Steroid binding sites in liver membranes: interplay between glucocorticoids, sex steroids, and pituitary hormones.

Steroid hormones activate target cells through specific receptors that discriminate among ligands based upon recognition of distinct structural features. For most known steroids, membrane and nuclear receptors co-exist in many target cells. However, while the structure of the nuclear receptors and their function as transcriptional activators of specific target genes is generally well understood, the identity of the membrane receptors remains elusive. Using pharmacological and biochemical approaches, we are beginning to characterize receptors for glucocorticoids and anabolic-androgenic steroids in male rat liver membranes. Male rat liver endoplasmic reticulum contains two steroid binding sites which are functionally related and associated with a 90-134 kDa oligomeric protein: (1) the low-affinity glucocorticoid binding site (LAGS), composed at least in part of two peptides (37 and 53 kDa) that bind glucocorticoids and (2) the stanozolol binding protein (STBP), composed at least in part of three peptides (22, 31, and 55 kDa) that bind the synthetic androgen stanozolol. These steroid binding proteins have many properties different from those of classical nuclear receptors, with the salient differences being a failure to recognize "classical" ligands for nuclear receptors together with marked differences in biochemical properties and physiological regulation. The mechanism of interaction of glucocorticoids with the LAGS can be clearly distinguished from that with STBP. Moreover, STBP shows an extremely narrow pharmacological profile, being selective for ST and its analog, danazol, among more than 100 steroids and non-steroidal compounds that were assayed, including those that are able to displace glucocorticoids from the LAGS. The level of LAGS activity undergoes dramatic variations following changes from the physiological serum levels of thyroid hormones, glucocorticoids, GH, vitamin A, and E2. However, neither thyroid hormones nor GH have a critical role on STBP activity. The STBP is functionally related to LAGS. We have suggested a novel mechanism for STBP whereby membrane-associated glucocorticoid binding activity is targeted by stanozolol (and 16beta-hydroxylated stanozolol): stanozolol modulates glucocorticoid activity in the liver through negative allosteric modulation of the LAGS resulting in an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.

High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability.

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

Low levels of cathepsin D are associated with a poor prognosis in endometrial cancer.

Total cytosolic cathepsin D (Cat D) levels were estimated by an immunoradiometric assay in a series of 156 consecutive patients with surgical stages I-III primary endometrial adenocarcinoma. Simultaneously, the tissue content of both oestrogen (ER) and progesterone (PR) receptors, and p185HER-2/neu, DNA content (ploidy), and the fraction of S-phase cells (S-phase) were also estimated. Tumoral Cat D content ranged from 0 to 243 pmol mg(-1) protein (median 44 pmol mg(-1) protein) and was not associated with any of the established clinicopathological and biological prognostic variables, with the exception of a weak positive correlation with the tumoral p185HER-2/neu levels. Univariable analysis performed on a subset of 97 patients, followed for a minimum of 2 years or until death, showed that patient age at diagnosis, high histological grade, advanced surgical stage, vascular invasion, positive peritoneal cytology, low levels of Cat D, negative ER and PR status, aneuploidy, and high S-phase were predictive of the presence of persistent or recurrent disease. However, multivariable analysis revealed that only histological grade, surgical stage, Cat D and PR were significantly associated with the patient's outcome. From these findings, we conclude that Cat D is an independent prognostic factor in endometrial adenocarcinoma, its low levels being associated with a worse clinical outcome.

Thyroid hormone regulation of rat hepatocyte proliferation and polyploidization.

