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The interpretation of non-coding variants still constitutes a major challenge in the application of whole-genome sequencing in Mendelian disease, especially for single-nucleotide and other small non-coding variants. Here we present Genomiser, an analysis framework that is able not only to score the relevance of variation in the non-coding genome, but also to associate regulatory variants to specific Mendelian diseases. Genomiser scores variants through either existing methods such as CADD or a bespoke machine learning method and combines these with allele frequency, regulatory sequences, chromosomal topological domains, and phenotypic relevance to discover variants associated to specific Mendelian disorders. Overall, Genomiser is able to identify causal regulatory variants as the top candidate in 77% of simulated whole genomes, allowing effective detection and discovery of regulatory variants in Mendelian disease., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Numerous disease syndromes are associated with regions of copy number variation (CNV) in the human genome and, in most cases, the pathogenicity of the CNV is thought to be related to altered dosage of the genes contained within the affected segment. However, establishing the contribution of individual genes to the overall pathogenicity of CNV syndromes is difficult and often relies on the identification of potential candidates through manual searches of the literature and online resources. We describe here the development of a computational framework to comprehensively search phenotypic information from model organisms and single-gene human hereditary disorders, and thus speed the interpretation of the complex phenotypes of CNV disorders. There are currently more than 5000 human genes about which nothing is known phenotypically but for which detailed phenotypic information for the mouse and/or zebrafish orthologs is available. Here, we present an ontology-based approach to identify similarities between human disease manifestations and the mutational phenotypes in characterized model organism genes; this approach can therefore be used even in cases where there is little or no information about the function of the human genes. We applied this algorithm to detect candidate genes for 27 recurrent CNV disorders and identified 802 gene-phenotype associations, approximately half of which involved genes that were previously reported to be associated with individual phenotypic features and half of which were novel candidates. A total of 431 associations were made solely on the basis of model organism phenotype data. Additionally, we observed a striking, statistically significant tendency for individual disease phenotypes to be associated with multiple genes located within a single CNV region, a phenomenon that we denote as pheno-clustering. Many of the clusters also display statistically significant similarities in protein function or vicinity within the protein-protein interaction network. Our results provide a basis for understanding previously un-interpretable genotype-phenotype correlations in pathogenic CNVs and for mobilizing the large amount of model organism phenotype data to provide insights into human genetic disorders.

Two antineoplastic agents, chlornaphazine (CN) and chlorambucil (CHL), were tested for the induction of dominant lethal mutations in male mice. Both compounds are nitrogen mustard derivatives and have been shown to be genotoxic in a variety of organisms. CN was administered intraperitoneally to DBA/2J male mice at a dosage of 0, 500, 1000, or 1500 mg/kg body weight (bw). Immediately following treatment, each male was mated at 4-day intervals to two virgin C57BL/6J females. CHL was administered intraperitoneally to C3H/HeJ and DBA/2J males at a dosage of 0, 2.5, or 5.0 mg/kg bw. These males were mated at weekly intervals to two virgin T-stock females. CN and CHL clearly induced dominant lethal mutations. CN induced dominant lethal effects in all post-meiotic germ-cell stages of treated DBA males, with a clear dose-response relationship. The results with CHL-treated DBA males indicated that all post-meiotic germ-cell stages, except late-spermatids, were affected by CHL treatment, while in C3H males, CHL induced dominant lethal effects in all post-meiotic germ-cell stages. A dose-response relationship was also observed with CHL in C3H male mice. In the present experiments, regardless of the agent or the mouse strain used, spermatids appeared to be the germ-cell stage most sensitive to dominant lethal induction., (Copyright 2003 Elsevier Science B.V.)

