Rescue from Dwarfism by Thyroid Function Compensation in rdw Rats.

The rdw rat was initially reported as having hereditary dwarfism caused by pituitary dysfunction. Subsequent studies on the rdw rat, however, have demonstrated that the primary cause of rdw dwarfism is present in the thyroid gland but not in the pituitary gland. The primary cause of rdw rat disorders is a missense mutation of the thyroglobulin (Tg) gene by a one-point mutation. In the present study, we attempted to rescue the dwarfism of the rdw rats using a diet supplemented with thyroid powder (T-powder) and a thyroid graft (T-graft). The infants of the rdw rat were successfully raised to a mature stage body weight, accompanied by elevation of serum growth hormone (GH) and prolactin (PRL), by the T-powder. Furthermore, the T-graft successfully increased the body weight with fertility. The serum GH and PRL levels in the T-graft rdw rat significantly increased. The serum thyroid-stimulating hormone (TSH) levels in the T-graft rdw rat were significantly decreased but were significantly higher than those in the control rat. The GH and PRL mRNA expression in the rdw rat with the T-graft was virtually the same as that of the control, but the TSH beta mRNA differed from that of the control rats. Thus, the dwarfism in the rdw rat is rescued by thyroid function compensation, such as that afforded by T-powder and T-graft.     

rdw rats, a new hereditary dwarf model in the rat

A new hereditary dwarf mutation was found in Csk: Wistar-Imamichi rat breeding colony. The mutant rats and control normal rats were studied with clinical, genetical and histological examination. The result showed that the present mutation was inherited in autosomal recessive trait (gene symbol; rdw) and was provoked with the hypoplasia of pars distalis (anterior pituitary), in particular the secretory cells of GH, PRL and TSH. The characteristics of the present mutant (rdw) was very similar to dw mouse, and was usefull for endocrinological research as an animal model of human pituitary dwarfism.     

Identification of N-acetyltransferase 2 genotypes by continuous monitoring of fluorogenic hybridization probes.

Three polymorphic sites in the N-acetyltransferase 2 (NAT2) gene were detected using rapid cycle DNA amplification with allele-specific fluorescent probes and melting curve analysis. Two fluorogenic adjacent hybridization probes were designed to NAT2*5A (C(481)T), NAT2*6A (G(590)A), and NAT2*7A (G(857)A). During amplification, probe hybridization is observed as fluorescence resonance energy transfer. The fluorescence increases every cycle as the product accumulates during amplification. A single base mismatch resulted in a melting temperature shift (T(m)) of 5 to 6 degrees C, allowing for the easy distinction of a wild-type allele from the mutant allele. The protocol is rapid, requiring 40 min for the completion of 45 cycles including the melting curves. It is also a simple and flexible method, since DNA templates prepared from different sources, including DNA from serum and paraffin-embedded tissue sections, could be used without adverse effects. Fluorescence genotyping of all three polymorphisms in a total of 155 DNA samples correlated perfectly with our previously validated genotyping by restriction enzyme digestion (PCR-RFLP). This new facile approach allows for the easy detection of NAT2 polymorphisms in hundreds of samples in only a day.     

Evaluation and characterization of congenital hypothyroidism in rdw dwarf rats

The rdw rat is a new strain of dwarf mutant that has decreased blood thyroxine (T4) and growth hormone (GH) concentrations and testicular enlargement during development and aging. To confirm whether this strain can be used as a new hypothyroid model, the experiments reported here were carried out, using adult rdw rats, rdw rats treated with thyroxine, and clinically normal (N) Wistar-Imamichi rats. Clinical parameters of deficient thyroid function in rdw rats were chosen for evaluation and characterization. Body weight, hemoglobin (Hb) concentration, hematocrit (Hct), glucose (GLU), and systolic blood pressure were significantly lower, and serum values for aspartate transaminase (AST), total cholesterol (TC), total protein (TP), and blood urea nitrogen (BUN) were higher in rdw than in N rats. Serum concentrations of total T4 and free triiodothyronine (FT3) were significantly lower, and serum thyroid-stimulation hormone (TSH) concentration was markedly higher in rdw than in N rats. Serum GH concentration was significantly lower in rdw than in N rats. Results of histologic examination indicated that the thyroid gland of rdw rats was markedly atrophied, compared with that of N rats. Results of clinical examination of organs and hematologic and biochemical values in rdw rats corresponded to those of the hypothyroid state in humans. Most organ weights (heart, kidney, spleen, and adrenal gland), hematologic and biochemical values (Hb, Hct TC, TP, BUN), blood pressure, and serum hormone (TSH and GH) values underwent substantial restoration (partial or complete) toward normal in response to replacement therapy. In conclusion, the rdw rat is a useful model of congenital hypothyroidism.     