The liver of adult mammals contains various classes of polyploid hepatocytes produced by a process that is partially regulated by hormones. However, it is not well understood how the hormones affect the rate of hepatocyte proliferation under physiological conditions. Here we have studied the specific roles of 3,5,3'-triiodothyronine (T3), growth hormone (GH), and sex steroids on the percentage of diploid nuclei in S phase and on the population of liver tetraploid (4C) cell nuclei in several rat model systems. Gonadal steroids had no effect on the S phase but account for gender differences in the 4C nuclei. Hypophysectomy in adult male rats produced a moderate decrease in 4C nuclei that was reversed by treatment with 25 micrograms T3. kg-1. day-1, whereas treatment with 200 micrograms human recombinant GH (hGH). kg-1. day-1 was ineffective. Rats made hypothyroid by methimazole treatment of dams and pups until death showed a low S phase and only 5% of 4C nuclei at 70 days of age. T3 significantly increased the S phase 24 h after administration and restored the adult normal level of 4C nuclei after 10 days of treatment. hGH did not affect the 4C nuclei or the S phase in the hypothyroid rats. These results suggest that the processes of hepatocyte proliferation and polyploidization of the rat liver are under endocrine control, with thyroid hormones playing the essential regulatory role.

Tamoxifen aziridine binding to cytosolic proteins from human breast specimens is negatively associated with estrogen receptors, progesterone receptors, pS2, and cathepsin-D.

[3H]Tamoxifen Aziridine ([3H]TAZ) is a derivative of the antiestrogen tamoxifen that covalently labels the Estrogen Receptor (ER), and perhaps other uncharacterized proteins. In a previous article we described that [3H]TAZ binds to a cytosolic protein from human uterine tissues that shares some, but not all, the ER properties. Here we have extended these studies to [3H]TAZ binding to cytosol proteins from human breast cancer specimens, and studied its quantitative association with other molecular markers and clinico-pathological variables. Cytosols were obtained in hypotonic buffer containing 20 mM molybdate and protease inhibitors, incubated with [3H]TAZ, and subjected to Sucrose Gradient Analysis (SGA). A [3H]TAZ labeled peak that consistently migrated with the 4S fractions was found in most of the assayed cytosols (range of 0 to 1278 fmol/ mg p.). The 4S peak of [3H]TAZ was partially inhibited by both estrogens and antiestrogens. When [3H]E2 was used instead of [3H]TAZ, only an 8S peak was detected. [3H]TAZ was covalently bound to a protein with an apparent MW of 65 kDa, as determined by SDS-PAGE and fluorography. The mean of [3H]TAZ binding was significantly higher in the subgroups of samples classified as ER-, PR-, pS2- or cathepsin D-, than in the respective positive subgroups (P < 0.01 in all the cases). [3H]TAZ binding was not associated with clinico-pathological variables, except that its mean was significantly larger in tumors larger than 5 cm than in smaller tumors. These results, and those previously reported, suggest that: 1) [3H]TAZ labels a cytosolic protein present in human breast cancers and uterine tissues that does not share all the ER properties, and 2) the [3H]TAZ binding by breast cancer cytosols is negatively associated with markers of estrogenic dependency, and its quantification may provide valuable information on antiestrogen responsiveness of a given tumor.

Quantitative analysis of p185(HER-2/neu) protein in breast cancer and its association with other prognostic factors.

The total cellular p185(HER-2/neu) protein (p185) content was measured by ELISA in 346 invasive primary breast cancers, and the results were compared with those of estrogen (ER) and progesterone (PR) receptors, pS2 and Cathepsin D (Cat D) content. At a cut-off level of 260 fmol/mg protein, 53 of the 346 tumors (15%) were p185-positive. A significant positive correlation was observed between p185 levels and those of Cat D, and a weaker, though significant, positive correlation with ER, and pS2 levels, but not with those of PR. However, when only the 293 p185-negative tumors were considered, the correlation between p185 and ER improved substantially, and statistical significance was reached for PR. p185-positive tumors exhibited lower ER and PR content and higher Cat D content than p185-negative tumors. The pS2 content, in contrast, did not undergo significant variation. Tumors considered to be p185-positive were significantly more frequently positive for Cat D at the cut-off of 45 pmol/mg protein, and were more frequently negative for ER and/or PR, but only significant at the cut-off of 15 fmol/mg or higher for both steroid receptors. Finally, p185 status was not associated with menopausal status, tumor size, axillary-lymph-node invasiveness or distant metastases. These results suggest that 260 fmol/mg protein as the cut-off for p185 allows the identification of a tumoral sub-population with a more aggresive phenotype.

Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer.

HER-2/neu oncogene status and total cellular p185HER-2 content were simultaneously analyzed in 415 invasive breast-cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut-off value of 1.7 for the ratio between the intensity of the HER-2/neu gene band and the reference gene band, to consider the HER-2/neu gene amplified, and of 260 fmol/mg protein, to consider p185HER-2 over-expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER-2/neu gene amplification. Of these tumors, 87% showed over-expression of the p185HER-2. Of the remaining 352 specimens that did not display HER-2/neu gene amplification, 97% showed no p185HER-2 over-expression (p < 0.0001). In 40 selected samples with a p185HER-2 level lower than 260 fmol/mg protein, the degree of p185HER-2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185HER-2 level higher than 260 fmol/mg protein exhibited a considerable degree of p185HER-2 phosphorylation (p < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER-2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER-2/neu oncogene is amplified or p185HER-2 is over-expressed.

Age-related changes in the induction of tyrosine aminotransferase by dexamethasone: correlation with the low-affinity glucocorticoid binding sites.

Rat liver membranes contain Low-affinity glucocorticoid binding sites (LAGS), capable of binding with low affinity (Kd approximately 100 nM) endogenous glucocorticoids. Unlike the glucocorticoid receptor (GR), the LAGS level undergoes abrupt changes throughout life. The investigation of these changes may be useful in determining whether the LAGS are involved in the cellular response to glucocorticoids. For this purpose, we have studied glucocorticoid induction of tyrosine aminotransferase (TAT), and its relationship with the LAGS level in adrenalectomized and fasted rats of different ages. No significant differences in the GR level, or in its Kd and activation, were observed among rats of 1, 3, and 12 months of age. On the other hand, the LAGS level showed an important variation with age, from almost undetectable in 1-month-old rats, to a maximum value in 3-month-old rats. With respect to TAT activity, an increase with age in the threshold of response to dexamethasone (DEX) administration was observed. The smallest dose of DEX capable of provoking a significant TAT induction rose from 0.1 microgram/kg body wt. in 1-month-old rats to 10 micrograms/kg body wt. in 12-month-old rats. However, the smallest dose of DEX able to elicit the maximal response was 10 micrograms/kg body wt. in all the assayed ages. This dose provoked a 40% decrease in the GR level, but did not significantly modify the LAGS content. From these results, we conclude that there is an age-related change in the threshold of response to DEX that cannot be explained by the GR-glucocorticoid interaction. The possibility that the LAGS modulate the cell response to glucocorticoids arises from the coincidence of this change with that observed in the LAGS concentration throughout life.

Ethinylestradiol interacts with liver microsomes and induces binding sites for steroid hormones in the male rat liver.

The present work focuses on the interaction of 17 alpha-ethinyl estrogen derivatives with the [3H]dexamethasone ([3H]DEX) binding site from male rat liver microsomes and the induction of this site by the in vivo administration of natural and synthetic estrogens. [3H]DEX binds to a single-saturating binding site (Kd = 100 nM; maximal binding = 13 pmol/mg of protein) in the liver microsomes. In competition experiments, ethinylestradiol (EE2) and mestranol were able to inhibit [3H]DEX binding to microsomes, whereas natural estrogens, tamoxifen or estrogen sulfates were ineffective. Saturation analysis performed by incubating [3H]EE2 with liver microsomes revealed the existence of a low-affinity (Kd = 280 +/- 30 nM) and high capacity (maximal binding = 16 +/- 2 pmol/mg of protein) binding site. Saturation, competition and dissociation experiments suggest that [3H]DEX and [3H]EE2 interact with the same microsomal entity. Synthetic and natural estrogens increased the hepatic expression of the [3H]DEX binding site in immature, hypothyroid and hypophysectomized male rats. This induction required at least 2 days of treatment, and could only be achieved by pharmacological doses of estrogens.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of growth hormone in regulation of low affinity glucocorticoid-binding sites from male rat liver microsomes.

