Arg Change Research Articles

Variations in antibiotic resistance genes and microbial community in sludges passing through biological nutrient removal and anaerobic digestion processes in municipal wastewater treatment plants

Antimicrobial resistance (AMR) represents a relentless, silent pandemic. Contributing to this are wastewater treatment plants (WWTPs), a potential source of antibiotic resistance genes' (ARGs) transmission to the environment, threatening public health. The presence of ARGs in pathogenic bacteria and their release into the environment by WWTPs threatens the public health. The current study investigated changes in ARGs' abundance in biological nutrient removal (BNR) processes and anaerobic digestion (AD) reactors of two WWTPs. Also, microbial community structure, which is known to shape the distribution and abundance of ARGs, was also analyzed. The relative abundance of eight ARGs (tetX, tetA, tetM, TEM, sul1, sul2, ermB and qnrD) was quantified as ARGs’ copies/16 S rRNA gene copies using quantitative polymerase chain reaction (qPCR). Microbial community composition was assessed by 16 S rRNA microbiome sequencing analysis. TetX was prevalent among the eight ARGs, followed by TEM and sul1. However, its abundance was decreased in the AD sludges compared to BNR sludges. Proteobacteria was the major bacterial phylum found in all the sludge samples, while Arcobacter, 12up and Acidovorax were the predominant genera. Acinetobacter and Flavobacterium were significantly more abundant in the BNR sludges, while 12up and Aeromonas were predominant in AD sludges. Principal component analysis (PCA) revealed a clear difference in dominant ARGs and bacteria between the sludges in the processes of BNR and AD of the two WWTPs. Clinically relevant bacterial genera, Klebsiella and Enterococcus, found in both the BNR and AD sludges, were significantly correlated with the tetX gene. Throughout this study, the relationship between microbial communities and specific ARGs was revealed, illustrating that the composition of the microbial community could play a vital role in the abundance of ARGs. These results will better inform future studies aimed at controlling the spread of ARGs and their potential hosts from WWTPs.

Mouse model suggests limited role for human mesotrypsin in pancreatitis

Mesotrypsin is a low-abundance human trypsin isoform with a unique evolutionary mutation that conferred resistance to trypsin inhibitors and restricted substrate specificity. Mesotrypsin degrades the serine protease inhibitor Kazal type 1 (SPINK1) and thereby might increase risk for pancreatitis. Here, we report a mouse model designed to test the role of mesotrypsin in pancreatitis. We introduced the human mesotrypsin evolutionary signature mutation into mouse cationic trypsinogen (isoform T7), resulting in a Gly to Arg change at the corresponding position 199. In biochemical experiments using purified proteins, the p.G199R T7 mutant recapitulated all salient features of human mesotrypsin. T7G199R mice developed normally with no spontaneous pancreatitis or other obvious phenotypic changes. Cerulein-induced acute pancreatitis in C57BL/6N and T7G199R mice showed similar severity with respect to inflammatory parameters and acinar cell necrosis while plasma amylase activity was higher in T7G199R mice. Neither SPINK1 degradation nor elevated intrapancreatic trypsin activation was apparent in T7G199R mice. The results indicate that in T7G199R mice the newly created mesotrypsin-like activity has no significant impact on cerulein-induced pancreatitis. The observations suggest that human mesotrypsin is unimportant for pancreatitis; a notion that is consistent with published human genetic studies.

Changes in antibiotic resistance genes and mobile genetic elements during cattle manure composting after inoculation with Bacillus subtilis

This study explored the effects of Bacillus subtilis at four levels (0, 0.5%, 1%, and 2% w/w compost) on the variations in ARGs, mobile genetic elements (MGEs), and the bacterial community during composting. The composting process had a greater impact on ARGs than Bacillus subtilis. The main ARG detected was sul1. The addition of Bacillus subtilis at 0.5% reduced the relative abundances of ARGs, MGEs, and human pathogenic bacteria (by 2–3 logs) in the mature products. Network and redundancy analyses suggested that intI1, Firmicutes, and pH were mainly responsible for the changes in ARGs, thus controlling these factors might help to inhibit the spread of ARGs.

