The safety of tranexamic acid administration in total knee arthroplasty: a population-based study from Taiwan.

Tranexamic acid is an effective treatment to reduce blood loss. We performed a retrospective observational study to evaluate safety in unilateral total knee arthroplasty. We utilised Taiwan's national health insurance database to identify relevant patients and to retrieve information on peri-operative blood transfusions and tranexamic acid administration within 60 days of follow-up. We examined changes in the rate of transfusions and adverse events with respect to tranexamic acid administration using logistic regression. We observed a total of 226,719 knee arthroplasty cases during 2010-2019. Transfusion and tranexamic acid administration rates were 38.9% (88,258) and 42.9% (97,237), respectively. Tranexamic acid was associated with a 50% decrease in blood transfusions (RR: 0.50, 95%CI: 0.48-0.51). After propensity-score matching, tranexamic acid was not associated with pulmonary embolism; deep vein thromboembolism; artery vein thromboembolism; acute myocardial infarction; ischaemic stroke; or in-hospital mortality, but was significantly associated with acute kidney injury. Patients with existing chronic kidney disease suffered a high absolute risk of kidney injury irrespective of tranexamic acid administration (832 per 10,000, 95%CI 797-869). Tranexamic acid was also associated with surgical site infection. There was strong interaction between blood transfusion; tranexamic aid administration; and development of surgical site infection. In conclusion, tranexamic acid use was associated with decreased blood transfusion and was not associated with thromboembolic events. However, careful consideration is required before use in patients with pre-existing renal disease. Further, our observed interaction between patients given tranexamic acid who subsequently require transfusion requires careful consideration with respect to enhanced prophylaxis against surgical site infection.

[The consistency of skeletal muscle mass measured by CT at L1 and L3 levels and the correlation of skeletal muscle density at L1 level with prognosis in dialysis patients].

Objective: To investigate the consistency of skeletal muscle mass by CT at 1st lumbar vertebrae (L1) and 3rd lumbar vertebrae (L3) levels and the correlation of skeletal muscle density (SMD) at L1 level with prognosis in dialysis patients. Methods: A total of 1 020 patients who underwent initial dialysis and had CT examination data in four centers (Zhongda Hospital Affiliated to Southeast University, the Third Affiliated Hospital of Soochow University, Taizhou People's Hospital Affiliated to Nanjing Medical University and the Affiliated Hospital of Yangzhou University) from January 2014 to December 2019 were retrospectively collected. The skeletal muscle index (SMI) and SMD at L1 and L3 CT images were measured and calculated in patients with both L1 and L3 level CT images. The consistency of SMI and SMD at L1 and L3 levels was analyzed, and the cut-off value of SMI and SMD at L1 level for predicting all-cause mortality and their correlation with the prognosis of dialysis patients were studied. Cox regression model was used to analyze the risk factors for all-cause death and cardiac death. Results: A total of 383 patients had both L1 and L3 level images, including 233 males and 150 females. The average SMD value of 16 samples (4.2%) exceeded the 95% consistency limit range (-8.71 to 7.75 HU), and the average SMI value of 15 samples (3.9%) exceeded the 95% consistency limit range (-20.45 to 9.53 HU). The optimal cut-off value of SMD at L1 level for predicting all-cause mortality was 36.46 HU and the area under curve (AUC) of receiver operating characteristic (ROC) curve was 0.658 (95%CI: 0.596-0.721, P<0.001), with the sensitivity and specificity of 83.8% and 57.5%, respectively. SMI at L1 level was not significantly associated with all-cause mortality (P=0.299). Multivariate Cox regression analysis showed that low SMD at L1 level was associated with all-cause mortality (HR=2.861, 95%CI: 1.576-5.193, P=0.001) and cardiac death (HR=3.771, 95%CI:1.462-9.724, P=0.006). Conclusions: SMD at L1 levelis consistent with SMD at L3 level and can be used to evaluate muscle mass. Low SMD is a risk factor for mortality in dialysis patients.

Current management practice for bladder cancer in Hong Kong: a hospital-based cross-sectional survey.