GH participates in the regulation of the expression of several hepatic proteins, some of which are subject to multihormonal control. We have previously shown the participation of glucocorticoids and thyroid hormones in the regulation of the hepatic low affinity glucocorticoid-binding sites (LAGS). Here, we provide evidence that also implicates GH in the endocrine control of the LAGS through the use of several animal models, all of them having a very low or undetectable plasma GH level: the hypothyroid (TX), the hypophysectomized, and the GH-deficient Lewis-derived dwarf rat. In dwarf rats, the level of LAGS was only 35% of that found in normal Lewis rats. Treatment of these rats with human (h) GH significantly increased the LAGS level in a dose-response manner. In TX rats, hGH treatment provoked a significant increase in the LAGS level (from 0.9 +/- 0.2 to 7.2 +/- 0.8 pmol/mg protein), so that it represented about 65% of the level found in intact animals. In both hypothyroid-adrenalectomized and hypophysectomized rats, the isolated effect of hGH was not as pronounced as in TX or dwarf rats; however, a potentiation of the effect of hGH was observed when this hormone was injected together with corticosterone acetate. On the other hand, when hGH, T3, and corticosterone acetate were given in combination to hypophysectomized rats, hGH and T3 behaved as agonists of the LAGS induction at T3 doses lower than or equal to 0.1 microgram/100 g BW and as antagonists at T3 doses higher than this. When T4 was used instead of T3, this hormone was capable of potentiating the effect of hGH at doses lower than or equal to 1.5 micrograms/100 g BW. From these results we conclude that 1) GH as well as thyroid and glucocorticoid hormones participate in the endocrine regulation of the LAGS; and 2) under physiological conditions, it is conceivable that GH, thyroid hormones, and glucocorticoids act synergistically in the endocrine regulation of the LAGS.

The estradiol induction of the microsomal low-affinity glucocorticoid binding sites (LAGS) in the male rat liver is independent of the endocrine status.

The low-affinity glucocorticoid binding sites (LAGS) are entities present in the microsomal fraction of the rat liver, capable of binding several glucocorticoids and progesterone with low affinity. The present work focuses on the demonstration that estradiol exerts a powerful stimulatory effect on the LAGS concentration. For this purpose, we studied the effect of this hormone in immature, hypothyroid, and hypophysectomized rats, three experimental models which present a very low level of LAGS. In all of them, estradiol showed ability to significantly increase the level of LAGS. The positive results obtained in hypophysectomized rats point to a direct action of estradiol on the liver. In immature rats, the estradiol induction of the LAGS was shown to be especially slow, 3-4 days after estradiol administration being necessary to obtain a significant rise in the level of LAGS. Moreover, the dose of estradiol necessary to obtain the LAGS induction in these rats (0.5 mg/100 g body weight) was clearly supraphysiological. From these data we concluded that: (A) estradiol is a powerful stimulator of the LAGS concentration, its effect probably being exerted directly on the liver; and (B) to elicit its effect, estradiol does not need the participation of other hormones known to be implicated in the endocrine regulation of the LAGS.

Thyroid hormones and glucocorticoids act synergistically in the regulation of the low affinity glucocorticoid binding sites in the male rat liver.

