Decreased cortical bone density and mechanical strength with associated elevated bone turnover markers at peri-pubertal peak height velocity: a cross-sectional and longitudinal cohort study of 396 girls with adolescent idiopathic scoliosis.

Decreased cortical bone density and bone strength at peak height velocity (PHV) were noted in girls with adolescent idiopathic scoliosis (AIS). These findings could provide the link to the previously reported observation that low bone mineral density (BMD) could contribute as one of the prognostic factors for curve progression that mostly occurs during PHV in AIS. INTRODUCTION: As part of the studies related to aetiopathogenesis of AIS, we assessed bone qualities, bone mechanical strength and bone turnover markers (BTMs) focusing at the peri-pubertal period and PHV in AIS girls. METHODS: 396 AIS girls in two separate cohorts were studied. Skeletal maturity was assessed using the validated thumb ossification composite index (TOCI). Bone qualities and strength were evaluated with high-resolution peripheral quantitative computed tomography (HR-pQCT) and finite element analysis (FEA). RESULTS: Cohort-A included 179 girls (11.95 ± 0.95 years old). Girls at TOCI-4 had numerically the highest height velocity (0.71 ± 0.24 cm/month) corresponding to the PHV. Subjects at TOCI-4 had lower cortical volumetric BMD (672.36 ± 39.07 mg/mm3), cortical thickness (0.68 ± 0.08 mm) and apparent modulus (1601.54 ± 243.75 N/mm2) than: (a) those at TOCI-1-3 (724.99 ± 32.09 mg/mm3 (p < 0.001), 0.79 ± 0.11 mm (p < 0.001) and 1910.88 ± 374.75 N/mm2 (p < 0.001), respectively) and (b) those at TOCI-8 (732.28 ± 53.75 mg/mm3 (p < 0.001), 0.84 ± 0.14 mm (p < 0.001), 1889.11 ± 419.37 N/mm2 (p < 0.001), respectively). Cohort-B included 217 girls (12.22 ± 0.89 years old). Subjects at TOCI-4 had higher levels of C-terminal telopeptide of type 1 collagen (1524.70 ± 271.10 pg/L) and procollagen type 1 N-terminal propeptide (941.12 ± 161.39 µg/L) than those at TOCI-8 (845.71 ± 478.55 pg/L (p < 0.001) and 370.08 ± 197.04 µg/L (p < 0.001), respectively). CONCLUSION: AIS girls had decreased cortical bone density and bone mechanical strength with elevated BTMs at PHV. Coupling of PHV with decreased cortical and FEA parameters could provide the link to the previously reported observation that low BMD could contribute as one of the prognostic factors for curve progression that mostly occurs during PHV in AIS.

Radial extracorporeal shock-wave therapy in patients with chronic rotator cuff tendinitis: a prospective randomised double-blind placebo-controlled multicentre trial.

The aim of this study was to determine the effect of radial extracorporeal shock-wave therapy (rESWT) on patients with chronic tendinitis of the rotator cuff. This was a randomised controlled trial in which 82 patients (mean age 47 years (24 to 67)) with chronic tendinitis diagnosed clinically were randomly allocated to a treatment group who received low-dose rESWT (three sessions at an interval 10 to 14 days, 2000 pulses, 0.11 mJ/mm(2), 8 Hz) or to a placebo group, with a follow-up of six months. The patients and the treating orthopaedic surgeon, who were both blinded to the treatment, evaluated the results. A total of 44 patients were allocated to the rESWT group and 38 patients to the placebo group. A visual analogue scale (VAS) score for pain, a Constant-Murley (CMS) score and a simple shoulder test (SST) score significantly improved in both groups at three and six months compared with baseline (all p ≤ 0.012). The mean VAS was similar in both groups at three (p = 0.43) and six months (p = 0.262). Also, the mean CMS and SST scores were similar in both groups at six months (p = 0.815 and p = 0.834, respectively). It would thus seem that low-dose rESWT does not reduce pain or improve function in patients chronic rotator cuff tendinitis compared with placebo treatment.