Pituitary and plasma levels of growth hormone (GH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) in hereditary dwarf rats (rdw/rdw)]

Koto et al found a new hereditary dwarf mutation from breeding colony of Wistar-Imamichi rat and named 'rdw'. To characterize endocrinological functions in rdw rats, pituitary and plasma levels of pituitary hormones including growth hormone (GH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were compared between rdw and normal rats. The hormone levels were estimated with radioimmunoassay (RIA). It was found that pituitary and plasma levels of GH of rdw were drastically decreased and those of FSH and LH were inclined to decrease but not remarkable as compared with normal. Rats of rdw were, therefore, considered to be useful as a model animal for endocrinological defects.     

A simple and rapid method for detection of the 106Gln mutation in Wilson-Konovalov disease

A simple and rapid method for detecting the 1069Gln mutation in gene ATP7B based on a PCR specific for this allele has been developed. The 1069Gln mutation is the main cause of Wilson disease (WD) in Russia and accounts for approximately 40% of all mutant alleles of gene ATP7B. Therefore, the method proposed makes the postnatal and prenatal diagnosis of Wilson disease in Russia considerably more rapid and less expensive.     

Oxidoreductase interactions include a role for ERp72 engagement with mutant thyroglobulin from the rdw/rdw rat dwarf

Newly synthesized thyroglobulin (Tg), the secretory glycoprotein that serves as precursor in thyroid hormone synthesis, normally forms transient covalent protein complexes with oxidoreductases of the endoplasmic reticulum (ER). The Tg-G2320R mutation is responsible for congenital hypothyroidism in rdw/rdw rats, in which a lack of secondary thyroid enlargement (goiter) implicates death of thyrocytes as part of disease pathogenesis. We found that mutant Tg-G2320R was retained within the ER with no detectable synthesis of thyroxine, had persistent exposure of free cysteine thiols, and was associated with activated ER stress response but incomplete ER-associated degradation (ERAD). Tg-G2320R associated with multiple ER resident proteins, most notably ERp72, including covalent Tg-ERp72 interactions. In PC Cl3 thyrocytes, inducible overexpression of ERp72 increased the ability of cells to maintain Tg cysteines in a reduced state. Noncovalent interactions of several ER chaperones with newly synthesized Tg-G2320R diminished over time in parallel with ERAD of the mutant protein, yet a small ERAD-resistant Tg fraction remained engaged in covalent association with ERp72 even 2 days post-synthesis. Such covalent protein aggregates may set the stage for apoptotic thyrocyte cell death, preventing thyroid goiter formation in rdw/rdw rats.     

Effect of thyroxine on abnormal pancreatic proteomes of the hypothyroid rdw rat

A mutation in the thyroglobulin (Tg) gene is the primary cause of hereditary dwarfism and hypothyroidism in the rdw rat. Despite the Tg mutation that causes a Tg shortage, rdw rats survive. The present study examines the influences of this condition on the pancreatic proteome. Normal control (group 1; n = 19) and rdw rats that did not receive L-thyroxine (T4) (group 2; n = 27) were sacrificed from 4 to 56 weeks after birth. The rdw rats were supplemented either with daily intraperitoneal injections of T4 from 3 to 28 days after birth (group 3; n = 4) or with normal thyroid tissues grafted at 4 weeks of age (group 4; n = 3). Groups 3 and 4 were sacrificed 12 weeks after birth. Pancreatic proteomes analyzed by two-dimensional gel electrophoresis showed that levels of at least four pancreatic proteins were higher in group 2 than in group 1, and that those of four were lower. Cluster decomposition and principal component analysis of the eight protein contents showed that groups 1 and 2 were separated into two clusters and that pancreatic proteomes of group 4 were better normalized than those of group 3. Injecting T4 into group 3 was temporarily effective, whereas the thyroid graft to group 4 provided a continuous positive effect, which concurred with the increased body weight of the other two groups of rdw rats that received grafts of normal thyroids.     

