Clinically relevant mouse models of severe spinal muscular atrophy with respiratory distress type 1.

Spinal Muscular Atrophy with Respiratory Distress (SMARD1) is a lethal infantile disease, characterized by the loss of motor neurons leading to muscular atrophy, diaphragmatic paralysis, and weakness in the trunk and limbs. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause a wide spectrum of motor neuron disease. Though mutations in IGHMBP2 are mostly associated with SMARD1, milder alleles cause the axonal neuropathy, Charcot-Marie-Tooth disease type 2S (CMT2S), and some null alleles are potentially a risk factor for sudden infant death syndrome (SIDS). Variant heterogeneity studied using an allelic series can be informative in order to create a broad spectrum of models that better exhibit the human variation. We previously identified the nmd2J mouse model of SMARD1, as well as two milder CMT2S mouse models. Here, we used CRISPR-Cas9 genome editing to create three new, more severe Ighmbp2 mouse models of SMARD1, including a null allele, a deletion of C495 (C495del) and a deletion of L362 (L362del). Phenotypic characterization of the IGHMBP2L362del homozygous mutants and IGHMBP2C495del homozygous mutants respectively show a more severe disease presentation than the previous nmd2J model. The IGHMBP2L362del mutants lack a clear denervation in the diaphragm while the IGHMBP2C495del mutants display a neurogenic diaphragmatic phenotype as observed in SMARD1 patients. Characterization of the Ighmbp2-null model indicated neo-natal lethality (median lifespan = 0.5 days). These novel strains expand the spectrum of SMARD1 models to better reflect the clinical continuum observed in the human patients with various IGHMBP2 recessive mutations.

Clinically relevant mouse models of Charcot-Marie-Tooth type 2S.

Charcot-Marie-Tooth disease is an inherited peripheral neuropathy that is clinically and genetically heterogenous. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause the infantile motor neuron disease spinal muscular atrophy with respiratory distress type 1 (SMARD1), and, more recently, juvenile-onset Charcot-Marie-Tooth disease type 2S (CMT2S). Using CRISPR-cas9 mutagenesis, we developed the first mouse models of CMT2S [p.Glu365del (E365del) and p.Tyr918Cys (Y918C)]. E365del is the first CMT2S mouse model to be discovered and Y918C is the first human CMT2S allele knock-in model. Phenotypic characterization of the homozygous models found progressive peripheral motor and sensory axonal degeneration. Neuromuscular and locomotor assays indicate that both E365del and Y918C mice have motor deficits, while neurobehavioral characterization of sensory function found that E365del mutants have mechanical allodynia. Analysis of femoral motor and sensory nerves identified axonal degeneration, which does not impact nerve conduction velocities in E365del mice, but it does so in the Y918C model. Based on these results, the E365del mutant mouse, and the human allele knock-in, Y918C, represent mouse models with the hallmark phenotypes of CMT2S, which will be critical for understanding the pathogenic mechanisms of IGHMBP2. These mice will complement existing Ighmbp2 alleles modeling SMARD1 to help understand the complex phenotypic and genotypic heterogeneity that is observed in patients with IGHMBP2 variants.

Overlapping spectrums: The clinicogenetic commonalities between Charcot-Marie-Tooth and other neurodegenerative diseases.

Charcot-Marie-Tooth (CMT) disease is a progressive and heterogeneous inherited peripheral neuropathy. A myriad of genetic factors have been identified that contribute to the degeneration of motor and sensory axons in a length-dependent manner. Emerging biological themes underlying disease include defects in axonal trafficking, dysfunction in RNA metabolism and protein homeostasis, as well deficits in the cellular stress response. Moreover, genetic contributions to CMT can have overlap with other neuropathies, motor neuron diseases (MNDs) and neurodegenerative disorders. Recent progress in understanding the molecular biology of CMT and overlapping syndromes aids in the search for necessary therapeutic targets.

Lack of nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2): consequences for mouse bladder development and function.

