Ribosomal genes in inbred mouse strains: Interstrain and intrastrain variation of copy number and extent of methylation

Quantitative dot hybridization was used to estimate the rDNA copy number in brain tissues of five inbred mouse strains (AKR/JY, NZB/B1OrlY, CBA/CaLacY, 101/HY, and 129/JY), which were obtained from the collection of the Research Center of Biomedical Technologies (Y). In each strain, 9–12 mice aged 1–2 months were examined. The rDNA copy number per diploid genome in strains AKR (range 105–181, mean ± SD 136 ± 27) and NZB (129–169, 148 ± 12) was significantly lower than in strains CBA (172–267, 209 ± 31), 101 (179–270, 217 ± 30), and 129 (215–310, 264 ± 33). Mice of strain NZB were relatively homogeneous in this trait (CV = 8.1%). Strains AKR, CBA, 101, and 129 displayed significant between-group differences, CV varying from 12.5 to 19.9%. The same DNA specimens were digested with MspI or HpaII and used to estimate the extent of methylation of the 28S rDNA region. Regardless of the strain, all mice could be classed into two groups. One group (20 mice) had a methylated fraction accounting for less than 8% of rDNA and included all nine mice of strain NZB, seven out of nine mice of strain 101, and three out of ten mice of strain 129. In the other group (29 mice of strains AKR, CBA, 101, and 109), the methylated fraction varied from 18 to 38%. A possible role of methylation and the genome dosage of ribosomal genes in phenotypic variation (quantitative trait variation) of inbred mouse strains is discussed.     

New copy number variations in schizophrenia

Genome-wide screenings for copy number variations (CNVs) in patients with schizophrenia have demonstrated the presence of several CNVs that increase the risk of developing the disease and a growing number of large rare CNVs; the contribution of these rare CNVs to schizophrenia remains unknown. Using Affymetrix 6.0 arrays, we undertook a systematic search for CNVs in 172 patients with schizophrenia and 160 healthy controls, all of Italian origin, with the aim of confirming previously identified loci and identifying novel schizophrenia susceptibility genes. We found five patients with a CNV occurring in one of the regions most convincingly implicated as risk factors for schizophrenia: NRXN1 and the 16p13.1 regions were found to be deleted in single patients and 15q11.2 in 2 patients, whereas the 15q13.3 region was duplicated in one patient. Furthermore, we found three distinct patients with CNVs in 2q12.2, 3q29 and 17p12 loci, respectively. These loci were previously reported to be deleted or duplicated in patients with schizophrenia but were never formally associated with the disease. We found 5 large CNVs (>900 kb) in 4q32, 5q14.3, 8q23.3, 11q25 and 17q12 in five different patients that could include some new candidate schizophrenia susceptibility genes. In conclusion, the identification of previously reported CNVs and of new, rare, large CNVs further supports a model of schizophrenia that includes the effect of multiple, rare, highly penetrant variants.     

DNA polymorphism of MHC III genes in inbred and wild mouse strains

Genes encoding the second component (C2), factor B, and complement protein C4 and Slp (sex-limited protein) are members of the major histocompatibility complex class III gene cluster. In this report we describe isolation of a mouse C2 cDNA clone and its use together with factor B and C4 cDNA clones to examine the S region in a panel of 42 haplotypes in laboratory and wild mice representing 5 species and subspecies of Mus. Conservation of the C2 factor B gene duplex was evidenced by relatively limited polymorphism associated with speciation and nucleotide sequence homology between mouse and human C2 and factor B The C4-Slp gene duplex, on the other hand, showed extensive polymorphism by DNA blot analysis. This polymorphism correlated poorly with the C2/factor B restriction fragment length polymorphism, suggesting independent evolution of these two segments of the S region. Taken together, these data will be of particular importance in studies of mouse strains with abnormal regulation of immune effector systems since the class III gene products are essential for activation of the complement cascade.     

Modeling genetic inheritance of copy number variations

Copy number variations (CNVs) are being used as genetic markers or functional candidates in gene-mapping studies. However, unlike single nucleotide polymorphism or microsatellite genotyping techniques, most CNV detection methods are limited to detecting total copy numbers, rather than copy number in each of the two homologous chromosomes. To address this issue, we developed a statistical framework for intensity-based CNV detection platforms using family data. Our algorithm identifies CNVs for a family simultaneously, thus avoiding the generation of calls with Mendelian inconsistency while maintaining the ability to detect de novo CNVs. Applications to simulated data and real data indicate that our method significantly improves both call rates and accuracy of boundary inference, compared to existing approaches. We further illustrate the use of Mendelian inheritance to infer SNP allele compositions in each of the two homologous chromosomes in CNV regions using real data. Finally, we applied our method to a set of families genotyped using both the Illumina HumanHap550 and Affymetrix genome-wide 5.0 arrays to demonstrate its performance on both inherited and de novo CNVs. In conclusion, our method produces accurate CNV calls, gives probabilistic estimates of CNV transmission and builds a solid foundation for the development of linkage and association tests utilizing CNVs.     

