ERK activation. The truncated protein resulting from the c.3182dupA variant also lacked the capacity to activate downstream signaling pathways. Conclusions We report the first patient with a novel multisystem syndrome disorder without congenital contracture resulting from biallelic loss-of-function variants of ERBB3.

scholarly journals Biallelic loss of function NEK3 mutations deacetylate α-tubulin and downregulate NUP205 that predispose individuals to cilia-related abnormal cardiac left–right patterning

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Yuan Zhang ◽  
Weicheng Chen ◽  
Weijia Zeng ◽  
Zhouping Lu ◽  
Xiangyu Zhou
Keyword(s):  
Stop Codon ◽  
Retinal Pigment ◽  
Premature Stop Codon ◽  
Nuclear Pore ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Tem Analysis ◽  
Protein Levels ◽  
Biallelic Mutations

AbstractDefective left–right (LR) organization involving abnormalities in cilia ultrastructure causes laterality disorders including situs inversus (SI) and heterotaxy (Htx) with the prevalence approximately 1/10,000 births. In this study, we describe two unrelated family trios with abnormal cardiac LR patterning. Through whole-exome sequencing (WES), we identified compound heterozygous mutations (c.805-1G >C; p. Ile269GlnfsTer8/c.1117dupA; p.Thr373AsnfsTer19) (c.29T>C; p.Ile10Thr/c.356A>G; p.His119Arg) of NEK3, encoding a NIMA (never in mitosis A)-related kinase, in two affected individuals, respectively. Protein levels of NEK3 were abrogated in Patient-1 with biallelic loss-of function (LoF) NEK3 mutations that causes premature stop codon. Subsequence transcriptome analysis revealed that NNMT (nicotinamide N-methyltransferase) and SIRT2 (sirtuin2) was upregulated by NEK3 knockdown in human retinal pigment epithelial (RPE) cells in vitro, which associates α-tubulin deacetylation by western blot and immunofluorescence. Transmission electron microscopy (TEM) analysis further identified defective ciliary ultrastructure in Patient-1. Furthermore, inner ring components of nuclear pore complex (NPC) including nucleoporin (NUP)205, NUP188, and NUP155 were significantly downregulated in NEK3-silenced cells. In conclusion, we identified biallelic mutations of NEK3 predispose individual to abnormal cardiac left–right patterning via SIRT2-mediated α-tubulin deacetylation and downregulation of inner ring nucleoporins. Our study suggested that NEK3 could be a candidate gene for human ciliopathies.

scholarly journals Identification of mutations that cooperate with defects in B cell transcription factors to initiate leukemia

2020 ◽  
Author(s):  
Lynn M. Heltemes-Harris ◽  
Gregory K. Hubbard ◽  
Rebecca S. La Rue ◽  
Sarah A. Munro ◽  
Todd P. Knudson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Cell Transformation ◽  
Lymphoblastic Leukemia ◽  
Critical Role ◽  
Sleeping Beauty ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Human B Cells

AbstractThe transcription factors EBF1 and PAX5 are frequently mutated in B cell acute lymphoblastic leukemia (B-ALL). We demonstrate that Pax5+/-x Ebf1+/- compound heterozygous mice develop highly penetrant leukemia. Similar results were seen in Pax5+/-x Ikzf1+/- and Ebf1+/-x Ikzf1+/- mice for B-ALL, or in Tcf7+/-x Ikzf1+/- mice for T cell leukemia. To identify genetic defects that cooperate with Pax5 and Ebf1 compound heterozygosity to initiate leukemia, we performed a Sleeping Beauty (SB) transposon screen that identified cooperating partners including gain-of-function mutations in Stat5 (∼65%) and Jak1(∼68%), or loss-of-function mutations in Cblb (61%) and Myb (32%). These findings underscore the role of JAK/STAT5 signaling in B cell transformation and demonstrate unexpected roles for loss-of-function mutations in Cblb and Myb in leukemic transformation. RNA-Seq studies demonstrated upregulation of a PDK1>SGK3>MYC pathway; treatment of Pax5+/-x Ebf1+/- leukemia cells with PDK1 inhibitors blocked proliferation in vitro. Finally, we identified conserved transcriptional variation in a subset of genes between human leukemias and our mouse B-ALL models. Thus, compound haploinsufficiency for B cell transcription factors likely plays a critical role in transformation of human B cells and suggest that PDK1 inhibitors may be effective for treating patients with such defects.

