Applicability of the SHBG androgen sensitivity test in the differential diagnosis of 46,XY gonadal dysgenesis, true hermaphroditism, and androgen insensitivity syndrome.

The sex hormone-binding globulin (SHBG) androgen sensitivity test has been used as a simple method to assess androgen receptor function in vivo. After a short term oral administration of the anabolic-androgenic steroid stanozolol the mean nadir serum concentration of SHBG is used as a measure of androgen response. We performed this test in order to evaluate its applicability in 16 patients with intersexual genital status: eleven with 46,XY gonadal dysgenesis and three with true hermaphroditism (group I), and in two patients with androgen insensitivity syndrome (AIS, group II). Ten healthy adult volunteers served as controls. In the two patients with AIS (group II) we found a diminished decrease of serum SHBG to 80.1 % and 80.7 %, respectively, indicating slight residual androgen responsiveness. In eleven patients of group I who were not on hormone replacement therapy, a mean nadir level of 51.7 +/- 8.7 % was found. In the controls the mean nadir serum SHBG level was significantly higher (62.7 +/- 5.2 %), probably due to interference of endogenous androgens and contraceptive medication with the stanozolol-induced SHBG decrease. In three gonadectomised patients who were on hormone replacement therapy the initial SHBG concentration was increased (513.5 +/- 239.1 nmol/l); the mean nadir SHBG concentration of 45.6 +/- 9.8 % of the initial level indicates an increased sensitivity of the test in patients in whom the counteracting ovarian androgens are absent. Our findings confirm that under standard test conditions the SHBG androgen sensitivity test is a simple diagnostic tool for the detection of androgen receptor malfunction.

Absence of Yq microdeletions in infertile men.

Microdeletions of the long arm of the Y chromosome (Yq) were described in men with idiopathic azoo- or oligozoospermia and seem to cause impairment of spermatogenesis. Deletion frequencies differ considerably among selected infertile men. The aim of this study was to investigate the prevalence of Yq microdeletions in patients with idiopathic infertility. Men with azoospermia or oligozoospermia resulting from endocrine or obstructive causes or with a constitutional cytogenetic anomaly were excluded. Ninety-seven patients presenting at infertility centers in Leipzig and Zurich were included in the study. Sixty-four (66%) of them were severely oligozoospermic (sperm concentrations < 5 x 10(6)/mL) and 33 (36%) were azoospermic. A sequence-tagged site (STS) PCR strategy was applied for the microdeletion screening. Thirteen STS markers spanning the whole euchromatic region of Yq were used. No Y-chromosomal microdeletion could be detected in these 97 infertile men. This result suggests a much lower Yq deletion frequency than previously thought, even among strictly selected patients with idiopathic azoo- or oligozoospermia.

Homozygous and heterozygous Arg614Cys mutations (1840C-->T) in the ryanodine receptor gene co-segregate with malignant hyperthermia susceptibility in a German family.

The determination of susceptibility to malignant hyperthermia (MH) by genetic investigation is a controversial issue because of the genetic heterogeneity of this disorder. The requirement for such an approach in MH diagnosis is a strong correlation between MH-associated genetic abnormalities and phenotypic findings in the in vitro contracture test (IVCT). After a severe clinical MH crisis during general anaesthesia a patient was diagnosed by the IVCT in which susceptibility to MH was confirmed. Genetic screening for MH-related mutations in the RYR1 gene revealed the presence of a homozygous 1840C-->T base exchange (Arg614Cys substitution) in this patient. A specific search for this defect in 20 relatives led to the identification of a total of 11 Arg614Cys mutations. Of these, 10 were heterozygous (including both parents) and one was homozygous (sister). Further IVCTs were subsequently performed on the parents of the index patient, the homozygous sister and all relatives who did not carry the Arg614Cys in order to determine the genotype/phenotype correlation. After analysing these data, and because of the strong correlation between clinical, phenotypic, and genetic results in the index patient, we assigned the diagnosis 'MHS' to all the remaining Arg614Cys mutation carriers of that family without performing the IVCT.

The p53 status in juvenile chronic arthritis and rheumatoid arthritis.

The aim of this study was to investigate the p53 status in two autoimmune diseases; juvenile chronic arthritis (JCA) and rheumatoid arthritis (RA). In a PCR-sequencing analysis of exons 4-9 of the p53 gene, no mutation was identified, except for the case of an RA synovectomy sample with two mutations of intron 7. p53 gene polymorphisms for codons 36, 47, and 213 were not detected. Codon 72 polymorphism showed an indication of an increased occurrence of the Pro/Pro allelotype in JCA. Expression of P53 protein was comparable for JCA and RA synovectomy samples. For all RA samples P53 protein was detectable, whereas one sample of a JCA patient failed to express P53 protein.

