Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11

In a large collaborative screening project, 370 men with idiopathic azoospermia or severe oligozoospermia were analysed for deletions of 76 DNA loci in Yq11. In 12 individuals, we observed de novo microdeletions involving several DNA loci, while an additional patient had an inherited deletion. They were mapped to three different subregions in Yq11. One subregion coincides to the AZF region defined recently in distal Yq11. The second and third subregion were mapped proximal to it, in proximal and middle Yq11, respectively. The different deletions observed were not overlapping but the extension of the deleted Y DNA in each subregion was similar in each patient analysed. In testis tissue sections, disruption of spermatogenesis was shown to be at the same phase when the microdeletion occurred in the same Yq11 subregion but at a different phase when the microdeletion occurred in a different Yq11 subregion. Therefore, we propose the presence of not one but three spermatogenesis loci in Yq11 and that each locus is active during a different phase of male germ cell development. As the most severe phenotype after deletion of each locus is azoospermia, we designated them as: AZFa, AZFb and AZFc. Their probable phase of function in human spermatogenesis and candidate genes involved will be discussed.      

Clinical profiling of recombinant follicle stimulating hormone (rFSH; Puregon): relationship between serum FSH and efficacy

Single-dose and multiple-rising dose studies of recombinantfollicle stimulating hormone (rFSH) in hypogonadotrophic maleand female volunteers demonstrated that the rate of FSH absorptionafter i.m. injection is higher in men than in women. In theabsence of endogenous FSH, a correlation between serum FSH andbody weight became apparent. The elimination half-life of rFSHwas not different between the sexes and was comparable withurinary FSH. However, the in-vitro bio:immuno ratio of serumFSH was significantly higher after the administration of rFSHthan after urinary FSH. When rFSH was administered daily witha fixed dose, steady state levels were reached within 3-5 days.Serum FSH concentrations increased in a dose-dependent mannerwhen the daily dose was increased weekly over 3 weeks from 75to 225 IU. In hypogonadotrophic women, rFSH induced normal folliculargrowth whereas oestrogen synthesis was impaired. In women pituitarysuppressed by a high-dose oral contraceptive, the daily administrationof 150 IU rFSH for 1 week induced more and larger antral folliclesthan the same regimen with urinary FSH, whereas the serum immunoactiveFSH concentrations measured 24 h after each dosing were similar.It is concluded that even though equal or lower serum immunoactiveFSH concentrations were obtained following the administrationof rFSH compared with urinary FSH, circulating bioactivity FSHconcentrations were higher. Therefore, the conventional ideathat serum immunoreactive FSH correlates positively with themagnitude of the ovarian response should be reconsidered.     

Gentamicin susceptibility in Escherichia coli related to the genetic background: problems with breakpoints

In total, 120 Escherichia coli isolates positive for one of the gentamicin resistance (GEN(R)) genes aac(3)-II, aac(3)-IV or ant(2')-I were tested for gentamicin susceptibility by the agar dilution method. Isolates positive for aac(3)-IV or ant(2')-I had an MIC distribution of 8-64 mg/L, whereas isolates positive for aac(3)-II had MICs of 32 to >512 mg/L, suggesting a relationship between the distribution of MICs and the specific GEN(R) mechanism. The MIC distribution, regardless of the GEN(R) mechanism, was 8 - >512 mg/L, which supports the clinical breakpoint of MIC >4 mg/L suggested by EUCAST and questions the breakpoint recommended by the CLSI (> or =16 mg/L).     

A novel SRY missense mutation affecting nuclear import in a 46,XY female patient with bilateral gonadoblastoma

Patients with disorders of sex development (DSD), especially those with gonadal dysgenesis and hypovirilization, are at risk of developing the so-called type II germ cell tumors (GCTs). Both carcinoma in situ and gonadoblastoma (GB) can be the precursor lesion, resulting in a seminomatous or non-seminomatous invasive cancer. SRY mutations residing in the HMG domain are found in 10–15% of 46,XY gonadal dysgenesis cases. This domain contains two nuclear localization signals (NLSs). In this study, we report a unique case of a phenotypical normal woman, diagnosed as a patient with 46,XY gonadal dysgenesis, with an NLS missense mutation, on the basis of the histological diagnosis of a unilateral GB. The normal role of SRY in gonadal development is the upregulation of SOX9 expression. The premalignant lesion of the initially removed gonad was positive for OCT3/4, TSPY and stem cell factor in germ cells, and for FOXL2 in the stromal component (ie, granulosa cells), but not for SOX9. On the basis of these findings, prophylactical gonadectomy of the other gonad was performed, also showing a GB lesion positive for both FOXL2 (ovary) and SOX9 (testis). The identified W70L mutation in the SRY gene resulted in a 50% reduction in the nuclear accumulation of the mutant protein compared with wild type. This likely explains the diminished SOX9 expression, and therefore the lack of proper Sertoli cell differentiation during development. This case shows the value of the proper diagnosis of human GCTs in identification of patients with DSD, which allows subsequent early diagnosis and prevention of the development of an invasive cancer, likely to be treated by chemotherapy at young age.     

Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance

Differences in the global methylation pattern, ie hyper‐ as well as hypo‐methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5‐ cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam‐2 before and after demethylation using 5‐azacytidine. Exposure to 5‐azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell‐specific marker VASA, showed increased expression. Following treatment with 5‐azacytidine, TCam‐2 cells were analysed using a high‐throughput methylation screen for changes in the methylation sites of 14 000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation

Yoo E‐H, Woo H, Ki C‐S, Lee HJ, Kim D‐K, Kang I‐S, Park P, Sung K, Lee CS, Chung T‐Y, Moon JR, Han H, Lee S‐T, Kim J‐W. Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation. Marfan syndrome (MFS) is an autosomal dominant disorder of the fibrous connective tissue caused by mutations in the fibrillin‐1 (FBN1) gene. Although clinical and genetic analyses have been performed in various populations, there have been few studies in Korea. The aim of this study was to investigate the clinical characteristics and genetic background of Korean patients with MFS. In 39 Korean patients with MFS who met the Ghent criteria, the most common clinical finding was aortic dilatation and/or dissection (94.9%), whereas only 35.9% of patients had ectopia lentis. The majority of MFS patients had fewer than four of the skeletal findings required to fulfill the major skeletal Ghent criterion for MFS. Only 21% of Korean patients had major skeletal abnormalities and most cases showed only minor skeletal involvement. FBN1 gene mutations were detected in 35 out of 39 patients (89.7%), which is similar to rates presented in the previous reports. These results suggest that some clinical features in Korean patients with MFS differed from those reported in Western MFS patients.     

Analysis of the relationship between age and treatment response in migraine

In migraine, headache severity varies with age. As a consequence, the effectiveness of medication may also depend on a patient's age. The purpose of this study was to assess the combined effect of age and drug treatment on headache characteristics. Using data from clinical trials of sumatriptan in adolescents and adults, we show how the interaction between age and drug exposure can be parameterised as a covariate on a Markov model that describes the decline of headache severity over three clinically defined stages (no relief, relief and pain-free status). The model explains important clinical observations: (i) the rates at which the pain relief and pain-free status were attained were found to be inversely related to age; (ii) in placebo-treated patients, the mean transit time from 'no relief' to 'relief' is 3 h for young adolescents and increases to 6 h for patients aged ≥ 30 years; and (iii) sumatriptan reduces the transit time to 2 h, irrespective of age. These findings indicate that the therapeutic gain over placebo increases with age. Prospective studies of antimigraine drugs should take this relationship into account when extrapolating efficacy data from adults to adolescents.