Tetsuo Kunieda

A mouse model of Waardenburg syndrome type 4 with a new spontaneous mutation of the endothelin-B receptor gene

Waardenburg syndrome (WS) is a hereditary auditory-pigmentary syndrome with hearing impairment and pigmentation anomaly of the skin and iris. In addition to these major symptoms, WS type 4 is associated with Hirschsprung disease. To date, three genes responsible for WS4 have been cloned: genes for a transcription factor SOX10, endothelin 3 (EDN3), and endothelin B receptor (EDNRB). We here describe a novel mutant mouse with a mutation of the Ednrb gene, and propose the mouse as an animal model of WS4. These mutants are with mixed genetic background of BALB/c and MSM (an inbred strain of Japanese wild mice) and have extensive white spotting. They died between 2 and 7 weeks after birth owing to megacolon: their colon distal to the megacolon lacked Auerbach’s plexus cells. Interestingly, these mutants did not respond to sound, and the stria vascularis of their cochlea lacked intermediate cells, i.e., neural crest-derived melanocytes. Since these symptoms resembled those of human WS4 and were transmitted in autosomal recessive hereditary manner, the mutants were named WS4 mice. Breeding analysis revealed that WS4 mice are allelic with piebald-lethal and JF1 mice, which are also mutated in the Ednrb gene. Mutation analysis revealed that their Ednrb lacked 318 nucleotides encoding Ednrb transmembrane domains owing to deletion of exons 2 and 3. Interaction between endothelin 3 and its receptor is required for normal differentiation and development of melanocytes and Auerbach’s plexus cells. We concluded that a missing interaction here led to a lack of these cells, which caused pigmentation anomaly, deafness, and megacolon in WS4 mice.

Cloning of bovine LYST gene and identification of a missense mutation associated with Chediak-Higashi syndrome of cattle.

Abstract. An inheritable bleeding disorder with light coat color caused by an autosomal recessive gene has been reported in a population of Japanese black cattle. The disease has been diagnosed as Chediak-Higashi Syndrome (CHS) of cattle which correspond to a human inheritable disorder caused by mutation in LYST gene. To characterize the molecular lesion causing CHS in cattle, cDNAs encoding bovine LYST were isolated from a bovine brain cDNA library. The nucleotide and deduced amino acid sequences of bovine LYST had 89.6 and 90.2% identity with those of the human LYST gene, respectively. In order to identify the mutation within the LYST gene causing CHS in cattle, cDNA fragments of the LYST gene were amplified from an affected animal by RT-PCR and their nucleotide sequences were completely determined. Notably, a nucleotide substitution of A to G transition, resulting in an amino acid substitution of histidine to arginine (H2015R) was identified in the affected animal. The presence of the substitution was completely corresponding with the occurrence of the CHS phenotype among 105 members of pedigrees of the Japanese black cattle and no cattle of other populations had this substitution. These findings strongly suggested that H2015R is the causative mutation in CHS of Japanese black cattle.

A locus responsible for hypogonadism (hgn) is located on rat Chromosome 10.