AIMS: To describe the morphological and functional consequences for bladder development and function when nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) is lacking or reduced. METHODS: The Bloated Bladder (Blad) mouse, lacking Nmnat2, and heterozygotes were utilized for this investigation. Morphology and development of the bladder were studied using immunohistochemistry against urothelial, smooth muscle, and nerve markers. Functional effects were assessed by organ bath experiments and cystometry. RESULTS: Homozygote mutants were malformed and died at birth, whereas heterozygotes survived and morphologically did not differ from wild-type controls. Morphological bladder changes appeared in the Blad mutants as early as embryonic day 15.5 (E15.5) with an extremely distended bladder at E18.5. Staining revealed that all the bladder layers were present and expressed mature markers in all three genotypes. No nerves could be demonstrated by immunohistochemistry in the Blad mutant bladder at E18.5. Organ bath analysis showed that bladders from Blad mutant showed signs of denervation supersensitivity in response to carbachol, and no response to electrical stimulation of nerves at E18.5. Adult heterozygotes, which have a reduced expression of Nmnat2 at E18.5, showed decreased responses to carbachol and electrical stimulation compared to wild-type controls. The latter also retained their ability to empty their bladders, but showed increased micturition pressures compared to controls. CONCLUSIONS: Complete loss of Nmnat2 leads to a mature but distended bladder in utero and is not compatible with survival. Moderate loss of Nmnat2 has no effect on bladder development, survival, and has only modest effects on bladder function later in life.

Nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) regulates axon integrity in the mouse embryo.

Using transposon-mediated gene-trap mutagenesis, we have generated a novel mouse mutant termed Blad (Bloated Bladder). Homozygous mutant mice die perinatally showing a greatly distended bladder, underdeveloped diaphragm and a reduction in total skeletal muscle mass. Wild type and heterozygote mice appear normal. Using PCR, we identified a transposon insertion site in the first intron of Nmnat2 (Nicotinamide mononucleotide adenyltransferase 2). Nmnat2 is expressed predominantly in the brain and nervous system and has been linked to the survival of axons. Expression of this gene is undetectable in Nmnat2(blad/blad) mutants. Examination of the brains of E18.5 Nmnat2(blad/blad) mutant embryos did not reveal any obvious morphological changes. In contrast, E18.5 Nmnat2(blad/blad) homozygotes showed an approximate 60% reduction of spinal motoneurons in the lumbar region and a more than 80% reduction in the sensory neurons of the dorsal root ganglion (DRG). In addition, facial motoneuron numbers were severely reduced, and there was virtually a complete absence of axons in the hind limb. Our observations suggest that during embryogenesis, Nmnat2 plays an important role in axonal growth or maintenance. It appears that in the absence of Nmnat2, major target organs and tissues (e.g., muscle) are not functionally innervated resulting in perinatal lethality. In addition, neither Nmnat1 nor 3 can compensate for the loss of Nmnat2. Whilst there have been recent suggestions that Nmnat2 may be an endogenous modulator of axon integrity, this work represents the first in vivo study demonstrating that Nmnat2 is involved in axon development or survival in a mammal.

The UDP-glucuronosyltransferase 2B17 gene deletion polymorphism: sex-specific association with urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronidation phenotype and risk for lung cancer.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone is a potent and abundant procarcinogen found in tobacco smoke, and glucuronidation of its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), by UDP-glucuronosyltransferases (UGT) including UGT2B17 is an important mechanism for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone detoxification. Both copies of the UGT2B17 gene are deleted in approximately 10% of Whites and the deletion is associated with a reduction in NNAL glucuronidation activity in vitro. In this study, we examined the effects of the UGT2B17 deletion (0/0) on NNAL glucuronidation rates in a sample of 82 healthy cigarette smokers and further examined its effects on lung cancer risk in a separate case-control study. In the healthy smokers study, a lower urinary ratio of NNAL-glucuronide to NNAL was observed in women with the UGT2B17 deletion (0/0) as compared with women with either the wild-type or heterozygous genotypes (P = 0.058). There were no significant differences in this ratio by genotype in men (P = 0.597). In the case-control study of 398 lung cancer patients and 697 community controls, the UGT2B17 deletion (0/0) was associated with a significant increase in risk of lung cancer in women (odds ratio, 2.0; 95% confidence interval, 1.01-4.0). The risk for the subset of women with lung adenocarcinoma was 2.8 (95% confidence interval, 1.2-6.3). The deletion was not associated with other lung histologic types in women and was not associated with the risk for any lung histologic types in men. The association of the UGT2B17 deletion with increased lung adenocarcinoma in women is consistent with its association with decreased NNAL glucuronidation rates in women and with studies showing that NNAL is a selective inducer of lung adenocarcinoma in experimental animals.