Key role of cyromazine in the distribution of antibiotic resistance genes and bacterial community variation in aerobic composting

The risks that have not been noted so far have come from the use of non-antibiotics. In this study, non-antibiotic drug (cyromazine) was used in composting to investigate its possible effects on the distribution of ARGs and changes of bacterial community. Results showed that cyromazine increased the abundances of highly-risky ARGs (blaCTX-M and blaVIM), and heavy metal resistance genes (MRGs). Low and high concentrations of cyromazine increased the abundance of Tn916/1545 by 18.27% and 64.26%, respectively, compared with the control treatment. Mobile genetic elements (MGEs) and MRGs were not the major cause of the dynamic changes in ARGs, but instead the bacterial community succession changed according to the moisture content, pH, and bio-Cu. Network analysis showed that Proteobacteria and Actinobacteria were the major hosts for ARGs, and there was a significant correlation between tcrB, sul1 and Tn916/1545.

Role of Bentonite on the Mobility of Antibiotic Resistance Genes, and Microbial Community in Oxytetracycline and Cadmium Contaminated Soil.

The effects of bentonite (BT), a commonly used heavy metal deactivator, on the ARGs and microbial communities in soils and lettuce systems contaminated by heavy metals and antibiotics are unclear. A study was conducted to investigate the effect of BT on the mobility of antibiotic resistance genes in oxytetracycline and cadmium contaminated soil. Results showed that the addition of BT reduced the accumulation of OTC and ARGs in the soil and lettuce roots, but increased the abundance of ARGs in lettuce leaves, and increase the risk of human pathogenic bacteria (HPB) transferring to lettuce leaves. Redundancy analysis showed that environmental factors (OTC, H2O, SOM, and pH) were the dominant factors that influence the distribution of ARGs and intI1. Network analysis showed that Proteobacteria and Bacteroidetes were the major host bacteria which caused changes in ARGs and intI1. There were significant positive correlations between ermX and ermQ, and a large number of HPB. The co-occurrence of intl1 with some ARGs (tetC, tetG, ermQ, sul1, and sul2), may threaten human health due to the dispersion of ARGs via horizontal gene transfer.

Impacts of addition of natural zeolite or a nitrification inhibitor on antibiotic resistance genes during sludge composting

Composting is commonly used for the treatment and resource utilization of sewage sludge, and natural zeolite and nitrification inhibitors can be used for nitrogen conservation during sludge composting, while their impacts on ARGs control are still unclear. Therefore, three lab-scale composting reactors, A (the control), B (natural zeolite addition) and C (nitrification inhibitor addition of 3,4-dimethylpyrazole phosphate, DMPP), were established. The impacts of natural zeolite and DMPP on the levels of ARGs were investigated, as were the roles that heavy metals, mobile genetic elements (MGEs) and the bacterial community play in ARGs evolution. The results showed that total ARGs copies were enriched 2.04 and 1.95 times in reactors A and C, respectively, but were reduced by 1.5% in reactor B due to the reduction of conjugation and co-selection of heavy metals caused by natural zeolite. Although some ARGs (blaCTX-M, blaTEM, ermB, ereA and tetW) were reduced by 0.3–2 logs, others (ermF, sulI, sulII, tetG, tetX, mefA and aac(6′)-Ib-cr) increased by 0.3–1.3 logs after sludge composting. Although the contributors for the ARGs profiles in different stages were quite different, the results of a partial redundancy analysis, Mantel test and Procrustes analysis showed that the bacterial community was the main contributor to the changes in ARGs compared to MGEs and heavy metals. Network analysis determined the potential host bacteria for various ARGs and further confirmed our results.

