Can T1-rho MRI detect acetabular cartilage degeneration in femoroacetabular impingement?: a pilot study.

Advanced MRI cartilage imaging such as T(1)-rho (T1ρ) for the diagnosis of early cartilage degradation prior to morpholgic radiological changes may provide prognostic information in the management of joint disease. This study aimed first to determine the normal T1ρ profile of cartilage within the hip, and secondly to identify any differences in T1ρ profile between the normal and symptomatic femoroacetabular impingement (FAI) hip. Ten patients with cam-type FAI (seven male and three female, mean age 35.9 years (28 to 48)) and ten control patients (four male and six female, mean age 30.6 years (22 to 35)) underwent 1.5T T1ρ MRI of a single hip. Mean T1ρ relaxation times for full thickness and each of the three equal cartilage thickness layers were calculated and compared between the groups. The mean T1ρ relaxation times for full cartilage thickness of control and FAI hips were similar (37.17 ms (SD 9.95) and 36.71 ms (SD 6.72), respectively). The control group demonstrated a T1ρ value trend, increasing from deep to superficial cartilage layers, with the middle third having significantly greater T1ρ relaxation values than the deepest third (p = 0.008). The FAI group demonstrated loss of this trend. The deepest third in the FAI group demonstrated greater T1ρ relaxation values than controls (p = 0.028). These results suggest that 1.5T T1ρ MRI can detect acetabular hyaline cartilage changes in patients with FAI.

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols.

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.

The molecular basis of phenylketonuria in Lithuania.

We report the spectrum of phenylalanine hydroxylase (PAH) gene mutations in patients with phenylketonuria (PKU) residing in Lithuania. A total of 184 independent chromosomes was investigated. R408W mutation was first analysed through restriction enzyme digestion of exon 12. The remaining uncharacterised PKU chromosomes were analysed by scanning the whole coding sequence of PAH gene by multiplex 'broad range' denaturing gradient gel electrophoresis. Mutations were identified by fluorescent automated sequencing or by restriction enzyme digestion analysis if an abnormal DGGE pattern was recognised. 21 different mutations were identified for 175 PKU chromosomes, with a mutation detection rate of 95%. The most common ones were R408W (73.5% chromosomes) and R158Q (7.0% chromosomes) whereas the remaining mutations appeared to be rare (relative frequencies 0.5%-2%). The high mutation detection rate obtained is an evidence of the efficiency of PAH genetic testing achieved in Lithuania. Moreover, the definition of the PKU mutation profile in the Lithuanian population will allow to perform a genotype-phenotype correlation study thus making feasible genotyped-based prediction of the biochemical phenotype in newborns with hyperphenylalaninemia. This may be useful for refining diagnosis and anticipating dietary requirements.

The molecular basis of phenylketonuria in Latvia.

Characterization of the molecular basis of phenylketonuria (PKU) in Latvia has been accomplished through the analysis of 96 unrelated chromosomes from 50 Latvian PKU patients. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed through a combined approach in which R158Q, R252W, R261Q, G272X, IVS10-11G>A and R408W mutations were first screened for by PCR or restriction generating PCR amplification of PAH gene exons 5, 7, 11 and 12 followed by digestion with the appropriate diagnostic enzyme. Subsequently 'broad range' denaturing gradient gel electrophoresis analysis of the 13 PAH gene exons has been used to study uncharacterized PKU chromosomes. A mutation detection rate of 98% was achieved. 12 different mutations were found, with the most frequent mutation, R408W, accounting for 76% of Latvian PKU alleles. Six mutations (R408W, E280K, R158Q, A104D, R261Q and P281L) represent 92% of PKU chromosomes. PAH VNTR and STR alleles have been also identified and minihaplotype associations with PKU mutations were also determined.

Genetic heterogeneity in five Italian regions: analysis of PAH mutations and minihaplotypes.