Validation of a Novel Semiautomated Segmentation Method for MRI Detection of Cartilage-Related Bone Marrow Lesions.

OBJECTIVE: To determine the relationship of bone marrow lesions (BMLs) with phenomena such as clinical symptoms, histological subchondral bone damage, and development of osteoarthritis, a reliable and reproducible method to localize and quantify BMLs accurately is indispensable. Therefore, the goal of the current study was to develop and validate a novel semiautomated segmentation method based on the KNN classification technique on T2-weighted (T2w) SPIR and proton density-weighted (PDw) magnetic resonance images (MRIs), as this would provide an accurate, reliable, and reproducible tool. MATERIALS AND METHODS: Twenty PDw and T2w SPIR MRIs were selected and manually segmented as a learning set for the software system. The manual segmentations were considered the gold standard. Automated segmentation based on the KNN classification technique was carried out on the same MRIs. To determine the accuracy and validity of the system, the automated segmentations were compared to the gold standard using the Dice Similarity Index (DSI). RESULTS: The KNN classification system resulted both visually and statistically in an accurate segmentation of BMLs on T2w SPIR MRIs with an excellent mean optimal DSI of 0.702 (±0.202; range, 0.409-0.908). Elimination of specific areas smaller than 10 voxels improved the accuracy. The accuracy was independent of BML size. The segmentation of BMLs on PDw MRIs was less reliable with a mean optimal DSI of 0.536 (±0.156). CONCLUSION: Although the applicability of this method is limited on PDw MRIs, the KNN classification system provides an accurate, reliable, and reproducible tool for semiautomated segmentation of BMLs in T2w SPIR MRIs of the knee.

Autologous interleukin-1 receptor antagonist improves function and symptoms in osteoarthritis when compared to placebo in a prospective randomized controlled trial.

INTRODUCTION: Incubation of blood with CrSO(4)-coated glass beads stimulates the synthesis of anti-inflammatory cytokines, such as interleukin-1 receptor antagonist (IL-1ra), IL-4, IL-10, and IL-13. As IL-1beta is thought to play a key role in the development of osteoarthritis (OA), this product, also known as Orthokin, might be a viable treatment for symptomatic knee OA. The aim of the current study was to evaluate the efficacy of Orthokin for treatment of symptomatic knee OA in a randomized, multicentre, double-blind, placebo-controlled trial. PATIENTS AND METHODS: One hundred and sixty-seven patients received six intra-articular injections either with Orthokin or physiological saline. The primary efficacy objective consisted of 30% superiority on the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) at 3, 6, 9, and 12 months post-treatment. Additionally, the patients completed the visual analogue scale for pain, the Knee injury and Osteoarthritis Outcome Score (KOOS) and Knee Society Clinical Rating System. RESULTS: Orthokin and placebo treatment resulted in similar improvements on the WOMAC (16.8% vs 16.5%, respectively; n.s.). Orthokin resulted in significantly more improvement for KOOS symptom (P = 0.002) and KOOS sport (P = 0.042) parameters as compared to placebo treatment. For most other outcome parameters, Orthokin-treated patients consistently showed higher improvement compared to placebo-treated patients, although none of these differences were statistically significant. Two serious adverse events were observed in the Orthokin group: one patient with repeated severe inflammatory reactions of the knee joint within hours after the injection and one patient with septic arthritis which was attributed to the injection procedure rather than the product. CONCLUSION: The statistically significant improvement of KOOS symptom and sport parameters together with the consistently higher, though non-statistically significant, improvement of most other parameters demonstrates that Orthokin clearly induces a biological response different from placebo treatment and warrant future investigations into the possible chondroprotective effect of Orthokin. However, in the current study the primary efficacy objective was not met and, therefore, the use of Orthokin currently cannot yet be recommended for the treatment of OA.

Validation of the short-form WOMAC function scale for the evaluation of osteoarthritis of the knee.