OBJECTIVES: To examine current practice in the management of bladder cancer in Hong Kong government and private hospitals. DESIGN: Cross-sectional survey. SETTING: All government hospitals and the major private institutions in Hong Kong, which provide urological services. PARTICIPANTS: Urologists responding to an anonymous, self-administered, web-based questionnaire regarding practices in smoking cessation, treatment of non-muscle invasive bladder cancer and muscle invasive bladder cancer, and research into bladder cancer. RESULTS: Of the 29 urologists from 11 government hospitals and eight private institutions who were invited, 18 from 11 (100%) government hospitals and seven from six (75%) private institutions responded, which amounted to an 86% response rate. In all, 88% of the respondents seldom or never referred their bladder cancer patients to smoking cessation programmes. Hong Kong urologists showed good compliance in the management of non-muscle invasive bladder cancer according to international guidelines. There was great variation with regard to regimens for maintenance of intravesical immunotherapy. There was underuse of perioperative systemic chemotherapy, despite wide acceptance of this practice; fewer than 10% of the patients received neo-adjuvant and adjuvant systemic chemotherapy for the treatment of muscle invasive bladder cancer. Of the surveyed urologists, 80% expressed an inadequacy of resources for bladder cancer research and 96% agreed that a local inter-hospital bladder cancer database was needed. CONCLUSIONS: This study demonstrated great diversity in the use of intravesical immunotherapy, perioperative systemic chemotherapy, and surgical treatment of bladder cancer among urology service providers. There is a need for clear recommendations in these areas.

Reliability and validity of the overactive bladder symptom score in Hong Kong Chinese.

OBJECTIVE: To validate the Hong Kong Chinese translation of the Overactive Bladder Symptom Score questionnaire (OABSS-HKC). DESIGN: Cross-sectional study. SETTING: Five urology clinics of different regional hospitals in Hong Kong. PARTICIPANTS: The Overactive Bladder Symptom Score questionnaire was translated and culturally adapted for Hong Kong Chinese, according to the Principles of Good Practice for the Translation and Cultural Adaptation Process for Patient-Reported Outcomes Measures. Chinese-speaking patients with overactive bladder symptoms were recruited from five urology clinics. The patients completed the OABSS-HKC, a 3-day micturition diary, International Prostate Symptom Scores, and the Patient Perception of Bladder Condition questionnaires (visit 1), and again after a 2-week interval (visit 2). Test-retest reliability was evaluated by the intraclass correlation coefficient and weighted Kappa coefficient. The relationship between OABSS-HKC total scores and items in the comparison measures was evaluated using Spearman's correlation coefficients. RESULTS: The OABSS-HKC was successfully translated and culturally adapted. Fifty-one patients completed the validation study. A high level of reliability was observed between the OABSS-HKC total score answered at visit 1 and 2 for all subjects (intraclass correlation coefficient, 0.82) and among the four items answered (weighted Kappa coefficients, 0.57-0.75). The OABSS-HKC total score correlated significantly with numbers of micturitions, incontinence and urgency episodes recorded in the 3-day micturition diary, as well as the total International Prostate Symptom Scores and the Patient Perception of Bladder Condition score. However, the OABSS-HKC total score was not significantly associated with nocturia episodes, total voided volume, or number of pads used. CONCLUSIONS: The OABSS-HKC total scores are reliable and moderately valid for the quantitative evaluation of overactive bladder symptoms in Hong Kong Chinese-speaking adults.

Age, tumour stage, and preoperative serum albumin level are independent predictors of mortality after radical cystectomy for treatment of bladder cancer in Hong Kong Chinese.

OBJECTIVES: To evaluate the association between patient age, other clinical factors and mortality following radical cystectomy for treatment of bladder cancer. DESIGN: Historical cohort study. SETTING: A urology unit in Hong Kong. PATIENTS: The outcomes of 117 patients who had radical cystectomies performed in one urological unit from 2003 to 2011 were reviewed. Demographic and perioperative data, including tumour stage, Charlson Comorbidity Index, and preoperative serum albumin levels were retrieved from computerised medical records. Risk factors for 30-day mortality, and cancer-specific, other-cause, and overall death rates at 5 years were calculated. The data were subsequently stratified and analysed according to age. RESULTS: Of the 117 patients, 83 (71%) were aged 75 years or below. The mean follow-up duration was 31 (standard deviation, 29) months. Age, tumour stage, and preoperative serum albumin level, but not the Charlson Comorbidity Index, were found to be predictors of survival following radical cystectomy. The overall 30-day mortality rate was 3% in the full sample, 1% in patients aged 75 years or below, and 10% in patients aged over 75 years. There was no significant difference in 5-year cancer-caused mortalities between patients aged 75 years or below and those aged over 75 years (33% vs 33%, P=0.956). In patients older than 75 years, the 5-year other-cause and overall mortality rates were 47% and 80%, respectively; such rates were higher than those for younger patients (13% and 46%, respectively). CONCLUSION: Age, tumour stage, and preoperative serum albumin level were predictors of survival after radical cystectomy. Non-cancer-related death played a crucial role in the overall mortality rate in elderly patients having radical cystectomy for bladder cancer.