Molecular analysis of 289 chromosomes has been performed in a cohort of phenylketonuria (PKU) patients whose ancestors lived in five Italian regions, Calabria, Campania, Piemonte, Puglia/Basilicata and Sicilia. Phenylalaninehydroxylase (PAH) gene mutations and minihaplotypes (combinations of PAH gene STR and VNTR systems) have been determined for 78.5 and 64%, respectively, of the chromosomes studied. 21 different minihaplotypes and 24 PKU mutations were found. Heterogeneity tests carried out for the frequencies of mutations and minihaplotypes show that the distribution of eight mutations and four minihaplotypes is statistically heterogeneous in the five Italian regions. Although the evolutionary rate of microsatellites or the age of these mutations is difficult to estimate with accuracy, our findings taken together show a genetic stratification of the Italian population. These results rule out allelic homogeneity of PKU at the molecular level between regions of Italy, yet minihaplotype data may be of practical use for a multistep approach to PAH gene genotyping.

Macromolecule and water magnetization exchange modeling in articular cartilage.

Magnetization exchange effects between the matrix macromolecules (e. g., collagen and proteoglycan) and water were examined in normal, deuterated, and proteoglycan-depleted articular cartilage. Relaxation results (T(2), T(1rho), and T(1)) suggested that a four-site exchange scheme provided an accurate model for articular cartilage relaxation and interspin group coupling details. Magnetization exchange within the collagen-bulk-water, proteoglycan-collagen, and collagen fibrillar water-collagen cartilage subsystems were quantified. Although collagen-bulk-water was the largest of the cartilage coupling subsystems ( approximately 90% signal) and is exploited in MRI, the rates of magnetization transfer (MT) within the latter subsystems were appreciably larger. Magnetization exchange rates for proteoglycan-collagen and collagen fibrillar water-collagen were 120 s(-1) and 4.4 s(-1), respectively. The observation of these latter two exchange subsystems suggested potential clinical MRI-MT applications in detecting molecular abnormalities associated with osteoarthritis.

Characterization of the CYP21 gene 5' flanking region in patients affected by 21-OH deficiency.

In order to test the hypothesis that mutations in the 5' non-coding region of CYP21 gene could contribute to the various spectrum of disease presentation due to 21-OH deficiency, the 400bp nucleotide sequence upstream of the ATG codon of CYP21 gene has been characterized in 28 CAH patients who have previously been genotyped by screening for the ten most frequent CYP21 mutations. Six specific sequence variations (-4C-->T, -73C-->T, -295T-->C, -294A-->C, -283A-->G, -281T-->G) have been identified in this region of CYP21 gene in 3 out of 28 21-OH deficient patients for whom the coding region mutations have been previously identified. Three of these mutations, -295T-->C, -294A-->C, -283A-->G, are apparently generated by a gene-conversion event, thus giving first evidence that this mechanism also applies to the 5' untranslated region of CYP21 gene in 21-OH deficiency. Four other sequence changes, identified at nucleotide position -279, -331, -350 and -353, could be referred to as normal since they are present also in healthy subjects. It may not be excluded that some of the newly-identified single nucleotide changes in the regulatory region could have a modulatory effect on the CYP21 gene transcriptional activity thus affecting the clinical outcome.

Effect of the posterior cruciate ligament in knee-joint proprioception in total knee arthroplasty.

The primary purpose of the study was to examine the role of the posterior cruciate ligament (PCL) in knee-joint proprioception after total knee arthroplasty (TKA). Knee-joint proprioception was measured in 10 patients with nonsacrificed PCL TKAs and 10 with sacrificed PCL TKAs. Knee-joint proprioception was evaluated through reproduction of static knee angles using a Penny and Giles electrogoniometer. The primary variable was absolute angular error (AAE). AAE was defined as the absolute value of the difference between the test angle and the patient's perceived version of the test angle. Proprioception deficit was compared to the WOMAC questionnaire which evaluates pain, stiffness, and physical function of the lower extremity. No significant difference was found between the nonsacrificed PCL TKA (4.33 degrees +/- 1.52 degrees) and sacrificed PCL TKA (4.38 degrees +/- 1.39 degrees) AAE values (P > .4). Furthermore, no significant differences were observed in the WOMAC questionnaire scores for all three parameters between the two types of knee prosthesis (P > .35). The current findings suggest that the preservation of the PCL in TKA may not improve knee-joint proprioception and subsequently may not improve TKA functional performance.