This study validates the short-form WOMAC function scale for assessment of conservative treatment of osteoarthritis of the knee. Data were collected before treatment and six and nine months later, from 100 patients with osteoarthritis of the knee to determine the validity, internal consistency, test-retest reliability, floor and ceiling effects, and responsiveness of the short-form WOMAC function scale. The scale showed high correlation with the traditional WOMAC and other measures. The internal consistency was good (Cronbach alpha: 0.88 to 0.95) and an excellent test-retest reliability was found (Lin's concordance correlation coefficient (rho(c)): 0.85 to 0.94). The responsiveness was adequate and comparable to that of the traditional WOMAC (standardised response mean 0.56 to 0.44 and effect size 0.64 to 0.57) and appeared not to be significantly affected by floor or ceiling effects (0% and 7%, respectively). The short-form WOMAC function scale is a valid, reliable and responsive alternative to the traditional WOMAC in the evaluation of patients with osteoarthritis of the knee managed conservatively. It is simple to use in daily practice and is therefore less of a burden for patients in clinical trials.

Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes.

Cartilage regeneration based on isolated and culture-expanded chondrocytes is studied in a variety of in vitro models, but with varying morphological quality of tissue synthesized. The goal of the present study was to investigate the extent of the influence of expansion and redifferentiation conditions on final tissue morphology by comparing 2 expansion and redifferentiation methods. Chondrocytes from 9 human donors were expanded in medium without growth factor supplementation (basic expansion condition [BEC]) or in medium with basic fibroblast growth factor (bFGF) supplementation (growth factor supplemented expansion condition [GFSEC]). After expansion, cells were either redifferentiated in pellet culture or seeded on collagen type II-coated filters. Post-expansion mRNA levels of collagen type I and II and Sox-5, -6, and 9, measured by semiquantitative real-time polymerase chain reaction (PCR), suggested that expansion in GFSEC results in increased dedifferentiation compared to BEC. However, after 28 days of redifferentiation culture, morphology of tissue synthesized by GFSEC-expanded chondrocytes scored significantly higher on the Bern scale compared to BEC (6.4 +/- 0.3 points vs. 4.5 +/- 0.3 points in pellet culture and 6.0 +/- 0.4 points vs. 4.5 +/- 0.3 points on collagen-coated filters; p < 0.05). Expansion in GFSEC compared to BEC increased proteoglycan (PG) synthesis rate at day 9 (4.0-fold in pellet culture and 1.9-fold on collagen-coated filters; p < 0.01), PG release (6.7-fold in pellet culture and 3.2-fold on collagen-coated filters; p < 0.001), and final PG content at day 28 (1.6-fold in pellet culture and 1.5-fold on collagen-coated filters; p < 0.05). Redifferentiation on collagen-coated filters compared to pellet culture increased PG synthesis rate at day 9 (5.2-fold in BEC-expanded chondrocytes and 2.6-fold in GFSEC-expanded chondrocytes; p < 0.01), PG release (4.2-fold in BEC-expanded chondrocytes and 3.1-fold in GFSECexpanded chondrocytes; p < 0.01), and final PG content (1.3-fold in BEC-expanded chondrocytes and 1.9- fold in GFSEC-expanded chondrocytes; p < 0.01). Moreover, as visualized via electron microscopy, chondrocytes and organization of extracellular matrix cultured on filters was more similar to those found for hyaline cartilage. In conclusion, chondrocyte expansion in GFSEC and redifferentiation on collagen-coated filters resulted in most optimal chondrogenesis.

Altered in vitro chondrogenic properties of chondrocytes harvested from unaffected cartilage in osteoarthritic joints.

