Factor X Frankfurt I: molecular and functional characterization of a hereditary factor X deficiency (Gla+25 to Lys).

A family with hereditary factor X deficiency is presented. One member, a 25-year-old man, showed a mild bleeding tendency. His factor X activity (extrinsic: 56%; intrinsic: 55%; Russell's viper venom: 57%) and his level of circulating factor X antigen (55% of normal) were markedly reduced. Analysis of his factor X gene revealed a single point mutation within exon II resulting in the substitution of +25 Gla (GAA) by Lys (AAA). The mutation was determined by gene analysis to be heterozygous in this patient, his mother and one of his brothers. Clotting assays of factor X purified from the plasma of the index patient revealed an activity of 89% of normal upon activation with Russell's viper venom, 77% of normal in the intrinsic and 81% of normal in the extrinsic coagulation pathway. The mutation responsible for the substitution of Lys for Gla+25 was introduced into an expression plasmid containing a wild type factor X cDNA and expressed in a mammalian cell line. Factor X antigen levels in the cell lysates and in the supernatant were identical in the mutant and wild type constructs. The specific activity of the factor X expressed from the mutant construct was 3% compared with the wild type construct. These data demonstrate that the substitution of Lys for Gla+25 results not only in a reduced level of factor X in the affected family members, but also in a substantial loss of specific factor X activity.

Primary treatment of endometrial carcinoma with high-dose-rate brachytherapy: results of 12 years of experience with 280 patients.

PURPOSE: This study aimed to evaluate the efficacy of high-dose-rate brachytherapy (HDRB) in the primary treatment of endometrial carcinoma. The results of 12 years of experience (1981-1992) covering 280 patients (mean age 72 years) and their follow-up over 10 years (mean 55 months) are reported. METHODS AND MATERIALS: Staging was based on clinical examination and fractionated curettage. There were 116 patients in clinical Stage Ia, 119 in Stage Ib, 37 in Stage II, and 8 in Stage III. HDRB was performed four to five times (8.5 Gy) with a one-channel intracavitary applicator and one to two times (7 Gy) with an intravaginal cylinder applicator. Overall and disease-specific survival, local control according to stage and histology, and late side effects were analyzed retrospectively (actuarial method). RESULTS: At 5 years, overall survival, disease-specific survival, and local control were 52.7%, 76.6%, and 75.4% (Stage Ia: 63.9%, 84.9%, and 86.0%; Stage Ib: 47.3%, 73.3%, and 68.8%; and Stage II: 40.2%, 68.6%, and 60.5%) according to histopathologic Grade 1: 65.1%, 83.5%, and 77.7%; for Grade 2: 44.7 %, 75.4%, and 75.8%; and for Grade 3: 37.7%, 63.9%, and 74.1%. Eight patients showed progressive disease, 64 developed recurrence after a median of 13 months (45 of whom had a local recurrence only, and 6 of whom had a local recurrence with distant metastases), 6 developed a lymph node recurrence only, and 7 developed distant metastases only. The calculated probability for developing a Grade III late side effect was 5.2% at 5 years. CONCLUSION: At Stages Ia, Ib, and II in endometrial carcinoma, HDRB is a very effective treatment modality with acceptable local control rates and disease-specific survival for patients who are not fit for surgery. During the time frame of 12 years and in 280 patients the method has proven to have a low risk of acute complications and an acceptable risk of long-term side effects.

[The results of primary HDR-afterloading brachytherapy in corpus carcinoma]. / Ergebnisse der primären HDR-Afterloading-Brachytherapie beim Korpuskarzinom.