Human tRNAGlu genes: their copy number and organisation

The tRNAGlu gene copy number, determined by genomic blot analysis of human placental DNA, is approximately thirteen. These studies, using several probes and DNA digested with several restriction enzymes singly or in combination, show that most of these tRNAGlu genes are flanked by DNA of very similar sequence for at least 5 kb. This conclusion is supported by the close similarity of the restriction maps of two lambda Charon-4A recombinants of human genomic DNA containing two different tRNAGlu genes.     

A map of copy number variations in Chinese populations

It has been shown that the human genome contains extensive copy number variations (CNVs). Investigating the medical and evolutionary impacts of CNVs requires the knowledge of locations, sizes and frequency distribution of them within and between populations. However, CNV study of Chinese minorities, which harbor the majority of genetic diversity of Chinese populations, has been underrepresented considering the same efforts in other populations. Here we constructed, to our knowledge, a first CNV map in seven Chinese populations representing the major linguistic groups in China with 1,440 CNV regions identified using Affymetrix SNP 6.0 Array. Considerable differences in distributions of CNV regions between populations and substantial population structures were observed. We showed that ～35% of CNV regions identified in minority ethnic groups are not shared by Han Chinese population, indicating that the contribution of the minorities to genetic architecture of Chinese population could not be ignored. We further identified highly differentiated CNV regions between populations. For example, a common deletion in Dong and Zhuang (44.4% and 50%), which overlaps two keratin-associated protein genes contributing to the structure of hair fibers, was not observed in Han Chinese. Interestingly, the most differentiated CNV deletion between HapMap CEU and YRI containing CCL3L1 gene reported in previous studies was also the highest differentiated regions between Tibetan and other populations. Besides, by jointly analyzing CNVs and SNPs, we found a CNV region containing gene CTDSPL were in almost perfect linkage disequilibrium between flanking SNPs in Tibetan while not in other populations except HapMap CHD. Furthermore, we found the SNP taggability of CNVs in Chinese populations was much lower than that in European populations. Our results suggest the necessity of a full characterization of CNVs in Chinese populations, and the CNV map we constructed serves as a useful resource in further evolutionary and medical studies.     

CNVDetector: locating copy number variations using array CGH data

CNVDetector is a program for locating copy number variations (CNVs) in a single genome. CNVDetector has several merits: (i) it can deal with the array comparative genomic hybridization data even if the noise is not normally distributed; (ii) it has a linear time kernel; (iii) its parameters can be easily selected; (iv) it evaluates the statistical significance for each CNV calling. AVAILABILITY: CNVDetector (for Windows platform) can be downloaded from http:www.csie.ntu.edu.tw/~kmchao/tools/CNVDetector/. The manual of CNVDetector is also available.     

Common copy number variations in fifty radiosensitive cell lines

Hypersensitivity to radiation exposure is a major challenge to radiotherapy in the treatment of cancer patients. Copy number variations (CNVs) are believed to identify genomic regions of functional significance for radiosensitivity (RS) but have yet to be systematically investigated. We used Affymetrix 6.0 SNP arrays to survey common CNVs in a cohort of 50 radiosensitive lymphoblastoid cell lines (RS-LCLs) derived from patients with undiagnosed diseases. A total of 317 CNVs that were present in at least 10% of the studied cell lines were identified. Three hundred and eight CNVs overlapped with polymorphic CNVs, 13 of which were significantly enriched in the RS-LCLs compared to the reference. The remaining 9 CNVs were novel. The majority of these enriched and novel CNVs were chromosomal gains. The dominance of the chromosomal gains over losses is inconsistent with the traditional concept of molecular basis of RS and suggests more complex genetic mechanisms for RS.     