OBJECTIVE: In vitro models of chondrogenesis often depart from chondrocytes harvested from less-affected areas of osteoarthritic joints. However, there are indications that these chondrocytes are phenotypically different from chondrocytes from healthy joints and thus might differ in their capacity to generate hyaline cartilage. The goal of this study was to compare the chondrogenic capacity of chondrocytes from healthy and OA joints. DESIGN: Chondrocytes isolated from nine healthy and nine OA knee joints were expanded in monolayer for two passages. Chondrocytes from passages 1 and 2 were analyzed for expression of (de)differentiation and hypertrophy markers and were seeded at passage 2 on collagen-coated filters for redifferentiation culture to study cartilage matrix formation. RESULTS: The collagen II/I mRNA ratio, reflecting differentiation, decreased from passage 1 to 2 in both chondrocytes from OA joints and chondrocytes from healthy joints (P<0.05), without a significant difference between the two donor types. At passage 1, levels of the cartilage transcription factors Sox-5, Sox-6 and Sox-9 appeared to be higher in chondrocytes from OA joints (n.s.), but this was not seen at passage 2. However, a clear difference was observed in collagen type X expression, which was high in chondrocytes from OA joints at both passages, while undetectable in chondrocytes from healthy joints (P<0.01). Tissue generated by chondrocytes from healthy joints redifferentiated for 28 days, showed a significantly better morphology, as assessed by histological scoring (P<0.01) and higher proteoglycan content (P<0.05), compared to chondrocytes from OA joints. Matrix turnover parameters, i.e., proteoglycan synthesis and degradation rate, were not significantly affected by donor tissue origin. CONCLUSIONS: These results suggest that clear differences between chondrocytes from healthy and OA joints exist and that these are not completely abolished during the process of de- and redifferentiation. Therefore, in vitro cartilage regeneration models, which use chondrocytes from OA joints, should be interpreted with care.

The effect of synovial fluid from injured knee joints on in vitro chondrogenesis.

Various in vivo and in vitro studies suggest that joint homeostasis may have a crucial effect on the quality of regeneration tissue resulting from cartilage tissue engineering techniques. The goal of the current study was to evaluate the effect of synovial fluid (SF) from injured knee joints on in vitro chondrogenesis. Chondrocytes were isolated from a healthy human femoral condyle (post-mortem) and expanded in monolayer for 2 passages. Subsequently, the chondrocytes were redifferentiated for 14 days on collagencoated filters, cultured either in the presence or absence of 10% SF. SF was obtained from 12 injured human knee joints. After 14 days of culture, SF supplementation resulted in a significant downregulation of final proteoglycan (PG) content (7.3 +/- 1.8 mg versus 15.6 +/- 1.3 mg; p = 0.0001), PG content normalized to DNA (0.7 +/- 0.5 mg/microg versus 3.0 +/- 0.6 mg/microg; p < 0.05), relative collagen type II mRNA levels normalized to GAPDH mRNA levels (0.2 +/- 0.3 versus 7.0 +/- 5.6; p < 0.001), and differentiation index (collagen type II/I mRNA ratio; 0.1 +/- 0.2 versus 6.0 +/- 2.9; p < 0.001) as compared to control culture conditions. Additionally, SF-supplemented media resulted in significantly increased cellularity, as reflected by DNA content, compared with control media (1,369 +/- 683 microg versus 514 +/- 72 microg; p < 0.0001). Morphology, and collagen type I, X, and aggrecan mRNA levels were not significantly affected. In conclusion, this study demonstrates that SF from injured human knee joints significantly affects in vitro chondrogenesis and therefore may provide a viable target for future improvement of ACT by refinement of culture techniques, patient selection, or pretreatment of affected joints to restore joint homeostasis.

The correlation and reproducibility of histological scoring systems in cartilage repair.

In recent years, the field of cartilage tissue engineering has seen a sharp increase in publications using many tissue engineering techniques and various analysis methods. Comparison between studies remains difficult, due to a lack of uniformity in methods used. A broad range of histological scoring systems is used to examine cartilage quality. Unfortunately, so far little is known on the reliability and correlation of these scoring systems. The objective of this study was to compare two frequently used cartilage repair grading scales, namely, the comprehensive O'Driscoll and the simple Pineda scale. We determined the intra- and interobserver variability of each score as well as the correlation between them. Thirty-eight joint section samples with variable cartilage quality were examined. Three observers documented their findings with both systems at two points in time. Statistical analysis showed very good intra- and interobserver reliability as well as a good correlation between the two scores. For the intraobserver variability of the O'Driscoll scale, we found an average difference of 0.05 with a SD of 0.93 in a 24-point score and a kappavalue of 0.87. For the interobserver reliability, the average difference was 0.001, SD 2.25, and a kappavalue of 0.92. The Pineda scale showed an average difference of 0.86 with a SD of 1.38 in a 14-point score and a kappavalue of 0.86 for the intraobserver reliability, whereas values for the interobserver reliability were average difference 0.82, SD 0.96, and a kappavalue of 0.89. The comparison between the two scales showed a high, inversely related correlation with a correlation coefficient of 0.71. From these results, we concluded that both the O'Driscoll and the Pineda scales are reliable semiquantitative cartilage scoring systems and that acceptance for general use of these two scores will benefit the reliability of literature on tissue engineering for cartilage repair. Thus, the added strength of comparison between published study results allows better understanding of cartilage repair publications and increases the impact of their results.