PURPOSE: In general the results of radiotherapy are regarded to be inferior compared to those of surgery, when it comes to the treatment of endometrial carcinoma, but some patients are elderly and have multiple medical problems, which make them inoperable. The risk of intracavitary radium therapy, caused by immobilisation, can be reduced by the use of fractionated high-dose-rate afterloading brachytherapy. With this method there are only very few results reported. PATIENTS AND METHODS: Treatment results of HDR brachytherapy (4 to 5 times 8.5 Gy, one-channel applicator, intracavitary and 1 to 2 times 7 Gy intravaginal) at the University Hospital Vienna were analysed retrospectively (actuarial method [Kaplan-Meier]) regarding overall survival and recurrence-free interval according to stage and histology. Over a period from April 1981 until December 1992 325 patients were treated by this technique alone or combined with external beam therapy. Two hundred and eighty patients could be evaluated. Staging based on clinical examination and fractionated curettage. RESULTS: Five-year overall survival was 58.1%, in stage Ia 68.5%, stage Ib 49.9%, stage II 48.7%, according to histopathologic grading 1 68.5%, grade 2 53.2%, grade 3 37.5%. 64 patients developed a recurrence after a median of 13 months, 45 of those a local recurrence only, 6 a local recurrence with distant metastases, 6 a lymph node recurrence only and 7 patients distant metastases only. CONCLUSION: These results are at least comparable to those of intracavitary radium therapy and low-dose-rate afterloading techniques. Better local control rates should be obtained by the Heyman packing method using Norman-Simon applicators based on individualised brachytherapy treatment planning, which optimises dose distribution according to the target volume based on computerised imaging.

Effects of sodium bromide on the biosynthesis of thyroid hormones and brominated/iodinated thyronines.

The influence of bromide on thyroid function was studied in iodine-deficient rats, fed on a diet containing 4-16 g/kg sodium bromide for 4 weeks. Measurement of total and free thyroxine and thyroid-stimulating hormone in blood, as well as the thyroid hormones in the thyroid gland, revealed typical signs of hypothyroidism, which were significantly enhanced by bromide intake. Special attention was paid to the possible formation of bromo/iodosubstituted thyronines in the thyroid. These measurements were performed by high-performance liquid chromatography with off-line radioimmunoassay detection. Such thyroid hormone analogues could be detected in all groups of animals with additional bromide intake, but the amounts were found to be too low to compensate adequately for the reduced amounts of thyroid hormones. The results of this study also indicate that bromide toxicity is dependent upon the state of the iodine supply, which should be taken into account for evaluation of acceptable daily intake values for bromide.

Androgen binding in cultured human fibroblasts from patients with idiopathic hypospadias.

Androgens stimulate development and growth of the external male genitalia. Since hypospadias represents the most common congenital abnormality in the male newborn and the mechanism of action in this disorder is still unclear, androgen binding was assessed in cultured fibroblasts from biopsies from genital skin of 10 patients with idiopathic hypospadias. For comparison, binding was determined in corresponding samples from 8 males with normal penile development and from 9 patients with known androgen resistance syndromes (testicular feminization, Reifenstein syndrome, pseudovaginal perineoscrotal hypospadias). Finally, binding was measured in 10 samples of nongenital skin. Maximum specific binding (Bmax) in idiopathic hypospadias varied from 3.2 to 15.5 (median 6.6) fmol.mg protein-1. Bmax in samples of persons with normal genital development was between 12.2 and 17.9 fmol.mg protein-1 (median 13.2). Bmax in samples of patients with known androgen resistance syndromes was exactly in the range reported previously in the literature. It is evident that Bmax in samples of patients with idiopathic hypospadias differs significantly (P less than 0.01), (Mann Whitney U-test) from those with normal genital development. Thus it seems reasonable to conclude that in some patients with idiopathic hypospadias the genital defect is caused by receptor deficiency.

Is heterozygosity for the steroid 21-hydroxylase deficiency responsible for hirsutism, premature pubarche, early puberty, and precocious puberty in children?