A Simple and Rapid Method for Detection of the 1069Gln Mutation in Wilson Disease

A simple and rapid method for detecting the 1069Gln mutation in gene ATP7B based on a PCR specific for this allele has been developed. The 1069Gln mutation is the main cause of Wilson disease (WD) in Russia and accounts for approximately 40% of all mutant alleles of gene ATP7B. Therefore, the method proposed makes the postnatal and prenatal diagnosis of Wilson disease in Russia considerably more rapid and less expensive.     

Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism

Accurate analysis of DNA sequence variation in not only humans and animals but also other organisms has played a significant role in expanding our knowledge about genetic variety and diversity in a number of different biological areas. The search for an understanding of the causes of genetic variants and mutations has resulted in the development of a simple laboratory technique, known as the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, for the detection of single nucleotide polymorphisms (SNPs). PCR-RFLP allows rapid detection of point mutations after the genomic sequences are amplified by PCR. The mutation is discriminated by digestion with specific restriction endonucleases and is identified by gel electrophoresis after staining with ethidium bromide (EtBr). This convenient and simple method is inexpensive and accurate for SNP genotyping and especially useful in small basic research studies of complex genetic diseases. The whole protocol takes only a day to carry out.     

A tetra-primer polymerase chain reaction approach for the detection of JAK2 V617F mutation

Recently, an acquired somatic point mutation (p.V617F) in a highly conserved residue of the pseudokinase domain of the JAK2 tyrosine kinase was shown to be associated with myeloproliferative disorders. Because of the clinical importance of this mutation in diagnosing myeloproliferative disorders and its relevance for disease progression, we have developed a tetra-primer polymerase chain reaction (PCR) assay to detect JAK2 p.V617F. Titration studies showed that the assay could reliably detect one copy of the mutant allele in a mix of 50 wild-type alleles suggesting that the lower detection limit of this assay is estimated to be 2%. This study demonstrates that genotyping and quantifying of the JAK2 V617F mutation can be performed by tetra-primer PCR using both freshly isolated and formalin-fixed tissues. Our tetra-primer PCR assay is sensitive, low-cost, and easy to use method for the detection of JAK2 p.V617F, which could be used even in low-tech laboratories.     

Two-color allele-specific polymerase chain reaction (PCR-SSP) assay of the leptin receptor gene (Leprdb) for genotyping mouse diabetes mutation.

An allele specific polymerase chain reaction (PCR-SSP) assay for genotyping the mouse leptin receptor (Leprdb) mutation and its wild type (Lepr+) gene was developed using two different fluorescent dye-labeled primers. First, we determined the Leprdb and Lepr+ allele by PCR-SSP assay with usual dye-unlabeled primers. However this method requires two separate PCR reactions because the amplified products specific for each allele are almost the same size. We further developed a simple and reliable two-color PCR-SSP method that uses a color complementation strategy to distinguish the Leprdb and Lepr+ alleles. Leprdb/Leprdb, Leprdb/Lepr+ and Lepr+/Lepr+ of mice (5 each) were clearly genotyped by the two-color PCR-SSP. We also performed PCR-direct sequencing for the same samples and confirmed the accuracy of this method. This method makes it possible to reduce the number of PCR reactions because both alleles are amplified in the same reaction mixture.     

High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.     

Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS).

We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.     

A novel genotyping method for detection of the CRHR1 (rs1396862: C>T) gene variation among North Indian population

Today, the genomic revolution in epidemiology, medicine and population based genetic association studies are results of on-going refocusing efforts toward development of inexpensive and accurate techniques for SNP genotyping. Despite this considerable gain, high throughput and routinely applicable newer SNP detection techniques are still needed. Therefore, aim of this study was to develop and validate a simple, rapid and inexpensive restriction enzyme based method for genotyping of corticotrophin-releasing hormone receptor1 (CRHR1; rs1396862: C>T) gene variant. This polymorphism has been investigated in a variety of psychiatric and association studies of asthma. A total of 250 healthy volunteers were recruited from same ethnicity and their blood DNA samples were employed for genotyping. Primers were designed using Batch primer3 Software. Specificity and functionality of primers were tested with BLAST database and UCSC In-silco PCR respectively. The lake of a PstI recognition site was seen with T allele. The allele frequencies for rs1396862: C>T were 0.88 (C allele) and 0.12 (T allele). We get 100 % concordant genotyping results for sequencing and PCR–RFLP. This newer genotyping approach lowers the cost and increased the speed. It is particularly useful for small basic research studies of complex genetic disorder.     