Risk factors for hip fracture in older adults: a case-control study in Taiwan.

UNLABELLED: We conducted a matched case-control study of hip fracture in older adults. Our findings suggest that hip fracture risk was determined by multiple factors. Older women characterized by low consumption of milk, peak flow rate, grip strength, and bone mineral density (BMD) had increased risk of hip fracture. Older men with impaired cognitive function and low BMD were also at higher risk of hip fracture. INTRODUCTION: Multiple factors contribute to low-trauma hip fracture in older adults. The aim of this study was to determine important characteristics of hip fracture in older population. METHODS: A total of 228 patients with first low-trauma hip fracture were matched with 497 controls. All 77 potential risk factors of hip fracture organized into 13 groups were analyzed using conditional logistic regression. RESULTS: Low milk intake, peak flow rate, hand grip strength, and bone mineral density in women and low mini-mental state examination score and bone mineral density in men were further identified to be independently associated with elevated hip fracture risk. CONCLUSIONS: The factors found in our study may help understand the etiology of hip fracture and be further adopted to evaluate the risk of hip fracture in community and clinical setting.

Skin and soft tissue infection caused by non-tuberculous mycobacteria.

SETTING: A medical centre in Taipei, Taiwan. OBJECTIVE: To investigate the trend and characteristics of patients with non-tuberculous mycobacteria (NTM) related skin and soft tissue infection. DESIGN: A total of 63 patients with culture-proven diseases were identified from January 1997 to December 2004. The medical records of all patients were reviewed. RESULTS: Twenty-seven patients were infected with rapidly growing mycobacteria (RGM), 19 with Mycobacterium marinum, six with M. avium complex (MAC), five with M. kansasii and six with other species. Most patients presented with a protracted cutaneous lesion without systemic symptoms, and two thirds of the patients had a history of exposure. Seventy-three per cent of the lesions involved the extremities. Underlying illness with suppressed immunity was documented in 30.2% of the patients, and was most prevalent in patients with MAC (100%) and M. kansasii (60%). Of the patients, 62% underwent at least one surgical intervention, and 77.8% received treatment with different antimicrobial combinations. Most patients (86%) recovered completely. Granulomatous inflammation was found in 88.9% of biopsied tissue associated with M. marinum infection, 31.8% with RGM and 25.0% with MAC. CONCLUSION: A combination of surgery and antimicrobials cured most patients with microbiologically proven localised NTM skin and soft tissue infection.

Adaptation and external validation of the European randomised study of screening for prostate cancer risk calculator for the Chinese population.

BACKGROUND: To adapt the well-performing European Randomized Study of Screening for Prostate Cancer (ERSPC) risk calculator to the Chinese setting and perform an external validation. METHODS: The original ERSPC risk calculator 3 (RC3) for prostate cancer (PCa) and high-grade PCa (HGPCa) was applied to a development cohort of 3006 previously unscreened Hong Kong Chinese men with initial transrectal biopsies performed from 1997 to 2015, age 50-80 years, PSA 0.4-50 ng ml-1 and prostate volume 10-150 ml. A simple adaptation to RC3 was performed and externally validated in a cohort of 2214 Chinese men from another Hong Kong hospital. The performance of the models were presented in calibration plots, area under curve (AUC) of receiver operating characteristics (ROCs) and decision curve analyses. RESULTS: PCa and HGPCa was diagnosed in 16.7% (503/3006) and 7.8% (234/3006) men in the development cohort, and 20.2% (447/2204) and 9.7% (214/2204) men in the validation cohort, respectively. The AUCs using the original RC3 model in the development cohort were 0.75 and 0.84 for PCa and HGPCa, respectively, but the calibration plots showed considerable overestimation. In the external validation of the recalibrated RC3 model, excellent calibration was observed, and discrimination was good with AUCs of 0.76 and 0.85 for PCa and HGPCa, respectively. Decision curve analyses in the validation cohort showed net clinical benefit of the recalibrated RC3 model over PSA. CONCLUSIONS: A recalibrated ERSPC risk calculator for the Chinese population was developed, and it showed excellent discrimination, calibration and net clinical benefit in an external validation cohort.