A study of human Kallikrein-2 gene polymorphism with special reference to prostate cancer patients in India

Background Prostate cancer is the third most common cancer among men in the world. The human kallikrein-2 gene (KLK2 gene) contains a mutant (T) for wild-type (C) allele substitution, resulting in a Trp to Arg change on the 250 amino acid at the 784 nucleotide of KLK2 cDNA. The reports on polymorphism in the KLK2 gene in relation to prostate cancer are contradictory among American and Korean population. Reports are not available regarding polymorphism in KLK2 gene in relation to prostate cancer in Indian population. The relationship between mutants (T) for wild-type (C) allele substitution of the KLK2, circulating human kallikrein-2 (hK2) levels and prostate cancer risk in Indian population has been examined.

A frequent Gene Polymorphism Affecting the Heart-rate Response to Carvedilol

b-adrenoceptor antagonists, known as b-blockers, are a cornerstone in the treatment of the most prevalent cardiovascular syndromes, including heart failure, ischemic heart disease, atrial fibrillation and hypertension. Their beneficial effects are explained by competitive antagonism at b1-adrenoceptors, the dominant adrenergic receptors expressed in cardiac myocytes. By contrast, antagonism at b2-adrenoceptors accounts for side effects (e.g., vasospasm [‘cold extremities’], bronchospasm and hypoglycemia) and contraindications, such as asthma bronchiale. Accordingly, the majority of currently prescribed second-generation b-blockers (e.g., metoprolol, bisoprolol and atenolol) preferentially act at b1-adrenoceptors, that is, they possess higher affinity towards b1than b2-adrenoceptors (‘b1-selective b-blockers’). Carvedilol is a thirdgeneration b-blocker, which is generally classified as nonselective, but, in fact, has a 13-fold-higher affinity for human b2than b1-adrenoceptors [1]. Carvedilol is also a potent a1-adrenoceptor antagonist [2], which balances the constrictive effect of b2-blockade, and has antioxidant effects of unknown relevance. Moreover, carvedilol directly blocks spontaneous diastolic Ca release from cardiac ryanodine receptors, an activity thought to suppress ventricular arrhythmias, for example in heart failure, where the ryanodine receptor is thought to be ‘leaky’ [3,4]. Finally, carvedilol has unusually slow dissociation kinetics, that is, it ‘sticks to adrenoceptors’, which causes a longer time of action than predicted from its plasma half-life [5]. The b1-adrenoceptor gene (ADBR1) exhibits a frequent SNP that causes a change of Arg at position 389 to Gly [6]. Approximately 40 and 7% of Caucasians are heterozygous and homozygous for the rarer Gly389 variant, respectively. The polymorphism has gained much attention because, when overexpressed at very high levels

Mutational analysis of p53 and PTEN in soft tissue sarcoma

p53 and PTEN are the two most frequently mutated tumor suppressors in human cancer. However, literature on the effect of the joint inactivation of tumor-suppressor genes in soft tissue sarcoma (STS) is lacking. The purpose of this study was to investigate whether p53 and PTEN mutations play a role in the carcinogenesis of STS, as well as to evaluate their mutual role in STS pathogenesis. We screened mutations of p53 and PTEN in 86 human STSs using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing, respectively. p53 mutations were detected in 25.6% (22 out of 86) of STSs: 6 cases of p53 mutations were detected in 46 cases of specific reciprocal translocations in STSs (13.0%), 16 cases were detected in 40 cases of nonspecific reciprocal translocations in STSs (40.0%); the majority of the mutations were point mutations in exon 6-7. Furthermore, PTEN mutations were observed in 2 out of 86 STSs (2.3%). Two out of 86 cases revealed a 130th codon G>A missense mutation in exon 8 of PTEN which resulted in an Arg change to Gln in the PTEN protein structure; and a 334th codon A>T missense mutation in exon 8 of PTEN, which resulted in an Asn change to Lys in the PTEN protein structure. All subjects were examined for p53 exon 5-9 mutations and for PTEN exon 5-9 mutations. However, no tumors contained an alteration of the two genes. The findings indicate that p53 mutations may be involved in the oncogenesis of STS and also suggest that p53 may function as a potential molecular marker for distinguishing between STSs with specific reciprocal translocations and nonspecific reciprocal translocations. Although the existence of PTEN mutations in STS was detected, the PTEN mutation frequency was quite low. We conclude that PTEN may have played a less prognostic role than p53 in the development and malignant transformation of STS in the patients examined.