Chinese in west Malaysia: the geography of beta thalassaemia mutations.

The overseas Chinese in West Malaysia are almost exclusively from the south-eastern provinces of China-Kwangtung, Fukien, and Kwangsi. To institute a comprehensive thalassaemia control programme for this region we have characterised the beta thalassaemia mutations in 16 Chinese patients from West Malaysia: 4 beta thalassaemia mutations were seen: a) an A----G substitution in the TATA box [-28 base pairs (bp)], an A----T substitution in codon 17 [17 A----T], c) a 4 base pairs - TCTT deletion in codon 41-42 [frameshift mutation (FSC 41-42)], and d) a C----T substitution at the second intervening sequence (IVS 11) position 654. Similar mutations have been described in patients from the south-eastern provinces of China. The delineation of the specific mutations present will enable effective prenatal diagnosis for beta thalassaemia of ethnic Chinese in West Malaysia to be instituted.

Two novel polyadenylation mutations leading to beta(+)-thalassemia.

In an ongoing effort to identify point mutations causing beta-thalassaemia, we have found two previously unreported mutations which are located in the Poly A site of the beta-globin gene. The screening programme used amplified DNA and dot-blot hybridization with several 32P-labelled oligonucleotide probes. DNA samples which remained unidentified by this methodology were subjected to sequencing with 32P-labelled primers and modified T7 DNA polymerase. The newly discovered mutations were confirmed by the dot-blot hybridization technique. One type concerned an AATAAA----AATGAA mutation in the polyadenylation site and was found in one family from Yugoslavia (including one patient with the C----T mutation at codon 29 in trans), one from Bulgaria (the patient had the G----A mutation at IVS-I-110 in trans), and one from Greece (this patient had the C----G mutation at IVS-II-745 in trans). Haematological data for three simple heterozygotes suggested a rather mild beta(+)-thalassemia. The second type involved an AATAAA----AATAGA mutation and was found in one family from Malaysia. The propositus had the beta E mutation on the other chromosome, was originally diagnosed as mild Hb E-beta(+)-thalassaemia, and had Hb A and Hb E percentages which were nearly the same.

GC-rich murine adenosine deaminase gene promoter supports diverse tissue-specific gene expression.

The murine adenosine deaminase (ADA) gene has a GC-rich promoter that is structurally typical of many mammalian "housekeeping" gene promoters. The ability of the ADA gene promoter to support diverse tissue-specific gene expression was investigated. Endogenous ADA gene expression in different mouse tissues was found to vary over a greater than 3000-fold range in a highly complex pattern. This range of expression was also observed in cultured human cell lines derived from different tissues. The ADA levels in all tissues and cell lines examined correlated closely with steady-state ADA mRNA levels. Several of the mouse tissues examined also showed stage-specific variation during postnatal development. In order to determine whether tissue-specific ADA expression was controlled by cis-acting sequences upstream of the coding region, constructs containing a reporter gene regulated by the ADA gene's 5' flanking sequences were used to generate transgenic mice. All transgene-expressing mice obtained showed diverse reporter gene expression in the tissues analyzed. Our results demonstrate that both in vivo and in the context of an integrated transgene this GC-rich promoter can support highly diverse gene expression in all tissues of the animal.