Phenylketonuria mutations and linked haplotypes in the Lithuanian population: origin of the most common R408W mutation.

A genealogical study was performed in Lithuanian phenylketonuria (PKU) families with the aim of tracing the origins of the R408W/haplotype 2/VNTR3 allele. The relative frequency of six phenylalanine hydroxylase (PAH) mutations (R408W, R158Q, R261Q, G272X, IVS10nt-11g --> a, and IVS12nt1g --> a) common in Eastern European populations and their association with variable number of tandem repeat (VNTR) and short tandem repeat (STR) sites in the PAH gene were examined in 130 PKU Lithuanian chromosomes, including 95 of Baltic, 28 of Slavonic and 7 of unknown origin. R408W was found to be the most frequent (70%) mutation in both Balts or Slavonians with a uniform frequency distribution. No statistically significant differences in the frequency distribution of the other mutations analysed were found. In Balts and Slavonians, the R408W mutation is strongly associated with the three-copy VNTR and the 240-bp STR allele. The frequency of this association is 68% in both ethnic groups. The genealogical data provided in this paper indicate that the most common R408W/VNTR3/STR240 allele arose in ancient times possibly among pre-Indo-Europeans and suggest that the high frequency of the R408W mutation and associated minihaplotype in Balts of Lithuania is due to a founder effect.

Effect of age and activity on knee joint proprioception.

Falls lead to significant morbidity and mortality in persons older than 65 years of age. Impaired proprioception may be a contributing factor to falls, and this may be influenced by the level of habitual physical activity. The primary purpose of this study was to investigate knee joint proprioception among young volunteers and active and sedentary elderly volunteers. Knee joint proprioception was measured through reproduction of static knee angles using a Penny and Giles electrogoniometer. The secondary purpose of this investigation was to test the reproducibility of the Penny and Giles electrogoniometer in measuring static knee angles. Sixteen young subjects (age range, 19-27 years) and 24 elderly subjects (age range, 60-86 years) participated. Subjects were given a screening history and physical examination to exclude neuromuscular or vestibular disorders or lower limb injuries. Knee joint proprioception was measured two times during one week. The elderly group was separated into active and sedentary subgroups based on their level of activity during the past year. The electrogoniometer was placed laterally across the dominant knee joint. From the prone position each subject attained one of ten randomly predetermined knee joint angles from 10 degrees to 60 degrees. The subject then returned to the starting position and reproduced the test angle. The absolute angular error (the absolute difference between the test angle and subject perceived angle of knee flexion) was determined. A positive correlation was found between control visits for all subjects (r = 0.88), and significant differences were observed between young (mean, 2.01 +/- 0.46 degrees) and active-fold (mean, 3.12 +/- 1.12 degrees; P < 0.001), young and sedentary-old (mean, 4.58 +/- 1.93 degrees; P < 0.001), and active-old and sedentary-old (P < 0.03). These findings demonstrate that the Penny and Giles electrogoniometer is a reproducible device for measuring knee joint angles in both young and elderly subjects. Furthermore, we found that proprioception is diminished with age and that regular activity may attenuate this decline. One strategy to reduce the incidence of poor proprioception and fall with ageing may be regular exercise.

Detection of microsatellites by ethidium bromide staining. The analysis of an STR system in the human phenylalanine hydroxylase gene.

The analysis of short tandem repeat (STR) systems usually relies on polyacrylamide gel electrophoresis analysis followed by visualization with silver staining or autoradiography. Both these techniques may not be suitable for clinical laboratories. We developed a simple procedure based on the visualization of STR alleles by ethidium bromide staining. The 4-bp STR system analysed is located in the human phenylalanine hydroxylase gene. Alleles differing by 4 bp are clearly separated independently of the size of the amplified fragments and homozygous samples are easily identified by comparison of the relative intensity of the electrophoretic bands. This method could be applied to the analysis of other STR systems located in different genetic loci by carefully changing the electrophoretic conditions.