Two novel HLA‐A alleles: HLA‐A*31:01:09 and HLA‐A*33:30

The HLA-A*31:01:09 differs from the closest allele HLAA*31:01:02 by a C to T silent substitution at position 447 in exon 3. The HLA-A*33:30 differs from the closest allele HLA-A*33:03:01 by a single nucleotide substitution at position 453 in exon 3, leading to a change of Arg 131 Cys.

Role of Mutations in Dihydrofolate Reductase DfrA (Rv2763c) and Thymidylate Synthase ThyA (Rv2764c) in Mycobacterium tuberculosis Drug Resistance

We would like to comment on a number of recent reports in this journal (6, 8, 12, 18) concerning Mycobacterium tuberculosis dihydrofolate reductase (DHFR), encoded by dfrA (Rv2763c). Around 36% of phenotypically para-aminosalicylic acid (PAS)-resistant M. tuberculosis strains harbor mutations in thyA (Rv2764c), which encodes a thymidylate synthase (20). In their effort to elucidate the remaining unknown resistance mechanism(s), Mathys et al. extended their sequence analysis to a number of additional genes, including dfrA (12). It was unclear whether the three dfrA mutations they identified in the PAS-resistant strains P-693 and P-3158 could contribute to PAS resistance on their own. Nonetheless, these findings are notable for two reasons. First, isoniazid (INH) has been shown to inhibit M. tuberculosis DHFR in vitro (1). Whether the same holds true for ethionamide, which shares a number of common resistance mechanisms with INH, was not tested (J. Blanchard, personal communication). In any case, the clinical relevance of DHFR-mediated INH resistance remains enigmatic. To date, only Ho et al. have addressed this question, but they did not identify any dfrA mutations in a screen of 127 INH-resistant clinical isolates (8). Consequently, Mathys et al. remain the first to describe mutations in this target (12). However, given that isolates with mutated DHFR are members of a cluster with baseline INH resistance, the importance of these mutations with respect to INH resistance remains unclear. Irrespective of their relevance in INH resistance, these dfrA mutations are noteworthy for a second reason. Contrary to previous wisdom, Forgacs et al. recently showed that M. tuberculosis is sensitive to the drug combination trimethoprim-sulfamethoxazole (TMP-SMX) (6, 18). DHFR is competitively inhibited by TMP, and consequently, mutations therein lead to resistance in a variety of organisms (9, 16, 19). The crystal structures of the wild-type M. tuberculosis DHFR in complex with NADPH alone and with NADPH combined with the inhibitor TMP, methotrexate, or an analogue of the antimalarial agent WR99210 have been solved (11). Interestingly, the wild-type 66Ser, which is mutated to Cys in strain P-693 (12), interacts directly with NADPH via an H bond and a hydrophobic interaction. Similarly, the wild-type 54Val, which is mutated to Ala in strain P-3158 (in addition to a second 110Cys→Arg change), has a hydrophobic interaction with BR-WR99210 (11). Should these amino acid substitutions alter NADPH interactions or inhibitor binding, this raises the possibility that these PAS-resistant strains might also be TMP-SMX resistant. To investigate this possibility, phenotypic susceptibility testing of both isolates, as described by Forgacs et al., is warranted (6, 12). In addition, the treatment history of both patients should be reviewed for evidence of treatment with TMP-SMX. We would now like to turn to the mutations in M. tuberculosis thyA, which were the focus of a number of studies (12, 13, 20). We note that the mutational loss of ThyA leads to TMP resistance in other organisms. Under these circumstances, tetrahydrofolate, the product of DHFR, is no longer required to regenerate N5,N10-methylenetetrahydrofolate, which would otherwise be oxidized by ThyA in the deoxyuridylate methylation process. Consequently, TMP-mediated competitive inhibition of DHFR can be tolerated by the cell (10, 13). In the case of Staphylococcus aureus, thyA mutations are also responsible for a small-colony-variant phenotype and are associated with hypermutability, which is thought to favor adaptation to long-term persistence and further antibiotic resistance (3-5, 14). Taken together, this raises the possibility that thyA mutations that account for PAS resistance in M. tuberculosis might also lead to cross-resistance to TMP-SMX (6, 18). In fact, in their effort to show the importance of thyA mutations for PAS resistance in M. tuberculosis, Rengarajan et al. showed that transposon-disrupted thyA from Mycobacterium bovis BCG was not able to rescue a ThyA− phenotype in Escherichia coli and restore TMP sensitivity (13). Moreover, they showed that these PAS-resistant M. bovis BCG strains harboring a transposon in thyA were resistant to the DHFR inhibitor 8710, which, unlike TMP on its own, is active against wild-type M. bovis BCG (13, 15). Should this hold true for TMP-SMX, this could have obvious implications for the utility of these antibiotics, comparable to the situation of isoniazid and ethionamide (17). To assess the possible clinical impact and the degree of cross-resistance, we must quantify the proportion of laboratory and clinical TMP-SMX resistance which might be due to thyA mutations rather than mutation in DHFR or due to a third resistance mechanism involving the dihydropteroate synthase FolP1 (Rv3608c) (2, 7, 10). This also has practical implications for the experiment we suggested above. Mathys et al. should include P strains from their collection that carry the same double thyA mutations as P-693 and P-3158 but have a wild-type DHFR as the control (12). We hope that these observations will spur a thorough preclinical examination of TMP-SMX, as called for by Young (18).