Hypogonadic rats of the HGN inbred strain show male sterility controlled by an autosomal single recessive allele (hgn). The testis weight of adult hgn/hgn rats is about 1% that of normal rats, and only a few thin seminiferous tubules surrounded by islet-like conformations of Leydig cells are present in the fibrous interstitial tissue of the testis (Suzuki et al. 1988). Primordial germ cells do exist in the neonatal hgn/hgn testis, but cannot differentiate into spermatogonia and degenerate before entering meiosis (Suzuki et al, 1998). hgn/hgn females have reduced fertility, including a small litter size, a reduced number of oocytes, and early reproductive senescence (Suzuki et al. 1992). hgn/hgn rats of both sexes also show bilateral hypoplastic kidney (HPK) with a reduced number (only one-quarter) of glomeruli (Suzuki et al. 1991; Suzuki and Suzuki 1995). Whether the phenotypes of hypogonadism and HPK are caused by a mutation in a single gene or defects in different genes located close to each other remains unclear. No other mutant animal or human disease showing a phenotype similar to that of the hgn/hgn rat has been reported. The hgn/hgn rat would, therefore, be a useful model for investigating the development of mammalian reproductive and urinary organs. In this study, linkage analysis was performed to determine the chromosome location of the hgn locus and to obtain information about candidate genes for hgn. Thirteen hgn/hgn females were selected from the population of the HGN inbred strain by laparotomy for detection of HPK at weaning (Suzuki et al. 1991). One hgn/hgn female was mated with a BN male rat (+/+), and the resulting F1 males were mated with the remaining 12 hgn/hgn females to produce backcross progeny. After the mating, the hgn/hgn females were autopsied to confirm the hgn/hgn phenotype (Suzuki et al. 1992). All male backcross progeny were autopsied at the age of 5 days to avoid postnatal loss, and their phenotypes were determined by macroscopic observation of testicular size. Relative testis weight of the affected rats (0.49 ± 0.06g, average ± S.D.) was about half that of normal rats (1.16 ± 0.09g). The histological appearance of the testes in the affected rats was similar to that of hgn/hgn rats of the HGN strain (Suzuki et al. 1993). Since it was difficult to clearly categorize females of the backcross progeny into affected and normal phenotypes by comparison of renal size, we did not use female rats for linkage analysis. Consequently, 29 affected and 26 normal males were obtained. The segregation ratio of the affected to normal rats did not deviate significantly from the expected 1 : 1 ratio (x 4 0.16, P > 0.20). Because of physical limitations of the PCR machine with a block of 48 samples, high-molecular-weight genomic DNA was isolated from livers of 24 affected and 24 normal rats by phenol extraction. The samples of remaining seven progeny were saved for the next mapping experiment. The total of 48 progeny were initially typed for 33 microsatellite markers on rat chromosomes. Primers for the microsatellite markers were purchased from Research Genetics Inc. (Huntsville, Ala.). The PCR was carried out in 10 m1 of a reaction mixture containing 25 ng of the genomic DNA, 1.0 pmol of each of the oligonucleotide primers, 2.0 nmol of each dNTP, and 0.26 U of Taq polymerase (TaKaRa) in the reaction buffer recommended by the manufacturer. After initial denaturation for 180 s at 92°C, 35 cycles of amplification, each consisting of denaturation for 15 s at 92°C, annealing 60 s at 55°C, and extension for 120 s at 72°C, were carried out with a Thermal Cycler TP2000 (TaKaRa). The PCR products were separated by nondenatured 10% polyacrylamide gel electrophoresis and visualized by silver staining (Silver Stain Plus Kit, Bio-Rad). The recombination fractions and the lod scores among the hgn and the other loci were calculated with Map Manager ver. 2.6.5 (distributed via the World Wide Web, URL: http://mcbio.med.buffalo.edu/mapmagr.html). The order of loci was determined so that the number of recombination events was minimized. By typing the 33 rat microsatellite marker loci in the backcross progeny, significant linkage was observed between the hgn locus and the D10Mgh10 locus on rat Chromosome (Chr) 10 with a lod score (z) of 5.1 and a recombination fraction (u) of 0.167. The D10Mgh3 locus also was linked to the hgn locus (z42.7, u40.25). No significant linkage was observed between the hgn locus and the other 31 loci. Therefore, the 48 progeny were further typed for the following seven loci located on rat Chr 10: D10Mit2, D10Mgh6, D10Mgh8 (Myh3), D10Wox14 (Asgr1), D10Wox6, D10Wox16 (Ngfr), and D10Wox12 (Abp). The highest lod score (z 4 14.4) was obtained for the D10Mit2 locus with a u value of 0. Segregation of alleles of the nine microsatellite loci as well as the hgn locus in the 48 progeny (Fig. 1) and a linkage map of rat Chr 10 with those loci (Fig. 2) are shown. The order and distances of the nine loci on the linkage map are basically identical with linkage maps reported previously (Jacob et al. 1995; Bihoreau et al. 1997). We therefore concluded that the hgn locus is located in the region close to the D10Mit2 locus on rat Chr 10. The Abp gene (Shbg in mouse) encodes androgen binding proteins expressed in Sertoli cells (Sallivan et al. 1991), which are apparently affected in the testis of hgn/hgn rats, suggesting that the gene is a candidate for hgh. As shown in Fig. 1, the presence of recombinations between the hgn and Abp loci, however, ruled out the Abp gene as a candidate. As shown in Fig. 2, the order of the functional genes (Myh3/Myhse, Abp/Shbg, Asgr1, and Ngfr) on rat Chr 10 is identical to that of the corresponding region of mouse Chr 11. Thus, the mouse gene corresponding to the hgn locus might be located in the region between map positions 37 (Asgr1) and 56 (Ngfr) of mouse Chr 11 (derived from the Mouse Genome Database; MGD). Possible candidate genes for the hgn mutation, including the Tex4, Fert2, Ube2b-rs2, Gsg2, and Lhx1 genes, have been mapped to this region. The Ube2b gene, which is implicated in postreplication repair, is located on either Chr 13 (Ube2b-rs1) or Chr 11 (Ube2brs2). Inactivation of the Ube2b gene of the mouse causes male sterility associated with chromatin modification, although homoCorrespondence to: H. Suzuki Mammalian Genome 10, 1106–1107 (1999).