scholarly journals Identification of novel FBN1 variations implicated in congenital scoliosis

2019 ◽  
Vol 65 (3) ◽  
pp. 221-230 ◽  
Author(s):  
Mao Lin ◽  
◽  
Sen Zhao ◽  
Gang Liu ◽  
Yingzhao Huang ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Missense Variant ◽  
Congenital Scoliosis ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Exact Test ◽  
Novel Variants ◽  
Burden Test

AbstractCongenital scoliosis (CS) is a form of scoliosis caused by congenital vertebral malformations. Genetic predisposition has been demonstrated in CS. We previously reported that TBX6 loss-of-function causes CS in a compound heterozygous model; however, this model can explain only 10% of CS. Many monogenic and polygenic CS genes remain to be elucidated. In this study, we analyzed exome sequencing (ES) data of 615 Chinese CS from the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) project. Cosegregation studies for 103 familial CS identified a novel heterozygous nonsense variant, c.2649G>A (p.Trp883Ter) in FBN1. The association between FBN1 and CS was then analyzed by extracting FBN1 variants from ES data of 574 sporadic CS and 828 controls; 30 novel variants were identified and prioritized for further analyses. A mutational burden test showed that the deleterious FBN1 variants were significantly enriched in CS subjects (OR = 3.9, P = 0.03 by Fisher’s exact test). One missense variant, c.2613A>C (p.Leu871Phe) was recurrent in two unrelated CS subjects, and in vitro functional experiments for the variant suggest that FBN1 may contribute to CS by upregulating the transforming growth factor beta (TGF-β) signaling. Our study expanded the phenotypic spectrum of FBN1, and provided nove insights into the genetic etiology of CS.

scholarly journals Functionally Confirmed Compound Heterozygous ADAM17 Missense Loss-of-function Variants Cause Neonatal Inflammatory Skin and Bowel Disease 1

2021 ◽  
Author(s):  
Issei Imoto ◽  
Masako Saito ◽  
Kenichi Suga ◽  
Tomohiro Kohmoto ◽  
Masanobu Otsu ◽  
...  
Keyword(s):  
Bowel Disease ◽  
Complete Loss ◽  
Whole Body ◽  
Compound Heterozygous ◽  
Functional Assay ◽  
Loss Of Function ◽  
Double Knockout ◽  
Missense Variants ◽  
Inflammatory Skin

Abstract A disintegrin and metalloprotease 17 (ADAM17) is the major sheddase that processes more than 80 substrates, including tumour necrosis factor-α (TNFα). The homozygous genetic deficiency of ADAM17 causing a complete loss of ADAM17 expression was reported to be linked to neonatal inflammatory skin and bowel disease 1 (NISBD1). Here we report for the first time, a family with NISBD1 caused by functionally confirmed compound heterozygous missense variants of ADAM17, namely c.1699T > C (p.Cys567Arg) and c.1799G > A (p.Cys600Tyr). Both variants were detected in two siblings with clinical features of NISBD1, such as erythroderma with exudate in whole body, recurrent skin infection and sepsis and prolonged diarrhoea. In a cell-based assay using Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17−/− mEFs) exogenously expressing each of these mutants, phorbol 12-myristate 13-acetate (PMA)-stimulated shedding was strongly reduced compared with wild-type ADAM17. Moreover, lysates from Adam10/17−/− mEFs exogenously expressing each mutant exhibited impaired basal and PMA-stimulated TNFα converting enzyme activity. Thus, in vitro functional assays demonstrated that both missense variants cause the complete loss-of-function of ADAM17, resulting in the development of NISBD1. Our study further expands the spectrum of genetic pathology underlying ADAM17 in NISBD1 and establishes functional assay systems for its missense variants.

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