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition.

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1(H) versus HA-1(R)) in the HMHA1 gene. The HA-1(H) peptide is restricted by HLA-A2 and is immunogenic in HA-1(R/R) into HA-1(H) transplants, while HA-1(R) has been suggested to be a "null allele" in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1(R) variant. To understand these findings, we determined the structure of an HLA-A2-HA-1(H) complex to 1.3A resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1(H)-specific T-cells bound HA-1(H) peptide with moderate affinity but failed to bind HA-1(R), indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition.

A novel R198H mutation in the glucose-6-phosphate dehydrogenase gene in the tribal groups of the Nilgiris in Southern India

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common red cell enzymopathy among humans. In India, G6PD Mediterranean, G6PD Orissa, and G6PD Kerala-Kalyan are the three common mutations which account almost 90% of G6PD deficiency. Here we describe G6PD Coimbra, an unreported variant from India, and a novel 593 G --> A mutation in exon 6 with an amino acid change of Arg 198 His, among the tribal groups of the Nilgiris in Southern India. Further, this novel mutation was structurally characterized and it was found that the mutation is located at the end of the coenzyme domain, which may cause enzyme instability.

Mutations in the ED1 gene in families with X-linked hypohidrotic ectodermal dysplasia

To detect mutations in the ED1 gene in two Chinese pedigrees and a sporadic case with X-linked hypohidrotic ectodermal dysplasia (XLHED) and provide evidences with the mutation analysis for genetic counseling, prenatal diagnosis and confirmation of carrier status. Peripheral blood samples were obtained from two pedigrees and the sporadic patient, and genomic DNA was extract by salting out method. Polymerase chain reaction (PCR) and direct sequencing were performed to screen mutations in ED1 gene. Three mutations were identified. In one of the pedigrees, a 1045G > A transition was evidenced in exon 9 that resulted in a change of Ala 349 Thr. In the other pedigrees and the sporadic patient, 467G > A and 466C > T transitions were demonstrated in exon 3 that resulted in change of Arg 156 His and Arg 156 Cys. These mutations were not found in 100 normal individuals. These mutations were responsible for the disease in the two families and the sporadic patient. All these mutations had been identified previously.