The low affinity glucocorticoid binding sites (LAGS) have been described and partially characterized in both the nuclei and microsomes of rat liver. The LAGS concentration is under endocrine regulation, as proved by their decrease after adrenalectomy and their almost complete disappearance after hypophysectomy. This article describes new data that also implicate the thyroid hormones in the endocrine regulation of LAGS. The LAGS were measured by [3H]dexamethasone exchange assay in crude microsome suspensions of rat liver. Propylthiouracil-induced hypothyroidism (TX) provoked a 90% reduction in the LAGS levels with respect to the control value. The administration of T3 to TX rats was able to completely restore the LAGS level. On the other hand, adrenalectomy (ADX) provoked a 50% decrease in LAGS levels, and this effect could be reverted by treatment with corticosterone acetate. TX rats that were also adrenalectomized (TX-ADX) showed a LAGS level similar to that of the TX rats. However, treatment of these rats with T3 was much less effective than in TX rats. A complete restoration of the LAGS level in TX-ADX rats could be achieved only with a combined treatment of corticosterone acetate plus T3. Similar results to those obtained in TX-ADX rats were also obtained in immature or hypophysectomized rats, two experimental models known to possess very low or undetectable levels of LAGS. From these findings we conclude that: 1) thyroid hormones, as well as glucocorticoids, play an important role in the regulation of the LAGS level; 2) glucocorticoids and thyroid hormones act synergistically in the endocrine regulation of LAGS; and 3) the results obtained in the hypophysectomized rats point to a direct action of glucocorticoids and T3 on the LAGS level of the rat liver.

Molecular heterogeneity of beta-thalassemia intermedia in Turkey.

A total of 38 patients with beta-thalassemia intermedia from 30 families were were studied. Twelve of the thirty unrelated patients had beta zero-thalassemia which was due to a homozygosity for one of two different thalassemia defects, namely the frameshift at codon 8, and the IVS-II-1 G----A mutation. Another mild variation, a beta +-thalassemia, was a homozygosity for the mutation of T----C at position 6 of IVS-1 (10 patients). Compound heterozygosities for mild thalassemic determinants or for one mild and one severe beta-thalassemic determinant were also found in some patients with beta-thalassemia intermedia. The mutations at beta-39 and IVS-I-110 were the most commonly occurring thalassemic determinants in these patients. Correlations between genotype and phenotype indicated significant differences in some of the hematological parameters among patients with the IVS-I-6 and the frameshift at codon 8, IVS-I-6 and IVS-II-1, and the frameshift at codon 8 and IVS-II-1 mutations.

Beta-thalassemia intermedia in two Turkish families is caused by the interaction of Hb Knossos [beta 27(B9)Ala----Ser] and of Hb City of Hope [beta 69(E13)Gly----ser] with beta (0)-thalassemia.

We have studied a few members of two Turkish families, who had a beta-thalassemia of the intermediate type. An abnormal hemoglobin was found in both families, which when present in association with beta(0)-thalassemia was considered to be the primary cause for the increased severity of the disease. In the first family this variant was Hb Knossos [beta 27(B9)Ala----Ser] which occurred together with the frameshift in codon #8 type of beta(0)-thalassemia. This compound heterozygosity, observed for the first time in the Turkish population was characterized by a considerable increase in Hb F production, mainly of the G gamma type, as expected for a chromosome with haplotype IV. In the second family, the variant was Hb City of Hope [beta 69(E13)Gly----Ser] which was present in combination with an unknown type of beta-thalassemia. The increase in Hb F production in the compound heterozygote was minimal. Reversed phase high performance liquid chromatography and the DNA amplification-synthetic oligonucleotide probe procedure were major tools in identifying the different abnormalities.

Monoclonal antibody characterization of progesterone receptors, estrogen receptors and the stress-responsive protein of 27 kDa (SRP27) in human uterine leiomyoma.