[Late diagnosis of Curschmann-Steinert myotonic dystrophy in a female patient with dilated cardiomyopathy and in her son]. / Späte Diagnose einer myotonen Dystrophie Curschmann-Steinert bei einer Patientin mit Dilatativer Kardiomyopathie und ihrem Sohn.

A 41 year old woman presented with dyspnoea at rest and swollen legs in the emergency room of our centre. She reported a history of slowly progressing dyspnoea and oedema in the legs. Physical examination showed signs of biventricular congestive heart failure and dysmorphia of the face. Routine laboratory examination revealed elevated CK levels without significant elevations of the CK-MB isoform. ECG showed complete left bundle branch block and first degree atrioventricular block. Echocardiography and angiography showed markedly reduced left ventricular systolic function, the ejection fraction was 25%. Coronary angiography excluded CAD and there was no evidence for congenital or valvular heart disease. The patient also reported a history of a serious complication during emergency general anaesthesia and cataracts of both eyes. Because of the clinical and chemical findings, the history of cataracts and complications during general anaesthesia, a systemic congenital disease of the muscular tissue was suspected. Molecular studies revealed a trinucleotide amplification at the myotonic dystrophy locus 19q 13.3, so the diagnosis myotonic dystrophy Curschmann-Steinert was established. The sixteen year old son of the patient suffered from an at this time unknown disease with retardation, muscular weakness and myotonia of the face. The diagnosis myotonic dystrophy was evident because of the clinical signs and the family history.

[In vitro contracture test and gene typing in diagnosing malignant hyperthermia. Each as an appropriate complement to the other method]. / In-vitro-Kontrakturtest und Gentypisierung in der Maligne Hyperthermie-Diagnostik. Ein Beispiel für die sinnvolle Ergänzung beider Methoden.

BACKGROUND: Malignant hyperthermia (MH) is an autosomal dominantly inherited disorder, triggered in susceptible individuals by inhalation anesthetics and depolarizing muscle relaxants such as succinylcholine. Because of its high sensitivity (97-99%) and specificity (93.6%) as well as the genetic heterogeneity of MH disorder, the in vitro contracture test (IVCT) following the European-MH-Group is considered to be the "Gold Standard" for phenotypical determination of predisposed patients. On the other hand mutations in the skeletal muscle ryanodine receptor gene (RYR1) are tightly linked with MH susceptibility. After detecting a C1840T-mutation (Arg614Cys) in the RYR1 gene in one individual of a large MH family, we searched for this mutation in the remaining family members and determined the concordance with IVCT. METHODS: According to the European standard protocol for MH, 43 individuals of a large MH pedigree were assigned the status of MH susceptible (MHS),--negative (MHN) or--equivocal (MHE). The genetic investigation of 44 family members for the Arg614Cys-mutation was carried out by restriction fragment analysis: Genomic DNA was prepared from EDTA whole blood followed by amplification of a 918 bp RYR1 gene fragment by polymerase chain reaction. In presence of the Arg614Cys-mutation digestion with the restriction endonuclease Rsal would result in different DNA fragments of the amplified sequence than in absence of mutation. RESULTS: According to the response to IVCT, 25 individuals phenotypically revealed MHS, 7 MHE and 11 MHN status. Out of the 44 family members screened genetically for the Arg614Cys-mutation, the mutation was detected in 23 individuals. Out of them 19 were MHS and one was MHEc. The mutation was absent in 9 predisposed individuals, but six of them were MHE and three MHS. The mutation was also present in three individuals who had no MH screening (IVCT) before. For these last mentioned individuals the diagnosis MHS was deduced from genetic results. CONCLUSION: Based on results of IVCT the identification of a MH associated mutation in a MH-family can make and support a correct MH diagnosis and can resolve MHE findings.

Pitfalls in prenatal diagnosis of DMD due to placental mosaicism of the X-chromosomes: prenatal and postnatal findings in a fetus with a deletion of exons 67-71 of the dystrophin gene.

Prenatal diagnosis of Duchenne and Becker muscular dystrophy (DMD) is performed as a routine procedure in many laboratories. The major potential problem is an incorrect diagnosis that could be obtained due to contamination with maternal tissue. We report a case of mosaicism of the X-chromosomes confined to the placenta as a possible source of confusing results in prenatal diagnosis of DMD. To the best of our knowledge, this is the first reported case of this problem in a prenatal DMD diagnosis.

Use of the phage display technique for detection of epitopes recognized by polyclonal rabbit gliadin antibodies.