Frequency of X-Y chromosome dissociation in mouse spermatocytes from interstrain crosses,recombinant inbred strains,and chimeras: Possible involvement of paternal genome imprinting

The frequency of dissociation of the X-Y chromosome bivalent in diakinesis-metaphase I spermatocytes differs significantly between two inbred mouse strains, CBA (29%) and KE (7%), that were used to obtain reciprocal F1 hybrids, and to develop recombinant inbred (R1) strains. The level of X-Y dissociation was significantly higher in (KExCBA)F1 hybrids sired by the CBA males (24%) than in reciprocal F1 hybrids (12%), revealing the inheritance after the father. Among 14 RI strains, nine were concordant with KE, one with CBA, and four had intermediate phenotype, significantly different from both progenitor strains. This shows that at least two genes are involved, and their possible linkage with agouti and Trf loci is suggested. The linkage with agouti was confirmed by testing additional 10 CBXE incipient RI strains. There was no significant difference in the level of X-Y dissociation between EXCB RI strains derived from the original cross sired by the CBA males and CBXE RI strains derived from the reciprocal cross. The involvement of the Y chromosome-linked factors was unlikely because it was found earlier (Krzanowska, 1989: Gamete Res 23:357–365) that two congenic strains, KE and KE.CBA, differing with respect to the source of the Y chromosome, had the same level of X-Y dissociation. Thus, the difference obtained between reciprocal F1 hybrids is interpreted in terms of paternal genome imprinting imposed by CBA males and propagated only in the presence of some alleles derived from this strain. Analysis of six KE ? CBA-T6 chimeras, among them three germ line chimeras, points to the conclusion that the tendency to low or high level of X-Y chromosome dissociation is expressed rather autonomously by KE or CBA-T6 spermatocytes (as recognized by a marker chromosome pair), respectively, and was not modified by the presence of somatic cells of the opposite strain. © 1994 Wiley-Liss, Inc.     

Genetic marker patterns and endogenous mammary tumor virus genes in inbred mouse strains of Japan

In order to establish the genetic relatedness of the inbred mouse strains kept in Nara, genetic marker patterns were determined in conjunction with a study on endogenous mammary tumor viral genes in these strains. Isoenzyme patterns combined with patterns of other genetic markers, show that the unrelatedness between various inbred strains of the dd stock is as high or even higher as between strains of known different origin and geneology. Based on endogenous viral gene patterns the dd stock derived mice can be subdivided into three group, DDD, DDN, DDO, KF and DD/Tbr. The DD/Tbr and its foster-nursed substrain (DD/Tbrf) have the lowest number of endogenous viral genes, i.e. two, while the other strains carry 4-6 such genes. The SLN and SHN strains, derived from a Swiss stock, have a similar pattern of viral genes different that of all other strains studied, also strains of Swiss origin from other sources, such as the NFS and the GR.     

Detection of copy number variations and their effects in Chinese bulls

Background

Copy number variations (CNVs) are a main source of genomic structural variations underlying animal evolution and production traits. Here, with one pure-blooded Angus bull as reference, we describe a genome-wide analysis of CNVs based on comparative genomic hybridization arrays in 29 Chinese domesticated bulls and examined their effects on gene expression and cattle growth traits.

Results

We identified 486 copy number variable regions (CNVRs), covering 2.45% of the bovine genome, in 24 taurine (Bos taurus), together with 161 ones in 2 yaks (Bos grunniens) and 163 ones in 3 buffaloes (Bubalus bubalis). Totally, we discovered 605 integrated CNVRs, with more “loss” events than both “gain” and “both” ones, and clearly clustered them into three cattle groups. Interestingly, we confirmed their uneven distributions across chromosomes, and the differences of mitochondrion DNA copy number (gain: taurine, loss: yak & buffalo). Furthermore, we confirmed approximately 41.8% (253/605) and 70.6% (427/605) CNVRs span cattle genes and quantitative trait loci (QTLs), respectively. Finally, we confirmed 6 CNVRs in 9 chosen ones by using quantitative PCR, and further demonstrated that CNVR22 had significantly negative effects on expression of PLA2G2D gene, and both CNVR22 and CNVR310 were associated with body measurements in Chinese cattle, suggesting their key effects on gene expression and cattle traits.

Conclusions

The results advanced our understanding of CNV as an important genomic structural variation in taurine, yak and buffalo. This study provides a highly valuable resource for Chinese cattle’s evolution and breeding researches.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-480) contains supplementary material, which is available to authorized users.     

The relationship between nephron number,kidney size and body weight in two inbred mouse strains

While some reports in humans have shown that nephron number is positively correlated with height, body weight or kidney weight, other studies have not reproduced these findings. To understand the impact of genetic and environmental variation on these relationships, we examined whether nephron number correlates with body weight, kidney planar surface area, or kidney weight in two inbred mouse strains with contrasting kidney sizes but no overt renal pathology: C3H/HeJ and C57BL/6J. C3H/HeJ mice had smaller kidneys at birth and larger kidneys by adulthood, however there was no significant difference in nephron number between the two strains. We did observe a correlation between kidney size and body weight at birth and at adulthood for both strains. However, there was no relationship between nephron number and body weight or between nephron number and kidney size. From other studies, it appears that a greater than 2-fold variation is required in each of these parameters in order to demonstrate these relationships, suggesting they are highly dependent on scale. Our results are therefore not surprising since there was a less than 2-fold variation in each of the parameters examined. In summary, the relationship between nephron number and body or kidney size is most likely to be demonstrated when there is greater phenotypic variation either from genetic and/or environmental factors.     