[Homozygote beta-thalassemia in 2 Dutch families]. / Homozygote beta-thalassemie in twee Nederlandse families.

beta-thalassaemia is a haematological disorder which is rare in The Netherlands although the influx of carriers from Mediterranean, West Indian, and South American countries has increased its frequency. Only a few homozygotes have been found among the original Dutch population. In this article, we describe the molecular abnormality observed in two such homozygotes. Both patients were mildly affected and had the same C-G mutation in the beta-globin gene promoter at position -87 relative to the Cap site of the beta gene. This mutation is known to cause a mild beta +-thalassaemia. Additional studies of the epsilon-gamma-delta-beta-globin gene complex on chromosome II, i.e. haplotype analyses, identified three different haplotypes in the two patients. However, crossovers might have been responsible for these differences because the 3' haplotype, which involves restriction sites within and 3' to the beta-globin gene, was the same for all four chromosomes in the two homozygotes.

First observation of haemoglobin Malay alpha 2B2 26 (B1) Asn----Ser--a case report.

A new haemoglobin, Haemoglobin Malay is described in a 22 year old Malay. Structural analysis showed a AAC----AGC mutation in codon 17, with the production of an abnormal beta chain (beta Malay) that has an Asn----Ser substitution at position beta 19. This haemoglobin variant could not be detected by conventional procedures.

Molecular characterization of beta-globin gene mutations in Malay patients with Hb E-beta-thalassaemia and thalassaemia major.

This study concerned the identification of the beta-thalassaemia mutations that were present in 27 Malay patients with Hb E-beta-thalassaemia and seven Malay patients with thalassaemia major who were from West Malaysia. Nearly 50% of all beta-thalassaemia chromosomes carried the G----C substitution at nucleotide 5 of IVS-I; the commonly occurring Chinese anomalies such as the frameshift at codons 41 and 42, the nonsense mutation A----T at codon 17, the A----G substitution at position -28 of the promoter region, and the C----T substitution at position 654 of the second intron, were rare or absent. Two new thalassaemia mutations were discovered. The first involves a frameshift at codon 35 (-C) that was found in two patients with Hb E-beta zero-thalassaemia and causes a beta zero-thalassaemia because a stop codon is present at codon 60. The second is an AAC----AGC mutation in codon 19 that was present on six chromosomes. This substitution results in the production of an abnormal beta chain (beta-Malay) that has an Asn----Ser substitution at position beta 19. Hb Malay is a 'Hb Knossos-like' beta +-thalassaemia abnormality; the A----G mutation at codon 19 likely creates an alternate splicing site between codons 17 and 18, reducing the efficiency of the normal donor splice site at IVS-I to about 60%.

Studies of beta-thalassemia mutations in families living in three provinces in southern China.

beta-Thalassemia is a common disease in Southern China and 10 different mutations or frameshifts are responsible for most types of beta-thalassemia in this area. We studied 126 chromosomes of 80 beta-thalassemia patients from the Guangxi, Guangdong, and Sichuan Provinces using the polymerase chain reaction followed by dot-blot hybridization with specific oligonucleotide probes. The most common mutation in the three provinces is the frameshift at codons 41-42, followed by the A----T mutation at codon 17. The A----G mutation at nt -29 of the promoter is common in Sichuan but not in the other two provinces. Three mutations (T----C at nt -30; G----T at IVS-I-1, and G----C at IVS-I-5) were not observed. These data were used to initiate a prenatal diagnosis program using the same techniques for identification. Eleven fetuses at risk for beta-thalassemia have been diagnosed.

Identification of base substitutions in the promoter regions of the A gamma- and G gamma-globin genes in A gamma- (or G gamma-) beta+-HPFH heterozygotes using the DNA-amplification-synthetic oligonucleotide procedure.

DNA amplification combined with hybridization with 32P-labeled synthetic oligonucleotide probes has been used to identify base substitutions in the 5' promoter region of the A gamma globin gene in members of eleven families from China, Sardinia, Canada, and the United States who had a heterozygosity for the A gamma-beta+-hereditary persistence of fetal hemoglobin (HPFH), and in members of six black families with a possible G gamma-beta+-HPFH heterozygosity. All three known A gamma types were observed, ie, the British type (-198, T----C), the Chinese type (-196, C----T), and the Green type (-117, G----A); the latter has been found in a black family. Of the six families with G gamma-beta+-HPFH, three had C----G at -202 and none T----C -175. Conditions for hybridization of amplified DNA with the specific probes are provided and the usefulness of the technique is discussed. The increase in numbers of A gamma(G gamma)-beta+-HPFH heterozygotes with specific base substitutions greatly enhances the probability of a direct correlation between these substitutions and the increase in the production of a specific gamma chain.