Hospital-based allogenic bone bank--10-year experience.

Bone banking in a hospital provides resources of allogenic bone grafts. However, they may transmit infection from donor to recipient. We found few reports discussing the infection rate and monitoring processes associated with bone banks. The discard rate using the screening test was 18.5% (309/1674) in this series. The leading cause was hepatitis B antigen (HBsAg) positive donor serum (67%), followed by Venereal Disease Research Laboratory (VDRL) positive donor serum (15%), and anti-hepatitis C virus (HCV) positive donor serum (12%). The overall infection rate in the recipients was 1.3% (17/1365). Among 1353 implanted allografts, 22 cases (1.6%) had a positive swab culture result after thawing. Only four out of these 22 cases (18.2%) developed infection. However, the wound cultures of the infected recipients were different from the swab culture of thawing allografts except in one case. Among the 1331 recipients with sterile allograft bones, 13 (1%) were found to have infection. In conclusion, our bone bank operates under a strict monitoring system which results in a low infection rate. The recipient's status, the aseptic technique and environment during operation is likely to be more critical in prevention of allograft-related infection.

Missense mutations and evolutionary conserved amino acids at the human hypoxanthine phosphoribosyl-transferase locus.

Molecular characterization of in vivo mutation at the human hypoxanthine phosphoribosyltransferase (hprt) locus has revealed a broad spectrum of mutation, both with regard to germ-line mutation in Lesch-Nyhan and gout patients, and somatic mutation in 6-thioguanine resistant T-lymphocytes from healthy individuals. The pattern of missense mutation shows a non-random distribution with a preferential location to codons for amino acids which are identical in human and the two parasites Schistosoma mansoni and Plasmodium falciparum. Although these 'evolutionary conserved' amino acids account for only 32% of the amino acids in the human hprt protein, they are involved in 76% of the missense mutations at the hprt locus in human T-lymphocytes, 67% in Lesch-Nyhan patients (with severe hprt-deficiency), but only 43% in gout patients (with partial hprt deficiency). This observation supports the notion that evolutionary conserved amino acids constitute functionally important sites in the hprt enzyme, and missense mutations affecting these amino acids will often lead to complete loss of enzyme activity. Substitutions of 'non-conserved' amino acids cause less severe hprt-deficiency (as seen in the gout patients), or may even escape clinical diagnosis. These considerations are important for the understanding of structure-activity relationships in the hprt protein, possible differences between hprt mutational spectra in germ-line and somatic cells, and the mutational spectra induced by specific exogeneous mutagens.

Glutathione S-transferase mu1 and N-acetyltransferase 2 genetic polymorphisms and exposure to tobacco smoke in nonsmoking and smoking lung cancer patients and population controls.

We conducted a case-control study to assess the risk of lung cancer in relation to genetic polymorphisms of the detoxifying enzymes glutathione-S-transferase mu1 (GSTM1) and N-acetyl transferase 2 (NAT2), focusing on never-smokers, women, and older people. The study base consisted of persons > or =30 years of age in Stockholm County from 1992 to 1995. We recruited never-smoking lung cancer cases and a sex- and age-matched sample of ever-smoking cases at the three county hospitals mainly responsible for diagnosing and treating lung cancer. A total of 185 cases (25.4% men; 47.6% never-smokers) and 164 frequency-matched population controls (28.7% men; 48.2% never-smokers) supplied blood for genotyping. Detailed information was collected by interview on active and passive smoking, occupations, residences, and diet. The overall odds ratio (OR) for lung cancer associated with the GSTM1 null (GSTM1-) versus GSTM1+ genotype was 0.8 [95% confidence interval (CI), 0.5-1.2], with an OR close to unity among smokers, and lower ORs suggested among never-smokers. For NAT2 slow versus rapid acetylator genotypes, the OR was 1.0 (95% CI, 0.6-1.5) overall, which broke down into an increased risk for slow acetylators among never-smokers but an increased risk for rapid acetylators among smokers. Among never-smokers, a gene interaction was suggested, with combined slow acetylator and GSTM1+ genotype conferring particularly high risk (OR = 3.1; 95% CI, 1.1-8.6), but no clear pattern emerged among smokers. A detailed analysis among smokers showed no interaction between pack-years of smoking and the GSTM1 genotype but suggested a steeper increase in risk with increasing pack-years of smoking exposure for rapid than for slow acetylators. Our results do not support a major role for the GSTM1 genetic polymorphism as a risk factor for lung cancer among smokers or nonsmokers. There was, however, some suggestion that the slow acetylator genotype may confer an increased risk among never-smokers and that the rapid acetylator genotype interacts with pack-year dose to produce a steeper risk gradient among smokers.