Localization of a locus responsible for the bovine chondrodysplastic dwarfism (bcd) on chromosome 6.

A hereditary chondrodysplastic dwarfism caused by an autosomal recessive gene has been reported in a population of Japanese Brown cattle. Affected calves show an insufficiency of endochondral ossification at the long bones of the limbs. In the present study, we mapped the locus responsible for the disease (bcd) by linkage analysis, using microsatellite markers and a single paternal half-sib pedigree obtained from commercial herds. Linkage analysis revealed a significant linkage between the bcd locus and marker loci on the distal region of bovine Chromosome (Chr) 6. The bcd locus was mapped in the interval between microsatellite markers BM9257 and BP7 or BMS511 with a recombination fraction of 0.05 and 0.06, and a lod score of 8.6 and 10.1, respectively. A comparison of genetic maps between bovine Chr 6 and human Chr 4 or mouse Chr 5 indicates possible candidate genes including FGFR3 and BMP3 genes, which are responsible for human chondrodysplasias and associated with bone morphogenesis, respectively.

Homozygosity mapping of the locus responsible for renal tubular dysplasia of cattle on bovine Chromosome 1

Abstract. Renal tubular dysplasia is a hereditary disease of Japanese black cattle showing renal failure and growth retardation with an autosomal recessive trait. In the present study, we mapped the locus responsible for the disease (RTD) by linkage analysis with an inbred paternal half-sib pedigree obtained from commercial herds. By analyzing segregation of microsatellite markers in the half-sibs, significant linkage was observed between the RTD locus and markers on bovine Chromosome (Chr) 1 with the highest lod score of 11.4. Homozygosity mapping with the inbred pedigree further defined the localization of the RTD locus in a 4-cM region between microsatellite markers BMS4003 and INRA119. Mapping of the RTD locus on bovine Chr 1 will facilitate cloning and characterization of the gene responsible for this disease.

A locus responsible for arrest of spermatogenesis is located on rat Chromosome 12

Department of Genetic Resources II, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan Division of Genetics and Breeding, National Institute of Animal Industry, Tsukuba, Ibaraki 305-0901, Japan Department of Genetic Resources I, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan Imamichi Institute of Animal Reproduction, Niihari, Ibaraki 300-0134, Japan Faculty of Agriculture, Okayama University, Okayama, Okayama 700-0082, Japan