Effects of fatigue on knee proprioception.

OBJECTIVE: To investigate the effects of muscular fatigue on knee joint proprioception. DESIGN: Prospective study. SETTING: Exercise physiology laboratory. PARTICIPANTS: Sixteen (eight men and eight women) healthy volunteers ages 19-27 years, with no history of neuromuscular disorders, vestibular disorders, or lower limb injuries (e.g., ligament/meniscus tear). INTERVENTION: Three separate fatigue protocols [ramp test (RT), continuous test (CT), and interval test (IT)] were performed. All tests consisted of lower limb cycling on a computer-driven cycle ergometer (Lode). The RT was used to calculate the maximal aerobic power (VO2max) and determine the work rates for the CT and IT. Work rate for the RT increased 20/25 W/min to maximal exhaustion. The CT consisted of cycling at 80% VO2max until maximal exhaustion. The IT consisted of cycling alternately at 120% VO2max and at 40% VO2max for 30 s each to the point of maximal exhaustion. MAIN OUTCOME MEASURE: In the standing position, subjects were instructed to perform a two-legged squat to specific knee flexion angles. The absolute angular error (AAE) was measured for each test angle using an electrogoniometer (Penny & Giles, Blackwood, Gwent, U.K.) placed laterally across the dominant knee joint. AAE was defined as the absolute difference between test angle and subject perceived angle of knee flexion. RESULTS: A statistically significant increase in AAE after the RT (1.0 +/- 0.66 degree, p < 0.01), CT (0.70 +/- 0.66 degree, p < 0.03), and IT (1.24 +/- 0.79 degrees, p < 0.01) protocols was observed in the male subjects. Female subjects reported a statistically significant increase in AAE after the CT (0.73 +/- 0.73 degree, p < 0.03) and IT (1.1 +/- 0.89 degrees, p < 0.01) protocols and a nonsignificant increase in AAE (0.19 +/- 0.70 degree, p > 0.5) after the RT protocol. CONCLUSION: These findings suggest that exercising to fatigue may produce a change in subjects' reproduction ability of knee joint angles. This may represent a decline in proprioceptive function after heavy exercise bouts. Whether this suggested proprioceptive decline is at the clinical significance level (e.g., significantly altering joint stability and motion) cannot be determined from the present findings.

Molecular screening of genetic defects with RNA-SSCP analysis: the PKU and cystinuria model.

RNA single-strand conformation polymorphism (rSSCP) is a recently developed method for detecting genetic defects. This technique requires DNA amplification with a polymerase chain reaction making use of one T7 promoter-containing primer. Amplification products are subsequently transcribed in vitro and the labelled transcripts are analysed for single-strand conformation changes. rSSCP has been applied to mutation screening of the phenylalanine hydroxylase gene and rBAT cDNA, from PKU and cystinuric patients, respectively. Experimental evidence shows that 83% and 86% of screened PKU and cystinuric mutations, respectively, give rise to detectable rSSCP signals. Thus, results obtained show that RNA single-strand conformation polymorphism analysis is generally applicable and is a suitable technique for detecting genetic disease causing mutations, both in basic research and in clinical practice.

Characterization of phenylketonuria alleles in the Italian population.

In order to identify the molecular basis of phenylketonuria (PKU) in Italy, we screened the entire coding sequence of the phenylalanine hydroxylase gene in 20 Italian PKU patients, whose origins are scattered throughout Italy. The frequency of each identified mutation and of 5 other European mutations was determined within a panel of 92 Italian PKU patients. This approach allowed us to identify 20 different PKU mutations and characterize 64% of the Italian PKU chromosomes. Eleven mutations (IVS10nt546, L48S, R158Q, R261Q, P281L, R261X, R252W, delta T55, IVS7nt1, IVS12nt1, Y414C) represent 55.4% of the Italian PKU alleles, the most common mutations being IVS10nt546 (12.4%) and L48S (9%). All the other mutations are very rare. These data confirm the great heterogeneity expected from previous RFLP haplotype studies. Genotype/phenotype correlation allowed for assessment of the clinical impact of the 20 identified mutations.