The use of compound heterozygotes and Hprt selection to analyze X-linked mottled alleles associated with prenatal lethality

X-linked mutant alleles associated with prenatal male lethality are difficult to analyze because only heterozygous females are readily available for study. Genomic analysis of the mutant allele is facilitated by the construction of somatic cell hybrids because this enables the segregation of the X Chromosomes (Chrs) that carry the mutant and wild-type alleles. We describe here a method that ensures that the X Chr carrying the mutant allele is retained in somatic cell hybrids in an active selectable state. This is achieved by mating heterozygous females to males that carry a mutation at the hypoxanthine phosphoribosyl transferase (Hprt) locus. The resultant F₁ females are compound heterozygotes, and when cells from these females are fused to HPRT− Chinese hamster cells and subjected to selection in HAT medium, the only survivors are those hybrid cells that retain an active X Chr carrying the mutant allele together with the wild-type Hprt allele. We use hybrids constructed by this method to demonstrate that there are no gross deletions or genomic rearrangements present in three mottled alleles associated with prenatal male lethality. Received: 8 January 1996 / Accepted: 29 February 1996     

Genotyping of single nucleotide polymorphisms in <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Decapoda,Penaeidae) by tetra-primer ARMA-PCR

This study introduced a kind of new SNP genotyping strategy in genetic analysis of Chinese shrimp (Fenneropenaeus chinensis). Twenty SNP tetra-primer ARMA-PCR primer sets were validated and used to investigate the genetic structure of six selected families of marine shrimp F. chinensis. The effective number of alleles ranged from 1.127 to 1.993, with an average value of 1.600. The average values of expected and observed heterozygosities of the SNPs ranged from 0.505∼0.609 and 0.373∼0.487, respectively. Polymorphism information content (PIC) values ranged from 0.145 to 0.373. Among 120 population-locus cases (six populations × twenty loci), only 8 (6.7%) showed significant deviation (P < 0.05), while the other 112 (93.3%) were in Hardy-Weinberg equilibrium (HWE) (P > 0.05). The frequencies of minor alleles ranged from 0.378 to 0.497. For the six families, the wild genotype and its allele frequency were significantly higher than that of the mutant genotype and its allele frequency. Fifty five point forty two percent of the population-locus showed heterozygosity. The results indicated that tetra-primer ARMA-PCR is a simple, rapid and efficient method for SNP genotyping which make it useful in a broad aspects of F. chinensis genetic and breeding studies.     

Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice

Ames dwarfism is caused by a homozygous single nucleotide mutation in the pituitary specific prop-1 gene, resulting in combined pituitary hormone deficiency, reduced growth and extended lifespan. Thus, these mice serve as an important model system for endocrinological, aging and longevity studies. Because the phenotype of wild type and heterozygous mice is undistinguishable, it is imperative for successful breeding to accurately genotype these animals. Here we report a novel, yet simple, approach for prop-1 genotyping using PCR-based allele-specific amplification (PCR-ASA). We also compare this method to other potential genotyping techniques, i.e. PCR-based restriction fragment length polymorphism analysis (PCR-RFLP) and fluorescence automated DNA sequencing. We demonstrate that the single-step PCR-ASA has several advantages over the classical PCR-RFLP because the procedure is simple, less expensive and rapid. To further increase the specificity and sensitivity of the PCR-ASA, we introduced a single-base mismatch at the 3' penultimate position of the mutant primer. Our results also reveal that the fluorescence automated DNA sequencing has limitations for detecting a single nucleotide polymorphism in the prop-1 gene, particularly in heterozygotes.     

An allele-specific genotyping method for rat lyst (lysosomal trafficking regulator) gene.

Two spontaneous mutant beige rats, with phenotypes resembling human Chediak- Higashi syndrome (CHS), were found independently in two inbred strains. Both beige mutations were identified to be recessive alleles in the Lyst locus on rat chromosome 17 and the alleles were denoted Lyst(bg) and Lyst(bg-Kyo). As it is almost impossible to discriminate these mutations phenotypically, we developed an allele-specific genotyping method for the Lyst gene. The nested PCR amplification was followed by restriction fragment length polymorphism (RFLP) analysis. By this method, we could discriminate the mutant Lyst(bg), Lyst(bg-Kyo) alleles, and the normal Lyst allele, easily and accurately.