Differential interactions between GSTM1 and NAT2 genotypes on aromatic DNA adduct level and HPRT mutant frequency in lung cancer patients and population controls.

We have studied the influence of GSTM1 and NAT2 genotypes on aromatic DNA adduct level (AL) and HPRT mutant frequency (MF) in smokers with newly diagnosed lung cancer and matched population controls. AL was analyzed in relation to genotypes in 170 cases and 144 controls (113 current/recent smokers and 201 former/never smokers), and MF in 157 cases and 152 controls (155 ever smokers and 154 never smokers). Both genotypes exhibited the a priori expected effects on AL and MF among controls only, especially among smoking controls [significantly lower pack-years (a pack-year is defined as 1 pack of cigarettes/day for 1 year) than among cases]. Among the 42 currently smoking controls, the NAT2 slow genotype [odds ratio (OR), 7.5; 95% confidence interval (CI), 1.5-38.4], in particular in combination with the GSTM1 null genotype (OR, 19.3, 95% CI, 1.1-338.6 for null/slow versus positive/rapid) was strongly associated with high AL. The null/slow combination was also significantly associated with high MF among ever smokers (cases and controls pooled) with lower pack-years (OR, 3.7; 95% CI, 1.3-10.7 versus all of the other genotypes; OR, 5.1; 95% CI, 1.2-22.4 versus positive/rapid). In contrast, an antagonistic gene-gene interaction was seen among smoking cases for both AL and MF. Only currently smoking cases with the combined GSTM1 null and NAT2 rapid genotype showed a positive correlation between InAL and InMF (r, 0.64; P = 0.1), and an increase of AL with both age and daily cigarette use. This genotype combination was also associated with high MF among ever-smoking cases (OR, 4.0; 95% CI, 0.9-17.7 versus positive/rapid). There was a significant interaction between NAT2 genotype and pack-years of smoking among cases, so that the rapid genotype was associated with high MF among ever-smoking cases diagnosed at higher pack-years, whereas the slow genotype was associated with high MF at lower pack-years. These findings suggest that the influence of NAT2 genotype on AL and MF and its interaction with GSTM1 genotype may be dose dependent. The NAT2 slow genotype, in particular when combined with the GSTM1 null genotype, may confer increased susceptibility to adduct formation, gene mutation, and lung cancer when the smoking dose is low.

Combination leflunomide and cyclosporine prevents rejection of functional whole limb allografts in the rat.

Whole rear limbs were transplanted from Brown Norway or Lewis rat donors to Lewis rat recipients (n=6 per group). One group of allograft recipients was treated with leflunomide (10 mg/kg/24 hr/orally) and cyclosporine (5 mg/kg24 hr/orally) starting 2 days before to surgery. Treatment continued for 60 days or until graft rejection. Untreated allografts were rejected over 6-8 days. After isograft transplantation, weight bearing began by day 17-25 after surgery. Sensory function was restored by 50 days after surgery. All allografts in the drug-treated group survived the 60-day period; survival in this group was significantly longer (P=0.0001) than the untreated controls. Weight bearing began by day 30, but was incomplete in two rats at 60 days. Peroneal nerve function was present in half the rats at 60 days after surgery. Leflunomide combined with cyclosporine prevented whole limb allograft rejection across a major histocompatibility barrier.

Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide, and ethylene oxide.