The relationship between nephron number, kidney size and body weight in two inbred mouse strains

While some reports in humans have shown that nephron number is positively correlated with height, body weight or kidney weight, other studies have not reproduced these findings. To understand the impact of genetic and environmental variation on these relationships, we examined whether nephron number correlates with body weight, kidney planar surface area, or kidney weight in two inbred mouse strains with contrasting kidney sizes but no overt renal pathology: C3H/HeJ and C57BL/6J. C3H/HeJ mice had smaller kidneys at birth and larger kidneys by adulthood, however there was no significant difference in nephron number between the two strains. We did observe a correlation between kidney size and body weight at birth and at adulthood for both strains. However, there was no relationship between nephron number and body weight or between nephron number and kidney size. From other studies, it appears that a greater than two-fold variation is required in each of these parameters in order to demonstrate these relationships, suggesting they are highly dependent on scale. Our results are therefore not surprising since there was a less than two-fold variation in each of the parameters examined. In summary, the relationship between nephron number and body or kidney size is most likely to be demonstrated when there is greater phenotypic variation either from genetic and/or environmental factors.     

Ribosomal proteins are encoded by single copy genes in Dictyostelium discoideum

Five recombinant plasmids which encode ribosomal proteins (r-proteins) from Dictyostelium discoideum have been isolated. Poly(A) + RNA was size-fractionated by preparative agarose gel electrophoresis and a fraction encoding proteins of less than 35 kDa was used to construct a cDNA library in the plasmid vector pBR322. Individual clones from the library were screened by hybrid-selected translation and those encoding r-proteins were identified by co-migration of the translation products in two-dimensional gel electrophoresis with marker proteins purified from Dictyostelium ribosomes. Initial characterization using the five cDNA plasmids indicates that these r-proteins are encoded by single copy genes and that they are not tightly clustered in the genome.     

Origins and breakpoint analyses of copy number variations: up close and personal

Array-based methods have enabled the detection of many genomic gains and losses. These are stated as copy number variants (CNVs) and comprise up to 13% of the human genome. Based on their breakpoints and modes of formation CNVs are termed recurrent or nonrecurrent. Recurrent CNVs are flanked by low copy repeats and are of a fixed size. They arise as a result of misalignment during meiosis by a mechanism named nonallelic homologous recombination. Several of such recurrent CNVs have been linked to human diseases. Nonrecurrent CNVs, which are not flanked by low copy repeats, are of variable size and may arise via mechanisms like nonhomologous end joining and replication-based mechanisms described by the fork stalling and template switching and microhomology-mediated break-induced replication models. It is becoming clear that most disease-causing CNVs are nonrecurrent and generally arise via replication-based mechanisms. Furthermore, it is now appreciated that genomic features other than low copy repeats play a role in the formation of nonrecurrent CNVs. This review will discuss the different mechanisms of CNV formation and how high resolution analyses of CNV breakpoints have added to our knowledge of their precise structure.     

Identification of genes with correlated patterns of variations in DNA copy number and gene expression level in gastric cancer

To identify DNA copy number changes that had a direct influence on mRNA expression in gastric cancer, cDNA microarray-based comparative genomic hybridization (aCGH) and gene expression profiling were performed using 17 K cDNA microarrays. A set of 158 genes showing Pearson correlation coefficients over 0.6 between DNA copy number changes and mRNA expression level variations was selected. In an independent gene expression profiling of 60 tissue samples, the 158 genes were able to distinguish most of the normal and tumor tissues in an unsupervised hierarchical clustering, suggesting that the differential expression patterns displayed by this specific group of genes are most likely based on the gene copy number changes. Furthermore, 43 statistically significant (P<0.01) genes were selected that correctly distinguished all of the tissue samples. The copy number changes detected by aCGH can be verified by fluorescence in situ hybridization and real-time polymerase chain reaction. The selected genes include those that were previously identified as being tumor suppressors or deleted in various tumors, including GATA binding protein 4 (GATA4), monoamine oxidase A (MAOA), cyclin C (CCNC), and oncogenes including malignant fibrous histiocytoma amplified sequence 1 (MFHAS1/MASL1), high mobility group AT-hook 2 (HMGA2), PPAR binding protein (PPARBP), growth factor receptor-bound protein 7 (GRB7), and TBC1 (tre-2, BUB2, cdc16) domain family, member 1 (TBC1D1).