We applied the ACTH-stimulation test developed in our laboratory for the detection of heterozygous carriers of the 21-hydroxylase deficiency gene to patients suffering from hirsutism (n = 89), premature pubarche (n = 75), early puberty (n = 37), and precocious puberty (n = 22). While, in the general population, this test is positive in less than 2%, we found in 33% of hirsute patients, in 41% of patients with premature pubarche, and in 33% of patients with early puberty a hormonal response similar to the one seen in heterozygous carriers for the 21-hydroxylase defect. In contrast, only 18% of patients with precocious puberty responded abnormally. Thus we speculate that, at least in some patients with hirsutism, premature pubarche, and early puberty, heterozygosity for the 21-hydroxylase defect plays a major role in the pathogenesis of these disorders.

Genetic differences between the salt-wasting, simple virilizing, and nonclassical types of congenital adrenal hyperplasia.

Human leukocyte antigen (HLA) alleles and plasma 17-hydroxyprogesterone levels after ACTH stimulation were studied in 134 German families of patients with the salt-wasting (SW), simple virilizing (SV), or nonclassical (NC) late-onset form of congenital adrenal hyperplasia (CAH). Unexpected hormonal evidence for CAH was found in 6 otherwise healthy members of the relatives' group, who, therefore, were considered to be NC cryptic cases. HLA typing revealed a genetic difference between the 2 classical disease forms; SW CAH was strongly associated with Bw47, whereas SV CAH was closely linked to B5(w51). It also confirmed the nearly complete connection of NC CAH with B14. These alleles, especially Bw47 and B14, are mostly components of normally rare haplotypes: A3,Bw47,DR7 and Aw33,B14,DR1, respectively. They do not occur in the families' disease-unaffected haplotypes. Thus, it may be that all or almost all individuals from the general population bearing 1 of them are in fact CAH heterozygotes. Moreover, it seems possible to predict the severity of an infant's disease from his genomic type. The HLA linkage data were consistent with those obtained from ACTH testing, which showed significantly higher 17-hydroxyprogesterone increases in the genetically defined heterozygous relatives of SW patients than in the respective members of SV families. Of the families, 2 were also informative for mapping of the CAH disease gene(s) within the HLA-B to Glo interval.

Different gene defects in the salt-wasting (SW), simple virilizing (SV), and nonclassical (NC) types of congenital adrenal hyperplasia (CAH).

HLA (human leucocyte antigens) alleles and plasma 17-hydroxyprogesterone levels after ACTH stimulation were studied in 134 German families of patients with salt-wasting (SW), simple virilizing (SV), and nonclassical (NC) late-onset forms of CAH. HLA typing revealed a genetic difference between the two classical disease forms. SW-CAH was strongly associated with Bw47 and SV-CAH was closely linked to B5. The nearly complete connection of NC-CAH with B14 was confirmed. Bw47 and B14 were mostly components of the normally rare haplotypes A3, Bw47, DR7 and Aw33, B14, DR1, respectively. They did not occur in the families' disease-unaffected haplotypes. The HLA linkage data were consistent with those obtained from the ACTH stimulation test which showed a higher 17-hydroxyprogesterone increase in the group of genetically defined heterozygous relatives of SW patients than in the groups of heterozygous members of SV and NC families.

Diagnosis of homozygosity and heterozygosity in congenital adrenal hyperplasia (CAH) and control of treatment.

In congenital adrenal hyperplasia the incidence of 21-hydroxylase deficiency is very high (approximately 1:7,000), whereas other enzyme defects such as 11-hydroxylase deficiency, 17-hydroxylase deficiency and 3 beta-hydroxysteroid dehydrogenase deficiency are much less frequent. The various forms of enzyme defects can be diagnosed by determining specific plasma steroids or specific urinary steroid metabolites. A new semi-automatic capillary gas liquid chromatography method has been introduced for the diagnosis of CAH and assessment of therapy. Heterozygous carriers of 21-hydroxylase deficiency can be detected in the general population by measuring 17-hydroxyprogesterone plasma levels after ACTH stimulation; however, results overlap with the general population. In families with a CAH index case, heterozygosity and homozygosity for the 21-hydroxylase deficiency gene can be detected by HLA-typing. 21-hydroxylase deficiency can be diagnosed prenatally by HLA-typing or by determining 17 OH-progesterone levels in the amniotic fluid.