Structure and function of sheep hemoglobin Chios: A novel allele at the HBBB locus with two Lys → Arg substitutions at positions β66(E10) and β144(HC1)

A novel hemoglobin variant was observed in pure sheep ( Ovis aries) breeds of the island of Chios (Greece), Egypt and Hungary. This silent variant was identified by gel electrophoresis and RP-HPLC of dissociated globin chains. Two Arg for Lys substitutions were detected, by means of MALDI TOF electrospray mass spectrometric analysis for the intact globins, at positions β66(E10) and β144(HC1) of a globin chain having the sequence of the β B chain. Sequencing of the β-globin gene confirmed the variant gene as being an allele of the HBBB locus having the AAG → AGG and the AAA → AGA mutations at codons 66 and 144, respectively, both corresponding to the Lys → Arg substitution. The intrinsic oxygen affinity of the variant Hb (log P 50 = 0.79 at pH 7.0) was found to be intermediate between that of the sheep Hb B (log P 50 = 0.92) and that of Cypriot mouflon ( O. a. ophion) Hb (log P 50 = 0.53), the latter having only the Lys → Arg change at β144, whereas nearly no differences were observed in the presence of the Cl − physiological effector. Result supports the indication that Arg at β144 enhances the role of the ligand in decreasing oxygen affinity, this effect being partially counteracted when Arg is at β66. Data also shows that the Lys → Arg change at β66 is responsible for 1.49 fold reduction in the intrinsic oxygen affinity. This hitherto undescribed variant increases to seven the number of alleles at the sheep HBBB locus. Following the nomenclature used for human Hb variants, the new allele was termed as the Hb Chios or [β B66(E10) Lys → Arg, 144(HC1)Lys → Arg], whereas the proposed genetic nomenclature of the locus is HBBK.

Polymorphism of exon 10 of prolactin receptor gene and its rela-tionship with prolificacy of Jining Grey goats

Prolactin receptor (PRLR) gene was studied as a candidate gene for the prolificacy of Jining Grey goats. Five pairs of primers were designed to detect single nucleotide polymorphisms of exon 10 and part of 3'untranslated region (UTR) of PRLR gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Liaoning Cashmere goat, Boer goat and Angora goat) by PCR-SSCP. The nucleotide sequence of exon 10 and part of 3'UTR of caprine PRLR gene was spliced in this study for the first time. The length of this sequence was 1,118 bp. This sequence shared 98.33%, 93.92%, 74.52% homology with the published mRNA of PRLR gene of sheep, cow and human separately, and shared 78.29% homology with the published partial genomic sequence of PRLR gene of the alpaca. Only the products amplified by primers P1, P2, P4 displayed polymorphisms. For primer P1, two genotypes (AA and AB) were detected in both Jining Grey goats and Liaoning Cashmere goats, two genotypes (AA and AC) were detected in Angora goats, and only genotype AA was detected in Boer goats. Sequencing revealed two mutations (186G-->A and 220T-->C) of PRLR gene in the genotype AB in comparison to the genotype AA. The former mutation resulted in an amino acid change of Asp-->Asn, and the latter mutation resulted in an amino acid change of Leu-->Pro. Only one mutation (140A-->G) was found in the genotype AC in comparison to the genotype AA, and this mutation did not cause any amino acid change. The difference of the least squares means (LSM) for litter size between AA and AB was non-significant (P>0.05) in Jining Grey goats. For primer P2, two genotypes (DD and DE) were detected in Jining Grey goats, Liaoning Cashmere goats and Boer goats, and only genotype DD was detected in Angora goats. Sequencing revealed two mutations (52G-->A and 122G-->A) of PRLR gene in the genotype DE in comparison to the genotype DD. The former mutation did not cause any amino acid change, and the latter mutation resulted in an amino acid change of Arg-->Gly. The difference of LSM for litter size between DD and DE was non-significant (P>0.05) in Jining Grey goats. For primer P4, two genotypes (FF and FG) were detected in Jining Grey goats, two genotypes (FF and GG) were detected in Liaoning Cashmere goats, only genotype FF was detected in Boer goats, and three genotypes (FF, FG and GG) were detected in Angora goats. Sequencing revealed one mutation (143A-->G) of PRLR gene in the genotype GG in comparison to the genotype FF, and this mutation resulted in an amino acid change of Met-->Val. The Jining Grey does with genotype FG had 0.76 (P< 0.05) kids more than those with genotype FF. These results preliminarily showed that the PRLR gene is either a major gene that influences the prolificacy of Jining Grey goats or a molecular marker in close linkage with such a gene.