The purpose of the study was to determine: 1) if male germ cells of Big Blue mice carrying newly induced mutations in the lacI transgene were effective in fertilization; 2) if offspring arising from such mutant sperm had the mutation in germ cells and multiple somatic tissues; and 3) how the frequency of mutants induced in the lacI transgene compared to the frequency induced in endogenous genes traditionally employed to study germ cell mutagenesis in mice. Male B6C3F(1) mice hemizygous for the lambda/lacI transgene were treated weekly with 100 mg/kg body weight of the mammalian germ cell mutagen N-ethyl-N-nitrosourea (ENU). The cumulative dose for each treated animal was 300 mg ENU/kg body weight. Ten weeks later the treated mice were mated to T stock females and the resulting offspring were screened for specific-locus mutations at six loci affecting external appearance, as well as for mutations in the lacI transgene in multiple somatic tissues and germ cells. Five offspring carrying recessive specific-locus mutations were observed among 597 offspring screened (mutant frequency = 139.6 x 10(-5) per locus). Four offspring carrying lacI mutations were observed among 280 offspring screened (mutant frequency = 35.7 x 10(-5) per locus (assuming 40 target loci)). Each of the four lacI mutant offspring carried a different mutation. Three of the mutations were A:T-->G:C transitions and one a G:C-->A:T transition. Consistent with the expectation that a mutation induced in a parental germ cell and transmitted to a conceptus would exist in every cell of the offspring, each mutant mouse had identical mutations in all somatic tissues sampled, as well as in its germ cells. These data provide preliminary evidence for the biological validity of assessing induced, heritable mutations using transgenic mice, without the need for generating an F(1) generation., (Copyright 2002 Wiley-Liss, Inc.)

A brief history of the developmental life cycle of the mammalian germ cell, from fertilization to gametogenesis in the mature gonad, is presented. The differences between gametogenesis in the mature gonad of males and females are also described with regard to properties that may affect their susceptibilities to mutation. It is emphasized that any historical control background rate of necessity will include mutations that occur in germinal tissue at all stages of development and differentiation, although it is not always possible to determine at what stage of germline development a spontaneous mutation has occurred. Studies of induced mutations suggest that the impact on the molecular level and the distribution of mutations among the F1 and F2 progeny may be partly determined by the stage and sex of germ cells in which spontaneous mutations occur. In summary, historical control rates should only be considered the sum total of mutations that occur during the entire life of the individual and cannot represent the control values of any individual germ cell stage. Nonetheless, it is certainly important and valid to use historical control data for calculating human risk, because the primary use of the estimation of mutant frequencies is to access the potential impact of agents in increasing the genetic load in the human population.

We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.

Male DBA/2J and C57BL/6J mice were treated with ethylnitrosourea (EtNU) at a dose of 250 mg/kg of body weight. After a sterile period of 11 weeks, the treated animals were mated with untreated females; treated DBA with C57 and treated C57 with DBA. A small control group of untreated males was established, and a larger control group from earlier experiments was also used for comparison. Tissue samples removed surgically from the parents and F1 offspring were examined by electrophoresis. Nine newly arisen mutants attributable to induction by EtNU were found in 22,512 loci tested for mutation. No mutations were encountered in 290,252 control locus tests. A preliminary study conducted concomitantly with the electrophoretic analyses for dominant lethal effects and physical malformation showed no increase over background due to EtNU treatment of spermatogonia, which suggests that mutations induced by EtNU tend not to be expressed in this way. The nature of the mutants identified by electrophoresis facilitates follow-up analyses leading to increased understanding of the molecular basis and the physiological effects of induced mutations in mammalian organisms.

Male mice of the DBA/2J strain were treated with the mutagen procarbazine and mated with C57BL/6J females. Offspring and parents were then analyzed for electrophoretically expressed mutations. A control group, not mutagen treated, was also examined. Two mutants probably due to induction were identified, and several of spontaneous origin were also found.

Rate calculations are described for germinal mutations detected by an electrophoretic system as applied to mice in a series of recent experiments. The utility of the electrophoretic system is examined from several comparative viewpoints. The electrophoretic approach has a number of features to recommend it for future applications concerned with genetic hazard assessment. Its advantages include sensitivity, versatility, cost effectiveness and relevance to humans.