Provisional mutational spectra at the hypoxanthine phosphoribosyl transferase (HPRT) locus in vitro have been worked out for acetaldehyde (AA) and benzo[a]pyrene diolepoxide (BPDE) in human (T)-lymphocytes and for ethylene oxide (EtO) in human diploid fibroblasts using Southern blotting and polymerase chain reaction (PCR)-based DNA sequencing techniques. The results indicate that large genomic deletions are the predominating hprt mutations caused by AA and EO, whereas BPDE induces point mutations that are mainly GC > TA transversions. The mutational spectra induced by the three agents are clearly different from the background spectrum in human T-cells. Thus, the hprt locus is a useful target for the study of chemical-specific mutational events that may help identify causes of background mutation in human cells in vivo.

Ultrastructural studies on myofibrillogenesis and neogenesis of skeletal muscles after prolonged traction in rabbits.

Little is known about the morphological response of muscle after long term traction. The purpose of this study was to investigate the morphological changes of skeletal muscle during limb lengthening. After application of mini-extraskeletal fixator, the hindlimb of New Zealand white rabbit was osteotomized and then slowly lengthened at the rate of 1 mm/day up to a 20 mm gain in length. The muscles of hindlimbs were perfused and dissected. Morphological studies were performed at electron microscopic level. Transmission electron microscopy revealed foci of microtrauma at the myotendinous junction. The distance between the muscle fibers and tendon parenchyma increased, with numerous primitive mesenchyme-like cells interposed within this gap. The cytoplasmic space of these cells was devoid of myofibril formation at the ends of stretched fibers. Within the satellite near the myotendinous junction myofilament production was observed in various gradations of maturation. It is concluded that myofibrillogenesis with traction neogenesis of skeletal muscle during limb lengthening does exist and occurs mainly near the myotendinous junction. The myotendinous junction in mature skeletal muscle actively participated in the process of limb lengthening.

Analysis of neogenesis in rabbit skeletal muscles after chronic traction.

After application of a modified Orthofix mini-extraskeletal fixator, the hind limb of New Zealand White rabbits was osteotomized and then slowly lengthened at a rate of 1 mm/day. After a 20 mm gain in length, the net weight and the length of muscular and tendinous portions were measured and histological examination was carried out in triceps surae muscles. Quantitative analysis showed a significant increase in the gained length of the muscular portion (28.05% to 30.65%). Histological studies of these lengthened muscles showed a generalized increase in cellularity with scanty inflammatory cell infiltration near the myotendinous junction. The increased cellularity is due to the presence of muscle precursor cells characterized by large, oval and pale-stained vesicular nuclei and two prominent nucleoli. The nuclei of these precursor cells were larger and more numerous near the myotendinous junction, and gradually changed into a flattened and more condensed form at a distance from the junction. Occasionally, chains of centrally-located nuclei of primitive myoblasts were also visible. It is concluded that traction neogenesis of the skeletal muscle during limb lengthening does exist and occurs mainly near the myotendinous junction.

Corticosteroid Reduces Blood Flow to Femoral Heads in Rabbits.

Avascular necrosis of the femoral head is one of the common problems in orthopedic practice in Taiwan. The subchondral bone loses its blood supply which weakens its biomechanical support. Steroid overuse is one of many possible etiologies in reducing blood flow to the femoral head. Laser Doppler velocimeter is a precise monitor of regional blood flow of bone which is expressed in perfusion units (PU). In the control group the rabbits were injected with normal saline and there were no statistical differences between blood flow to the right hip (39.26 +/- 5.64 PU) and left hip (38.58 +/- 4.35 PU). In group B a weekly injection of methylprednisolone into rabbits for 6 weeks demonstrated the reduction of blood flow of femoral head (24.74 +/- 3.13 PU) by the laser Doppler velocimeter. The flow decreased further (15.93 +/- 2.33 PU) by 12 weeks of steroid treatment. In group C after a weekly injection of steroid for 6 weeks the flow became 31.63 +/- 4.79 PU. The steroid was then discontinued for 3 weeks and the flow was 34.6 +/- 1.34 PU. In group D the blood flow was 25.89 +/- 4.01 PU after 6 weeks of steroid treatment and we stopped the steroid for 6 weeks, the blood flow became 29.86 +/- 2.59 PU. The merit of our experiment established a model of study in avascular necrosis of the femoral head in rabbits. Copyright 1994 S. Karger AG, Basel

Splicing mutations at the HPRT locus in human T-lymphocytes in vivo.