Polymorphisms of the DNA polymerase β gene in breast cancer

DNA polymerase beta (Polbeta) provides most of the gap-filling synthesis at apurinic/apyrimidine sites of damaged DNA in the base excision repair pathway. Mutations in the gene encoding DNA polbeta have been identified in various carcinomas. We performed a case-control study to test the association between two polymorphisms in the polbeta gene: a Pro --> Arg change at codon 242 (the Pro242Arg polymorphism) and a Lys --> Met change at codon 289 (the Lys289Met polymorphism) and breast cancer risk and cancer progression. Genotypes were determined in DNA from peripheral blood lymphocytes of 150 breast cancer patients and 150 cancer-free, age-matched women (controls) by PCR-RFLP. A strong association between breast cancer occurrence and the Met/Met phenotype of the Lys289Met polymorphism [odds ratio (OR) 3.67; 95% confidence interval (CI) 1.87-7.56] and the Pro/Arg phenotype of the Pro242Lys polymorphism (OR 1.96; 95% CI 1.15-3.34) was found. Polymorphism-polymorphism interaction between the Met/Met phenotype of the Lys289Met and the Pro/Arg phenotype of the Pro242Arg variants increased the risk of breast cancer (OR 3.05; 95% CI 1.31-7.09). We did not observe any correlation between studied polymorphisms and breast cancer progression evaluated by node-metastasis, tumor size and Bloom-Richardson grading. In conclusion, Polbeta may play a role in the breast carcinogenesis and the Lys289Met polymorphism of the polbeta gene may be considered as an independent, early, molecular diagnostic marker in breast cancer. The Pro242Arg polymorphism may contribute to the carcinogenesis through the interaction with the Lys289Met and therefore may be regarded as a dependent, auxiliary marker.

The mismatch repair gene hPMS2 is mutated in primary breast cancer

Mismatch repair (MMR) genes play a fundamental role in the correction of replication errors and their mutation leads to cancer development. In the present study we have analyzed the hPMS2 MMR gene for mutation using 20 primary breast cancers and seven breast tissues obtained from areas adjacent to breast cancer. For this purpose we have used cDNA sequence analysis and Western blotting using the specific antibody against the amino-terminal domain E-19. In primary breast cancers we found that the hPMS2 gene had 9 missense mutations [codons: 513 (by change of Ser x Asp) in 14 tumors, 520 (Ala x Val) in 8 tumors, 573 (by change of Thr x Ser in 19 tumors), 578 (by change of Arg x Leu in 9 tumors), 587 (by change of Ser x Asp in 7 tumors), 590 (by change of Ile x Leu in 12 tumors), 598 (by change of Gln x His in 5 tumors), 601 (by change of Ser x Leu in 13 tumors), 608 (by change of Ala x Ser in 9 tumors. Nine out of 20 breast cancers had a non-sense mutation in nucleotide 1862 by changing Adenine by Thymine (AAG x TAG), which corresponded with a change in codon 613 by a change of Lys by stop codon. This non-sense mutation is responsible for the premature truncation of the protein hPMS2, which is reflected in the Western blotting by two bands, one corresponding with the wild-type form (100 kDa) and a lower one (75 kDa) corresponding with the truncated form of the hPMS2 MMR protein. This truncated protein and the mutations in the hPMS2 gene were also detected in two samples of normal-appearing tissue adjacent to their corresponding cancerous lesion. Altogether the present report demonstrates that primary breast cancers harbor mutations in this MMR gene and that normal-appearing breast tissue adjacent to the primary lesion also harbors the same mutations before the neoplastic process is manifested.