A random phage heptapeptide library was screened with rabbit antibodies against wheat flour proteins comprising gliadins and a small amount of low molecular weight glutenins (gli/glu). Gli/glu antibodies isolated from the sera selected different consensus sequences (CS). All CS contained tri- to pentapeptide stretches homologous to gli/glu sequences (proposed epitopes). In alpha- and gamma-type gliadins, these sequences are clustered in the N-terminal region recently suspected to be toxic for humans with celiac disease. Peptides with CS were synthesized and checked for reactivity. Only immune and no control rabbit sera reacted with synthetic peptides. One of eight human sera containing gliadin antibodies was reactive as well (4/8 peptides) but control sera were negative. Thus the phage display technique is useful for epitope screening of polyclonal antibodies even in the case of a group of homologous but diverse antigens.

[Frequency, distribution and prognostic relevance of p53 mutations in soft tissue sarcomas]. / Häufigkeit, Verteilung und prognostische Relevanz von p53-Mutationen in Weichteilsarkomen.

Soft tissue sarcomas although rarely occurring (about 1% of malignant tumors), are because of their histo-morphological diversity and often similar appearance to tumor-like lesions difficult to characterize and estimate in their tumor biological behaviour. Analysis of molecular characteristics as alterations in tumor-suppressor and oncogenes may allow insight in STS genesis. We have chosen the in carcinomas well, but in STS not comprehensively investigated tumor-suppressor gene p53 for mutational analysis. In 16 out of 146 STS patients we could identify p53-mutations. In a multivariate Cox-regression analysis prognosis was correlated with the p53-mutation type. However, only patients with non-frameshift mutations possessed a poorer prognosis (RR = 2.42; p = 0.014) in comparison to patients without mutations, but frameshift-mutations didn't seem to affect prognosis negatively. Compiling our results and those of the literature an overall frequency of 16.3% of p53-mutations in STS, with various frequencies in different entities is detectable. STS specific hotspots are not recognizable. Rather mutational hotspots in codons 175, 245, 248 and 273 well known from studies in carcinomas are also apparent in STS. Summarizing, we want to state that the occurrence of p53-mutations (non-frameshift mutations) is of prognostic importance in STS. Combination of histo-pathological, clinical and molecular characteristics may allow to distinguish in future different groups of patients for an individual treatment.

Molecular and immunohistochemical p53 status in liposarcoma and malignant fibrous histiocytoma: identification of seven new mutations for soft tissue sarcomas.

BACKGROUND: p53 mutations are the most frequently observed tumor-related genetic changes. Mutational analysis concerns mostly carcinomas and is not comprehensive for soft tissue sarcomas. Among soft tissue sarcomas, malignant fibrous histiocytoma (MFH) and liposarcoma represent the most frequent tumor types. Most of the few identified mutations for soft tissue sarcomas are localized in the core domain of p53. A correlation between p53 positive immunoreactivity, missense mutations, and a poor prognosis is generally assumed. However, the character of p53 mutations and their functional importance for the clinical process is still unknown. METHODS: Sixty-two soft tissue sarcoma samples were investigated for the presence of p53 mutations and for p53 immunoreactivity. Exons 4-9 of the p53 gene were amplified from genomic DNA with the polymerase chain reaction. A prescreen for mutations was performed by nonradioactive single strand conformation polymorphism analysis; striking cases were sequenced directly. For an evaluation of the immunohistochemical status, five p53 antibodies were used. RESULTS: In 10 tumor samples 7 new p53 mutations and one polymorphism were identified. Mutations were detected for five liposarcomas (four patients) and four MFHs (three patients). Of the seven mutations, three were missense point mutations, three were deletions, and one was a complex conversion. All mutations but one were localized in the core domain of p53. Of 62 tumor samples, 56% (14 of 32 liposarcomas and 21 of 30 MFHs) were positive for p53 immunostaining. CONCLUSIONS: The mutations identified in the core domain affect codons that are structurally or functionally involved in DNA binding. A relation between p53 positive immunoreactivity and a poor prognosis, but not with an exclusively high tumor grade, is evident. p53 mutations in soft tissue sarcomas have a similar spectrum to those in carcinomas.

[Multiple abnormalities in a child with male karyotype due to familial partial Xp duplication]. / Multiple Fehlbildungen bei einem Kind mit männlichem Karyotyp infolge einer familiären partiellen Duplikation Xp.

A child with multiple abnormalities and intersexual external genitals is presented. The child has a male karyotype with a partial duplication Xp. Using additionally methods of molecular genetics the SRY-region was detected. We reviewed the only few persons described in the literature with male karyotype and partial duplications Xp. There are similarities in phenotype including sex inversion or intersexual genitals if the duplication is localised in the region Xp 21-->Xp 22. Whether these affected patients represent a new syndrome is a moot point. We also found the aberrant X-chromosome in the mother of the child. In the present pregnancy of the mother there is, therefore, a risk of recurrence.