We studied 58 splicing mutations originating in vivo at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in T-cells of 30 nonsmoking males. A nonrandom distribution of skipped exons was seen after cDNA sequence analysis, with 71% involving exons 2-3 (15), 4 (11), and 8 (15). The mutations likely to have caused the aberrant splicing were identified in 36 mutants by genomic sequencing. The most frequently observed mutations were simple base substitutions (27) and small deletions (7). Among the base substitutions, 23 occurred in the splice consensus sequences, mainly at the highly conserved dinucleotides (21), and preferentially in the acceptor sites (15). The remaining four base substitutions occurred in the coding sequence where one tandem base substitution, one single bp insertion, and two single bp deletions were also observed. The predicted change in three of the base substitutions would be a stop codon. The tandem mutation (CC --> TT) occurred at position 550-551, a possible hotspot for splicing mutations (five of nine previously reported base substitutions at position 551, all C --> T, resulted in abnormal splicing). Four of the base substitutions were new HPRT mutations, two in splice sites (IVS7-3T --> G and IVS8 + 3A --> C) and two in the coding sequence (307A --> T and 594C --> G). All the small deletions (> 1 bp) affected the acceptor sites. The only three identified mutations related to skipping of exons 2 and 3 were located within exon 3, suggesting a frequent involvement of unknown splicing elements distant from these exons.

Hprt mutant frequency and GSTM1 genotype in non-smoking healthy individuals.

The T-cell cloning assay which combines mitogen- and growth factor-dependent expansion of lymphocyte clones with thioguanine selection of hypoxanthine-guanine phosphoribosyl transferase (hprt)-negative cells has been extensively used for studying human somatic gene mutation in vivo. However, large interindividual variations in the hprt mutant frequency (MF), much of which is not explained by donor attributes such as age and smoking habit, and interlaboratory variations in the experimental methodology, including cloning efficiency (CE), call for further developments of the cloning protocol and additional population studies. Using an improved T-cell cloning method, we have studied in vivo hprt MF of 76 non-smoking healthy males aged 23-77 years. The addition of 5% human serum to the growth medium was found to produce a consistently high CE of 61% in average. The MF, ranging from 1.4 to 22.6 x 10(-6) with a mean of 8.6 x 10(-6), increased significantly (P < 0.0001) with age, by 2% per year. A significant (P = 0.002) inverse relationship between MF and CE was observed. Using a PCR-based technique for GSTM1-genotyping, we also studied the relationship between MF and GSTM1 polymorphism. The 38 (50%) GSTM1-negative individuals showed a 20% higher mean MF than the 38 (50%) GSTM1-positive individuals. The difference was however not significant, neither before (P = 0.1) nor after (P = 0.5) correction for CE and the significantly (P = 0.04) higher mean age in the GSTM1-negative group. This study shows that age contributes more than GSTM1 polymorphism to the large interindividual variation in the hprt MF of non-smokers. The relationship between GSTM1 polymorphism and hprt MF in smokers remains to be investigated.

Mutations causing defective splicing in the human hprt gene.

Ten intron mutations and one exon mutation giving rise to defective splicing in the human gene for hypoxanthine phosphoribosyl transferase (hprt) in T-lymphocytes have been characterized. The splicing mutants were detected by PCR amplification of hprt cDNA and direct sequencing. Nine of the mutants showed skipping of whole exons or parts of exons in the cDNA, one mutant had an inclusion of an intron sequence into the cDNA, and one mutant showed both inclusion of an intron sequence and skipping of exons as well as a normal cDNA. Genomic PCR and direct sequencing of the splice sites involved showed one deletion of three base pairs and 10 different single base alterations to be responsible for these splice alterations. One mutation in the last base pair of exon 6 causing skipping of the entire exon 6 was found, whereas an identical mutation in the last base pair of exon 2 caused no aberrant splicing. It was also found that a deletion mutation in the pyrimidine rich stretch of the acceptor site of intron 7 caused skipping of the entire exon 8, whereas a base substitution in the last base of intron 7 caused exclusion of only the first 21 base pairs of exon 8 as a result of the activation of a cryptic acceptor site in exon 8. The results show that many different types of mutations at several different sites can cause splicing errors in the hprt gene and that the sequence differences between the splice sites influence the possible spectrum of mutations in each site.