Electrophoretic screening of (C57BL/6J x DBA/2J)F1 progeny of male mice treated with N-ethyl-N-nitrosourea revealed a mouse that lacked the paternal carbonic anhydrase II (CA II). Breeding tests showed that this trait was heritable and due to a null mutation at the Car-2 locus on chromosome 3. Like humans with the same inherited enzyme defect, animals homozygous for the new null allele are runted and have renal tubular acidosis. However, the prominent osteopetrosis found in humans with CA II deficiency could not be detected even in very old homozygous null mice. A molecular analysis of the deficient mice shows that the mutant gene is not deleted and is transcribed. The CA II protein, which is normally expressed in most tissues, could not be detected by immunodiffusion analysis in any tissues of the CA II-deficient mice, suggesting a nonsense or a missense mutation at the Car-2 locus.

A null mutation at the phosphoglucomutase locus (Pgm-1) was discovered by electrophoretic analysis of the inbred mouse strain C57BL/6J. The null allele (Pgm-1n) was shown to segregate as a Mendelian unit alternative to the Pgm-1a and Pgm-1b alleles. Mice expressing the Pgm-1n allele, either in the heterozygous or homozygous state, are viable, healthy, and fertile. The occurrence of the Pgm-1n mutant revealed a previously unreported genetic locus (Pgm-3) that controls the expression of a third phosphoglucomutase. Two electrophoretically expressed alleles of Pgm-3 (inherited without dominance) are found in the inbred mouse strains C57BL/6J and DBA/2J. Linkage observed between the Pgm-3 locus, the dilute locus (d) and the cytoplasmic malic enzyme locus (Mod-1) has allowed assignment of the Pgm-3 locus to chromosome 9. A striking tissue specific expression of Pgm-1 and Pgm-3 was observed. Products of the Pgm-3 locus were detected in kidney, testes, brain, and heart. In contrast, Pgm-1 controlled isozymes were present in kidney, spleen, ovaries, and erythrocytes.

A new neonatal lethal mutation in the mouse with pleiotropic effects, edematous (oed), arose spontaneoutosomal recessive mode of inheritance. The external phenotypic characteristics include a generally bloated appearance, shiny celophane-like skin, and distal hematomata of the extremities. Internally, no gross or histological abnormalities could be identified, with the exception of a striking leukocytosis. Biochemical analysis revealed a severe disturbance of lipid metabolism. Deficiencies in the VLDL, LDL, and the HDL lipoprotein fractions have been found in the mutant plasma. In addition, all serum lipids are markedly decreased. However, in the mutant liver, only triglycerides are significantly decreased; total liver cholesterol and phospholipid values are within normal limits. The primary biochemical defect as well as the causal relationship between the striking abnormalities of lipid metabolism and those of skin and blood are unknown at this time.

A mouse with a variant hemoglobin was discovered during electrophoretic screening of (C57BL/6J X DBA/2J)F1 progeny of females treated with ethylnitrosourea. The variant trait was transmitted as a simple Mendelian alternate at the Hbb locus in all crosses except those involving the original carrier of the mutation. The proband mouse which received the mutation directly from the mutagen-treated parent was a germinal mosaic for the mutant and normal Hbbs alleles. The mutant allele was designated Hbbs2. The mutant haplotype specifies both an electrophoretically fast hemoglobin band and a hemoglobin band in the normal beta single hemoglobin position. Thus, the mutation has altered one of the tandemly duplicated genes at the Hbbs locus. A comparison of the relative concentrations of the two hemoglobins in Hbbs2 mice demonstrates preferential expression of the mutant gene, possibly analogous to the enhanced expression of Hbbdmaj in the Hbbd haplotype. Analysis of the amino acid sequence of the variant beta-globin revealed that the valine at position 60 was changed to glutamic acid. The simplest mutation mechanism for such an alteration is an A X T----T X A transversion.