Uterine leiomyoma occurs in one of every four or five women during their reproductive life. Its origin is unknown but it is accepted that estrogens play a significant role in its development. In order to learn more about the estrogen dependency of leiomyoma, the biochemical and immunological properties of two markers of estrogen response in target cells (the progesterone receptor (PR) and the stress-responsive protein of 27 kDa (SRP27)) were studied in leiomyoma. The ER (estrogen receptor) and PR content were determined by conventional DCC exchange assays. Specific anti-ER, anti-PR and anti-SRP27 monoclonal antibodies were used in immunoblots and immunohistochemical (IHC) studies. The binding properties of PR from cytosol of leiomyoma showed a Kd of 0.8-1.3 nM, which is in the range described for other human tissues. 80% of all studied leiomyoma contained PR, in a range of 805-2000 fmol/mg protein. The Kd for leiomyoma ER was 0.1-0.9 nM, and 84% of the samples were positive for ER. The PR of leiomyoma has the two A and B forms of 120 and 94 kDa, as shown in the immunoblot using the AB52 anti-PR monoclonal antibody. The IHC study revealed that the PR is concentrated in the cell nuclei, in the form of perinuclear bodies, with a homogeneous staining pattern from cell to cell. The leiomyoma fibres contain SRP27 in a higher concentration than the healthy myometrium. The leiomyoma SRP27 shows a typical doublet of 24 kDa and 27 kDa in immunoblot, the same as in MCF-7 cells. The IHC study revealed a high degree of organization of SRP27 in leiomyoma cells, suggesting that this protein may be part of the cytoskeleton. The results obtained show that human leiomyomas contain ER, PR and RSP27 with similar immunological and biochemical properties to those of other human tissues, including the MCF-7 breast cancer cell line.

Characterization of a newly discovered alpha-thalassaemia-1 in two Spanish patients with Hb H disease.

A new deletion of more than 27 kb, removing the psi zeta 1, psi alpha 2, psi alpha 1, alpha 2, alpha 1 and theta 1 globin genes has been found in four members of a Spanish family, including two patients with Hb H disease. The 5' end point of the deletion is located between the zeta and psi zeta genes, and the 3' end of the deletion is downstream of the 3' hypervariable region.

Inclusion body beta-thalassemia trait in a Swiss family is caused by an abnormal hemoglobin (Geneva) with an altered and extended beta chain carboxy-terminus due to a modification in codon beta 114.

We have analyzed the sequence of the beta globin gene of a chromosome that is linked to the occurrence of an inclusion body beta-thalassemia characterized in the heterozygote by moderate anemia, severe red cell abnormalities, splenomegaly, inclusion body formation, elevated Hb A2 levels, and an increased in vitro alpha/beta chain synthetic ratio. The data indicate a change in codon 114 from CTG (Leu) to -GG that resulted in a frameshift and the presumed synthesis of an abnormal beta chain that is 156 residues long with a completely different C-terminal amino acid sequence. The change in codon 114 gives a -GGGCCC- sequence that creates a new ApaI site; the resulting 2.6-kilobase fragment has been observed in all subjects with this thalassemia condition. Protein structural analyses failed to demonstrate any trace of the abnormal beta chain, even in reticulocytes and nucleated red cells that were isolated by density gradient centrifugation. The inclusion bodies appear to contain mainly normal alpha chains. It is assumed that the structure of the beta-Geneva chain prevents it from combining with normal alpha chains; this results in a rapid breakdown of the abnormal protein during the early stages of red cell maturation and an accumulation of free alpha chains.

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.

Two new large deletions resulting in epsilon gamma delta beta-thalassemia.

Detailed gene mapping data are provided for members of a Yugoslavian and Canadian family with a thalassemia heterozygosity characterized by mild anemia with severe microcytosis and hypochromia, normal levels of Hb A2 and slightly raised Hb F levels. The condition in both families results from large deletions (minimally approximately 148 kb in the Yugoslavian family and minimally approximately 185 kb in the Canadian family), which include all functional and psi genes of the beta globin gene cluster. The Canadian propositus was a newborn baby who has been followed for nearly 2 years; severe anemia developed some 30-40 days after birth when the Hb F level was still 70%; recovery was evident at the age of 90 days when the Hb F level had decreased to 40%.