Mutations that modulate receptor–hormone congruency as a cause of the primate GH receptor species specificity

Peptide hormones depend on reliable recognition by their receptors. Any mutation that compromises recognition of hormone and receptor molecules is dangerous, the carrier animal would not procreate and the mutation would be lost. Although, most of the hormones from one mammalian species are active when injected into another, the incompatibility of human GH receptor toward nonprimate GHs is a notable exception. It is reported that the coevolution of GH and GHR in primates includes two crucial steps (Mol. Biol. Evol. 18 (2001) 945). The first was mutation of GH His→Asp at position 171 that happened before the split of Old world and New world monkeys. The second event was Leu→Arg change at position 43 in the GH receptor molecule that happened in the ancestor of Old world monkeys. The proposed model is based on the possibility that certain mutations can modify the surface of one of interacting molecules to form a confined empty space, a niche in the otherwise congruent hormone/receptor interface. Although affinity between molecules is probably slightly reduced, recognition and function are not compromised in this special case. Further mutations of hormone and receptor molecules are allowed under the condition that they remain confined to the niche space. Mutations that do not compromise hormone function can be passed to offsprings. If the consequent mutation of one molecule change its shape to fill the niche space, further mutations without function loss will become less probable. Without the niche space, the phase of fast evolution is closed and both genes become conserved. In this setting, accumulated mutations before the niche closing mutation are the cause of species specificity. To become a dominant variety, carrier animals must possess survival advantage in comparison to the carriers of other less advantageous mutations.

Association Testing in 9,000 People Fails to Confirm the Association of the Insulin Receptor Substrate-1 G972R Polymorphism With Type 2 Diabetes

The insulin receptor substrate (IRS)-1 is an important component of the insulin signal transduction cascade. Several reports suggest that a Gly-->Arg change in codon 972 is associated with type 2 diabetes and related traits, and a recent meta-analysis reported a modest but nominally significant association with type 2 diabetes (odds ratio [OR] 1.25 in favor of carriers of the Arg allele [95% CI 1.05-1.48). To test the reproducibility of the model in a recent meta-analysis, we examined genotype-phenotype correlation in three large Caucasian samples (not previously reported for this variant) totaling 9,000 individuals (estimated to have >95% power to obtain a P < 0.05 for the OR of 1.25 estimated in the meta-analysis). In our combined sample, comprising 4,279 case and 3,532 control subjects, as well as 1,189 siblings discordant for type 2 diabetes, G972R was not associated with type 2 diabetes (OR 0.96 [0.84-1.10], P = 0.60). Genotype at G972R had no significant effect on various measures of insulin secretion or insulin resistance in a set of Scandinavian samples in whom we had detailed phenotypic data. In contrast, the well-documented associations of peroxisome proliferator-activated receptor gamma P12A and Kir6.2 E23K with type 2 diabetes are both robustly observed in these 9,000 subjects, including an additional (previously unpublished) confirmation of Kir6.2 E23K and type 2 diabetes in the Polish and North American samples (combined OR 1.15 [1.05-1.26], P = 0.001). Despite genotyping 9,000 people and >95% power to reproduce the estimated OR from the recent meta-analysis, we were unable to replicate the association of the IRS-1 G972R polymorphism with type 2 diabetes.