Variation in clinical severity among patients with Hb Lepore-Boston-beta-thalassaemia is related to the type of beta-thalassaemia.

Clinical and haematological observations, made for 10 Yugoslavian patients with the Hb Lepore-beta-thalassaemia condition, suggested a considerable variation from severe disease and complete blood transfusion dependency to a moderate, compensated, anaemia without major complications and without a need for regular blood transfusions. As the type of Hb Lepore was the same in all patients (Lepore-Boston-Washington) and an alpha-globin gene deficiency was absent, it was concluded that the type of beta-thalassaemia determined the severity of the disease. Six patients with severe disease had one of the following three beta-thalassaemia determinants: IVS-1 position 110 G----A, exon 2 codon 39 C----T, and IVS-1 position 1 G----A, while the three patients with mild disease had the Portuguese type of thalassaemia which is caused by the T----C substitution at position 6 of the IVS-1. In one patient with severe disease the beta-thalassaemia determinant remained unknown. Our observations are consistent with those made for thalassaemia patients with a homozygosity for these determinants.

Newer developments in the identification of beta-thalassemia.

One of the easiest and most sensitive methods of detecting mutations in the beta-globin gene leading to beta-thalassemia is by the use of oligonucleotide probes. The current method involves digestion of 5-10 micrograms of genomic DNA followed by gel electrophoresis, and blotting onto nitrocellulose. The membrane is then hybridized with a 32P-radiolabeled oligonucleotide probe containing the specific point mutation of interest. Finally, the membrane is subjected to X-ray film for 3-10 days. We wish to report a method for detecting these mutations which involves 1 microgram of genome DNA or less. The method involves the use of a gene amplification technique. A series of primers are synthesized which span the beta-globin gene. In each primer set, one primer is complementary to the beta-gene and the other primer is complementary to the non-coding strand. The suspected mutation point is located between these two primers. With the use of this primer set, the beta-globin gene region is amplified by denaturing, annealing, and DNA synthesis. The amplification cycle is repeated 25 to 30 times. The amplification is conducted using the Klenow fragment of DNA polymerase I or Taq polymerase in the presence of all four deoxynucleotide triphosphates. The resulting amplified DNA is applied to a nylon membrane with the aid of a dot-blot apparatus and directly hybridized with normal and mutant deoxynucleotide probes. The entire process requires one to two days. More than 300 beta-thalassemia homozygotes have been identified in our laboratories; over 20 different mutations have been observed.

Biochemical and molecular aspects of beta-thalassemia types in northern Sardinia.

Forty-three patients with beta-thalassemia from Northern Sardinia (31 severe and polytransfused, six follow-up babies, five adults with mild thalassemia who were not transfusion dependent, and a young transfused patient was also affected by a disease of intermediate severity) were studied in order to establish the fetal hemoglobin composition, restriction fragment length polymorphism haplotypes at the beta-globin gene cluster, and the type(s) of mutation. Haplotype II was prevalent, [56/86 chromosomes (65%)], haplotype I was also fairly common, [22/86 chromosomes (25%)], while other types were relatively rare. The nonsense mutation at codon 39 was nearly exclusive, [76/80 chromosomes (95%)]. Other beta-thalassemia mutations occurred on chromosomes with haplotypes III, IX, X, and perhaps V, and a new type related to II. The mutated A gamma T gene was associated with type II, X, and the new type. Type IX was linked to a beta(0) gene and to an Xmn I site 5' to the G gamma gene, to a high G gamma globin level, and to a disease of mild severity. Type III was associated with a beta(+)-thalassemic gene. The (0)39 mutation linked to type II was associated with thalassemia intermedia in three patients.